Canadian Society of Plant Biologists / Société canadienne de biologie végétale Edmonton, June 25-27, 2012 Program & Abstracts

Sponsors …………………………......……………… 2 General Information ………………………………… 3 Venue Maps …………………………......………….. 4 Brief Schedule…………………………...…………... 6 Detailed Schedule …………………………………... 7 Abstracts Plenary Sessions …………………………..………. 14

Education Session ………………………………… 21 Parallel Oral Sessions …………………………….. 23 Posters……………………………………………… 60

Index ………………………………………………… 80 CSPB 2012 Logo designed by Peter Mankowski. Cover photo by Joshua McDill.

 

  CSPB 2012 p. 2  

CSPB 2012 – Sponsors

We thank the all of the sponsors of CSPB 2012 for their support.

Gold Level Genome Alberta Genome Prairie/TUFGEN Total Utilization of Flax Genomics University of Alberta Faculty of Science Silver Level Agilent Technologies BioChambers Botany (An NRC Research Press Journal) CID Bio-Science Hoskin Scientific Integrated DNA Technologies University of Alberta Faculty of Agricultural, Life & Environmental Sciences University of Alberta Department of Renewable Resources University of Alberta Department of Agricultural, Food & Nutritional Science Bronze Level Biochemical Journal Fisher Scientific Regent Instruments

CSPB 2012 – General Information Food: Coffee and other beverages are available during scheduled morning and afternoon breaks. Fast food is available in SUB (Students’ Union Building), HUB, and CAB (see the campus map for directions; ). There are also other coffee vendors in CCIS (Second Cup and Starbucks) and in CAB (Tim Horton’s). Bars can be found on-campus (Dewey’s; open until 8PM), and just off-campus (Hudsons, Earl’s 111 Street & 87 Avenue; Earl’s ), and many bars and full-service restaurants are on Whyte (82nd) Avenue, or on 109th Street (near 87th Avenue). Oral presentations: Please bring a USB memory stick to the session room 15 minutes before the session start time to load your presentation on the computer. Poster presentations: Please remove posters during the lunch break (11:20AM-12:50PM) on Wednesday, June 27. Any remaining posters will be discarded at 1:00PM. Poster reception: Snacks and drinks will be provided (cash bar also available). Presenters with even-numbered posters (e.g. P24) please attend your posters from 5:00PM to 6:15PM. Presenters with odd-numbered posters (e.g. P25) please attend your posters from 6:15PM to 7:30PM. Wireless internet: Connect to the Guest@UofA network. A password is not required.

 

  CSPB 2012 p. 4  

CSPB 2012 – Campus Map

 

  CSPB 2012 p. 5  

CSPB 2012 – CCIS Map

 

  CSPB 2012 p. 6  

CSPB 2012 – Brief Schedule  

Monday, June 25 Tuesday, June 26 Wednesday, June 27 8:15 AM

Parallel Sessions D1, E1

Education Symposium

9:30 AM

BREAK BREAK 10:00 AM

Plenary III Carbon Flux

11:20 AM

Plenary I Genetic Adaptation

Plenary II Water Relations

12:00 PM LUNCH

12:50 PM LUNCH LUNCH

1:30 PM Plenary IV

Keeping Time

2:10 PM BREAK 2:30 PM

Parallel Sessions A1, B1, C1

Parallel Sessions F1, G1, H1

3:00 PM BREAK BREAK 3:30 PM

Plenary V Awards

4:15 PM Parallel Sessions

A2, B2, C2 Parallel Sessions

F2, G2, H2

5:00 PM

Business Meeting

7:00 PM

Poster Reception

7:30 PM Banquet

   

 

 

Monday, June 25th

8:30 Registration Desk opens 9:30 Botany Journal Update  9:45 C

CIS

1-

440

Conference Opening 10:00 Genetic Adaptation Chair: C. Douglas

I.1 Loren Rieseberg University of British Columbia and University of Indiana Genomics of plant speciation

10:40 I.2 Kirsten Bomblies Harvard University Genome-wide patterns of selection in autotetraploid A. arenosa suggest adaptation to whole genome duplication

11:20

Plen

ary

I C

CIS

1-4

40

I.3 Kenneth Olsen Washington University, St. Louis Tracing the molecular origins of adaptive cyanogenesis variation in white clover

12:00 LUNCH Outgoing Executive Committee Meeting, CCIS 6-003 1:30 A1.1 M. Dastmalchi Soybean Chalcone

Isomerase gene family and their role in isoflavonoid biosynthesis

B1.1 S. Zhan Protein coding cis natural antisense transcripts have high and broad expression across diverse stress conditions in A. thaliana

C1.1 T. Baylis SHI/STY transcription factors have highly dynamic expression patterns during leaf development and act non-cell-autonomously on vein patterning

1:45 A1.2 J. Stout Genomic analysis of C. sativa reveals the recruitment of an acyl-activating enzyme for cannabinoid biosynthesis through a change in subcellular localization

B1.2 G. Owttrim RNA helicase alteration of small RNA metabolism

C1.2 C. Stewart Ribosomal Protein Mutants and Their Effect on Plant Development

2:00 A1.3 H. Yang Nutrient, fibre and bioactive component profiles in lentil, pea and faba bean seeds

B1.3 A. Rosana Driving RNA metabolism: Autoregulation of RNA helicase expression during cold acclimation

C1.3 S. Rood Building small trees: transgenic modification of gibberellin metabolism or action produces semi-dwarf poplars

2:15 A1.4 G. Chen Four B. napus TT16 homologs have different genome origins and broadly regulate gene transcription related to seed coat morphology

B1.4 O. Tarassova Proteolytic regulation of cyanobacterial RNA helicase accumulation in response to abiotic stress

C1.4 W. El-Kayal Molecular events of apical bud formation in white spruce

2:30 A1.5 T. Quilichini The Arabidopsis ABCG26 transporter: a tool for investigating the nature of sporopollenin

B1.5 L. Capo-Chichi Quantifying responses of seed germination and seedling performance to low temperature: Variation among genotypes of Brassica

C1.5 A. El-Mezawy Developing canola lines with reduced pod shattering

2:45 Nat

ural

Pro

duct

s B

ioSc

i M-1

45 C

hair

: J. O

zga

A1.6 P. Uzuegbu Identification of ABC transporters involved in secondary metabolite trafficking E

nvir

onm

enta

l Res

pons

es C

CIS

1-1

40 G

. Ow

ttrim

B1.6 K. Griegoschewski Acadian Ascophyllum nodosum extract (AANE) helps plants overcome salinity stress

Dev

elop

men

t CC

IS 1

-160

Cha

ir: S

tew

art R

ood

C1.6 M. Pliner Visual marker technology

3:00 BREAK

 

 

     

Monday, June 25th cont’d 3:30 A2.1 V. Qu Reverse genetic studies of

natural rubber biosynthesis in lettuce (Lactuca sativa)

B2.1 M. Akhter Role of phytochelatins and their precursors in differential Cd accumulation in barley and lettuce

C2.1 K. St. Onge Natural variation in response to flooding and shading: A GWAS study in Arabidopsis

3:45 A2.2 C. Copeland The effect of water deficit and fungal pathogen on the expression of terpene synthase genes in jack pine and lodgepole pine

B2.2 M. Sonier Two distinct pathways for iron acquisition by iron-limited cyanobacterial cells: experiments using siderophores and synthetic chelators

C2.2 A. McKown Gene discovery through SNP analysis in adaptive traits of black cottonwood poplar (Populus trichocarpa)

4:00 A2.3 T. Nguyen Adaptive evolution of sesquiterpene lactone biosynthesis in the sunflower (Asteraceae) family

B2.3 N. Harris TtHMA3-B1, a P1B-ATPase, is a tonoplast-localized cadmium transporter that associates with grain cadmium concentration in durum wheat

C2.3 L. Galindo Identification, characterization and influence of transposable elements in the flax (Linum usitatissimum L.) genome

4:15 A2.4 S. Mahmoud robing isoprenoid metabolism in Lavandula

B2.4 C. Chang Effects of elevated temperature and elevated CO2 on autumn acclimation in Pinus strobus

C2.4 G. Perry Sequencing the Bean Genome: Using Comparative Genomics to Examine Interspecific Introgressions

4:30 A2.5 Z. Demissie Cloning, functional characterization and genomic organization of a monoterpene synthase gene from three Lavandula (lavenders) species

B2.5 S. Schreiber Sixteen years of winter stress: an assessment of cold hardiness, growth performance and survival of hybrid poplar clones at a boreal planting si

C2.5 C. Daigle Evolution and expansion of the MAPK family in solanaceous species by using Next Generation Sequencing technologies

4:45

Nat

ural

Pro

duct

s B

ioSc

i M-1

45 C

hair

: Dae

-Kyu

n Ro

A2.6 L. Sarker Cloning and functional characterization of two monoterpene synthases from Lavandula E

nvir

onm

enta

l Res

pons

es C

CIS

1-1

40 N

. Har

ris

B2.6 I. Major Suppression of photosynthesis in response to biotic stress: fine-scale temporal mapping of jasmonate-induced transcriptional programs G

enom

ics &

Ada

ptat

ion

CC

IS 1

-160

Cha

ir:

K. O

lsen

C2.6 C. Cullingham Adaptive genomics of lodgepole and jack pine populations

5:00 –

7:30

Poster Reception CCIS Atrium Presenters of even-numbered abstracts, please stand by your poster from 5:00 – 6:15 PM Presenters of odd-numbered abstracts, please stand by your poster from 6:15 – 7:30 PM

 

 

   

Tuesday, June 26th

8:15 D1.1 M. Greer Improved production of recombinant Brassica napus type 1 diacylglycerol acyltransferase in a yeast strain deficient in triacylglycerol synthesis

E1.1 M. Fujita The Arabidopsis anisotropy1 (any1) encodes CesA1 subunit of cellulose-synthase-complexes that affects growth anisotropy and cell wall properties.

8:30 D1.2 R. Silito Using directed evolution to increase oil accumulation in oilseed crops

E1.2 A. Hannoufa Analysis of DIMINUTO 1 reveals a role for brassinosteroids in secondary cell wall formation in Arabidopsis

8:45 D1.3 E. Mietkiewska Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in a high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid

E1.3 M. Arif Genotype Effects on Physical Properties of Corn Stem Residues in Composite Materials

9:00 D1.4 X. Pan A systematic characterization of TAG-synthesizing enzymes from flaxseed

E1.4 N. Hobson Roles of beta-galactosidase and arabinogalactan proteins in flax bast fiber cell wall development

9:15

Lip

id B

ioyn

thes

is C

CIS

1-1

40 H

ugo

Zhen

g

D1.5 A. Wickramarathna Identification and functional characterization of genes encoding phosphatidylcholine diacylglycerol cholinephosphotransferase from flax (Linum usitatissimum).

Cel

l Wal

ls C

CIS

1-1

60 L

acey

Sam

uels

E1.5 A. Geitmann Statolith-less plant cells respond to gravity-induced stress by modulating cell wall assembly

9:30 BREAK 10:00 Water Relations Chairs: Uwe Hacke, Janusz Zwiazek

Sponsored by University of Alberta Faculty of Agriculture, Life & Environmental Sciences and Department of Renewable Resources II. 1 Christophe Maurel Université Montpellier Aquaporins: molecular bases of hydraulic regulations in response to hormonal and environmental stimuli

10:40 II. 2 Matthew Reynolds CIMMYT (Centro Internacional de Mejoramiento de Maíz y Trigo) Impacts of physiological breeding in water stressed environments

11:20

Plen

ary

II

CC

IS 1

-440

II. 3 Barbara Lachenbruch Oregon State University Linking xylem structure to plant performance during drought and wind

12:00 LUNCH

 

 

 

Tuesday, June 26th cont’d 1:30 F1.1 J. Laur From shoot to root: How do

changes in the leaf environment affect whole-tree hydraulic properties?

G1.1 M. Shane PEPC of proteoid ‘cluster’ roots of an Australian native plant (H. prostrata) is post-translationally controlled by phosphorylation and monoubquitination

H1.1 E. Indriolo The role of ARC1 in the self-incompatibility pathway in A. lyrata

1:45 F1.2 A. Jacobsen Xylem vessel length of woody plants: A global analysis

G1.2 P. Beatty The effect of AlaAT over-expression on the transcriptome of O. sativa grown hydroponically with various concentrations of ammonium

H1.2 D. Safavian An Investigation of the cellular events during compatible and self-incompatible pollinations in Brassicaceae

2:00 F1.3 R. Pratt Xylem vulnerability to cavitation can be accurately characterized in long vesselled species with a centrifuge

G1.3 R. Begam Arabidopsis L-type Amino acid Transporter 4 (LAT4) is a green tissue specific transporter that exports amino acids under low carbon

H1.3 S. Shahidi The microtubule plus-END-BINDING1 (EB1) protein modulates endocytosis in plant root cells

2:15 F1.4 M. Arshad Ranking of drought tolerance in Canadian poplar hybrids and identification of candidate genes for drought tolerance breeding

G1.4 C. McAllister Alanine aminotransferase: a kinetic analysis

H1.4 J. Park Bacterial- and plant-type PEPC isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria

2:30 F1.5 K. Ayton Growth and Physiology of Hybrid Poplars in Northern Alberta

G1.5 D. Plett Understanding the response of the maize nitrate transport system to nitrogen supply and demand

H1.5 J. Chen From root hairs to spinal neurons: tubular ER network and directional cell growth

2:45

Wat

er R

elat

ions

Bio

Sci M

-145

Cha

ir: U

we

Hac

ke

F1.6 L. Plavcová Hydraulic acclimation to drought, shade and nitrogen fertilization in hybrid poplar saplings C

&N

Met

abol

ism

CC

IS 1

-140

Cha

ir: A

llen

Goo

d

G1.6 Z. Ma Metabolism of reactive oxygen and nitrogen species during germination of barley seeds with different levels of dormancy C

ell B

iol.

& B

ioch

emis

try

CC

IS 1

-160

Cha

ir: D

. Mue

nch

H1.6 R. Uhrig Biochemical characterization of two ancient bacterial-like serine /threonine PPP-family protein phosphatases from Arabidopsis thaliana

3:00 BREAK

 

 

   

Tuesday, June 26th cont’d 3:30 F2.1 M. Equiza Leaf senescence, dieback

and abscission during severe drought in Populus: the interplay with stem cavitation, stomatal conductance and nutrient remobilization efficiency

G2.1 E. Fedosejevs In vivo phosphorylation of SUS1, a cytosolic sucrose synthase, at Serine-11 in developing castor oilseeds

H2.1 D. Pinzon Role of pectinesterases (PME) and pectinesterase inhibitors (PMEI) on fiber development in flax (Linum usitatissimum)

3:45 F2.2 S. Lee Methyl jasmonate decreases cell water permeability in Arabidopsis

G2.2 S. Mukherjee Analysis of Sucrose to Starch Metabolism Pathway in Developing Grains of Common Wheat (Triticum aestivum L.)

H2.2 K. Bender Physiological analysis of an Arabidopsis Calmodulin-Like protein involved in seedling establishment

4:00 F2.3 U. Hacke Aquaporins contribute to recovery from drought-induced xylem embolism in rice leaves

G2.3 S. Khan DUF642, a putative galactan-binding protein from Arabidopsis thaliana involved in cell elongation

H2.3 M. Cvetkovska Mitochondrial alternative oxidase modulates leaf levels of superoxide and nitric oxide

4:15 F2.4 M. Grzesiak Influence of soil compaction on the growth, daily changes of leaf water potential and gas exchange in maize and triticale seedlings exposed to flooding stress

G2.4 F. Liu Binding of multi-enzyme complexes to starch granules is determined by the glucan-binding capacity of Starch Synthase iia

H2.4 K. Neimanis An investigation of the alternative oxidase in the moss Physcomitrella patens

4:30 F2.5 A. Arango-Velez Effect of water limitation on lodgepole and jack pine defenses against the mountain pine beetle symbiotic fungus G. clavigera

G2.5 K. Ellens Completing the Methionine Salvage Pathway: A Comparative Genomics Approach

H2.5 H. Zhuang Functional investigations of two intrinsically disordered proteins (IDPs) in Arabidopsis thaliana

4:45 Wat

er R

elat

ions

Bio

Sci M

-145

Cha

ir: J

anus

z Zw

iaze

k

F2.6 M. Meents Microarray analysis of lodgepole and jack pine responses to water deficit and inoculation with the mountain pine beetle fungal associate G. clavigera

C&

N M

etab

olis

m C

CIS

1-1

40 C

hair

: And

rew

Han

son

G2.6 Lai To Flax stem transcriptomics: GASA gene expression during fiber development

Cel

l Bio

logy

& B

ioch

emis

try

CC

IS 1

-160

Cha

ir:

Jim

Mat

tsso

n

H2.6 W. Lyzenga A CBL-interacting protein kinase (CIPK) interacts with Keep On Going (KEG), a RING-type E3 ligase, as part of the ABA signal transduction network.

5:00 Annual Business Meeting CCIS 1-140 7:00

Conference Banquet & Awards Presentations University of Alberta Faculty Club

 

 

 

Wednesday, June 27th

8:15 Education Symposium Organizer: Anja Geitmann 1. Emily Indriolo University of Toronto An Introduction to Planting Science – An opportunity for education and outreach for CSPP

8:40 2. Shona Ellis University of British Columbia Integrating Sustainability into a Biology Program: a work in progress 9:05 3. Santokh Singh University of British Columbia Evaluating the impact of new instructional strategies and assessment methods

on student learning in an undergraduate plant physiology laboratory 9:15

Edu

catio

n C

CIS

1-1

40

4. Lacey Samuels University of British Columbia First Year Seminar in Science: an introduction to the writing and culture of science

9:30 BREAK 10:00 Carbon Flux Chair: William Plaxton

III.1 Robert Turgeon Cornell University What drives phloem transport? Mechanistic and ecological perspectives  

10:40

Plen

ary

III

CC

IS 1

-440

III.2 Andrew Hanson University of Florida Metabolite damage and repair: Under-recognized carbon and energy fluxes

11:20 LUNCH **Posters must be removed before 1:00PM** Incoming Executive Committee Meeting, CCIS 6-003

12:50 Keeping Time Chair: Janice Cooke IV.1 Ove Nilsson Umeå  Plant  Science  Centre Photoperiodic regulation of flowering and life cycle adaptation in sugar beet  

1:30

Plen

ary

IV

CC

IS 1

-440

IV.2 C. Robertson McClung Dartmouth College As easy as ABC: Applying the circadian clock model developed in Arabidopsis to Brassica crops

2:10 BREAK 2:30 C.D. Nelson Address

Charles Despres Brock University The NPR1 disease-resistance signaling pathway - a decade or so later

3:15 Gold Medal Address Fathey Sarhan Université du Quebec à Montreal Freezing Tolerance: A Four-Decade Obsession

4:00

Aw

ards

C

CIS

1-4

40

Students Awards and Conference Closing  

 

 

 

CSPB2012 p. 14

Plenary Session I: Genetic Adaptation I.1 Genomics of plant speciation Loren H. RIESEBERG1,2, Sébastien RENAUT1 1. Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver, BC, Canada 2. Department of Biology, Indiana University, Bloomington, IN, USA Levels of genetic differentiation among populations can be highly variable across the genome. During speciation in geographically isolated populations (allopatry), divergence should accumulate across the genome due to the action of both drift and selection. In contrast, during speciation with gene flow (sympatry or parapatry), accentuated divergence should restricted to loci (and linked sites) under divergent natural selection. Here, we report on high resolution genomic scans of differentiation among several pairs of sunflower (Helianthus) species that vary in their divergence time and degree of geographic separation. We find that in all comparisons, genomic islands of differentiation are numerous and small (< 1 centiMorgan). However, in sympatric species pairs, divergent loci tend to be more clustered in the genome as compared to allopatric pairs. In addition, the proportion of divergent sites fixed by selection (alpha) was higher in sympatric (43%) than allopatric species pairs (24%), thus confirming the more prominent effect of selection in shaping genomic divergence in sympatry. Lastly, we also find that among independent species pairs, patterns of genomic divergence are surprisingly repeatable, especially in highly differentiated regions. In conclusion, we find that the extent of genomic divergence and the spatial clustering of fixed loci is greatly influenced by the timing of speciation and the degree of geographic separation. I.2 Genome-wide patterns of selection in autotetraploid A. arenosa suggest adaptation to whole genome duplication Kirsten BOMBLIES1, Jesse D. HOLLISTER1,2, Brian ARNOLD1, Katherine XUE1

1. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 2. Present address: Department of Ecology and Evolutionary Biology, University of Toronto

Genome duplication, resulting in polypoidy, can be very disruptive to basic biological processes. Genome duplications occur spontaneously in a range of taxa and problems such as sterility, aneuploidy and gene expression aberrations are common in newly formed polyploids. Nevertheless, stable polyploids occur in both plants and animals. Understanding how natural polyploids overcome early difficulties can provide important insights into mechanisms by which core cellular functions can adapt to sudden and drastic perturbations of the genomic environment. Arabidopsis arenosa is a stable tetraploid related to well-characterized diploids A. lyrata and A. thaliana and thus provides a rare opportunity to leverage genomic tools to investigate the genetic basis of polyploid stabilization. We sequenced twelve A. arenosa individuals and found signatures suggestive of selective sweeps throughout the genome. These are primarily in genes implicated in meiosis, transcriptional regulation, chromatin structure, and cell growth. For a critical meiosis gene, ASYNAPSIS1, we identified a non-synonymous mutation that is highly differentiated by cytotype, but present as a rare variant in diploid A. arenosa, indicating selection may have acted on standing variation already present in the diploid. Several sister chromatid cohesion genes we identified are homologous to genes shown in a yeast mutant screen to be necessary for survival of polyploid cells, and are also implicated in genome instability in human diseases including cancer. This points to commonalities across kingdoms, and strongly supports the hypothesis that selection has acted on genes controlling genome integrity in A. arenosa, likely as an adaptive response to genome doubling.

 

CSPB2012 p. 15

I.3 Tracing the molecular origins of adaptive cyanogenesis variation in white clover Kenneth M. OLSEN, Nicholas J. KOOYERS, and Linda L. SMALL Department of Biology, Washington University, St. Louis, MO Understanding the genetic basis of adaptation is a major goal of modern evolutionary biology. In this talk I describe work that capitalizes on an ecologically well-characterized system to study the molecular mechanisms underlying adaptive variation. White clover (Trifolium repens) is polymorphic for cyanogenesis (cyanide release with tissue damage); both cyanogenic and acyanogenic plants occur in natural populations. This polymorphism has been the subject of extensive ecological study for over sixty years, making it one of the best documented cases of an adaptive polymorphism in plants. Ecological studies have established that cyanogenesis serves as protection against herbivores, but that the production of the cyanogenic components creates fitness tradeoffs for vegetative growth and reproduction. Here I discuss recent work characterizing the molecular genetic basis and molecular evolution of the cyanogenesis polymorphism, the relationship between this molecular variation and natural selection in wild populations, and the processes shaping the long-term molecular evolution of cyanogenesis polymorphisms in white clover and related Trifolium species.

 

CSPB2012 p. 16

Plenary Session II: Water Relations II.1 Aquaporins: molecular bases of hydraulic regulations in response to hormonal and environmental stimuli Christophe MAUREL Université Montpellier Uptake of soil water by roots and its delivery from xylem vessels to inner leaf tissues are crucial for maintaining the plant water status. The present talk will discuss how the integrated function and regulation of plant aquaporins and their role in water transport throughout the whole plant body can be addressed by a combination of genetic, molecular, cellular and physiological approaches. Knock-out mutants of Arabidopsis thaliana for Plasma membrane Intrinsic Proteins (PIPs) were used to dissect the osmotic and/or hydrostatic modes of water uptake in roots and leaves. Water permeability measurements in protoplasts isolated from various leaf tissues and specific expression of aquaporins in these tissues was also used to establish the limiting role of veins in leaf hydraulics. In addition, the variation of root hydraulic architecture and of aquaporin expression in natural accessions of Arabidopsis provided complementary insights into the role of specific cell layers and aquaporin isoforms in root water transport. Aquaporins are also crucial for adjusting cell and tissue hydraulics in response to various environmental stimuli. Previous studies have highlighted the role of Reactive Oxygen Species (ROS)-dependent signalling in the response of roots and leaves to water stress, anoxia or changes in irradiance. We recently dissected the mechanisms and routes of ROS-dependent trafficking of PIPs in root cortical cells and evidence for an enhanced cycling of PIPs between the plasma membrane and endosomes in response to salt stress and ROS will be presented. Our recent studies also establish light-dependent quantitative changes in aquaporin phosphorylation in leaves and, for the first time, genetic evidence for a role of this modification in plant tissue hydraulics. Besides environmental stimuli, auxin was found to regulate aquaporin expression and hydraulic conductivity in roots. An integrated model will be presented, showing how this hormonal regulation can be connected to a novel role for a PIP isoform in lateral root emergence.

 

CSPB2012 p. 17

II.2 Impacts of physiological breeding in water stressed environments Matthew P REYNOLDS1,2, Marta LOPES 1, Carolina SAINT PIERRE1 1. Global Wheat Program, International Maize and Wheat Improvement Centre, Mexico 2. Australian Centre for Plant Functional Genomics A new generation of drought-adapted wheat lines has been distributed worldwide by the International Maize and Wheat Improvement Centre based on physiological trait (PT) based breeding. Lines were derived from crosses designed to combine complementary PTs to generate cumulate gene action for agronomic performance under water deficit. For example, to increase water uptake, lines with deeper roots were selected using as a selection criterion cooler canopy temperatures expressed under drought. Water use efficiency was measured indirectly by estimating -under well watered conditions- poor discrimination against the heavy 13C isotope of CO2 which indicates conservative expression of stomatal conductance. A trait associated with the ability to partition assimilates to grain yield under stress is the capacity for accumulation in the stem and subsequent remobilization to the grain of water soluble carbohydrates. One of the challenges of applying plant physiology in crop improvement is scale, therefore, in addition to thermal screening, other remote sensing approaches were applied in parental and progeny selection such as spectral radiometry to screen for hydration status of leaves and their concentration of photo-protective pigments. The same kinds of tools are used to screen among genetic resources -such as landraces- for high levels of expression of desirable stress adaptive traits, as well as in phenotyping mapping populations. The latter has led to the discovery of QTL associated with cooler canopies as well as the identification of a common genetic basis for adaptation to drought and heat stress, most probably associated with improved root capacity in both environments. Gene constructs designed to improve the coordinated response of wheat to abiotic stress have also been tested in field trials in Mexico. II.3 Linking xylem structure to plant performance during drought and wind Barbara LACHENBRUCH Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR Studies are increasingly elucidating the significant adaptive role played by the wood anatomical features that work in conjunction with each other and with the tree’s whole form—it’s stature, leaf and sapwood area relative to fine root area—to maintain the tree alive in its environment. A volume of xylem provides a plethora of physiological services, including those considered hydraulic (e.g., water transport during drought, water storage), those considered biomechanical (e.g., providing strength, stiffness, and toughness), and those related to other functions such as defence or protection. The services required, however, depend greatly on the tree’s environment and shape, and will also change over the plant’s lifetime. By studying xylem structural variation in a variety of settings (among species, life stages, environments, and plant parts), people are deducing balance of services provided by a tree’s xylem at a given time and space. They can then test these hypotheses by evaluating the physiological or biomechanical responses to manipulations of wood structure (e.g. ,low-lignin vs. normal genotypes), plant structure (e.g., pruned plants vs. controls), or environment (e.g., wet vs. dry sites). These two approaches provide robust means to understand plant survival in the face of stresses in terms of the balance of the different services provided simultaneously by xylem. This talk will discuss examples of the strong linkages between xylem structure, woody plant form, and performance during hydraulic and mechanical stress using examples from different growth forms, genetically modified hybrid poplar, and young vs. old conifers.

 

CSPB2012 p. 18

Plenary Session III: Carbon Flux III.1 What drives phloem transport? Mechanistic and ecological perspectives. Robert TURGEON Department of Plant Biology, Cornell University, Ithaca, NY It is generally assumed that active phloem loading evolved to drive long-distance nutrient transport from leaves to sinks, such as reproductive tissues. However, studies on the mechanisms of phloem loading in a number of herbaceous and woody plants cast this paradigm into doubt. To date, three loading mechanisms have been described. Two, apoplastic loading and polymer trapping, are metabolically active in the sense that energy is used to propel carbohydrates into the phloem against a concentration gradient. Apoplastic loading is driven by the proton motive force and employs plasma membrane transporters. Polymer trapping, common to many plant families, uses metabolic energy for the synthesis of raffinose and stachyose in the companion cells of minor vein phloem. The third phloem loading mechanism is thermodynamically passive; sucrose, or in some cases sugar alcohol, diffuses into the phloem from the mesophyll. Passive loading is restricted almost entirely to trees. The existence of passive loading in species that have the longest transport routes conflicts with the concept that an active loading step is needed to drive phloem transport. We suggest that the primary advantage to active loading is that it allows plants to maintain low foliar carbohydrate levels and still transport nutrients in the phloem. The advantages to maintaining low sugar concentrations in leaves are probably several, but one of these is apparently to reduce inventory costs, improving return on investment in carbohydrate synthesis and thus stimulating rapid growth. These concepts will be discussed in the context of herbaceous and woody growth patterns.  III.2 Metabolite damage and repair: Under-recognized carbon and energy fluxes Andrew D. HANSON Horticultural Sciences Department, University of Florida, Gainesville, FL Of the carbon fixed by photosynthesis, a large fraction – perhaps one-quarter – is lost in ‘maintenance’ respiration, i.e. respiration that supplies the energy and reducing power needed to maintain the status quo. Despite their significance, maintenance processes and their carbon and energy costs are ill defined. Historically, maintenance has been understood to include protein turnover, ion gradient upkeep, and – least characterized of all – repair or replacement of damaged metabolites. Advances in chemical biology and genomics are now shedding light on metabolite damage and repair. It has become clear that metabolites suffer different kinds of damage, that cells have dedicated metabolite repair systems, and that damage and repair always cost energy. Thus, chemical biology is demonstrating that diverse metabolites are damaged by side-activities of ‘promiscuous’ enzymes or by spontaneous reactions, that the products are useless or toxic, and that their unchecked buildup can be devastating. Genomics and genetics are uncovering a network of novel, conserved enzymes that repair damaged metabolites. Metabolite repair is analogous to DNA and protein repair, appears to be equally widespread, and could be the function of many conserved protein families lacking known activities. Classical biochemistry overlooked metabolite damage and repair, possibly because of a paradigm that saw metabolism as a flawless set of linear pathways with exquisitely specific enzymes. A better paradigm is of a messy, error-prone network whose smooth operation requires a battery of unobtrusive repair enzymes. Identifying these under-recognized enzymes and their operating costs in terms of carbon and energy are frontiers in biochemistry and physiology.    

 

CSPB2012 p. 19

Plenary Session IV: Keeping Time

IV.1 Photoperiodic regulation of flowering and life cycle adaptation in sugar beet

Pierre A. PIN1,2, Reyes BENLLOCH1, Dominique BONNET3, Elisabeth WREMERTH-WEICH2, Thomas KRAFT2, Jan J.L. GIELEN2, and Ove NILSSON1

1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901-83 Umeå, Sweden 2. Syngenta Seeds AB, box 302, 261-23 Landskrona, Sweden 3. Syngenta Seeds SAS, 12 chemin de l’hôbit, 31790 Saint-Sauveur, France Cultivated beets (Beta vulgaris ssp. vulgaris), such as many other biennial flowering plants, are not able to form reproductive shoots during the first year of their life cycle. Transition only occurs if plants pass through the winter and are subsequently exposed to the increasing photoperiod gradually taking place in spring. The most important determinant of the difference between annual and biennial beet varieties is the B locus, where the dominant B allele is associated with the annual growth behaviour. We will describe the cloning and characterization of the B gene from beet. We will also show how the B gene controls flowering time in sugar beet through regulation of an antagonistic pair of Arabidopsis FLOWERING LOCUS T (FT) homologs. One of the genes (Bv FT2) is functionally conserved with FT and is necessary for the flowering of annual sugar beet plants. In contrast, the other FT-like gene (Bv FT1), is preferentially expressed in juvenile biennial plants and is down-regulated in response to vernalization. The Bv FT2 gene is positively regulated by increasing photoperiod, while Bv FT1 is negatively regulated. Bv FT1-overexpressing annual and biennial plants are non-flowering and do not respond to vernalization suggesting that Bv FT1 is indeed acting as a repressor of bolting and flowering. We conclude that the regulation of flowering time in beets is controlled by the interplay between two FT-like gene paralogs that have evolved antagonistic functions and that the down-regulation of the Bv FT1 repressor is crucial for the vernalization response in beet.   IV.2 As easy as ABC: Applying the circadian clock model developed in Arabidopsis to Brassica crops C. Robertson McCLUNG1. Brent E. EWERS2, Cynthia WEINIG2 1. Department of Biological Sciences, Dartmouth College, Hanover, NH 2. Department of Botany, University of Wyoming, Laramie, WY Arabidopsis thaliana has served as a model species for the study of the plant circadian clock. Experimental disruption of the clock via large-effect mutations reduces fitness. Therefore, it is of interest to explore the extent to which the circadian clock model developed in Arabidopsis can be generalized, and to ask whether manipulation of the circadian clock can enhance agricultural yield. Accordingly, we have extended our studies to Brassica rapa. Using a recombinant inbred population derived from cultivated oil seed and rapid cycling varieties we have mapped Quantitative Trait Loci for circadian period, water use efficiency (WUE), shade avoidance, and other morphometric traits. There is striking colocalization of QTL for circadian period with QTL for shade avoidance and WUE. Current efforts emphasize the exploration of the possible mechanistic relationship underlying this colocalization. With regard to crop accessions, history of domestication appears to affect the evolution of net carbon assimilation and stomatal conductance among replicates grown in either controlled or field settings, Specifically, oil seed varieties have higher values of both traits than do cabbage and turnip vegetable crops and take longer to recover from drought. B. rapa has undergone triplication through two whole genome duplication events since its separation from Arabidopsis. This triplication has been followed by considerable fractionation (gene loss). Dosage sensitive genes, such as those that are highly networked or those whose products contribute to protein complexes, are preferentially retained during fractionation and we show that circadian clock genes, which are dosage sensitive, are preferentially retained in B. rapa.  

 

CSPB2012 p. 20

Plenary V: Awards V.1 C.D. Nelson Award: The NPR1 disease-resistance signaling pathway - a decade or so later Charles DESPRÉS Department of Biological Sciences, Brock University, St. Catharines, Ontario Localized exposure of plants to certain microbes can induce subsequent resistance to a broad range of otherwise virulent pathogens in distant, non-infected tissues. One such type of systemic disease-resistance has been described and coined Systemic Acquired Resistance (SAR). SAR is triggered by non-virulent pathogens that cause cell death at the site of infection and is associated with the activation of pathogenesis-related (PR) genes and the accumulation of PR proteins. Genetic and biochemical studies have identified Salicylic Acid (SA) as a mandatory metabolite for the activation of PR genes and the deployment of SAR. In 1997, fifteen years ago, two groups independently reported the identification of a key SAR regulator called NPR1 (a.k.a NIM1). This presentation will be given as part of the 2011 CD Nelson Award. As such, I will reflect on my work for the past ten years as a faculty member at Brock University where my goal has been to decipher the NPR1 signaling pathway in an attempt to engineer crop plants with enhanced disease-resistance. V.2 Gold Medal Address: Freezing tolerance: a four-decade obsession Fathey SARHAN Département des Sciences Biologiques, Université du Québec à Montréal Winter cereals sense the upcoming winter and delay flowering by postponing the transition from the vegetative to the cold-sensitive reproductive growth phase. This delayed transition is associated with the development of the high degree of freezing tolerance required for winter survival. This important biological process fascinated me throughout the forty years of my career. In this presentation, I will review the progress made and the difficulties encountered in studying the complexity of low temperature responses in plants using a combination of approaches including genome-wide expression studies, bioinformatics predictions, structural analyses, subcellular localization, protein-protein interactions, genetic mapping and epigenetic. These studies conducted by my dedicated research team resulted in several important discoveries, including the freezing tolerance associated proteins, the CBF and vernalization genes and cold regulated miRNAs. Some of these genes are used to improve low temperature tolerance, to manipulate flowering time and in biomedical applications. I will also discuss the fascinating strategies of how plants retain the cellular memory of prolonged winter cold and how this memory is reset in the next generation to ensure plants have the capacity to respond to both cold acclimation and vernalization.

 

CSPB2012 p. 21

Education Symposium: Oral Presentations EDU1. An Introduction to Planting Science – An opportunity for education and outreach for CSPP Emily INDRIOLO Department of Cell & Systems Biology, University of Toronto, Toronto, ON Planting Science is an interactive research program for a wide range of students (grades 6-12). This program is structured so that a plant biologist is able to mentor teams of students in their own research projects. This program has been incredibly successful for students and teachers as it gives them the chance to perform real science and meets the standards of the National Research Council (USA) including; inquiry-based science with hands on experiments, working as a scientific team as well as having input from an expert – a real plant biologist. Therefore, this program allows for the students design, test and analyze their own experiments. All of this is possible by the use of an interactive website where teams and mentors as well as teachers interact. As a Planting Science mentor since 2007, I would like to introduce this program to CSPP as a potential education and outreach activity. There are several ways for CSPP and its members to become involved in this program either as active mentors or as a sponsor to the program. EDU2. Integrating Sustainability into a Biology Program: a work in progress Shona M. ELLIS

Department of Botany, University of British Columbia, Canada The University of British Columbia is committed to providing all students the opportunity to study sustainability. Implementation requires initiative from within departments and programs. In the new biology specialization, we are developing an advising tool to help students identify ways they can incorporate sustainability into their program. At one end of the spectrum we will help them identify aspects of sustainability in coursework and other activities while at the other end provide a more formal concentration with accreditation. The concentration curriculum will include foundational electives, courses and community-base experiential learning activities selected from sustainability themes, and culminate in a multidisciplinary capstone course. Coordination and collaboration with other campus units is necessary to help students build interdisciplinary competence and provide them balanced coverage of sustainability concepts, skills, and values. I will present the challenges faced with developing a sustainability pathway in the context of a new biology curriculum and the progress we have made to date.   EDU3. The impact of new instructional strategies and assessment methods on student learning Santokh SINGH

Department of Botany, University of British Columbia, Canada In an attempt to transform plant physiology laboratory experience for undergraduate students, I have introduced new research project-based instructional strategies to replace demonstration- and observation-based exercises. Students actively engage in the various aspects of their research project from project development to the execution of research experiments, data analysis, and communicating their research findings. Recently, I have introduced a rapid short presentation method based on the "Three-Minute Thesis (3MT) competition" to assess its effectiveness in student learning. The results on the assessment of these undergraduate research practices will be discussed.      

 

CSPB2012 p. 22

 EDU4. First Year Seminar in Science: an introduction to the writing and culture of science Lacey SAMUELS

Department of Botany, University of British Columbia, Canada  First year science students in research-intensive Canadian universities encounter large classes and limited contact with faculty. The UBC Vancouver First Year Seminar in Science was designed to give students an introduction to the nature of science, building a scientific argument, and scientific evidence, in a small class setting. Building a sound argument with claim, reasons and evidence is linked to writing a thesis statement, with coherent paragraphs and supporting evidence. Student assessment is primarily through writing and participation as students interview researchers, investigate scientific claims in daily life and attend a science in society lecture series.

 

CSPB2012 p. 23

Parallel Sessions: Oral Presentations A1.1 Soybean Chalcone Isomerase gene family and their role in isoflavonoid biosynthesis Mehran DASTMALCHI1,2, Sangeeta DHAUBHADEL1,2 1. Department of Biology, University of Western Ontario, London ON, Canada 2. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London ON, Canada Isoflavonoids are plant secondary metabolites produced via a legume-specific branch of the phenylpropanoid pathway. They are actors in signaling for nitrogen fixation and plant response to biotic and abiotic stress. Isoflavonoids are noted for their human health benefits. Chalcone Isomerase (CHI) catalyzes the reaction producing flavanones, the skeletal backbone for isoflavonoids. There are eight CHI genes in the soybean genome including the novel CHI3B. We identified CHI3B through in silico analysis and confirmed expression in root tissue of four different soybean cultivars. CHI3B has high levels of similarity, at the amino acid level, with CHI3A, but resides on a different chromosome. Characterization of the gene family has included tissue-specific gene expression and the subcellular localization of each member. CHI genes show different temporal and spatial expression. CHI isozymes, previously confirmed to have catalytic capabilities to produce flavanones, were localized to the nucleus and cytoplasm. Quantitative gene expression analysis of the CHI family, in soybean roots with different isoflavonoid levels, showed CHI2 expression corresponds with higher isoflavonoid content. The function of CHI2 in planta has been further studied through silencing of CHI2 using soybean hairy root transformation, and subsequent analysis of isoflavonoid levels. Our results suggest that CHI2 is the isoflavonoid-specific member of the family. Identification of factors regulating CHI will help our understanding of the genetic and molecular basis of isoflavonoid biosynthesis, allowing future manipulation of isoflavonoid content in legumes, and introduction of the pathway into non-legumes. A1.2 Genomic analysis of Cannabis sativa trichomes reveals the recruitment of an acyl-activating enzyme for cannabinoid biosynthesis through a change in subcellular localization Jake M. STOUT1,2, Steve AMBROSE1, Randy PURVES1, Jonathan E. PAGE1,2 1. Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9 2. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, S7N 5E2 Canada Cannabis sativa is a multi-use plant that has been used for millennia as a source for food, fibre, and medicine. The medicinal properties of cannabis are in part due the accumulation of over seventy prenylated polyketides collectively known as the cannabinoids. The biosynthesis of hexanoyl-CoA, a precursor molecule used to form the polyketide core of cannabinoids, is unclear. We first showed that the hexanoyl-CoA pool size is high in female cannabis flowers that actively produce cannabinoids. We identified eleven genes encoding acyl-activating enzymes (AAEs) in the transcriptome of glandular trichomes using a combination of Sanger and 454 sequencing. Of these, two (CsAAE1 and CsAAE3) encode enzymes that possess hexanoyl-CoA synthetase activity. CsAAE1 was shown to function as the cannabinoid pathway synthetase based upon its high transcript abundance in glandular trichomes, its cytosolic localization, and the catalytic properties of the recombinant enzyme. Phylogenetic analysis shows that CsAAE1 belongs to the Clade II group of AAEs, which are peroxisomally localized. In order to broaden our understanding of the structure and regulation of the cannabinoid metabolic pathway, we then produced a draft genome and a full transcriptome assembly using a combination of Illumina and 454-based sequencing. The genome data allowed us to show that CsAAE1 likely neofunctionalized through a tandem duplication event, followed by the loss of peroxisomal targeting and subsequent recruitment to cannabinoid biosynthesis. Finally, we show that these genes can be used in a synthetic biology context to generate cannabinoid pathway intermediates in heterologous systems.

 

CSPB2012 p. 24

A1.3 Nutrient, fibre and bioactive component profiles in lentil, pea and faba bean seeds Han YANG, Alena JIN, Jocelyn OZGA and Dennis REINECKE Department of Agricultural, Food and Nutritional Science. University of Alberta, Edmonton AB The protein, starch, and fibre components of faba bean (‘Fatima’ and ‘Snowbird’), lentil (‘CDC Lemay’, ‘CDC Plato’, and ‘CDC Redberry’) and pea (‘Courier’, ‘Solido’, and ‘Canstar’) have been investigated in raw and cooked seed fractions to determine their nutritional and potential bioactive component (PAs) value. The protein content of embryos the ranged from 25 to 35%, and seed coats contained 7% to 12% protein. Total seed starch content ranged from 34 to 42 % and resistant starch ranged from 7 to 13% among these grain legume seeds. The cooking treatment (boiling in water for 30 minutes) increased total seed starch content by 1 to 6%, and decrease the resistant starch content by 4 to 9%. For the embryos, the total, insoluble, and soluble fibre content ranged from 6-8%, 5-7% and 1-3% respectively; for the seed coat, the range was from 39-72%, 40-67% and 0-8% respectively. For pea seed coat fractions, the cooking treatment raised the average mean soluble fibre content by about 3%, but this was not statistically significant. Seed coat total soluble proanthocyanidin (PA) content was determined to characterize the potential bioactive components (flavonoids) derived from these grain legume seeds. Seed coat total soluble PA content ranged from 4.5 to 9% in these grain legume seeds, and the cooking treatment did not affect seed coat PA content. These data show that grain legume seeds are a good dietary source for protein, carbohydrate, fibre, as well as potential bioactive components (PA-type flavonoids). A1.4 Four Brassica napus TT16 homologs have different genome origins and broadly regulate gene transcription related to seed coat morphology Guanqun CHEN, Wei DENG, Fred PENG, Martin TRUKSA and Randall J. WESELAKE

Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB In Arabidopsis, the transcription factor, TRANSPARENT TESTA 16 (TT16), encodes a Bsister MADS-box protein regulating endothelial development and proanthocyanidin (PA) accumulation. The function of TT16 in crops, however, has not been explored. In this study, four TT16 homologs were identified from Brassica napus, an allotetraploid (AACC) formed from Brassica rapa (AA) and Brassica oleracea (CC), in which genes are present as multiple homologs due to polyploidy and genome-wide rearrangement. Phylogenetic analysis of the TT16 genomic DNA sequences revealed that three BnTT16 homologs originated from the C genome and one from the A genome. Analysis of tt16 RNA interference (RNAi) lines of B. napus revealed abnormal endothelial development and decreased PA content. Microarray analysis showed that 1723 genes were differentially expressed in the tt16 RNAi lines. Among them, 5 PA-associated genes and 28 seed coat-associated genes were identified as candidates being closely regulated by TT16. Further analysis by qRT-PCR showed that TT16 positively regulated the transcript abundance of PA biosynthetic genes, exerting strong control at an early development stage. Among seed coat-related genes, TT16 strongly up-regulated the expression of TTG2, UGT72E2, and δ-VPE and down-regulated SUS1, SEC8, and GAE5 at both early and middle seed coat developing stages. Overall, our results revealed the genome origins of BnTT16 homologs and their broad and complex regulatory function on the transcript abundance of seed coat- and PA-related genes. A1.5 The Arabidopsis ABCG26 transporter: a tool for investigating the nature of sporopollenin Teagen D. QUILICHINI, A. Lacey SAMUELS, Carl J. DOUGLAS Department of Botany, University of British Columbia, Vancouver BC Canada Sporopollenin, a tough biopolymer in spore and pollen walls, protects these structures from environmental stresses. From the data available, sporopollenin is a polymer of fatty acids and oxygenated aromatic compounds. Analyses of Arabidopsis male sterile mutants defective in pollen wall formation, have revealed genes required for sporopollenin biosynthesis and/or deposition, including MS2, ACOS5, PKS-A/PKS-B, TKPR1 and ABCG26. Based on genetic and biochemical analysis of these genes and the corresponding enzymes, it has been proposed that an aliphatic polyketide sporopollenin monomer is synthesized in the tapetum. ABCG26, an ABC transport protein, is thought to function in sporopollenin export from

 

CSPB2012 p. 25

tapetum cells. However, the substrate transported by ABCG26 is unknown. In the abcg26 mutant, sporopollenin precursors are predicted to accumulate in the tapetum. Through the analysis of abcg26 by two-photon microscopy, lipidic and autofluorescent compounds in tapetum cells can be visualized. No differences between wild type and abcg26 tapetum lipids were observed. Conversely, abcg26 exhibits autofluorescence in tapetum vacuoles, not observed in wild type. Transmission electron microscopy supports these findings, with enlarged, debris-filled vacuoles in the tapetum of abcg26 mutants. Identification of the autofluorescent components accumulating in abcg26 tapetum cells by biochemical methods will provide an opportunity to examine the composition of sporopollenin in planta. Using live-cell imaging and biochemical methods, we are also investigating the nature of sporopollenin precursors that accumulate in double mutants of abcg26 and acos5, pks-a/pks-b, and tkpr1. A1.6 Identification of Adenosine Triphosphate Binding Cassette (ABC) transporters involved in secondary metabolites trafficking Perpetua UZUEGBU, David A. BIRD Department of Biological Sciences, University of Calgary, Calgary, Alberta A vast majority of secondary metabolites (alkaloids, terpenoids etc.) produced by plants have strong biological activity and are thought to perform some ecophysiological roles. Plants containing alkaloids and its derivatives are used as folk and modern medicine respectively. Usually, they are synthesized in very small amount and at different developmental stages. Their biochemical pathways are often spread out across different cell layers. Hence, they accumulate in plant cell different from the cells harboring the biosynthetic enzymes, suggesting that a type of transport mechanism is involved in their production pathway. A number of Adenosine Triphosphate Binding Cassette (ABC) transporters have been implicated in secondary metabolites transport. In plants for instance, a multidrug-resistant protein from Coptis japonica (CjMDR1) has been identified as a potential ABC transporter involved in the transport of berberine in C.japonica cells. We used Eschscholzia californica suspension cultures as a model system to test the involvement of ABC transporters in the transport of secondary metabolites using ABC transporter inhibitor drugs. Results obtained show that elicitation with yeast extract can induce accumulation of alkaloid in the media two hours after eliciting. However, treating the elicited cells with micromolar concentrations of thioridazine hydrochloride, cyclosporine A and millimolar concentrations of vanadate inhibited the accumulation of alkaloids in the media following two hours of incubation. These results suggest the involvement of ABC transporter (s) in alkaloid trafficking into the media. To study the functions of putative ABC transporter candidates, RNA interference (RNAi) technique will be used. A2.1 Reverse genetic studies of natural rubber biosynthesis in lettuce (Lactuca sativa)

Vince QU, Romit CHAKRABARTY, Dae-Kyun RO Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada Natural rubber (poly-cis-isoprene) is one of the most important raw industrial materials. Its extremely large molecular weight (>1 million Da) and exclusive cis-configuration have made it irreplaceable by synthetic rubber and provide the unique physical properties of natural rubber (e.g., elasticity, heat and abrasion resistance). However, the biosynthesis of natural rubber has yet to be understood, and the genes involved in natural rubber biosynthesis have been also debated for decades. Here we report that the down-regulation of one unique cis-prenyltransferase (CPT) directly reduced more than 95% of the natural rubber content in lettuce through RNAi. Meanwhile, a family-wide silencing of Rubber particle Associated Proteins (RAPs), proposed to be important components in natural rubber biosynthesis, did not induce any observable change of natural rubber content in lettuce. The use of this new CPT brings the possibility of manipulating natural rubber content in plant and inspires the research of natural rubber. On the other hand, the research on RAP questioned the decades-long idea of it being the synthase itself. Not only did our work identify the key player in natural rubber biosynthesis, but we also developed the compact and easy plant system, lettuce, for the ongoing research of natural rubber.

 

CSPB2012 p. 26

A2.2 The effect of water deficit and fungal pathogen on the expression of terpene synthase genes in jack pine and lodgepole pine Charles COPELAND, Adriana ARANGO, Miranda MEENTS, Walid EL KAYAL, Janice COOKE Department of Biological Sciences, University of Alberta, Edmonton, AB Mountain pine beetle has caused severe damage to the pine forests of British Columbia, where the dominant pine species is lodgepole pine (Pinus contorta). Now in Alberta, the mountain pine beetle outbreak has spread to jack pine (P. banksiana). Beetles carry symbiotic fungi, such as Grosmannia clavigera, between trees; the fungal symbionts are thought to serve as sources of nutrition for the beetles. As the fungi grow within the tree, they may induce the tree’s defense response until it is exhausted, allowing colonization of the tree by the beetle. Pine trees constitutively produce a number of defensive compounds to defend themselves against attack, and this production is induced further by herbivores and pathogens. Terpenes are a group of secondary metabolites, many of which have defensive functions, which have become very diverse during the evolution of conifers. We are testing the hypothesis that lodgepole pine and jack pine exhibit different responses to attack by mountain pine beetle and G. clavigera. We are also testing the hypothesis that drought conditions reduce the ability of the trees to defend themselves, since it has been shown that stressed trees are more likely to be attacked during the endemic phase of an outbreak. Stomatal conductance and photosynthetic capacity are significantly reduced in water-limited lodgepole and jack pine seedlings, suggesting that reduced carbon gain could affect allocation to carbon-based defensive secondary metabolites. Quantitative reverse-transcription PCR is currently underway to examine expression profiles of terpene synthase genes in response to wounding, G. clavigera inoculation and water deficit. A2.3 Adaptive evolution of sesquiterpene lactone biosynthesis in the sunflower (Asteraceae) family Trinh-Don NGUYEN and Dae-Kyun RO Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Sunflower family (or Asteraceae) is the largest plant family, comprised of more than 24,000 species. One characteristic metabolite in Asteraceae is sesquiterpene lactone (STL) which is synthesized by coordinated reactions of terpene synthase and cytochrome P450s (P450s). Several structurally distinctive subfamilies of STLs have evolved in Asteraceae, and therefore the studies of related STL subfamilies in the Asteraceae could provide an insight into the adaptive evolution of enzymes that has led to the chemical diversification in Asteraceae. We focused on the biochemical evolution of the P450s, catalyzing the three-step oxidation of germacrene A (sesquiterpene) to germacrene A acid (sesquiterpene carboxylic acid). We experimentally proved that this activity appeared at the base lineage of the Asteraceae family dated back to 50 million years ago and has been conserved in three major Asteraceae subfamilies (~90% of Asteraceae species). However, in one recently diverged species, Artemisia annua, this activity has rapidly evolved to accommodate a new sesquiterpene substrate, amorphadiene. Through this process, the newly diverged P450 lost its original activity for germacrene A oxidation. Intriguingly, the P450s from ancient species have promiscuous activities for amorphadiene, even though this substrate was not available at that time. When this primordial P450 enzyme was tested for several different sesquiterpene substrates, many uniquely oxidized sesquiterpenes could be synthesized and some of them were not present in nature. Such promiscuous activities of primordial P450 support the hypothesis that ancient enzymes have innate promiscuous activities, with which specific catalytic activities can be rapidly evolved in new environments. A2.4 Probing isoprenoid metabolism in Lavandula

Soheil S. MAHMOUD Biology,  University  of  British  Columbia,  Okanagan  campus,  Kelowna,  BC   The isoprenoids, or terpenoids, encompass over 50,000 structurally and functionally diverse natural products. Because of their physiological importance to plants (for example, as pigments, growth regulators, and defensive agents), and their tremendous impact on human lives (for example as cosmetics, pharmaceuticals, medicines and industrial raw materials), isoprenoids have been the subject of extensive recent research. We have generated extensive genomics resources to

 

CSPB2012 p. 27

facilitate investigating the regulation of isoprenoid formation in Lavandula (lavenders). Several members of this genus are economically important crop plants that produce and store large quantities of low molecular weight terpenoids, in particular monoterpenes (C10), as essential oils (EO) in glandular trichomes. Because glandular trichomes, or oil glands, are specialized for EO production, they strongly express the related structural and regulatory genes. We generated cDNA libraries from flowers, leaves and glandular trichomes, and assessed the expression of over 22,000 ESTs in various tissues of three lavender species by microarrays. Our investigations have resulted in the cloning and functional characterization of four key EO biosynthetic genes (terpene synthases), and highlighted putative genes that control trafficking of EO constituents in plants. Further, our results indicate that monoterpene metabolism is regulated at multiple levels, including transcriptional control of the terpene synthase genes. Manipulation of the expression of terpene synthases through metabolic engineering resulted in improvement of EO yield and composition in mints, and will most likely lead to enhanced EO production in lavenders. A2.5 Cloning, functional characterization and genomic organization of a monoterpene synthase gene from three Lavandula (lavenders) species Zerihun A. DEMISSIE, Lukman S. SARKER, Mark R. RHEAULT and Soheil S. MAHMOUD Biology, The University of British Columbia - Okanagan campus, Kelowna, British Columbia, Canada. Despite the economic importance of their essential oils (EOs), so far only a few EO biosynthetic genes have been described from Lavandula (lavenders). Here, we report the cloning, functional characterization and genomic organization of a monoterpene synthase (mTPS) gene and its orthologs from L. x intermedia, L. angustifolia and L. latifolia. The ORF, excluding the transit peptide, of the L. x intermedia cDNA was over-expressed in E. coli, purified by Ni-NTA agarose affinity chromatography and functionally characterized in vitro. The ca. 63 kDa bacterially over-expressed recombinant protein converted geranyl diphosphate (GPP) primarily to one of the major lavender monoterpenes with Km and kcat values of 5.75 µM and 8.8 x 10-3 s-1, respectively. Like previously characterized Lamiaceae mTPSs, the relative abundance of the gene and accumulation of its product across L. x intermedia flower ontology and in the three lavender species examined was directly proportional. However, unlike other lavender mTPSs, the transcriptional activity of this gene was not developmentally up-regulated. The genomic architecture of the three genes was identical and belongs to the class III TPS clade. Further, the three mTPSs have identical coding regions, except for a single polymorphic nucleotide in L. angustifolia that altered the encoded amino acid without affecting the product specificity of the protein. The direct correlation between the expression of this gene and its end product accumulation across species and developmental stages indicates its potential use in metabolic engineering to improve EO quality and yield in lavenders and other economically important EO producing plants. A2.6 Cloning and functional characterization of two monoterpene synthases from Lavandula Lukman S. SARKER, Zerihun A. DEMISSIE and Soheil S. MAHMOUD Department of Biology, University of British Columbia, Okanagan campus, Kelowna, B.C, Canada In search of genes responsible for the production of essential oil (EO) constituents in Lavandula (lavenders), we obtained two putative terpene synthase (TPS) clones from cDNA libraries derived from L. x intermedia and L. angustifolia floral tissues. These clones – tentatively designated LiTPS and LaTPS, respectively - were expressed in E. coli, and the corresponding recombinant proteins were purified by Ni-NTA agarose affinity chromatography and functionally characterized in vitro. LiTPS, which had a molecular mass of 28kDa, produced a major lavender EO constituent with Km and Kcat values of 53.6 µM and 3.79 x10-4 s-1, respectively. On the other hand, LaTPS - which lacked an internal stretch of 73 amino acids including the signature DDxxD metal binding motif of plant terpene synthases, and had a reduced molecular mass of 54.3kDa - did not produce detectable amounts of mono- or sesquiterpenes from standard substrates GPP, NPP and FPP. Although transcripts corresponding to both gene(s) were strongly expressed in flowers compare to leaves, LiTPS was substantially less abundant and LaTPS was found at the same level of expression compare to linalool synthase, catalyzes the formation of one of the major lavender EO constitute-linalool, in glandular trichome. Given its role in EO synthesis, LiTPS could be an important candidate for metabolic engineering to improve the quality of the EO in lavenders and other plants.

 

CSPB2012 p. 28

B1.1 Protein coding cis natural antisense transcripts (cis-NATs) have high and broad expression across diverse stress conditions in Arabidopsis thaliana Shuhua ZHAN, Lewis LUKENS

Department of Plant Agriculture, University of Guelph, Guelph, ON

Pairs of genes within eukaryotic genomes are often located on opposite DNA strands such that transcription generates cis natural antisense transcripts (cis-NATs). Here, we report that protein coding (PC) genes are highly represented among Arabidopsis thaliana cis-NAT gene pairs. PC cis-NATs are present in more stress and control growing conditions than are non-cis-NATs. cis-NATs transcribed from the same overlapping gene pair are present in the same condition more often than are PC non-cis-NATs. Finally, PC cis-NATS have high transcript levels and rare instances of trimethylation of histone 3 lysine 27 (H3K27), a mark of transcriptionally repressive heterochromatin. Some cis-NAT gene pairs generate natural antisense small RNAs, nat- siRNAs, that target one cis-NAT of a gene pair for silencing. We find that sRNAs match PC cis-NATs less frequently than PC non-cis-NATs in both abiotic stress and control conditions. sRNAs sequenced from nat-siRNA biogenesis mutants (dcl1, dcl2, dcl3, and rdr6) also map to PC cis-NATs at a similar frequency as sRNAs sequenced from wild-type plants. Our results suggest that cis-NAT gene orientations may contribute to high and broad transcript abundances, and nat-siRNAs play a small role in cis-NAT regulation.

 B1.2 RNA helicase alteration of small RNA metabolism Danuta CHAMOT1, Jens GEORG2, Albert Remus R. ROSANA1, Wolfgang R. HESS2 and George W. OWTTRIM1

1. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 2. Institut f. Genetik, Universität Freiburg, Germany The activity of many prokaryotic sRNAs is mediated by the RNA chaperone Hfq, however, other proteins may perform similar functions. Potential candidates for regulatory RNA-target mRNA interaction are RNA helicases, ubiquitous enzymes associated with a diverse range of functions involving RNA metabolism. CrhR is a cyanobacterial RNA helicase capable of altering RNA secondary structure by catalyzing RNA unwinding, annealing and strand exchange. A diverse range of small RNAs are encoded in the model cyanobacterium, Synechocystis sp. PCC 6803. Whole transcriptome tiling arrays were utilized to identify transcripts whose abundance is altered in response to crhR mutation. A limited number of mRNAs and small RNAs accumulate differentially in the crhR mutant. We are employing molecular and biochemical approaches to determine which effects are a direct result of crhR mutation. Biochemically, CrhR can anneal predicted mRNA-regulatory RNA duplexes but cannot unwind them. The functional categories of genes whose transcript levels change in the crhR mutant directly correlate with alterations in cellular physiology and morphology. The data suggest that CrhR functions as an RNA chaperone, associated with the metabolism of a specific subset of RNAs, i.e. a CrhR regulon. Overall, the results indicate RNA helicases perform unexpected role(s) in prokaryotic RNA metabolism.

 B1.3 Driving RNA metabolism: Autoregulation of RNA helicase expression during cold acclimation Albert Remus R. ROSANA and George W. OWTTRIM Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Cyanobacteria are ubiquitous microorganisms with species capable of surviving in extremely cold environments and still carry out oxygenic photosynthesis. In the model cyanobacterium Synechocystis sp. PCC 6803, expression of the cyanobacterial RNA helicase gene, crhR, is upregulated in response to temperature downshifts. A CrhR knockout strain was used to investigate CrhR expression kinetics in response to cold stress (20oC) with a profile suggesting autoregulated expression. crhR mutant analysis indicates that transcript and protein stabilization involve CrhR-independent and CrhR-dependent mechanisms, respectively. CrhR abundance is regulated by temperature alteration of transcript and protein half-life. Basal levels of mRNA transcript and protein accumulate at 30oC, which are significantly enhanced at 20oC. crhR

 

CSPB2012 p. 29

inactivation showed unregulated accumulation of both transcript and protein in the cold, suggesting a requirement for CrhR RNA helicase activity in autoregulation of expression during temperature acclimation. Furthermore, multiple transcripts accumulate in the mutant at prolonged cold stress associating the helicase with its own mRNA maturation and/or degradation. CrhR does not, however, appear to be involved in transcript degradation, as crhR transcript half-life is not altered in the crhR mutant. Temperature regulates the half-life of both crhR transcript and protein which are stabilized at 20oC. The data indicate that CrhR activity autoregulates its own expression through a complex network acting at the transcriptional and post-transcriptional levels.

B1.4 Proteolytic regulation of cyanobacterial RNA helicase accumulation in response to abiotic stress Oxana S. TARASSOVA, George W. OWTTRIM Department of Biological Sciences, University of Alberta, Alberta, Canada The proteolytic regulation of protein levels enables cells to adjust their physiology to rapidly changing environmental conditions. The proteolytic mechanism controlling differential expression of a cyanobacterial RNA helicase is the basis of my thesis. RNA helicases rearrange RNA secondary structure and have been shown to participate in almost every aspect of RNA metabolism. CrhR is an RNA helicase in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803, whose expression is regulated by the redox status of the electron transport chain and therefore can be affected by various abiotic stresses, including temperature. A basal level of CrhR protein is observed at 30°C, which is significantly increased at 20°C. In vitro and in vivo experiments indicate that proteolysis is a major player in the temperature regulation of CrhR accumulation. The proteolytic mechanism is functional at 30°C and inactive at 20°C or at any temperature in the absence of functional CrhR. Microarray analysis indicated that expression of two genes involved in proteolysis, ftsH and clpC, is reduced in the crhR mutant. Western analysis indicates that while ClpC levels are not affected, FtsH levels decrease in the mutant. Experiments investigating whether FtsH protease degrades CrhR and if functional CrhR is required for the presence or activation of this degradation machinery are in progress. B1.5 Quantifying responses of seed germination and seedling performance to low temperature: Variation among genotypes of Brassica species Ludovic J.A. CAPO-CHICHI1, Isobel PARKIN2, Jan SLASKI1, Ralph LANGE1, Limin WU1 1. Alberta Innovates – Technology Futures, P.O. Bag 4000, Vegreville, Alberta T9C 1T4 2. Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, Saskatchewan S7N 0X2 Frost during seedling development in the late spring and seed maturation in the early fall is one of the major factors affecting spring canola production in Canada, resulting in significant yield reduction and low seed quality. In order to investigate the potential of chlorophyll fluorescence to reveal plant tolerance to low temperature, we used a collection of Brassica species and compared their fluorescence response. Untreated seeds of 234 genotypes including Brassica napus, Brassica rapa, Brassica juncea, and Brassica oleracea were sown in pots containing field soil and placed in a germination chamber with temperatures ranging from 1°C to 15°C. Light conditions were set at 16/8 h light/dark. Germination was determined by counting the number of seed with 2 mm or more of radicle growth. Seedlings at the cotyledon stage were freeze-shocked at -5°C for a given period of time after dark adaptation for 60 min. Frost injury was visually assessed as well as being inferred from the measurement of the chlorophyll fluorescence parameter [Fv/Fm = (Fm – F0)/Fm]. Genotypes were significantly different (P < 0.05) for seed germination and emergence, depending on the tested temperature. At 15°C, seed germination and emergence ranged from 2 to 4 days after planting. At 5°C, the variability in seed germination and emergence rates among genotypes was higher, ranging from 13 to 20 days after planting. Upon freeze-shock at -5°C, the distribution of the ratio of variable to maximum fluorescence (Fv/Fm) indicated that leaves of tolerant genotypes maintained higher rates of electron transport than leaves of sensitive genotypes.

 

CSPB2012 p. 30

B1.6 Acadian Ascophyllum nodosum extract (AANE) helps plants overcome salinity stress Katherine A. GRIEGOSCHEWSKI, Dayna M. TITUS, Laurel G. SHISHKOV, Will G. NEILY, Alan T. CRITCHLEY Acadian Seaplants Limited, Dartmouth, NS Commercial extract of the brown seaweed Ascophyllum nodosum (Rockweed), from the Scotian Shelf of Eastern Canada, has been shown to aid plants overcome salinity stress. However, it was not clearly understood whether or not treated plants are able to prevent sodium from entering their tissues or, if treated plants were just more tolerant of higher salt concentrations within their tissues. This replicated study, demonstrated that when exposed to the same concentration of sodium chloride, plants treated with AANE had the lower concentration of sodium in the shoot tissues, as compared to the control. However, between control and treatments, the root tissue contained roughly equal sodium concentrations, thus indicating that the sodium was able to enter the root but had not been translocated through the plant to the salt-sensitive leaf tissues. B2.1 Role of phytochelatins and their precursors in differential Cd accumulation in barley and lettuce Mst F. AKHTER, Sheila M. MACFIE Department of Biology, Western University, London, ON Cadmium (Cd) is a non-essential trace element and its environmental concentrations are approaching toxic levels, especially in some agricultural soils. Understanding how and where Cd is stored in plants is important for ensuring food safety. Previous experiments have determined that lettuce (Lactuca sativa) and barley (Hordeum vulgare) seedlings can grow in hydroponic nutrient solution containing 1.0 µM Cd without showing toxicity symptoms and, at this concentration, approximately 80% of the total Cd is translocated to leaves of lettuce, whereas only 20% of the total Cd is translocated to barley leaves. Preferential retention of Cd with phytochelatins (PCn’s, n=2-4) or their precursor peptides (Cys, γ-Glu-Cys and Glutathione) in barley roots could explain the reduced Cd translocation to barley leaves. Plants were grown in nutrient solution under two conditions: chronic (4 week) exposure to a low, environmentally relevant concentration (1.0 µM) of Cd and acute (1 hour) exposure to a high concentration (5 mM) of Cd, and the total amounts of PCs and their precursor peptides in the roots and shoots were measured using HPLC. Each of PC2-4 were synthesized in the barley root upon chronic exposure to Cd and did not increase further upon acute exposure. In the case of lettuce, no PCs were detected in the root. The high amounts of PCs produced in barley root could have contributed to greater Cd-tolerance in barley relative to lettuce as well as the observed preferential retention of Cd in barley roots. B2.2 Two distinct pathways for iron acquisition by iron-limited cyanobacterial cells: evidence from experiments using siderophores and synthetic chelators Mathew B. SONIER1,3, Daniel A. CONTRERAS1, Ron G. TREBLE2, Harold G. WEGER1 1. Department of Biology, University of Regina, Regina, SK 2. Department of Chemistry & Biochemistry, University of Regina, Regina, SK 3. Present address: Novozymes BioAg Ltd., Saskatoon, SK Iron-limited cyanobacterial cells are generally considered to acquire extracellular iron through a siderophore-dependent system, although evidence has started to accumulate that other, as yet poorly-characterized, iron acquisition systems may also play a role. Iron-limited cells of the cyanobacterium Anabaena flos-aquae are well-known to produce the relatively low Fe(III) affinity dihydroxamate siderophore schizokinen. In this set of experiments we show that iron-limited A. flos-aquae cells: 1) acquired iron at substantial rates in the absence of the schizokinen, and 2) acquired iron from a bacterial siderophore (the trihydroxamate molecule desferrioxamine B [DFB]), and also a synthetic chelator (DTPA), with substantially higher affinities for Fe(III) than schizokinen, indicating that a schizokinen-independent iron acquisition pathway was operating. We suggest that there exists a siderophore-independent iron acquisition system that is able to acquire Fe(III) from high stability Fe(III)-chelates that are not accessible to iron-limited cells via the schizokinen-based

 

CSPB2012 p. 31

system. As well, we present two possible models for iron acquisition by iron-limited A. flos-aquae cells. Both of these models suggest that there are two major routes for Fe(III) entry into the periplasm of iron-limited cells: 1) the well-characterized siderophore (schizokinen)-dependent process and 2) a siderophore-independent process that is able to access Fe(III) sources not biologically available to the schizokinen system. B2.3 TtHMA3-B1, a P1B-ATPase, is a tonoplast-localized cadmium transporter that associates with grain cadmium concentration in durum wheat Neil S. HARRIS1, Krysta WIEBE2, Curtis J. POZNIAK2, Ron MACLACHLAN2, John M. CLARKE2, Gregory J. TAYLOR1 1. Department of Biological Sciences, University of Alberta, Edmonton, AB 2. Crop Development Centre, University of Saskatchewan, Saskatoon, SK Cadmium (Cd) is an environmental hazard that can negatively affect human health. The dominant source of human exposure to environmental Cd is through contaminated food. Among cereals, some durum wheat cultivars have a genetic propensity to accumulate Cd in grain to levels exceeding international trade limits. A single major gene on chromosome 5B (Cdu-B1) regulates grain Cd levels but the genetic factor conferring the low Cd phenotype was previously not known. We have isolated and functionally characterized an allelic gene, TtHMA3-B1 (Triticum turgidum Heavy-Metal ATPase 3), from durum wheat genotypes contrasting in grain Cd accumulation. HMA3-B1 encodes a P1B-type ATPase that is predicted to have Cd transport activity based on sequence similarity with Arabidopsis and rice HMA3. Allelic variation was assessed in lines isogenic for Cdu-B1, and a 17 bp disruptive insertion that generates a premature stop codon in the first exon of HMA3-B1 was identified in high Cd accumulating lines. Using a fine-mapping population of over 5200 F2 plants, HMA3-B1 and Cdu-B1 co-segregated with phenotypic variation in grain Cd concentration. Sequence analysis of bacterial artificial chromosomes spanning Cdu-B1 confirmed the presence of HMA3-B1. Heterologous expression of the functional allele, HMA3-B1a, phenotypically complements the Cd-hypersensitive yeast strain Δycf1 that lacks the ability to transport Cd into vacuoles. In contrast, the disrupted allele did not complement Cd sensitivity in Δycf1. The fusion protein HMA3-B1a::GFP localizes at the tonoplast, consistent with a role for HMA3-B1 in the influx of Cd into the vacuole. In planta characterization is currently ongoing. B2.4 Effects of elevated temperature and elevated CO2 on autumn acclimation in Pinus strobus Christine CHANG, Emmanuelle FRÉCHETTE, Tarek BIN YAMEEN, Ingo ENSMINGER Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada Autumn cold hardening in evergreen conifers includes downregulation and acclimation of metabolic processes and enhanced frost defense in response to two environmental signals, shortened daylength and decreasing temperature. Temperature and CO2 levels are predicted to rise over the next century, which will impact long-lived species unable to quickly adapt to changing conditions. To evaluate conifer response to future warmer climate and CO2-enriched atmosphere, we assessed the effects of photoperiod, elevated temperature and elevated CO2 on photosynthesis and cold hardiness in 2-year old white pines (P. strobus) via gas exchange and chlorophyll fluorescence. Seedlings subjected to both short daylength and low temperature (cold autumn) for six weeks showed decreased maximum photosynthetic quantum yield (Fv/Fm) and carbon assimilation compared to summer control plants; light response and CO2 response also decreased. When only given a short daylength signal (warm autumn), seedlings maintained high Fv/Fm and unchanged carbon assimilation compared to summer controls. The combination of short daylength and elevated CO2 (warm autumn +CO2) enhanced photosynthetic efficiency of seedlings compared to summer controls. However, freezing tests revealed that only seedlings given normal autumn signals were able to survive freezing episodes (-20°C) without incurring damage; only cold autumn seedlings recovered to maximum Fv/Fm values within one month past freezing. Warm autumn seedlings were able to partially recover whereas warm autumn +CO2 seedlings were unable to recover. These results suggest that in a future climate, increased productivity due to lengthened growing season may be offset by increased susceptibility to autumn frost damage in white pine seedlings.

 

CSPB2012 p. 32

B2.5 Sixteen years of winter stress: an assessment of cold hardiness, growth performance and survival of hybrid poplar clones at a boreal planting site Stefan G. SCHREIBER1, Andreas HAMANN1, Uwe G. HACKE1 & Barb R THOMAS1,2 1. Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada 2. Alberta Pacific Forest Industries Inc., P.O. Box 8000, Boyle, AB, Canada In recent years, thousands of hectares of hybrid poplar plantations have been established in Canada for the purpose of carbon sequestration and wood production. However, boreal planting environments pose special challenges that may compromise the long-term survival and productivity of such plantations. In this study, we evaluated the effect of winter stress, i.e. frequent freeze-thaw and extreme cold events, on growth and survival of 47 hybrid poplar clones in a long-term field experiment. We further assessed physiological and structural traits that are potentially important for cold tolerance for a selected set of seven clones. We found that trees with narrow xylem vessels showed reduced freezing-induced embolism and showed superior productivity after 16 growing seasons. With respect to cold hardiness of living tissues, we only observed small differences among clones in early autumn, which were nonetheless significantly correlated to growth. Maximum winter cold hardiness and the timing of leaf senescence and budbreak were not related to growth or survival. In conclusion, our data suggests that reduction of freezing-induced embolism due to small vessel diameters is an important adaptive trait to ensure long-term productivity of hybrid poplar plantations in boreal planting environments.    B2.6 Suppression of photosynthesis in response to biotic stress: fine-scale temporal mapping of jasmonate-induced transcriptional programs Ian MAJOR, Bruce ROSA, Elham ATTARAN, Jin CHEN, Sheng Yang HE, and Gregg HOWE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA It is well established that photosynthesis is perturbed by environmental stress. In particular, insect herbivory and pathogen infection have been shown repress the expression of photosynthesis-associated genes (PAGs) and decrease photosynthetic efficiency. However, the underlying molecular mechanisms responsible for these negative effects are unknown. It is likely that some of the changes are mediated by the plant hormone jasmonate (JA), which is a key regulator of defense responses and is also associated with the suppression of photosynthesis. We are pursuing several complementary approaches to elucidate the molecular nature of JA-mediated repression. Here, we describe the use of RNA-sequencing and qRT-PCR-based techniques to develop a fine-scale temporal map of transcriptional changes occurring during the JA response. RNA-sequencing offers unique perspectives for transcriptome analysis compared with conventional microarray analysis, including a higher dynamic range, which is crucial for analysis of the highly expressed PAGs, and the ability to determine gene expression changes in absolute terms rather than fold change, which can be misleading. Our approach has highlighted clusters of functionally related PAGs with distinct expression profiles, which has provided some clues as to the nature of their repression. Finally, network analysis of these fine-scale temporal profiles should allow us to identify candidate regulatory genes associated with the suppression of photosynthesis by stress. C1.1 SHI/STY transcription factors have highly dynamic expression patterns during leaf development and act non-cell-autonomously on vein patterning Tammy BAYLIS1, Izabela CIERLIK2, Eva SUNDBERG2, and Jim MATTSSON1 1. Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada 2. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden Leaves have evolved complex venation systems to distribute water throughout the leaf blade and to transport fixed carbon to the rest of the plant. While it has been known for decades that auxin can induce vascular differentiation, the molecular mechanisms behind leaf vein patterning are not well understood. This is especially the case for aspects of auxin biosynthesis. Here we have investigated the roles of the SHORT INTERNODES/STYLISH (SHI/STY) gene family of transcription factors in leaf vein pattern formation. Characterized primarily in gynoecium development, SHI/STY members

 

CSPB2012 p. 33

have been previously linked to auxin homeostasis through the transcriptional regulation of YUCCA auxin biosynthesis genes. We have found that four SHI/STY genes are expressed primarily at the apical, basal, and marginal domains of Arabidopsis cotyledonary and rosette leaf primordia at different developmental time points, with very little expression at sites of vein formation. Still, shi/sty mutants display venation pattern defects ranging from mild single mutant anomalies to more severe multiple mutant phenotypes. We have also found that the spatial expression patterns of some SHI/STY genes shift with auxin transport inhibition and exogenous auxin treatments. Overall, our data suggest that SHI/STY genes play a role in leaf vein patterning, likely through the localized regulation of auxin biosynthesis and transport from the leaf blade margins. C1.2 Ribosomal protein mutants and their effect on plant development Chad STEWART, Peta BONHAM-SMITH

Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada Ribosomes are large, ubiquitous, two-subunit ribonucleoprotein particles, responsible for protein synthesis. In Arabidopsis, the cytoplasmic ribosome, comprised of a large 60S and small 40S subunit, contains 4 rRNAs and 81 ribosomal proteins (RPs) encoded by multigene families (254 genes) of two to five actively expressed members. Expression of each RP isoform from each family, of which only one is incorporated into any one ribosome, may be related to stress, developmental or environmental pressures. A reduced amount of a single RP can result in embryo lethality (reduction of the dominant paralog), pointed first rosette leaves (pfl) or no effect (reduction of the non-dominant paralog). Double mutants involving RPs are typically lethal or result in exaggerated pfl phenotypes. Here we show for the first time a novel RP mutant phenotype resulting from the concomitant reduction of RPS18A (pfl1) and overexpression of RPL18B (35S:HF/RPL18B). F1 heterozygotes display a late flowering phenotype reminiscent of flowering locus c or frigida mutants, flowering anywhere from 33-44 days post sowing compared to 21-24 days for wildtype. Upon transition from vegetative to reproductive growth, the primary inflorescence grows approximately 2.6 fold larger in diameter (similar to enhanced ethylene response1 mutants) and overall plant height is 21% taller then wildtype. Development stage specific mRNA expression profiles (Genevestigator) and protein cellular localization (transient expression in tobacco epidermal cells) of each member of the RPS18 and RPL18 gene families reveals no abnormalities when compared to a variety of large and small subunit RPs. We suggest a novel extraribosomal function for RPS18A in vegetative growth and primary inflorescence development.  

C1.3 Building small trees: transgenic modification of gibberellin metabolism or action produces semi-dwarf poplars Stewart B. ROOD1, Ani A. ELIAS2,3, Victor B. BUSOV4, Kevin R. KOSOLA2,5, Cathleen MA2, Elizabeth ETHERINGTON2, Olga SHEVCHENKO2, Harish GANDHI2,6, David W. PEARCE1, Steven H. STRAUSS2 1. Department of Biological Sciences, University of Lethbridge, AB 2. Department of Forest Ecosystems and Society, Oregon State University, OR 3. Department of Agronomy, Purdue University, IN 4. School of Forest Resources and Environmental Science, Michigan Technological University, MI 5. Monsanto Company, MO 6. Syngenta Seeds Inc., IA While tall trees are often favored in forestry, shorter trees with vigorous root systems should better withstand some environmental stresses and could be useful for applications such as reclamation and phytoremediation, carbon sequestration, and in some fuel and fiber plantations. Subsequently, mimicking the Green Revolution cereal semi-dwarfs, we developed semi-dwarf hybrid poplar trees (Populus tremula x P. alba) through transgenic modification of gibberellin (GA) metabolism or signaling. We subsequently characterized the endogenous GAs, and identified 15 GAs within an interlinked oxidative network of pathways involving 2-, 3-, 12-, 13- and 17- hydroxylations. The GA catabolic gene GA2-oxidase (GA2ox) increased levels of the biologically inactive 2-hydroxylated GAs, while the bioeffector GA1 was relatively unaffected and its precursor GA20 was elevated, indicating metabolic compensation. Insertion of GA insensitive (GAI) elevated levels of the bioeffector GA1 and its precursor, and the Repressor of GAI-Like (RGL) substantially elevated most GAs analyzed (~4-fold). Associated with the GA alterations, the three transgenes produced semi-dwarf saplings with heights reduced by about

 

CSPB2012 p. 34

one-third. Stem dry weights were less substantially reduced and leaf weights were relatively unaltered, while root weights were initially increased, elevating the root-to-shoot ratios. Leaf areas of some semi-dwarfs were reduced while chlorophyll concentrations were increased and correlated with leaf δ13C (~ -25o/oo), indicating altered photosynthesis and water use efficiency. Along with prospects for various field applications, these semi-dwarf poplars also provide insight into phytohormone regulation and the allometric coordination of growth and development in broad-leaved trees.    C1.4 Molecular events of apical bud formation in white spruce Walid EL KAYAL1, Chelsea J.-T. JU2, Janice E. K. COOKE1 1. University of Alberta, Department of Biological Sciences, Edmonton, Alberta 2. University of California at Los Angeles, Computer Science Department, Los Angeles, CA 90095, USA Bud development associated with the annual activity-dormancy cycle is a complex physiological and developmental process comprising bud formation, cold acclimation and dormancy acquisition. However, the molecular basis for terminal bud formation is still poorly understood. We have characterized transcriptome-level changes that occur during bud formation of white spruce (Picea glauca) in order to identify genes whose products play roles in this developmental process. Firstly, we used microarrays to compare gene expression profiles for distinct developmental stages of bud formation during a short day time course, corresponding these changes in expression with anatomical phases of bud development. Secondly, we took advantage of the ability of white spruce to initiate bud formation in the absence of short day cues to compare expression profiles in developing buds under long day and short day conditions. This identified a core set of genes that are differentially expressed during bud formation under both photoperiods that are likely important for bud formation, as well as possible photoperiod-responsive genes that may not be essential for bud development. Moreover, a comparison of gene expression profiles during bud formation in different tissues revealed a small set of genes differentially expressed only in developing buds. Altogether, these findings provide a temporal roadmap of bud formation in white spruce.    C1.5 Developing canola lines with reduced pod shattering Aliaa EL-MEZAWY1, Limin WU1, Mohammad AL-FORKAN1,2, Habibur RAHMAN2, Saleh SHAH1 1. Biotechnology and Genomics, Alberta Innovates-Technology Futures, Vegreville, Alberta 2. Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta Brassica plants disperse their seeds by a pod-shattering mechanism. Although this mechanism is an advantage in nature, unsynchronized pod shatter constitutes one of the biggest problems for canola farmers. Just before harvest, the pods are prone to shatter, causing a 10-25% loss of seeds and up to 70% in some cases. We are developing canola (B. napus) lines with reduced pod shattering using transcription factors involved in fruit opening. Mutations in one such gene, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), in Arabidopsis caused specific loss of secondary walls at fruit valve margins. We have down regulated activity of this gene in canola using Arabidopsis NST1 fused in the antisense direction downstream of a weak promoter from Macfadiana plant. Among first generation transformed canola plants, many produced normal abundant fruits and showed 10-80% pod shattering in laboratory test compared to 60-70% in non-transgenic control. Lines that displayed ≤40% pod shattering were grown to second generation and they showed 0-56% pod shattering. Four second generation lines that showed <20% pod shattering will be tested in field trial during summer of 2012.    C1.6 Visual marker technology Margarita R. PLINER, Susan KOZIEL, Jurgen QUANDT, John VIDMAR Alberta Innovates - Technology Futures, Vegreville, AB A reliable and trustful system for visual marker selection, by genetically variegation of tobacco plants was developed. Tobacco leaf disks were transformed by Agrobacterium tumefacience, carrying RNAi Chaperonin 21 gene under different promoters – 35SCaMV, rbc and alcohol inducible promoter. Variegated regenerants were distinguished as early as the stage

 

CSPB2012 p. 35

of shoot development and transformed plants were able to be selected visually on antibiotic free selection media. Genetically transformed plants were confirmed to be cpn21 positive by PCR. Variegated plants were grown vigorously and produced viable seeds by controlling the expression of cpn21 under tissue specific (rbc) and alcohol inducible promoter.  C2.1 Natural variation in response to flooding and shading: A GWAS study in Arabidopsis Kate R St.ONGE1, D. VREUGDENHIL2, J. KEURENTJES2, R. SASIDHARAN1, L.A.C.J VOESENEK1, and R. PIERIK1 1. Plant Ecophysiology, Utrecht University, Utrecht, the Netherlands 2. Laboratory of Plant Physiology, Wageningen University, Wageningen, the Netherlands The aim of my project is to elucidate genes and molecular mechanisms involved in crosstalk between shade avoidance and submergence escape responses. Plants perceive these two stresses differently but display similar responses: hyponasty and elongation. The first step of my genomic approach to this problem is to survey natural variation in responses to flooding and shading in a collection of 349 genotyped Arabidopsis thaliana accessions and perform a genome-wide association (GWA). These accessions were screened for hypocotyl length under 2 control treatments and 2 stress treatments: control light conditions, complete dark control, 1 ppm ethylene to simulate flooding and green filtered light to simulate shading. Considerable variation among accession was observed under all treatments. Hypocotyl length varied from ~70-250% and ~125-430% of the control light treatment under ethylene and green shade treatments respectively. Compared with the complete dark treatment hypocotyl length varied from ~15-35% and ~25-60% of the length under total dark, demonstrating that although nearly all accession elongate under our stress treatments they only elongate to 60% or less of their potential length. GWA discovered both a prior and de novo candidate genes which are associated with these phenotypes. Follow-up work leading from this screening and GWAS include: investigating potential causative mutations in candidate genes, comparing the transcriptomes of accessions with opposite responses, and exploring how different alleles have evolved using molecular evolution approaches to give insight to how plants can adapt to local flooding and shade conditions.  

 C2.2 Gene discovery through SNP analysis in adaptive traits of black cottonwood poplar (Populus trichocarpa) Athena D. McKOWN, Jaroslav KLAPSTE, Rob GUY

Department of Forest Sciences, University of British Columbia, Vancouver, BC Black cottonwood poplar (Populus trichocarpa) is a widespread, deciduous tree in western North America that shows selection for adaptive traits relating to local growing season and climate. In our collaborative research program at UBC, we have studied over 2000 trees grown in a common garden at Vancouver (representing replicate plantings of 456 collected genotypes within 150 provenance localities from Alaska to Oregon). Our studies investigated inherent trait variation within P. trichocarpa focusing on seasonal (phenological) events, biomass accumulation and growth rates, nutrient isotopes, and gas exchange. Our findings showed that common garden trees demonstrated substantial variation in traits, many of which were repeatable year to year, and most traits correlated with latitude (ie. photoperiod) and climate parameters. Genetic analyses for each genotype included assessment of ~34k single nucleotide polymorphisms (SNP) in 7000 candidate genes using Illumina sequencing. Many traits showed high narrow sense heritability, supporting the correlations observed with geography. Although genetic population structure was generally weak, some structure was observed between northern and southern groups. In our SNP association analyses, including the population structure model provided the best fit to trait data based on BIC comparison. The SNP association analyses, strengthened by replication in genotype and repeated measures of traits, revealed a large number of genes involved with each trait. Some genes or pathways were found repeatedly; especially, genes involving hormones were often found with photoperiod related traits and growth. Numerous traits and SNP associations point to local adaptation, with selection becoming especially strong in northern P. trichocarpa genotypes.

 

CSPB2012 p. 36

C2.3 Identification, characterization and influence of transposable elements in the flax (Linum usitatissimum L.) genome Leonardo M. GALINDO, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, AB Transposable elements (TEs) are powerful engines for gene evolution and regulation. An unmasked, whole-genome shotgun assembly of flax (www.linum.ca) was searched to identify transposable elements. Similarity and de-novo analyses showed that complete and partial transposable elements covered more than 20% of the full genome. The LTR-Copia and Gypsy elements were the most prevalent elements while DNA transposons constituted a smaller proportion. Distribution of TEs and genes showed a range of abundance of TEs, from TE-rich regions to regions where abundances were similar to gene-rich regions, but TEs do not seem to be associated with genes more than expected by chance in any of the sections analyzed. However, the proportion of genes with putative expression was negatively correlated with the proportional coverage of TEs. Selected predicted agronomical important genes that contained TEs either as insertions, chimeras or in close proximity to them, were further characterized and confirmed by PCR and sequencing. The analyzed regions showed that TEs inside genes are mostly located in introns. Several genome sections showed evidence of TEs related to gene families like disease resistance genes, and also of TE clustering. Some elements, mainly of the Copia superfamily, were found to be recently active as shown by their high LTR similarity; phylogenetic analyses show that this group of TEs is also more diverse when compared to Gypsy retrotransposons. A large proportion of small RNAs maps to TEs and may constitute an important regulatory mechanism. C2.4 Sequencing the bean genome: Using comparative genomics to examine interspecific introgressions Gregory E. PERRY1, William CROSBY2, Kangfu YU3, Alireza NAVABI3, Zou XAIOLU3, Krista POWER3, Frederic MARRSOLAIS4, Weilong XIE1, Alfons WEERSINK1, Rong CAO3, Denise COOPER1 and K. Peter PAULS1* 1. Department of Plant Agriculture, University of Guelph, ON 2. Department of Biology, Windsor University, ON 3. Agriculture and Agri-Food Canada, ON 4. Department of Biology, University of Western Ontario, ON Common bean (Phaseolus vulgaris) is an important crop for Canadian farmers and consumers for both its economic and health benefits. The Applied Bean Genomics and Bioproducts project aims to sequence and annotate the genome of OAC-Rex, a registered variety of white bean of Mesoamerican origin. This variety was developed from an interspecific cross between P. vulgaris and Phaseolus acutifolius, which is resistant to common bacterial blight. International sequencing efforts are also underway to sequence an Andean (G19833) and a second Mesoamerican line (Bat93). Current assemblies are working towards chromosome-length scaffolds, which opens the possibility comparative genomics between these lines. Efforts to identify the blight-resistance genes in OAC-Rex have been frustrated by the limited sequence information, and sparse molecular marker coverage in the introgressed regions. By comparing the genome sequence between these three lines, we hope to be able to identify genes introduced through the interspecific cross, and screen these for potential resistance genes. Initial analyses of the OAC-Rex scaffolds have identified contigs which contain sequences homologous with those from an P. acutifolius EST library. These contigs were annotated in-silico and the sequences compared with the G19833 and Bat93 sequences. Between the three lines, gene order and orientation is largely preserved, with only small regions surrounding the identified P. acutifolius gene sequences not being shared between lines. These interspecific islands will be further characterized to identify potential resistance genes. C2.5 Evolution and expansion of the MAPK family in solanaceous species by using Next Generation Sequencing technologies Caroline DAIGLE and Daniel P. MATTON Institut de Recherche en Biologie Végétale, Université de Montréal

 

CSPB2012 p. 37

Plant MAPK signaling cascades are well known for their role in biotic and abiotic stress responses. In recent years their involvement in numerous developmental processes including male and female gametophytes development, cytokinesis and embryogenesis have been uncovered. Although well established throughout the eukaryotes, it is in plants that they have flourished the most. For example, the Arabidopsis genome contains around sixty genes coding for MAPKKKs, ten for MAPKKs and twenty for MAPKs, but relatively little is known for others species. We have recently showed that a small pMEKK subfamiliy in Solanum chacoense, the FRK family (FRK1, 2 and 3) plays various roles in gametophytes and ovule development. In Arabidopsis, three members of this family are also present but do not display similar phenotypes as the ones observed in S. chacoense, suggesting that this family might have evolved new functions in solanaceous species. With the aim of finding full MAPK modules acting in plant reproduction, a deep transcriptomic analysis was performed on pollen and ovule samples. This unveiled a much larger FRK family in the three solanacesous species analyzed (S. chacoense, S. tuberosum, and S. lycopersicum), confirming that expansion of this family has probably given rise to new functions in Solanaceous species compared to Arabidopsis, poplar and rice. Interacting partners for FRK1, 2 and 3 and were sought through yeast two-hybrid screening and new signalling networks working in plant reproduction will be discussed. C2.6 Adaptive genomics of lodgepole and jack pine populations Catherine CULLINGHAM, Sophie DANG, Stephanie BOYCHUK, Janice COOKE, David COLTMAN

University of Alberta, Department of Biological Sciences, Edmonton, AB The most recent outbreak of the mountain pine beetle has devastated lodgepole pine forests in both Canada and the United States. The outbreak has now spread eastwards across the Rocky Mountains into Alberta, and we have recently shown that it can successfully invade jack pine of the boreal forest, a naive host that ranges across northern North America. This has prompted us to develop genomic resources to identify regions under selection. We have identified over 1500 SNPs primarily in silico using 454 transcriptome sequences from lodgepole and jack pine, generated using pooled individuals harvested at various stages of stress response. We are using lodgepole pine as a point of reference because these species readily hybridize and lodgepole pine is a co-evolved host for the beetle. Populations of lodgepole, jack pine and their hybrids with varied historic and recent exposure to the beetle have been characterized for these SNPs from Alberta, British Columbia, Ontario and Saskatchewan. We have identified over 500 SNPs that were variable in either lodgepole or jack pine. Of these, approximately 50% appear to be variable in both species. We will analyze these data using Fst outlier approaches to identify loci that show signs of selection, then use these loci to test for associations between beetle attack status, climatic and environmental variables as a means to investigate adaptive variation. Finally, we will do a comparative analysis between lodgepole and jack pine that investigates genes showing high levels of introgression to understand variation of evolutionary importance. D1.1 Improved production of recombinant Brassica napus type 1 diacylglycerol acyltransferase in a yeast strain deficient in triacylglycerol synthesis Michael S. GREER1, Martin TRUKSA,2 Shiu-Cheung LUNG,1 Randall J. WESELAKE1 1. Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB 2. Alberta Advanced Education and Technology, Edmonton, AB Diacylglycerol acyltransferase (DGAT) 1 catalyzes the acyl-CoA-dependent acylation of sn-1, 2 diacylglycerol to produce triacylglycerol (TAG). The level of DGAT activity during seed development in Brassica napus may have a substantial effect on the flow of carbon into seed oil. Four DGAT1 genes were isolated from the B. napus (DH12075) genome. The polymorphisms of these genes are predominantly located in their first exon, encoding a relatively hydrophilic, cytosol facing N-terminal domain that is non-essential for catalysis. Based upon homology, these genes can be divided into two sub-groups. Expression of the coding sequences of the DGAT1s in the TAG-deficient quadruple knock-out yeast (Saccharomyces cerevisiae) strain H1246 revealed that the enzymes of one sub-group produced twice the quantity of TAG in comparison to the other sub-group. Further investigation using chimeric DGAT1s demonstrated that high and low TAG- producing phenotypes could be interchanged by replacing the native N-terminal region from one sub-group with the N-terminal region from the other sub-group. Western blotting of microsomal proteins from cultures expressing the DGAT1s showed that the high and low TAG-producing phenotypes result from differential accumulation of the enzymes. Analysis

 

CSPB2012 p. 38

of DGAT1s with mutations in their second amino acid residue suggests the altered accumulation of the enzymes results from different turnover rates, dictated by the “N-end rule”. Placement of an N-terminal epitope at the 5’ end of the DGAT1 coding sequences resulted in more stable production of the enzymes irrespective of original high or low TAG- producing phenotype. D1.2 Using directed evolution to increase oil accumulation in oilseed crops Rodrigo M.P. SILOTO and Randall J. WESELAKE Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB There is currently a rising demand for vegetable oils, which is largely attributed to their utilization as biofuels. This is a cause of great concern worldwide, especially with the expansion of cultivated land and the consequent environmental impact. Increasing seed oil content is an alternative to help meet this demand. This is major challenge, especially since traditional breeding has already maximized the natural capacity of oilseed crops to accumulate oil. Metabolic engineering is an effective means of increasing seed oil accumulation through overexpression of genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs). In the present study we increased activity of a Brassica napus DGAT1 (BnDGAT1) through directed evolution. A high throughput screening method was developed based on functional selection and fluorescence detection of TAG using a yeast recombinant expression system. With this method we were able to screen thousands of DGAT variants from mutagenized libraries created by error-prone PCR. Variants with significant increases in activity were detected as reflected by TAG accumulation in yeast. Among these, a single amino acid substitution produced a 7-fold increase in yeast TAG production compared to the natural BnDGAT1 enzyme. This result was validated using site-directed mutagenesis, and the effect of this modified DGAT in seed oil content is currently being tested in Arabidopsis. Through a continuous improvement of this and other BnDGAT1 variants, we expect to produce engineered enzymes that are highly active, and contribute to seed oil production in oilseed crops. D1.3 Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in a high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid Elzbieta MIETKIEWSKA, Robin MILES and Randall J. WESELAKE Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science; University of Alberta, Edmonton, AB Punicic acid (18:3Δ9cis,11trans,13cis) is a conjugated linolenic acid naturally produced in the oil of Punica granatum (pomegranate) seeds. Numerous studies have indicated that punicic acid is a nutraceutical fatty acid with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bond in punicic acid is synthesized by the catalytic action of ‘‘fatty acid conjugase’’ (FADX; AY178446). This occurs through the conversion of the Δ12 double bond of linoleic acid (18:2Δ9cis,12cis) into two conjugated and trans-cis configurated double bonds at the 11 and 13 positions. Previous attempts to over-express P. granatum FADX in A. thaliana resulted in limited accumulation of punicic acid accompanied by increased accumulation of oleic acid (18:19cis), suggesting that production of punicic acid in some way inhibits the desaturation of oleic to linoleic acid. In the current study, we developed a new strategy to enhance accumulation of punicic acid in a high linoleic acid A. thaliana fad3/fae1mutant background using combined expression of P. granatum: FADX and FAD2. This resulted in the highest accumulation of punicic acid reported so far in transgenic A. thaliana. Detailed analysis of transgenic A. thaliana plants carrying P. granatum FADX+FAD2 genes will be presented.

 

CSPB2012 p. 39

D1.4 A systematic characterization of TAG-synthesizing enzymes from flaxseed Xue PAN, Rodrigo M. P. SILOTO, Aruna D. WICKRAMARATHNA, and Randall J. WESELAKE Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.

Flaxseed oil has outstanding amounts of α-linolenic acid (18:3) and is one of the richest sources for omega-3 polyunsaturated fatty acids. Triacylglycerol (TAG) is assembled through sequential reactions catalyzed by membrane-bound acyltransferases. Type-1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) catalyzes the final acylation reaction, and is proposed to exert substantial control over TAG accumulation. But other enzymes, including phospholipid:diacylglycerol acyltransferase (PDAT), type-2 DGAT (DGAT2), and a soluble DGAT (DGAT3), can also catalyze TAG synthesis. Here we present a systematic characterization of TAG-synthesizing enzymes from flaxseed. A single DGAT1 gene was identified in the flax genome, compared to three DGAT2 and six PDAT genes. Phylogenetic analyses indicated that PDATs are divided into three families, each containing two genes. Analysis of expression profile indicated that DGAT1, two DGAT2s and three PDATs are preferentially expressed in developing flaxseed embryos, while transcripts of two PDATs are present at higher levels in vegetative tissues. Functional expression in Saccharomyces cerevisiae showed that DGAT1 is the most active enzyme. Flax DGAT2, DGAT3 and PDAT displayed low activity in yeast compared to DGAT1; however, two flax PDATs displayed higher levels of TAG accumulation in response to 18:3 feeding in yeast. We are currently investigating possible mechanisms of PDAT activation by 18:3. Overall, this study provides substantial insight into the TAG biosynthetic machinery in flaxseed and valuable information which can be utilized towards improving oil quality of flaxseed and other oilseeds. D1.5 Identification and functional characterization of genes encoding phosphatidylcholine diacylglycerol cholinephosphotransferase from flax (Linum usitatissimum) Aruna D WICKRAMARATHNA, Rodrigo M P SILOTO, Xue PAN, Randall J WESELAKE Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Flax seed oil is a rich source of α-linolenic acid, an essential dietary fatty acid. Understanding different biochemical processes involved in the biosynthesis of triacylglycerol (TAG) and its precursors in flax is critical to improve the production and quality of flax seed oil. Several novel enzymes involved in TAG biosynthesis were identified in Arabidopsis. Among these, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT) appears to play a key role in determining the fatty acid composition of TAG. PDCT catalyzes a symmetrical inter-conversion between sn-1,2-diacylglycerol (DAG) and phosphatidylcholine (PC) through phosphocholine headgroup exchange acting as a gatekeeper enzyme that provides a major path through which oleoyl moieties enter PC for desaturation, while linoleoyl and linolenoyl moieties appear as DAG available for further acylation to form TAG. Two flax PDCT homologous cDNAs (LuPDCT1 and LuPDCT2) were isolated, sharing 70.0% and 71.0% identity, respectively, with the corresponding cDNA from Arabidopsis. qRT-PCR studies revealed that the transcript levels of LuPDCT1 and LuPDCT2 increase substantially during early embryo development while low expression is observed in vegetative tissues. Topology prediction programs identified LuPDCT1 and LuPDCT2 as integral-membrane proteins with five putative transmembrane regions. Functional expression of LuPDCT1 or LuPDCT2 in the yeast mutant strain BY4742, which lacks cholinephosphotransferase activity, demonstrated that the LuPDCTs could restore the organism’s ability to synthesize PC from DAG. These results indicate that LuPDCT1 and LuPDCT2 encode active PDCTs in flax, mainly produced during embryo development. These enzymes likely play a role in the accumulation of high levels of unsaturated fatty acids in in the TAG of flax embryos.

 

CSPB2012 p. 40

E1.1 The Arabidopsis anisotropy1 (any1) encodes CesA1 subunit of cellulose-synthase-complexes that affects growth anisotropy and cell wall properties Miki FUJITA1, Regina HIMMELSPACH2, Juliet WARD 2, Angela T. WHITTINGTON2, Christine LIU1, Thy TRUONG3, Charles HOCART3, Moira E. GALWAY4, Shawn D. MANSFIELD5, Geoffrey O. WASTENEYS1 1. Department of Botany, University of British Columbia, Canada 2. Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, Australia 3. Research School of Biology, Australian National University, Australia 4. Department of Biology, St. Francis Xavier University, Canada 5. Department of Wood Science, University of British Columbia Cellulose is synthesized by multiple-enzyme complexes consisting of cellulose synthase (CesA) located at the plasma membrane. Among 10 CesAs in Arabidopsis, CesA1 is known to be involved in primary cell wall formation. We isolated the anisotropy1 (any1) CesA1 allele from a screen of an ethyl methane-sulfonate (EMS) mutagenized population and determined that the phenotype is caused by the substitution of aspartic acid residue 604 with asparagine. Unlike other CesA1 mutant alleles that show severe phenotypes such as embryo lethality or reduced cellulose production at restrictive temperature (rsw1-1 mutant), the any1 plants are viable and produce seeds. The mutant is dwarf with short root and shoot elongation zones and has abnormal cellular morphology such as swollen epidermal cells of aerial tissues. Cellulose content was not altered but cell wall crystallinity was reduced in any1. Cellulose microfibrils aligned transversely to the cell growth axis in root cells, dark-grown hypocotyl cells, and most cell types of any1 inflorescence stems. Live-cell imaging of YFP-CesA6 in any1 demonstrated that cellulose-synthase-complexes (CSCs) moved slowly relative to those in wild type. From the complementation cross with rsw1-1 (CesA1 allele), CesA1any1 is likely to be incorporated into CSCs to produce relatively normal cellulose.    E1.2 Analysis of DIMINUTO 1 reveals a role for brassinosteroids in secondary cell wall formation in Arabidopsis Abdelali HANNOUFA1, Zakir HOSSAIN1, Brian McGARVEY1, Lisa AMYOT1, Margaret GRUBER2, Jinwook JU 1. Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 4T3 2. Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2 Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38% and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.  

 

CSPB2012 p. 41

 E1.3 Genotype effects on physical properties of corn stem residues in composite materials Muhammad ARIF1, Muhammad RIAZ1, Joe MARTIN1, Yarmilla REINPRECHT1, Leonardo SIMON2 and K. Peter PAULS1 1. Plant Agriculture, University of Guelph, Ontario, Canada 2. Chemical Engineering, University of Waterloo, Ontario, Canada This study was initiated to investigate the influence(s) of plant genotype on the performance characteristics of fibres extracted from corn stems after incorporation into a polypropylene (PP) matrix. Corn stem fibres from a recombinant inbred (RI) population (from a CG62 x CO387 cross) of 40 lines grown in two environments were chemically analysed and incorporated into PP at 20% (wt/wt) by extrusion at 190°C and 40 rpm. Test samples were injection moulded at a barrel temperature of 190°C and a tool temperature of 50°C with an injection period of 15 sec at 100 psi, and annealed at 150°C for 10 min and cooled to room temperature. Corn stem fibres from different genotypes were significantly different for their cellulose, hemicellulose, lignin, para-coumeric acid, ferule acid and free phenolic contents. Corn stem fibres improved flexural strength up to 12%, flexural modulus up to 31%, tensile strength up to 7%, and tensile modulus up to 49%; but reduced impact strength up to 49% compared to pure PP. On a genetic linkage map we identified 59 Quantitative Trait Loci (QTL) that affected fibre (26 QTL) and composite (33 QTL) traits that explained from 8 to 41% of the variation. The tensile strength of the composites was negatively correlated with the hemicellulose content of the fibres and the tensile modulus values were negatively affected by the cellulose, hemicellulose and para-coumeric acid contents of the fibres. This study will provide information required to identify fibre-related genes and to breed corn hybrids with fibres that also produce superior composites.   E1.4 Roles of a β-galactosidase and arabinogalactan proteins in flax bast fiber cell wall development Neil HOBSON1, Mary DEPAUW1, Melissa J. ROACH1,2, Michael DEYHOLOS1 1. Department of Biological Sciences, University of Alberta, Edmonton, AB 2. Umeå Plant Sciences Center, Umeå, Sweden Flax (Linum usitatissimum) bast fibres have industrial applications in textiles, composites, and papermaking. The suitability of fibres for various industries is depends on a number of metrics, including cell length, cell diameter, colour, strength, and surface polarity, most of which are in turn derived from cell wall composition. Recent work has identified a number of genes suspected to be involved in cell wall development, as inferred by their expression in tissues containing developing bast fibres. Specifically, transcripts for beta-galactosidases (BGALs) and fasciclin-like arabinogalactan proteins (FLAs) were found to be enriched in tissues containing bast fibres undergoing cell wall expansion. Here, we examine the prevalence of the BGAL and FLA gene families in the flax genome, and further characterize the expression patterns of their more fibre-enriched members, using promoter-GUS fusions to examine tissue specificity. We also describe ongoing experiments that will examine the effects of gene over-expression and RNAi downregulation on fibre quality and cell wall composition.    E1.5 Statolith-less plant cells respond to gravity-induced stress by modulating cell wall assembly Youssef CHEBLI, Lauranne PUJOL, Anja GEITMANN Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, Montréal, Québec, Canada Plants are able to perceive gravity stimulation and to respond to this trigger. Several mechanisms have been invoked to explain how plant cells perceive the direction and strength of the gravity vector and statolith based perception is well characterized. However, most plant cells are not equipped with statoliths and it is poorly understood, how they perceive and respond to gravity related signals. Modulation in the synthesis and deposition of the cell wall is a common response to

 

CSPB2012 p. 42

gravity-induced stress in plant cells, and this response is particularly pertinent as it is directed to mechanically counteract the effect of the gravity-induced compression or bending load. To understand how altered gravity acts on cell wall assembly proper we investigated one of the most rapidly growing plant cells, the pollen tube. This cellular protuberance is formed by the pollen grain to deliver the sperm cells to the ovules and it can easily be cultivated in vitro. The main metabolic activity of the pollen tube is the synthesis and the deposition of cell wall precursors. Cellular expansion occurs only at the tip and at extremely high rates allowing for short term experiments. We monitored the effect of hyper- and micro-gravity on pollen tube growth, cell wall assembly, and intracellular transport using brightfield and epi-fluorescence illumination. Live cell imaging at different hyper-gravity levels was enabled by placing the remote controlled microscope in the Large Diameter Centrifuge facility operated at the labs of the European Space Agency. Micro-gravity conditions were simulated in a random positioning machine.    F1.1 From shoot to root: How do changes in the leaf environment affect whole-tree hydraulic properties? Joan LAUR, Uwe G. HACKE Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6E 2E3 In the prospect of global climate warming, the sustainable management of forest ecosystems is a challenge for Canada which is home to 10% of the world’s forests. There is a need to understand physiological and molecular aspects of how trees respond to different and changing environments. We studied how hybrid poplar saplings responded to changes in their leaf environment (changes in light and relative humidity) to maintain an adequate water balance. The control of water loss through stomata and water uptake through roots is influenced by many intrinsic and environmental factors and there is accumulating evidence for a complex role of water channel proteins (aquaporins) in the regulation of these processes. We monitored physiological adjustments of leaves (leaf area, water potential, transpiration, stomatal conductance, stomata properties) and roots (root mass, whole root system hydraulic conductance), and measured transcriptional levels of selected aquaporins. When saplings growing in high humidity were suddenly exposed to a decrease in relative humidity, they exhibited a rapid 35% increase of root hydraulic conductance while stomatal conductance remained high and unchanged. We also found that the responsiveness of stomata to ABA was impaired in high humidity-acclimated plants. Our findings underline the importance of dynamic adjustments of root properties for contributing to water potential homeostasis in the light of a changing above-ground environment.    F1.2 Xylem vessel length of woody plants: A global analysis Anna L. JACOBSEN Department of Biology, California State University, Bakersfield, CA Long distance water transport in the majority of flowering plants predominantly occurs via xylem vessels. Xylem vessels are long conduits that are composed of many specialized cells, vessel elements, which are connected end-to-end to form long pipes. The length of vessels within the xylem is a key trait that determines plant hydraulic function, yet relatively little is known about this xylem feature. Previously published studies were examined to generate a new global data set of vessel length in woody plants. Mean vessel length data were available from similarly sized stem samples from 120 species. Liana species had longer vessels than tree or shrub species. For all species, including long vesselled lianas, most vessels within measured samples were short and there were very few long vessels. Globally, mean vessel length did not vary between species that differed in growth ring porosity (i.e. ring porous species did not generally have longer vessels than diffuse porous species). Vessel diameter and length varied independently and may represent independent axes of vessel structural and functional variation. The decoupling between vessel diameter and length may be particularly important in areas that experience freezing. Many traits often assumed to be indicative of vessel length, such as organ, porosity, environment, or vessel diameter, are not generally predictive of vessel length. Vessel data are limited and increased knowledge of vessel length is needed in order to increase our understanding of the structure and function of the plant hydraulic pathway.      

 

CSPB2012 p. 43

F1.3 Xylem vulnerability to cavitation can be accurately characterized in long vesselled species with a centrifuge Robert B. PRATT, Michael F. TOBIN, Anna L. JACOBSEN Department of Biology, California State University Bakersfield, Bakersfield, CA Plants transport water in a metastable state under negative pressure making the transport system vulnerable to cavitation. Study of cavitation is important for understanding water stress resistance in plants. A widely used and efficient means of measuring cavitation resistance (CR) uses centrifugal force to generate negative xylem pressures in excised organs (stems or roots). Two recent studies found disagreement between centrifuge based measurements and other methods. These studies hypothesized that long vessels prematurely drain in the centrifuge leading to artifact in measured CR in long vesselled species. We tested this hypothesis in stems and found no evidence of a long vessel artifact using long and short vesselled species. For example, in an oak species we found that CR measurements generated using short segments (14 cm) that had on average 10% of their vessels open were not different than segments that were 27 cm and had 0.2% of their vessels open (most had 0% open). Moreover, centrifuge CR measurements agreed well with those generated using benchtop dehydration and to native-state cavitation. We suggest that not all centrifuge techniques are the same and this likely explains part of the disagreement found in previous studies. In addition, we show that non-centrifuge CR measures can be affected by clogging of vessels by gels over relatively short time frames leading to errors. We conclude that long open vessels alone are not the sole cause of artifacts and that the centrifuge technique we used is generally robust for long vesselled species. F1.4 Ranking of drought tolerance in Canadian poplar hybrids and identification of candidate genes for drought tolerance breeding Muhammad ARSHAD, Kamal BISWAS, Sherryl BISGROVE, Jim MATTSSON, Aine PLANT Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada Poplar hybrids are cultivated in Canada as a fiber source for the pulp and paper industry primarily because of their fast growth and for the same reason, have potential to be used for carbon sequestration as well as a feedstock for carbon-neutral production of energy. Poplars are generally regarded as drought sensitive, which poses a problem for large-scale cultivation, particularly in the light of predicted droughts on the Canadian prairies in response to global warming. To approach this problem, we tested nine Canadian poplar hybrids with respect to a series of physiological responses to drought, resulting in a ranking of drought tolerance. To identify differences on the molecular level correlating with tolerance, we assessed the least and the most resistant varieties with respect to the expression of 26 drought-responsive genes. For the majority of genes, expression levels correlated with levels of physiological stress. However, the expression of two genes defied the trend, and may therefore be part of the molecular basis of resistance. Orthologs of these genes in other species encode enzymes with known functions in drought tolerance, providing additional evidence that they are suitable substrate for targeted breeding of drought resistance in poplar.    F1.5 Growth and physiology of hybrid poplars in northern Alberta Kelsey AYTON1, Janusz ZWIAZEK1, Barbara R. THOMAS1,2 1. Department of Renewable Resources, University of Alberta, Edmonton, AB 2. Alberta-Pacific Forest Industries Inc., P. O. Box 8000, Boyle, AB, Canada T0A 0M0 Climate variables are a growing concern and changing the way in which industries that depend upon natural resources are conducting their operations. Water availability is among the most significant factors affecting the survival and growth of trees. Hybrid poplars are fast growing and highly water-demanding trees which are known to be relatively susceptible to drought. The present project had three main objectives: 1.) Identify whether water availability is limiting the growth of selected Populus clones grown under field conditions in northern Alberta. 2.) Identify the clones which are the least affected by drought and examine the physiological strategies that contribute to their drought tolerance, and 3.) Identify the clones which have the highest productivity and the physiological strategies that contribute to their productivity. During the

 

CSPB2012 p. 44

2010 and 2011 growing seasons, eight poplar clones and three irrigation treatments were used to assess these objectives. It was found that, although the examined hybrid poplar clones had growth and survival strategies and produced biomass at different rates, water availability was not a factor restricting their growth under the study conditions. F1.6 Hydraulic acclimation to drought, shade and nitrogen fertilization in hybrid poplar saplings Lenka PLAVCOVÁ, Uwe G. HACKE Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada Variation in xylem structure and function has been extensively studied across different species with a wide taxonomic, geographical and ecological coverage. In contrast, our understanding of how xylem of a single species can adjust to different growing condition remains limited. Therefore, we studied hydraulic acclimation of xylem traits in hybrid poplar (Populus trichocarpa x deltoides). Clonally propagated saplings were subjected either to experimental drought, shade or nitrogen fertilization for ~30 days. Xylem hydraulic and anatomical traits were subsequently examined in stem segments taken from two different vertical positions along the plant’s main axis. The experimental treatments affected hybrid poplar growth and development and induced changes in xylem phenotype. Across all treatments and stem segment positions, the amount of leaf area supported by the stem segments scaled linearly with their maximal hydraulic conductivity (kh), suggesting that the area of assimilating leaves is constrained by the xylem transport capacity. In turn, kh was mainly driven by the size of xylem cross-sectional area. Moreover, xylem structural and functional properties varied significantly. Xylem vulnerability to cavitation, measured as the xylem pressure inducing 50% loss of conductivity, ranged from -1.71 MPa to -0.15 MPa in saplings subjected to drought and nitrogen fertilization, respectively. Xylem transport efficiency also varied reflecting differences in vessel diameters and overall xylem network structure. Our results provide insights into the developmental and phenotypic plasticity of xylem traits in hybrid poplar and have potential implications for the propagation and nursery management of poplars. F2.1 Leaf senescence, dieback and abscission during severe drought in Populus: the interplay with stem cavitation, stomatal conductance and nutrient remobilization efficiency Maria A. EQUIZA1, Janusz J. ZWIAZEK1, Melvin T. TYREE1, 2 1. Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada 2. United States Forest Service, Northern Research Station, Burlington, Vermont, USA Leaf senescence, dieback and abscission represent a suite of drought-avoidance mechanisms that contribute to plant survival by reducing water loss, thus improving plant water status. When leaf mortality is preceded by a controlled senescence process, an important fraction of the nutrients accumulated during the life span of the leaf can also be recovered. Leaf senescence and abscission have been observed among different Populus species under drought stress. However, the interplay of these responses with stem cavitation, stomatal conductance and nutrient remobilization efficiency (NRE) remains to be understood. Three Populus species with different habitat distribution, vulnerability to cavitation and stomatal control (P. tremuloides, P. trichocarpa and P. deltoides) were grown in a greenhouse during 4 months and subjected to a progressive drought cycle that lasted 25-30 days. In P. tremuloides, leaf dieback started only after stomatal conductance (gs) reached minimum values and stem hydraulic conductivity was reduced by 60%. Leaf dieback was not preceded by any of the signs observed in seasonally-driven senescence and NRE was negligible. Observation of the fracture plane in petioles using scanning electron microscopy showed that formation of the abscission layer was completed only after plants had been rewatered. In contrast, leaf abscission in P. trichocarpa and P. deltoides started before gs had reached its minimum and stem hydraulic conductivity had been reduced by only 8%. Abscission was preceded by a pattern similar to that of seasonally-driven senescence, which led to significant levels of nutrient recovery.

 

CSPB2012 p. 45

F2.2 Methyl jasmonate decreases cell water permeability in Arabidopsis

Seong Hee LEE, Ji Young JANG, Janusz J. ZWIAZEK Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6G 2E3 Jasmonates are stress-induced hormones regulating a wide range of processes in plants. In this study, we examined the effects of methyl jasmonate (MeJA) on aquaporin-mediated water transport in Arabidopsis thaliana. When the roots of plants were subjected to 10, 20, 50 and 100 µM MeJA treatments, shoot growth was inhibited by two- to four-fold, but root growth was reduced by only 30-37% resulting in increased root to shoot ratios. Cell hydraulic conductivity (Lp) in roots of MeJA-treated and control plants was examined with the cell pressure probe. The lowest, 10 µM MeJA, concentration had no effect on Lp. However, 20, 50, and 100 µM MeJA quickly (0.5 – 1 h) and sharply decreased Lp compared with controls. The reductions in Lp in MeJA-treated plants were accompanied by decreases in the expression levels of PIP1;3, PIP1;5, PIP2;2 and PIP2;8 aquaporins suggesting their significant involvement in cell water transport in Arabidopsis roots. F2.3 Aquaporins contribute to recovery from drought-induced xylem embolism in rice leaves Uwe HACKE1, Adriana ALMEIDA-RODRIGUEZ1,2, Joan LAUR1 1. Department of Renewable Resources, University of Alberta, Edmonton, Canada 2. Current address: Department of Biological Sciences, University of Montreal, Montreal, Canada Xylem cavitation reduces hydraulic conductivity and water supply to leaves. Some plants are able to repair embolism even at negative xylem pressures, but the molecular mechanism of this process is not well understood. We studied embolism repair in rice leaves, combining hydraulic measurements with gene expression studies and immunofluorescence microscopy. Refilling at negative pressure could be important in rice plants that are subjected to unpredictable drought periods and may reduce drought-associated yield loss. Profound changes in gene expression were found while plants went through a drying-rehydration cycle. Many aquaporin genes were up-regulated during a moderate drought before xylem conductivity recovered to pre-drought values. Increased transcript abundance corresponded with higher PIP2 protein levels in drought-stressed leaves. Aquaporins accumulated in groups of xylem parenchyma cells adjacent to metaxylem vessels, phloem cells, and in parenchyma cells adjacent to protoxylem lacunae. Several genes related to trans-membrane transport processes were up-regulated during drought. Among these were genes encoding plasma membrane H+-ATPases and a sucrose transporter, suggesting a role of sucrose in creating osmotically driven water flow into embolized vessels. The gene expression and immunolabeling patterns suggest a role for aquaporins in directing water to vessels during embolism repair. Water used for refilling may originate from phloem and other sources including protoxylem lacunae. Identifying candidate genes paves the way for a more complete understanding of the refilling process. F2.4 Influence of soil compaction on the growth, daily changes of leaf water potential and gas exchange in maize and triticale seedlings exposed to flooding stress Maciej T. GRZESIAK The Institute of Plant Physiology, PAS, Cracow, Poland Seedlings of maize and triticale genotypes differing in tolerance to the flooding stress were grown in two levels of soil compaction (L-1.11 and S-1.41 g cm-3) and were flooded (F) after 4 weeks of growth. A study was carried out to determine seedlings dry matter after 3 weeks of growth under flooding and daily changes in leaf water potential (ψ) and gas exchange (Pn, E, gs) during first 3 days of flooding. Seedlings of treatments L+F and S+F comparing to treatments L and S showed a decrease in dry matter of shoot and root and an increase in the shoot to root ratio. Also in S+F treatment, a drastic decrease number and biomass of roots in the soil profile from 15 to 40 cm than L+F treatment was observed. Differences between L and S treatments in ψ Pn, E, and gs were, in most cases statistically insignificant for maize, and for triticale were statistically significant. In morning and evening hours in treatments L+F and S+F differences in ψ, Pn E and gs between resistant and sensitive genotypes were statistically insignificant. Distinct and significant differences were observed in treatments L+F and S+F, in particular for measurements taken at 12:00 and 16:00 and for maize changes were greater than for triticale.

 

CSPB2012 p. 46

Stomatal conductance (gs) was closely related to changes in water potential (ψ) which indicates the close association between stomata behavior and changes in leaf water status. The research was supported by National Science Centre (NCN) Project No. N N310 782540. F2.5 Effect of water limitation on lodgepole and jack pine defenses against the mountain pine beetle symbiotic fungus G. clavigera Adriana ARANGO-VELEZ, Charles COPELAND, Miranda MEENTS, Leonardo GALINDO, Jean LINSKY, Stephanie BOYCHUCK, Inka LUSEBRINK, Janice COOKE. University of Alberta, Department of Biological Sciences, Edmonton AB Canada T6G 2E9 The ongoing outbreak of mountain pine beetle (MPB; Dendroctonus ponderasae) and its associated pathogenic fungi (e.g. Grosmannia clavigera) had impacted more than 15 millions of hectares of pine forests, mainly lodgepole pine (Pinus contorta) in British Columbia. Since 2006, MPB has spread across northern Alberta into lodgepole x jack pine (Pinus banksiana) hybrid and pure jack pine forests. Given that lodgepole pine and MPB share a co-evolutionary history whereas jack pine is a new host for MPB, we hypothesized that there are differences in defense responses between species. Some regions affected by this outbreak have also experienced drought conditions. Accordingly, we further hypothesized that water limitation will reduce tree carbon gain, altering carbon partitioning into constitutive and induced defenses. We evaluated structural, biochemical and transcriptional responses in G. clavigera-inoculated lodgepole and jack pine seedlings in growth chambers, and in mature hybrids in field conditions, subjected to well watered or water deficit conditions. Water limitation decreased stomatal conductance and photosynthesis in both species, leading to reduced photosynthates. G. clavigera induced stem lesions in both species, however lesions developed slower in jack pine. Lesion development was altered by water limitation. Total monoterpene content increased in inoculated lodgepole pine under well-watered conditions, with individual monoterpene levels also affected. Transcript abundance of genes showed that both constitutive and induced defense responses are modulated in pines by water deficit, and this response appears to be gene-specific, differing between species. This study shows evidence of cross talk between water stress and defense responses of pine trees. F2.6 Microarray analysis of lodgepole and jack pine responses to water deficit and inoculation with the mountain pine beetle fungal associate Grosmannia clavigera Miranda J. MEENTS, Adriana ARANGO, Charles COPELAND, Walid EL KAYAL, Janice COOKE Department of Biological Sciences, University of Alberta, Edmonton, AB The ongoing outbreak of mountain pine beetle (MPB) in western Canada has already caused huge losses of forest and the beetle continues to spread beyond its historic range. Lodgepole pine (Pinus contorta) has been a preferred target of MPB in British Columbia, but as the beetle has spread eastward it has encountered new tree hosts in the form of lodgepole pine x jack pine (Pinus banksiana) hybrids and pure jack pine. It is not yet clear whether, as an MPB host, jack pine is comparable to lodgepole pine. Additionally, stressed trees are preferentially attacked by MPB at lower densities, and the years of drought experienced in Alberta over the past decade may also affect outbreak dynamics in these newly affected areas. We hypothesize that water stress decreases the ability of pine trees to defend against MPB attack in part because of reduced carbon gain. Furthermore, we theorize that jack and lodgepole pine will respond differently when faced with water stress and MPB attack. To test this, we conducted experiments on lodgepole and jack pine seedlings (in growth chambers) and mature trees (in the field; Smoky Lake, AB). To simulate MPB attack, lodgepole and jack pine subjected to either well-watered or water deficit conditions were inoculated with the MPB fungal symbiont Grosmannia clavigera. A microarray experiment is in progress to examine the effect of these abiotic and biotic stresses on transcript abundance profiles in lodgepole and jack pine seedlings, and first results from this large factorial experiment will be presented.

 

CSPB2012 p. 47

G1.1 Phosphoenolpyruvate carboxylase of proteoid ‘cluster’ roots of an Australian native plant (Hakea prostrata) is post-translationally controlled by phosphorylation and monoubiquitination Michael W. SHANE1, William C. PLAXTON2 1. School of Plant Biology, The University of Western Australia, Australia 2. Department of Biology, Queen’s University, Kingston, ON, Canada Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PEPC protein kinase in plant acclimation to nutritional phosphate (Pi) deprivation. This includes controlling the production of organic acids (e.g., malate, citrate) that are excreted in copious amounts by proteoid roots of non-mycorrhizal species such as Hakea prostrata; this enhances the bioavailability mineral-bound Pi by solubilizing calcium, iron, and aluminum phosphates in the soil. H. prostrata thrives in the extremely P-impoverished, ancient soils of S.W. Australia. Proteoid roots from hydroponically cultivated, Pi-deprived H. prostrata were analyzed over their time-course of development (~20 d) to correlate developmental changes in organic acid exudation with the activity, post-translational modifications (inhibitory monoubiquitination vs. activatory phosphorylation), and kinetic/allosteric properties of PEPC. Immunoblotting using anti-(phosphosite specific PEPC)-IgG, PEPC purification, and kinetic studies were augmented by in vitro deubiquitination and dephosphorylation experiments (using ubiquitin-specific protease and phosphoprotein phosphatase, respectively). Proteoid-root development and organic acid excretion were paralleled by in vivo deubiquitination and consequent kinetic activation of a phosphorylated ~440-kDa native PEPC tetramer. Our discovery that H. prostrata PEPC is in vivo activated by transforming from a monoubiquitinated (young roots) to a deubiquitinated state (mature roots), while maintaining its phosphorylation status across development is unprecedented in the plant PEPC literature. These results are relevant to long-term applied efforts to engineer Pi-efficient transgenic crops, needed to minimize the input of non-renewable and polluting Pi fertilizers in agriculture. (supported by Australian Research Council & NSERC). G1.2 The effect of alanine aminotransferase over-expression on the transcriptome of Oryza sativa grown hydroponically with various concentrations of ammonium Perrin H BEATTY1, Rebecka CARROLL,1,2 Ashok SHRAWAT1,3 and Allen G GOOD1 1. Department of Biological Sciences, University of Alberta 2. Current location, TEC Edmonton 3. Current location, Monsanto Biotechnology Cereal plants are poor at nitrogen use efficiency (NUE), yet they require fixed nitrogen (N) for nucleic acids, amino acids and secondary metabolism, ultimately affecting both plant biomass and grain yield. To compensate for low NUE, farmers in developed countries tend to apply excess N fertilizers, however this leads to N loss to the environment via leaching, soil microbial competition and volatization. Developing high NUE cereal crops would reduce the need for excess N fertilizer application, in turn reducing farmer costs and environmental N pollution. Tissue specific alanine aminotransferase over-expression (AlaAT-ox) confers a NUE phenotype in rice. We analyzed the transcriptomes from vegetative stage wild-type and OsANT1:AlaAT transgenic rice grown at low, sufficient and luxurious concentrations of ammonium. Using stringent comparison parameters, the numbers of differentially expressed transcripts (DET) were higher from N concentration variation than for genotype (transgenic versus wildtype) variation, although in no condition was the total DET higher than 2%. The differentially expressed transcripts from each experimental condition were compared using MapMan and PageMan freeware. The up and down regulated genes from the same genotype but different N concentrations or from the same N concentration but different genotype were mapped onto rice metabolism pathways to visualize transcriptome effects. As well, the data from multiple comparisons were analyzed for the holistic view of AlaAT overexpression and N concentration affects to the respective transcriptomes. From this analysis, and in conjunction with other data, a working model of how AlaAT over-expression results in an NUE phenotype will be discussed.

 

CSPB2012 p. 48

G1.3 Arabidopsis L-type Amino acid Transporter 4 (LAT4) is a green tissue specific transporter that exports amino acids under low carbon Rowshon A. BEGAM, Allen G. GOOD Department of Biological Sciences, University of Alberta, Edmonton, AB The distribution of amino acids (AAs) in plants is a complex process which in Arabidopsis involves more than sixty amino acid transporters (AATs). To date, approximately half of the annotated AATs have been functionally characterized. Arabidopsis L-type amino acid Transporter (LAT) family was primarily classified and named based on a phylogenetic analysis with animal LATs. In animals, LATs mediate exchange of intracellular AAs for extracellular AAs at a 1:1 stoichiometry, while in plants LATs remained uncharacterized. This report presents the functional characterization of LAT4, a member of the Arabidopsis LAT family. In addition to a basic characterization through in silico studies, heterologous expression in yeast, organ and tissue specific expression and subcellular localization, a LAT4 knock-out mutant line (lat4-1) was characterized phenotypically in various growth conditions and using radio-labelled AA-uptake studies. In Arabidopsis, LAT4 is a widely expressed AAT in the whole plant with high levels of expression in the green tissues. The functionality of LAT4 is detectable under low carbon conditions. Mutant lat4-1 shows reduced growth in normal growth medium, increased sensitivity to exogenous AAs and increased accumulation of free AAs. In planta AA-uptake studies using mesophyll cell protoplasts as well as whole plants provided evidence that LAT4 mediates AA export through a mechanism yet to be resolved. G1.4 Alanine aminotransferase: a kinetic analysis   Chandra H. McALLISTER, Michelle FACETTE, Andrew HOLT and Allen G. GOOD Department of Biological Sciences, University of Alberta, Edmonton, AB Alanine aminotransferase (AlaAT), also known as glutamic:pyruvic transaminase (GPT), is an enzyme that catalyzes a reversible reaction, transferring the amino group from alanine to 2-oxoglutarate to produce both pyruvate and glutamate. While this enzyme has been well characterized in a variety of organisms, including from eukarya, archaea and eubacteria, new interest in this enzyme has developed in agricultural sciences due to finding that tissue-specific over-expression of this enzyme in canola and rice increases nitrogen use efficiency (NUE) in these cereals in greenhouse and field trials. In order to better understand the enzymatic properties of AlaAT that produce an NUE phenotype in plants, a kinetic analysis of a variety of AlaAT enzymes was carried out. The KM values for ten AlaATs and two GGTs (glutamate:glyoxylate transaminases) were obtained for all four enzyme substrates. Furthermore, changes in growth of E. coli over-expressing the various AlaAT enzymes was analyzed under conditions of varying concentrations of 2-oxoglutarate with ammonium as the nitrogen source, in order to observe phenotypic changes as a result of AlaAT activity. These results, and the implications they may have on the overall NUE response in cereal plants are discussed.

 

CSPB2012 p. 49

G1.5 Understanding the response of the maize nitrate transport system to nitrogen supply and demand Darren PLETT1, Juergen ZANGHELLINI2, Luke HOLTHAM1, Ute ROESSNER3, Antoni RAFALSKI4, Kanwarpal DHUGGA5, Mark TESTER1 , Brent N. KAISER6 Trevor GARNETT1 1. Australian Centre for Plant Functional Genomics, Waite Research Institute, University of Adelaide, Adelaide, South Australia, 5064, AUSTRALIA 2. Austrian Centre of Industrial Biotechnology, Vienna, A1190, AUSTRIA 3. Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, Parkville, Victoria, 3010, AUSTRALIA 4. DuPont Crop Genetics, Wilmington, Delaware, 19803, USA 5. Pioneer HiBred, Johnston, Iowa, 50131, USA 6. School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, South Australia, 5064, AUSTRALIA An understanding of nitrate (NO3

-) uptake throughout the lifecycle of plants and how this process responds to nitrogen (N) availability is an important step towards the development of plants with improved nitrogen use efficiency. We characterised the nitrate transport system across the lifecycle of dwarf maize (Zea mays) plants grown with adequate and reduced N. Regardless of treatment, plants showed major changes in high affinity NO3

- uptake capacity across the lifecycle which varied with changing relative growth rates of root and shoots during growth. Plants grown with reduced NO3

- had increased high affinity NO3

- uptake capacity. Changing uptake capacity showed good correlation with amino acid levels within the plant. Our observations are being used to develop a model to describe the response of the maize nitrate uptake system to N supply and demand. G1.6 Metabolism of reactive oxygen and nitrogen species during germination of barley seeds with different levels of dormancy Zhenguo MA, Natalia V. BYKOVA and Abir U. IGAMBERDIEV Department of Biology, Memorial University of Newfoundland, St. John’s, NL, A1B 3X9, Canada Metabolism of ascorbate, glutathione, and nitrosylation process were investigated during germination of two barley (Hordeum vulgare L.) cultivars: Harrington (non-dormant) and Sundre (more dormant). The content of ascorbate was similar in embryo of Harrington and Sundre seeds and decreased slightly from 0 to 48 h after imbibition. The content of reduced glutathione (GSH) in embryo declined dramatically after imbibition. Compared to the level of reduced species (ascorbate and GSH), the content of corresponding oxidized forms (dehydroascorbate and GSSG) was very low and its fluctuation pattern was different in embryo from germinated and non-germinated seeds. The reduction potential of the GSSG/2GSH half-cell increased dramatically in embryos of Harrington and Sundre seeds within first 3 h after imbibition and then continued to increase slowly in germinating seeds, while no more increase was observed in non-germinated Sundre seeds. The activity of S-nitrosoglutathione reductase (GSNOR) in embryo of both Harrington and Sundre seeds declined gradually during 15 h after imbibition and then started to increase but not reached the level observed in dry seeds. The content of nitrosothiols in proteins extracted from whole Harrington seeds rose up four folds during 15 h after imbibition and then descended to 48 hours, a similar dynamics was observed in germinated Sundre seeds; no increase took place in non-germinated seeds. It is concluded that germinating seeds exhibit dramatic changes in metabolism of reactive oxygen and nitrogen species and these changes strongly depend on the level of dormancy.

 

CSPB2012 p. 50

G2.1 In vivo phosphorylation of SUS1, a cytosolic sucrose synthase, at Serine-11 in developing castor oilseeds Eric T. FEDOSEJEVS, William C. PLAXTON Department of Biology, Queen’s University, Kingston, Ontario Seed development requires a large influx of carbon and energy in the form of sucrose, which must be cleaved enzymatically as an initial step in the biosynthesis of storage end products. Sucrose synthase (SUS) is a key player in this process, catalyzing the UDP-dependent cleavage of sucrose into UDP-glucose and fructose. However, little is known about SUS’s role or regulation during oilseed development. The castor plant is well suited for oilseed SUS studies, as it produces large, oil-rich seeds (>60% oil by weight) and has a sequenced genome containing 5 SUS genes. RT-PCR indicated that SUS1 was the most transcriptionally-active SUS gene in castor oil seeds (COS), and that SUS1 transcripts peaked during early COS development. Levels of SUS activity and immunoreactive SUS polypeptides maximized during mid-development but became undetectable in fully mature COS. A SUS homotetramer composed of phosphorylated 93-kDa subunits was purified 170-fold to homogeneity (Sp. Act. = 3.5 units mg-1) from developing COS using FPLC, and identified as SUS1 by LC MS/MS. Immunoblots probed with an anti-(SUS1 phosphosite-specific)-IgG revealed that relative in vivo SUS1 phosphorylation at Ser11 was maximal during the early phase of COS development, whereupon it showed a progressive decline. Incorporation of 32Pi from [γ-32P]ATP into a SUS1 synthetic peptide substrate revealed abundant Ca2+-dependent SUS1 (Ser11) kinase activity in developing COS. Work is in progress to characterize the: (i) influence of Ser11

phosphorylation on SUS1’s kinetic and regulatory/allosteric properties, and (ii) Ca2+-dependent protein kinase that in vivo phosphorylates SUS1 in developing COS. (supported by NSERC) G2.2 Analysis of sucrose to starch metabolism pathway in developing grains of common wheat (Triticum aestivum L.) Shalini MUKHERJEE, Konstantin KULICHIKHIN, Anita BRULE-BABEL, Claudio STASOLLA, Belay T. AYELE

Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada Starch comprises about 65% of grain dry weight in wheat, making it the major storage reserve in this crop. During the reproductive growth of wheat, majority of the sucrose synthesized in the source tissues is transported into the developing grains, where it is used as a substrate for the synthesis of starch. To better understand the coordination of sucrose-starch metabolic pathways, this study investigates the dynamics of sucrose catabolism and starch synthesis in common wheat at both molecular and metabolite levels. Our data show that a gene encoding sucrose synthase (TaSuSy2) is highly expressed during the rapid phase of starch accumulation; reflecting that sucrose hydrolysis in developing wheat grains is catalyzed mainly by the TaSuSy2 isoform. ADP-glucose pyrophosphorylase (AGPase) has been shown previously to be a regulatory starch biosynthetic enzyme in plants. Thus, the high expression of AGPL1 and AGPS1b, encoding the large and small subunits of AGPase, respectively, during the same period of wheat grain development, suggest that these two genes contribute the majority of AGPase activity required for converting the hexose sugars to starch. Furthermore, the upregulation of TaSuSy2, AGPL1 and AGPS1b was coordinated with increased expression of specific members of genes encoding granular bound and soluble starch synthases. G2.3 DUF642, a putative galactan-binding protein from Arabidopsis thaliana involved in cell elongation. Shanjida KHAN, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, Canada The DUF642 domain consists of approximately 262 amino acids and is mainly plant specific. We previously found that DUF642 transcripts are enriched in stems of hemp (Cannabis sativa) with actively developing fibers. In Arabidopsis thaliana there are 10 proteins that contain DUF642 domains. We are characterizing two of these A. thaliana genes: AT5G25460 and AT5G11420, and have designated them as DUF642a and DUF642b, respectively. These genes show a

 

CSPB2012 p. 51

high degree of sequence similarity (86.2% identity) as well as showing similar expression patterns. These proteins are also predicted to contain a galactose-binding domain-like fold and hydrolase fold. Expression analysis of DUFa and DUFb showed that these proteins are highly expressed in hypocotyls, where no cell division occurs and where growth occurs only by cell elongation. They are also highly expressed in the top of the stem where cell elongation occurs. DUF642 contains a predicted secretory signal peptide. We hypothesize that DUF642 domain-containing proteins hydrolyze Gal-containing polymers in plant cells as a mediator of cell elongation. Characterization of the catalytic activity of a heterologously expressed DUF642 is underway. G2.4 Binding of multi-enzyme complexes to starch granules is determined by the glucan-binding capacity of starch synthase IIa Fushan LIU, Michael J. EMES, and Ian J. TETLOW Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada The present study reports a biochemical and molecular analysis of an allelic variant of maize sugary-2 mutation which expresses a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. Molecular analysis of the mutant SSIIa revealed two amino acid substitutions, one of which is a highly conserved residue and responsible for loss of catalytic activity and the inability of the mutant SSIIa to bind starch or amylopectin. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBEIIb found in wild-type amyloplasts. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. Measurement of the catalytic capacity of the soluble protein complexes showed that the total activity of SS and SBE in the sugary-2 complex was reduced to 10-20% of the respective activities of the wild-type complex. The sugary-2 mutant showed normal levels of granule-bound starch synthase I (GBSSI) but, unlike the wild-type, was devoid of amylopectin-synthesising enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix. G2.5 Completing the methionine salvage pathway: a comparative genomics approach Kenneth W. ELLENS1, Lynn G. L. RICHARDSON2, Robert T. MULLEN2, Andrew D. HANSON1 1. Horticultural Sciences Department, University of Florida, Gainesville, FL 2. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON A major fate of methionine (Met) is synthesis of S-adenosylmethionine (SAM), the universal methyl donor and the precursor of polyamines and ethylene. During polyamine and ethylene synthesis, SAM is converted to methylthioadenosine, which contains the methylthio group of methionine that can be recycled to methionine in a salvage pathway that is common to plants, animals, and bacteria. Genes have been definitively assigned to all steps except the last, a transamination that ‘closes’ the salvage cycle. In mammals, biochemical evidence implicates a specific type of transaminase (glutamine transaminase K, GTK) that forms the potentially toxic α-keto acid α-ketoglutaramate (αKGM), which is then hydrolyzed by an ω-amidase to α-ketoglutarate. Further biochemical evidence implicates the nitrilase-like protein Nit2 as an ω-amidase that acts upon αKGM. Nit2, working in concert with GTK, is thought to drive the transamination reaction irreversibly towards Met. Comparative genomics analysis reveals that Bacillus subtilis and other bacteria have genes encoding homologs of GTK and Nit2 that cluster on the chromosome with genes of the Met salvage pathway. Such clustering makes GTK and Nit2 strong candidates for the main enzymes mediating the transamination step. Plant genes encoding GTK and Nit2 homologs have been identified, and the recombinant proteins have demonstrated the expected enzymatic activities. The subcellular localization of these candidate genes is being investigated as both genes encode proteins with predicted plastid targeting sequences. Plant GTK and Nit2 genes will also be used to attempt to complement the metabolic phenotypes of GTK and Nit2 knockouts in B. subtilis.

 

CSPB2012 p. 52

G2.6 Flax stem transcriptomics: GASA gene expression during fiber development Lai TO1, David PINZON1, Yongguo CAO2, Raju DATLA2, Michael DEYHOLOS1

1. Department of Biological Sciences, University of Alberta 2. NRC Plant Biotechnology Institute, Saskatoon Bast fibers from flax phloem tissues are notable for their length (which is derived from intrusive growth) and tensile strength (derived from a high proportion of crystalline cellulose with low microfibrillar angle). To investigate bast fiber development, transcript abundance was compared at five different stages of early stem development using a CombiMatrix based microarray platform that contained oligonucleotide probes for essentially all of the predicted protein coding genes of flax. A group of genes of the GASA (GIBBERELLIC ACID-STIMULATED IN ARABIDOPSIS) family showed differential enriched expression during stages of stem development. The members of this GASA family were selected for further characterization based on their observed expression patterns in our microarray studies and previous evidence for their role in fiber development. Cross-validation of the microarray result was performed by quantitative qRT-PCR. The gene expression level of each target gene was compared and showed a significant positive correlation between microarray and qRT-PCR results in all biological replications. Further experiments will be carried out to determine the sub-cellular localization of these gene products in order to predict their function in cell wall formation during bast fiber development. H1.1 The role of ARC1 in the self-incompatibility pathway in A. lyrata Emily INDRIOLO, Pirashaanthy THARMAPALAN, Daphne GORING Department of Cell & Systems Biology, University of Toronto Self-incompatibility is a complex process in flowering plants that facilitates genetic diversity by preventing self-fertilization. In the Brassicaceae, this process is regulated by a signalling pathway initiated by the stigma-specific, S Receptor Kinase (SRK), following binding of a pollen-specific ligand, SCR/SP11. In Brassica species, downstream signalling components of the SRK pathway have been identified as the M Locus Protein Kinase, the ARC1 E3 ubiquitin ligase, and the Exo70A1 subunit of the exocyst complex. While the functions of SCR/SP11 and SRK are conserved in the related Arabidopsis species, nothing is known about the downstream signalling pathway. As a result, we set out to investigate if the role of ARC1 is conserved in regulating pollen rejection in the naturally occurring Arabidopsis lyrata self-incompatibility system. We have identified an A. lyrata ARC1 homologue to Brassica ARC1, and have generated transgenic A. lyrata with an AlARC1 RNAi construct. These plants are being tested to determine what effect knocking down AlARC1 expression has on the self-incompatibility trait. Thus, we have developed a transgenic system to investigate the self-incompatibility signalling pathway in A. lyrata. H1.2 An investigation of the cellular events during compatible and self-incompatible pollinations in Brassicaceae Darya SAFAVIAN, Daphne GORING Cell and Systems Biology Department, University of Toronto This study aims to elucidate the molecular and cellular mechanisms of polarized exocytosis in Brassicaceae. Polarized secretion has been shown in yeast and animal systems to be promoted by the eight-subunit exocyst complex which tethers secretory vesicles to the plasma membrane for fusion and exocytosis. Previously, we have found that Exo70A1, a predicted subunit of the exocyst complex, is essential in the stigma for compatible pollen-pistil interactions in Arabidopsis thaliana and Brassica napus. Based on this discovery, we hypothesize that Exo70A1 functions as part of the exocyst complex to tether secretory vesicles to the plasma membrane at the pollen attachment site to deliver essential stigmatic resources for the compatible pollen. This is thought to result in water transfer to the pollen grain for hydration as well as the expansion of the papillar cell wall to promote pollen tube penetration for the subsequent fertilization. We are interested in observing the proposed secretory activity in the stigmatic papillae following compatible pollinations at the ultrastructural level using the transmission electron microscope. To date, we have examined the presence of secretory vesicles at the plasma membrane for self-compatible pollinations in A. thaliana and B. napus, and cross-compatible pollinations in A. lyrata. In contrast, this

 

CSPB2012 p. 53

polarized secretion is expected to be absent in self-incompatible pollinations correlating with self-pollen rejection, and this was examined using self-incompatible pollinations in A. lyrata and B. napus. As well, Arabidopsis thaliana exocyst mutants were studied to determine if there are altered patterns of secretory vesicle accumulation next to the papillae cell membrane at the pollen contact site. H1.3 The microtubule plus-END-BINDING1 (EB1) protein modulates endocytosis in plant root cells Saeid SHAHIDI, Sherryl BISGROVE Department of Biological Sciences, Simon Fraser University, Burnaby, BC EB1 is an evolutionarily conserved protein in eukaryotes that preferentially binds to the fast growing ends of microtubules where it regulates microtubule dynamics. In addition to microtubules, EB1 also interacts with several additional proteins and through these interactions modulates a variety of cellular processes. Plants carrying T-DNA insertions in the EB1b, one of three EB1 genes encoded in the Arabidopsis thaliana genome, have roots that exhibit exaggerated responses to mechanical cues. In addition, the sensory columella cells in mutant root caps have vacuoles that are larger than those found in wild type root cap cells. We are investigating a possible relationship between the aberrant root responses to mechanical cues and the vacuolar defects in eb1b mutants. To determine whether there are additional architectural defects in mutant columella cells, the arrangement of the microtubule cytoskeleton and the positions of the  endoplasmic  reticulum  and  nuclei  were  examined. No differences between mutants and wild type were observed, suggesting that the defects in eb1b columella cells may be limited to the vacuole. We assessed whether the increase in vacuole size may be due to defects in the endomembrane recycling pathway. By analyzing uptake of the lipophilic dye, FM4-64, we found that the number of FM4-64-labeled compartments per cell were greater in eb1b mutants than in wild type plants. Analyses aimed at determining how the defect in the endomembrane recycling pathway in eb1b mutants could affect root responses to mechanical cues are underway. H1.4 Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria Joonho PARK1, Nicholas KHUU2, Robert T. MULLEN2, William C. PLAXTON3 1. Department of Biology, Queen’s University, Kingston, ON 2. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON Phosphoenolpyruvate carboxylase (PEPC) is a multifaceted and tightly regulated enzyme situated at a critical branchpoint in plant C-metabolism. PEPC from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical Class-1 PEPC homotetramer consists of 107 kDa plant-type PEPC (PTPC) subunits, whereas the unusual, allosterically-desensitized 910 kDa Class-2 PEPC hetero-octamer arises from Class-1 PEPC’s association with 118 kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension cells. BTPC-FP mainly localized to mitochondria, as revealed by co-immunostaining of cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP. FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal [NLS]) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. TEM of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as cytosolic. Thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Our results demonstrate that: (1) COS BTPC and PTPC interact in vivo as a Class-2 PEPC complex that associates with the surface of mitochondria, (2) BTPC’s unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (3) the PTPC-containing Class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated Class-2 PEPC facilitates rapid refixation of respiratory CO2 while sustaining a large anaplerotic flux to replenish TCA cycle C-skeletons withdrawn for biosynthesis.

 

CSPB2012 p. 54

H1.5 From root hairs to spinal neurons: tubular ER network and directional cell growth Jun CHEN, Caitlin DOYLE, Maria LIU, and HUGO ZHENG Department of Biology, McGill University, Montreal, QC, H3A 1B1 The endoplasmic reticulum (ER) is a highly conserved cellular organelle where proteins and lipids are synthesized. In eukaryotic cells, the cortical ER is organized into a tubular network, which undergoes drastic rearrangements in response to developmental cues and outside influences. However, the mechanisms underlying the ER rearrangement are not well understood. We showed that Arabidopsis RHD3 (ROOT HAIR DEFECTIVE3), originally identified in a genetic screen for mutants defective in root hair tip growth, plays an essential role in the generation of interconnected ER tubules. RHD3 is a plant member of dynamin-like atlastin GTPases. Interestingly, improper alterations in Atlsatin1 in humans cause a medical condition called Hereditary spastic paraplegia (HSP), in which the morphology of the long axons of corticospinal motor neurons is affected. Atlastin1 has also been recently shown to be primarily involved in the generation of the tubular ER network in humans. Thus Arabidopsis rhd3 has been considered as a model system for further dissection of how atlastin GTPases, including RHD3 and Atlastin1 may work in tip growth of root hairs and axonal growth of corticospinal neuron cells, respectively. Our recent progress in 1) how the function of RHD3 in the ER is regulated, and 2) how a defective formation of tubular ER network induced by altered RHD3 could cause developmental defect in directional cell growth, will be presented. H1.6 Biochemical Characterization of Two Ancient Bacterial-Like Serine / Threonine PPP-family Protein Phosphatases from Arabidopsis thaliana R. Glen UHRIG, Keaton COLVILLE, Greg B. MOORHEAD Department of Biological Sciences, University of Calgary, Calgary, Alberta, CAN Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Previously, an unique group of phosphatases, designated the Shewanella-like (SLP) phosphatases, was annotated in Arabidopsis thaliana. Amino acid sequence alignments indicated they were of bacterial origin and distantly related to modern day eukaryotic phosphoprotein phosphatase (PPP)-family protein phosphatases. Here, the bioinformatic, biochemical, and cellular characterization of this ancient bacterial-like subclass of PPP-family protein phosphatases, the SLP phosphatases, will be discussed. Results indicate SLP phosphatases are conserved across all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. In Arabidopsis thaliana (At) there exists two nuclear-encoded SLP phosphatases (AtSLP1 and AtSLP2), that when phylogenetically examined along with other plant SLP phosphatases, distinctly cluster into two groups: chloroplast targeted SLP1 phosphatases and cytosol targeted SLP2 phosphatases. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined and AtSLP phosphatase specific antibodies generated. Enzymatic analysis revealed a number of unique biochemical properties, while anti-AtSLP1 and AtSLP2 western immunoblotting demonstrated opposing tissue expression profiles. The novel biochemical properties uncovered here coupled to the conservation of SLP phosphatases across plants, renders the continued characterization of these new PPP-family protein phosphatases of particular interest. H2.1 Role of pectinesterases (PME) and pectinesterase inhibitors (PMEI) on fiber development in flax (Linum usitatissimum) David PINZON, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, Edmonton, AB L. usitatissimum is an annual eudicot for which two types are cultivated: linseed and fiber flax. The stem fibers of linseed are not generally used commercially because they are of lower quality and yield than those obtained from fiber flax. Moreover, the extraction of fibers by dew-retting is not possible in the climate of Canada. Flax fibers elongate intrusively.

 

CSPB2012 p. 55

This, the middle lamella between fibers and between fibers and surrounding tissues affects fiber elongation and also fiber extractability. The degree and pattern of methylesterification of galacturonic acid (GalA) residues in homogalacturonan (HG) influences the rigidity of the middle lamella and cell wall. Pectinesterases (PME) mediate the de-esterification of GalA in muro, in a linear or random fashion, resulting in wall rigidification, or wall loosening, respectively. Using transcript profiling assays (e.g. qRT-PCR), we confirmed the expression patterns of 66 PMEs and 76 PMEIs in different tissues and developmental stages. Based on these expression data, and phylogenetic relationships with other pectinesterases in Arabidopsis and poplar, we selected a subset of flax PMEs for further study. The overall pectinesterase activity from cortical peel tissues at different developmental stages was assessed, finding the highest expression before secondary cell wall deposition starts. The degree of methylesterification of the fibers cell wall was also assessed using pectin specific antibodies. H2.2 Physiological analysis of an Arabidopsis Calmodulin-Like protein involved in seedling establishment Kyle W. BENDER, Daniel M. ROSENBAUM, Wayne A. SNEDDEN Department of Biology, Queeen's University, Kingston, ON As sessile organisms, timely detection and response to environmental changes is of paramount importance for plant survival. Thus, plants have evolved complex signal transduction pathways to co-ordinate development and reproduction in a dynamic environment. It has become clear in recent years that the calcium ion (Ca2+) plays a particularly important role in plant response to environmental cues, and this idea is underscored by the observation that myriad external stimuli evoke cytosolic Ca2+ transients as well as the expansion of Ca2+-sensor protein families encoded by plant genomes. Our lab is interested in understanding the physiological roles of one such family, the Calmodulin-Like (CML) proteins, in Arabidopsis. Using a reverse-genetics approach, we have identified one CML which plays an important role in seedling establishment. Insertional knockout seedlings undergo non-permanent developmental arrest when grown in the absence of exogenous carbon. Furthermore, developmental arrest occurs only in light-grown seedlings, although dark-grown seedlings do exhibit a moderate impairment in hypocotyl elongation, indicating that this CML might function in a light dependent signaling pathway. Collectively, our data suggest the existence of a Ca2+-dependent signal transduction pathway mediating seedling establishment, a critical time-point in the plant life-cycle. H2.3 Mitochondrial alternative oxidase modulates leaf levels of superoxide and nitric oxide Marina CVETKOVSKA, Greg C. VANLERBERGHE Department of Biological Sciences and Department of Cell and Systems Biology, University of Toronto Scarborough, Toronto, Ontario, Canada Alternative oxidase (AOX) is a non-energy conserving branch of the plant mitochondrial electron transport chain hypothesized to modulate the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by mitochondria, but there remains sparse direct in planta evidence to support this. We used fluorescent confocal microscopy and biochemical methods to directly estimate leaf levels of superoxide (O2

-), nitric oxide (NO) and H2O2 in wild-type (Wt) tobacco and transgenic tobacco with altered levels of AOX. We found that leaves lacking AOX have increased levels of both NO and mitochondrial-localized O2

- in comparison to Wt, while levels of H2O2 were similar or lower in the AOX-suppressed plants. We also found that, upon addition of antimycin A (a Complex III inhibitor), the levels of O2

- and NO increased dramatically in Wt but not in plants overexpressing AOX. Interestingly, plants lacking AOX showed a delayed increase in O2

- and NO following antimycin A, but later the levels were again well above Wt. Based on our results, we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of single electrons from the electron transport chain to O2 or nitrite. This may represent a universal role for AOX in plants.

 

CSPB2012 p. 56

H2.4 An investigation of the alternative oxidase in the moss Physcomitrella patens Karina I. NEIMANIS, Allison E. McDONALD Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada, N2L 3C5 Alternative oxidase (AOX) is an inner mitochondrial membrane protein that introduces a branch point at ubiquinone within the respiratory electron transport chain (ETC). The AOX protein bypasses two sites of proton translocation within the ETC and as a result the yield of ATP is significantly reduced. Although AOX appears to be energetically wasteful, recent studies have revealed that AOX has a wide taxonomic distribution and is most extensively observed in the plant kingdom. AOX multigene families, gene expression, protein levels, and enzymatic activity have been thoroughly studied in a number of angiosperm species including Arabidopsis, soybean, tobacco, and rice. Although much is known about angiosperm AOXs, evidence of non-angiosperm AOXs in the primary literature is scarce and therefore is a logical starting point for comparative studies. Our bioinformatics work indicates that AOX is present within a moss, liverwort, lycopods, ferns, and many species of conifers. An analysis of these sequences indicates that the amino acids that are essential for AOX activity are conserved in non-angiosperms. Further investigation of the moss Physcomitrella patens indicates that it likely has a single AOX gene and we are currently validating this by Southern blot. To confirm the expression of AOX gene(s), we have designed specific primers and are using both reverse transcriptase PCR and PCR. We are currently measuring moss AOX protein levels by performing mitochondrial isolations, SDS-PAGE and immunoblotting techniques. We anticipate that moss will provide an advantage over other systems as future knockout experiments can examine the physiological role of AOX in these plants. H2.5 Functional investigations of intrinsically disordered proteins (IDPs): KIN1 in Arabidopsis thaliana Haiyan ZHUANG, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, Edmonton, AB Intrinsically disordered proteins (IDPs) lack a rigid three-dimensional structure and are thus known also as natively unfolded proteins. Despite their lack of regular structure, IDPs participate in various structural, catalytic, and regulatory activities within cells. Previous bioinformatics analyses have predicted that at least 25% of eukaryotic proteins are comprised mostly of intrinsically disordered regions. We used a range of software tools to evaluate the diversity of predicted IDPs within transcriptomes of more than 500 species of plants including the data accumulated to date from the1KP (One Thousand Plants) project. Among the predicted IDPs, we identified KIN1, an Arabidopsis thaliana gene that is a well-known marker of certain types of abiotic stress. KIN1 transcripts have been previously reported to increase in response to dehydration, osmotica, ABA, and low temperature. But beyond the fact KIN1 transcripts are induced by stress conditions, very little is known about this gene’s function. We expressed A. thaliana KIN1 in E.coli and purified the heterologously expressed protein. Functional characterization of this IDP, including investigation of molecular chaperone activities, is underway. H2.6 A CBL-interacting protein kinase (CIPK) interacts with Keep On Going (KEG), a RING-type E3 ligase, as part of the abscisic acid signal transduction network Wendy J. LYZENGA, Hongxia LIU, Andrew SCHOFIELD, Alexandria MUISE-HENNESSEY, and Sophia L. STONE  Department of Biology, Dalhousie University, Halifax, NS Normal cellular processes depend on the selective degradation of numerous proteins through the ubiquitin 26S proteasome system. The target specificity of the ubiquitination system is controlled by E3 ubiquitin ligases which bind to select proteins and coordinate the transfer of ubiquitin onto a lysine residue of the target protein. Proper abscisic acid (ABA) signal transduction requires the RING-type E3 ligase Keep On Going (KEG). Under normal growth conditions, KEG targets an

 

CSPB2012 p. 57

ABA-responsive transcription factor, Abscisic Acid Insensitive -5 (ABI5), for ubiquitin-mediated degradation thereby preventing transcription of ABA-responsive genes. In contrast, when ABA accumulates it induces a molecular signalling event that causes KEG to change its substrate specificity. Rather than ubiquitinating ABI5, KEG ubiquitinates itself and promotes its own self-ubiquitination and subsequent degradation. The upstream ABA induced signalling events that regulate KEG function and target specificity remain to be characterized. We have identified a CBL-interacting protein kinase (CIPK) as a KEG interacting protein. Overexpression of CIPK using a 35S promoter rendered transgenic plants hypersensitive to ABA suggesting that CIPK promotes ABA signal transduction. Results of a cell-free degradation assay demonstrated that constitutively active CIPK can promote KEG degradation. We have also demonstrated that protein phosphatase type 2Cs (PP2C), Abscisic Acid Insensitive-1 and 2 (ABI1, ABI2), can interact with CIPK. This interaction suggests that CIPK is part of the signalling cascade that connects KEG to the ABA receptor family (PYR1/PYL/RCAR) and PP2Cs which represent the core network of ABA signalling. We are currently investigating the interplay and regulation mechanisms between CIPK and KEG.

 

CSPB2012 p. 58

 

CSPB2012 p. 59

 

CSPB2012 p. 60

Poster Presentations  P01. Chemical genetic approaches to studying a putative network of microtubule-associated signalling proteins in Arabidopsis thaliana Caitlin DONNELLY1,2, Sylwia WOJAS1, Brian ELLIS2, and Geoffrey WASTENEYS1 1. Department of Botany, University of British Columbia, Vancouver, BC 2. Michael Smith Laboratories, University of British Columbia, Vancouver, BC The plant microtubule cytoskeleton is involved in directing formation of the cell wall, allowing for controlled expansion of the cell along one or more axes. The ability of the microtubule array to direct expansion is influenced by its organisation, and by the stability and dynamicity of microtubule polymers; these properties are themselves controlled by interactions with various microtubule-associated proteins. We are studying propyzamide, which is a microtubule-destabilising drug with the property of causing cells to expand in such a way that they rotate helically (“twist”) around the axes of plant organs. Twisting is also observed in plants with mutations in tubulin isoforms and in microtubule-associated proteins. Interestingly, a subset of these mutants exhibit more severe, or directionally reversed, twisting when grown in the presence of propyzamide. These include mutations in a-tubulin, in a microtubule-binding protein (MOR1), and in two proteins with a poorly-understood connection to microtubules (MAP kinase 18 and an associated phosphatase). The involvement of signalling proteins in a microtubule-related process, as well as the common responsiveness to propyzamide, suggests that these twisting mutants represent disruptions to a common signalling pathway involved in microtubule regulation. We describe the various approaches that we have used to identify new proteins that could be potential links between the signalling aspect of the pathway and microtubules. These include a microarray to study gene expression following propyzamide treatment, a mutagenesis project to create new twisting mutants, and reverse genetic experiments to study the function of MAP kinase-interacting genes identified through biochemical and bioinformatic means. P02. Elucidation of promoter elements governing a subfamily of Calmodulin-Like Genes in Arabidopsis David MAJ, Wayne A SNEDDEN

Department of Biology, Queens University, Kingston, ON Observations of stimulus-specific [Ca2+]cyt oscillations during signal transduction suggest that Ca2+ signals directly encode information. These stimulus-specific oscillations, known as Ca2+ signatures, can be interpreted by an array of Ca2+-binding sensors and effectors which subsequently regulate appropriate cellular responses. While progress has been made regarding the classic Ca2+ sensor calmodulin, less research has been directed towards the calmodulin-like family of Ca2+ sensors (CMLs). This family, unique to plants, is suspected to regulate a multitude of stress and developmental pathways; however, to date very few members of this family have had their functions elucidated by the identification of downstream targets and upstream regulators. We have focused our research upon a subfamily of CMLs (CML37, CML38, CML39), which have been shown to be significantly upregulated in response to multiple stress and developmental cues in Arabidopsis thaliana. A series of reporter-fused promoter deletion constructs in A. thaliana has been analyzed to identify regions of wound and jasmonic acid (a stress hormone) responsiveness in CML37 and CML39 respectively. Intriguingly, bioinformatic analysis shows that the CML37 promoter lacks identified wound-responsive motifs. In contrast, while the CML39 promoter is predicted to have multiple MeJA motifs empirical evidence suggests that only a handful are responsive. Ongoing work aims to identify the specific cis-elements within the CML37 and CML39 promoters, as well as, ascertain why only a few of many identical motifs is functional within the CML39 promoter.

 

CSPB2012 p. 61

 P03. Identification of serine:glyoxylate aminotransferase as an asparagine aminotransferase in Arabidopsis thaliana Qianyi ZHANG, Frédéric MARSOLAIS Asparagine is an important form of nitrogen transported to sink tissues. A previous study has shown that an Arabidopsis thaliana mutant lacking asparaginase activity develops normally, which highlights a possible compensation by another type of catabolic enzyme that is asparagine aminotransferase. Research in barley and tobacco has associated asparagine aminotransferase activity with the photorespiratory enzyme serine:glyoxylate aminotransferase. This enzyme is encoded by AGT1 in Arabidopsis thaliana. Ten-day-old wild-type seedlings were subject to a 20 mM asparagine treatment or no asparagine for 2 hours. Quantitative RT-PCR assays were used to measure the change in expression levels of AGT1 involved in nitrogen assimilation in roots. The result shows AGT1 transcript was elevated by 2-fold and the internal asparagine concentration was raised by 5-fold. In addition, recombinant N-terminal His-tagged AGT1 was purified from Escherichia coli was characterized with serine, alanine and asparagine as the amino acid donor and glyoxylate, pyruvate and hydroxypyruvate as organic acid acceptors. The enzyme activity of AGT1 with asparagine was increased by 10-fold, which is compared with the activity of AGT1 with serine or alanine under the same organic acid acceptor and conditions. Therefore, those results indicate a substrate preference for asparagine over the two other amino acids.    P04. What is the biological role of AtMPK20 in Arabidopsis? Siyu CHENG, Brian ELLIS Michael Smith Laboratories, University of British Columbia, Vancouver, CA Mitogen-activated protein kinase (MAPK) cascades form an important signal transduction system in eukaryotic organisms. The cascade module consists of three protein kinase super-families: MAPK kinase kinases (MAPKKKs), which activate MAPK kinases(MKKs), which in turn phosphorylate MAPKs (MPKs). In Arabidopsis, 20 MAPKs (MPKs) have been found which belong to two subtypes: the TEY subtype (MPK Groups A, B, and C) and the TDY subtype (MPK Group D). Very little is known about the biological roles of Group D MPKs, but the expression of one group D MPK, AtMPK20, is correlated across many microarray experiments with expression of primary cell wall CesAs – CesA1, 3, and 6 (Persson et al. 2005). This suggests that AtMPK20 may be involved in primary cell wall synthesis or associated biochemical processes. We are therefore using forward genetics and recombinant protein assays to elucidate the biological functions of AtMPK20 and help us understand signalling associated with primary cell wall formation. P05. Role of Arabidopsis RAD4/XPC in UV-damaged DNA repair Triparna LAHARI, Dana F. SCHROEDER Department of Biological Sciences, University of Manitoba, Winnipeg, MB Ultraviolet (UV) radiation is an important extrinsic genotoxic agent that poses a threat to the integrity of genomic DNA. Cells have inherent repair mechanisms that identify and repair UV-damaged DNA. These DNA repair mechanisms are either light dependent, such as photoreactivation by photolyase enzymes, or light independent, such as nucleotide excision repair (NER). The absence of such mechanisms can result in serious disorders, for instance, mutation of the human nucleotide excision repair gene, XPC, can cause the serious disease Xeroderma pigmentosum. In yeast, mutation of the XPC homologue RAD4 results in radiation sensitivity. Therefore, studying the repair of UV damage by NER will provide helpful insight into the mechanism and pathways involved in DNA repair. In our current research we are studying the role of the Arabidopsis (Arabidopsis thaliana) homolog of RAD4/XPC in nucleotide excision repair. The objectives of this research are: (i) to study the role of Arabidopsis RAD4 in NER by means of genetic loss and gain of function; (ii) to determine the effect of UV on RAD4 transcript level; (iii) to determine the interactions of AtRAD4 with other Arabidopsis NER components at both the genetic and biochemical levels.

 

CSPB2012 p. 62

P06. Screening a cDNA library for protein-protein interactions directly in planta Lan-Ying LEE1*, Fu-Hui WU2*, Chen-Tran HSU2, Shu-Chen SHEN3, Hsuan-Yu YEH2, De-Chih LIAO2, Mei-Jane FANG4, Nien-Tze LIU2, Stanton B. GELVIN1, and Choun-Sea LIN2

1. Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392 2. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan 3. Scientific Instrument Center, Academia Sinica, Taipei, Taiwan 4. Core Facilities, Institute of Plant & Microbial Biology, Academia Sinica, Taipei, Taiwan Screening cDNA libraries for genes encoding proteins that interact with a particular bait protein is usually performed in yeast. We have developed a method, based on bimolecular fluorescence complementation (BiFC), to screen a plant cDNA library against a bait protein directly in plants. In this report, we separately used nVenus-tagged Agrobacterium VirE2 and VirD2 as baits to screen an Arabidopsis cDNA library encoding proteins tagged at their amino termini with cYFP. Colonies containing approximately 2x105 cDNAs were individually arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf protoplasts eventually identified single cDNA clones encoding proteins that interact with either of the two bait proteins. T-DNA insertions in the genes represented by some of these cDNAs revealed two novel Arabidopsis proteins important for Agrobacterium-mediated plant genetic transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid. Thus, this technology can be applied to different plant species. P07. The Alberta Innovates Phytola Centre – adding value to Canada’s oilseeds Stacy SINGER, E. Chris KAZALA, Nancy CRANSTON, Randall J. WESELAKE

Alberta Innovates Phytola Centre, University of Alberta, Edmonton, AB The Alberta Innovates Phytola Centre is a multidisciplinary research program based at the University of Alberta whose vision is to be a leader in the development of novel oilseeds and specialty oils. The program is based on four research themes serving a variety of nutritional and industrial markets: 1) increasing seed oil content in canola using biotechnological approaches, 2) developing high-value bioactive oils for human and animal nutrition, 3) developing renewable, oilseed-based alternatives to petrochemicals, and 4) developing novel technologies for oilseed improvement. Currently, one of our major focus areas is applying high-throughput technology to engineer high-performance enzymes for increasing seed oil content. Phytola brings together a full spectrum of complementary expertise in molecular biology and lipid biochemistry as well as business development expertise in intellectual property issues relevant to plant biotechnology. Together with excellent laboratory facilities – a newly renovated 3000 sq. ft. space dedicated to oilseed biotechnology – the Phytola Centre is well-positioned to develop market-responsive specialty oil products in collaboration with industry. P08. Abscisic acid regulation of gibberellin metabolism in pea (Pisum sativum L.) Courtney D. NADEAU1, Jocelyn A. OZGA1, Leonid V. KUREPIN2, Richard P. PHARIS2, and Dennis M. REINECKE1

1. Plant BioSystems Group, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada, T6G 2P5 2. Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, T2N 1N4 Gibberellins (GAs) have important roles in the regulation of fruit growth and seed development. The role of abscisic acid (ABA) as a GA antagonist has been well documented in several plant processes, but the interaction between ABA and GA during fruit and seed development has not been extensively studied. In pea (Pisum sativum L.) GAs are metabolized primarily through the early 13-hydroxylation pathway (GA12→GA53→→GA19→GA20→GA1) to bioactive GA1. To understand the effects of ABA on GA metabolism, seeds were treated at two development stages with ABA using a split-pericarp technique. The expression patterns of four GA biosynthesis genes (PsGA3ox1, PsGA3ox2, PsGA20ox1, and PsGA20ox2) and two GA catabolism genes (PsGA2ox1 and PsGA2ox2) were monitored by qRT-PCR. Concentrations of extractable ABA [applied + endogenous], GA20, GA1 and their immediate catabolites (GA29 and GA8, respectively) were quantified by the isotope dilution method using GC-MS-SIM. During early seed development, which is characterized by

 

CSPB2012 p. 63

both rapid expansion of the seed coat and a pre-storage phase embryo (10-12 days after anthesis; DAA), applied ABA altered the expression levels of several GA biosynthesis genes as well as GA concentrations. This suggests that ABA may influence GA biosynthesis in both the seed coat and actively growing embryo. At a later developmental stage, one which is characterized by nutrient storage in the embryo (16-18 DAA), data suggest that ABA decreases GA biosynthesis in the embryo axis. Overall, these data suggest that ABA can regulate GA biosynthesis in a developmentally- and tissue-specific manner during pea seed development. P09. System for multiplication of Jerusalem artichoke (Helianthus tuberosus L.) Margarita R. PLINER, Limin WU, John VIDMAR, Jan SLASKI Alberta Innovates - Technology Futures, Vegreville, AB

A highly efficient system for multiplication of Jerusalem artichoke was developed by combination of different prior art methods. This system comprises steps of producing disease-free Jerusalem artichoke plants through the apical meristem isolation and regeneration; mini tuber production in vitro or propagation by vegetative multiplication in vitro; hardening in small growing environment (mini-pots).

The effects of various growth conditions and plant growth regulators upon the production of micro-tubers have been studied. As a result, it has been found that: i) In vitro tuberisation and multiplication via shoot organogenesis is genotype depending; ii) Composition of cultivation medium is essential factor for tuber production in vitro. Most important factors for mini tuber development in vitro were found to be - dithiotreitol (DTT) in cultivation media, essential for inhibiting phenol secretion by the explants; Ancymidol - a growth retardant, which inhibits gibberellins biosynthesis); BA (6- benzil amino purin) important for Jart tuberisation and organogenesis. MS media, supplemented with 9% sucrose was optimal for tuber production and growth; iii) Cultivation medium could be ether solidified medium or double layer - solid / liquid medium.

Growth conditions for each stage of mini tuber initiation, development and germination were optimized accordingly. The virus-free artichoke micro tubers were harvested in 60 to 90 days.

P10. Hormonal regulation of senescence and longevity of lily flowers: Development of a new research project for undergraduate plant physiology students Rosy SHARMA, Jarnail MEHROKE, Santokh SINGH

Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada Lilies are one example of ornamental flowers that have a very short shelf life and can be quite expensive. Attempting to increase the life span of lilies would help increase their post harvest quality. We analyzed the effects of various plant hormones, e.g. cytokinin (6-benzylamino purine, BAP), epibrassinolide (epiBL), gibberellin (GA3), auxin (IAA), thiadiazuron (TDZ) and abscisic acid (ABA) on senescence and longevity of cut flowers of Oriental hybrid lily (Lilium spp.) cultivar Sorbonne. In addition, we investigated the effects of ethephon (2-chloroethylphosphonic acid), an ethylene-producing compound and ethylene inhibitors, aminooxyacetic acid (AOA) and silver nitrate AgNO3 on senescence and abscission of cut lily flowers. Ethephon induced a rapid senescence and abscission of petals of cut lily flowers. BAP alone or in combination with epiBL delayed senescence of cut lily flowers. These experiments have led to the development of a new undergraduate research project on floral biology.

 

CSPB2012 p. 64

P11. Introduction of new experimental methods and assessment tools to improve undergraduate research experience for plant physiology students Santokh SINGH Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada It is important to introduce new experimental methodologies and biological tools to improve learning and research experience of undergraduate students in a plant physiology laboratory. I have introduced Arabidopsis thaliana mutants impaired in cellulose synthesis (rsw1-1) and microtubule assembly dynamics (mor1-1) as tools for students to develop their research projects to investigate root growth. Furthermore, students developed new research project using the Agrobacterium tumefaciens strain to introduce a gene specifying resistance to hygromycin B and a proteinase inhibitor gene (PIN) promoter fused to the β-glucuronidase (GUS) reporter gene to examine the developmentally regulated expression of the transgene in tobacco. I have also investigated the assessment of undergraduate research practices, such as open-ended research projects and short presentations in two third year plant physiology courses. The results of this project will be discussed. P12. Discovery of the COLLAPSED VESSEL gene by a reverse genetic approach in Arabidopsis thaliana Etienne GRIENENBERGER and Carl J. DOUGLAS University of British Columbia, Vancouver, BC, Canada. The appearance of secondary cell walls was a major adaptation in land plant evolution, providing crucial structural, physiological and defensive innovations. Secondary wall formation is tightly regulated by developmental and environmental cues and recently, several transcription factors have been characterized defining a basic regulatory network that controls these processes. However, our understanding of the complexity of secondary wall formation and its regulation is far from complete. In order to identify potential new transcriptional regulators of secondary wall biosynthesis and deposition, we used co-expression, phylogenetic, and reverse genetic approaches. Based on their specific conservation amongst tracheophytes and angiosperms and their specific expression patterns, we selected 24 Arabidopsis gene candidates for their putative involvement in the regulation of secondary cell wall formation. Analysis of the corresponding mutants for cell wall and growth related phenotypes led to the identification of the COLLAPSED VESSEL (COVE) gene. The cove mutant displays an irregular xylem phenotype with collapsed vessels, but lacks growth defects. COVE belongs to a small family of unknown genes in Arabidopsis and is highly conserved as a small family amongst the angiosperms. The protein lacks known domains and does not share similarity with other known proteins, but proteins encoded by all COVE homologs exhibit high structural conservation. Interestingly, conserved COVE domains are found in certain bHLH transcription factors. RT-PCR analysis showed high expression of COVE in Arabidopsis inflorescence stems while transient expression of the COVE-YFP fusion in tobacco leave revealed dual nuclear and cytosolic subcellular localization. Taken together these data suggest a role for COVE in the regulation of secondary cell wall formation.

P13. The Role of Gibberellin in Regulating Preharvest Sprouting in Barley (Hordeum vulgare L.) Lingwei LIU, Kirandeep DEOL, Zhen YAO, Belay T. AYELE Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada Grain dormancy is an important trait that prevents preharvest sprouting, germination of grains while still on the mother plant, in cereal crops. It is controlled at least partly by the balance between two phytohormones: gibberellin (GA), which promotes seed germination; and abscisic acid (ABA), which enhances the induction and maintenance of dormancy in grains. To gain insight into the role of GA in regulating grain dormancy (preharvest sprouting), this study characterized the expression of GA metabolism genes in developing, and imbibing dormant and non-dormant (after-ripened) barley grains. Our results show that HvGA20ox3, HvGA3ox1 and HvGA2ox5 are the major genes expressed in developing grains with differential temporal expression pattern, suggesting their role in modulating the level of GA during this developmental process. Imbibing dormant grains exhibited increased expression of GA inactivating genes (HvGA2ox1 and 4), whereas

 

CSPB2012 p. 65

after-ripening upregulated the expression of a GA biosynthesis gene (HvGA3ox2), implying that these GA metabolic genes are involved in controlling the amount of GA produced during imbibition, thereby inhibiting or enhancing the germination of grains, respectively. P14. Uncoupling intrinsic growth potential and phenology in intra-specific hybrids of Populus balsamifera L. Natalie RYAN, Robert D. GUY Department of Forest Sciences, University of British Columbia, Vancouver BC The geographically widespread Populus balsamifera L. exhibits large intra-specific variation in photosynthetic rates and phenology. Northern trees of the species have been shown to exhibit higher photosynthetic carbon assimilation rates (A) than trees from the south. High A in these trees has also been partially attributed to high internal conductance (gm). However, because bud set occurs earlier in northern trees they accomplish far less height growth than do southern trees. If there exists no physiological constraints for the combination of high A and long growing season in nature, the progeny of intra-specific crosses between northern and southern populations may accomplish more growth in one growing season than their parents. These trees would be considered good candidates for a tree breeding program and for use in agro-forestry projects. Full reciprocal crosses were conducted between individuals from two northern (N) and two southern (S) populations found at the extremes of the P. balsamifera range. Representative selections of progeny and parental material were planted in a greenhouse and in the field and characters including shoot elongation rate, phenology, photosynthetic rates, leaf mass per area (LMA), and internal conductance were evaluated. High variance in LMA and a relationship between A, LMA, and gm was observed. Higher variation in growth and phenological traits was found in families of progeny from N x S crosses than in representative parents. Although N x S family means show no evidence of heterosis, a number of individuals possess trait combinations useful for a variety of potential deployment areas. P15. Temperature affects the spectral properties of Pinus strobus during the autumn downregulation of photosynthesis Emmanuelle FRÉCHETTE1, Christine CHANG1, and Ingo ENSMINGER1

Department of Biology, University of Toronto at Missississauga, Ontario. In summer, dissipation of excess light energy in evergreen conifers is facilitated by dynamic changes in photoprotective xanthophyll pigments. This quickly reversible process allows for rapid adjustment of non-photochemically-quenched energy (NPQ). Changes in xanthophylls are reflected in the spectral signature of leaves, and are closely related to the reflectance-based photochemical reflectance index (PRI). In autumn, as conifers transition to winter dormancy and build-up frost-hardiness, flexible energy dissipation via the xanthophyll-cycle (qE) is gradually replaced by sustained energy quenching (qI) as the photosynthetic apparatus reorganizes. Our objective was to investigate the relationship between xanthophyll-cycle dynamics, PRI, and NPQ during the autumn transition from the qE to the qI energy quenching mode. We also aimed to investigate how elevated autumn temperature and concomitant reduced frost-hardiness might affect this relationship. In climate-controlled experiments, summer-adapted 3-yr-old Pinus strobus seedlings were subjected to either control autumn conditions with low growth temperature (11°C), or an elevated temperature autumn treatment (22°C). For a 36-day period, we followed PRI calculated from spectral measurements, as well as quantum yield of photosystem II (ΦPSII) and NPQ from chlorophyll fluorescence. We show strong correlation between PRI and ΦPSII under summer conditions, which weakens under elevated autumn temperature and disappears completely under low autumn temperature. Low temperature induced qI development, which represented a large proportion of NPQ after only 8-day cold exposure. Our results suggest that xanthophyll-cycle dynamics alone cannot explain autumn variations in photosynthetic capacity due to qI, and that there is a clear mismatch between xanthophyll-cycle dynamics, PRI and NPQ during the autumn transition.

P16. Role of nitrogen metabolism in plant salt tolerance Natalia AVERINA, Rostislav SHERBAKOV, Zahra BEYZAEI

 

CSPB2012 p. 66

Institute of Biophysics and Cell Engineering of National Academy of Sciences, Belarus

Role of the nitrate reductase (NR) in salt tolerance of barley and wheat seedlings grown for 7 – 8 days on 150 mM NaCl in presence of 20 mM KNO3 and exogenous 5-aminolevulinic acid (ALA) was studied. Salinity insignificantly (by 10 %) inhibited total NR activity, stimulated accumulation of free proline 3 times, carotenoids by 30 % and increased rate of superoxide radical (SOR) generation by 50 %. Addition of KNO3 to NaCl solution increased NR activity 2 times, stimulated proline accumulation 1.9 – 2.5 times, decreased capacity to form SOR and showed tendency to increase content of chlorophyll a and b. Plants grown on NaCl+KNO3 solution had wider and unfolded leaf blades and were higher than plants grown under salt alone. Addition of ALA (40 and 60 mg/l) to NaCl solution increased NR activity in plants by 44 and 30 % accordingly. Addition of KNO3 to NaCl+ALA solution increased NR activity, content of proline and plant growth. In such plants a value of NR activity was sum of individual effects of KNO3 and ALA on the enzyme (additive effect). This could denote independent (but possibly identical) mechanisms of KNO3 and exogenous ALA action on NR. Thus under salt stress common action of KNO3 and exogenous ALA increases activity of NR, proline content, decreases a rate of SOR generation and promote plant salt tolerance.

P17. Nitrogen regulation of RNA helicase expression Denise S. WHITFORD, George W. OWTTRIM Department of Biological Sciences, University of Alberta, Edmonton, AB Expression of the stress-regulated RNA helicase of Synechocystis sp. PCC 6803, CrhR, is altered by three known stresses, temperature, light and salt concentration. Altering the source of fixed nitrogen, either urea, ammonia, nitrate or lack of fixed nitrogen, also results in differences in the expression of the CrhR protein. In particular, CrhR expression is induced transiently by transfer to nitrate-based media at both warm (30°C) and cold (20°C) temperatures. When grown in nitrogen limited conditions, CrhR protein levels decrease rapidly in cold-stressed wild type cells. The protein levels in a CrhR mutant grown under nitrogen limited conditions do not decrease. These results indicate that nitrogen source regulates CrhR expression via a mechanism that involves proteolytic degradation. P18. RNA helicase CrhR drives temperature adaptation in cyanobacteria Albert Remus R. ROSANA1, George S. ESPIE2 and George W. OWTTRIM1 1. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 2. Department of Biology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6 The cyanobacterium Synechocystis sp. PCC 6803 encodes for an RNA helicase, crhR, whose expression is regulated by the redox poise of the electron transport chain. Here, we reported the dramatic effects of crhR knockout on the cellular morphology and photosynthetic functionality and gene expression profiles during cold stress. Mutant cells undergo rapid cessation of photosynthesis upon cold shock, showing capability of accumulating Ci but not able to fix it. Such defects resulted in a cold-sensitive phenotype, cell growth ceased, which was reflected in decreased cell size, cellular viability and DNA content. Microscopic analyses showed structural abnormalities resulting from the mutation that are further aggravated by cold stress. Both strains showed similar cold-sending response but a transient mRNA accumulation was abolished in the absence of a functional CrhR. Native CrhR protein accumulates and was stabilized in the cold and rapidly degraded at ambient temperature. Surprisingly, the truncated CrhR polypeptide produced in the mutant was expressed at all temperatures indicating the absence of regulation and possibly suggesting that CrhR is causing feedback regulation on its own expression, most prominently through proteolytic degradation. Using translational inhibitors, we have shown that the de novo synthesis of CrhR protein is not required for the cold shock response. Interestingly, CrhR protein was differentially detected in translation initiation- or elongation-inhibiting treatments, a mechanism not yet established. These data link CrhR RNA helicase activity to pathway(s) involved in photosynthesis more importantly during cyanobacterial cold adaptation. The physiological function performed by CrhR to generate these observations is under investigation.

 

CSPB2012 p. 67

P19. Influence of metal contamination on plant-water relationships: a lysimeter study Fallon M. KIRKEY, Peter RYSER Biology Department, Laurentian University, ON Plants live with multiple stressors. Due to anthropogenic sources such as metal mining, soils are contaminated with heavy metals. Usually these ecosystems are severally damaged and have poor soil structure. Meaning that they are more prone to drought. Most investigations on metal toxicity only look at the effect of the one stress and rarely investigate multiple stressors simultaneously. A factorial lysimeter study was established to investigate the effect of multiple metal contamination of 5%-slag substrate mixture (Cu and Ni , p<0.001) by mining activity in the Sudbury region on soil-plant water interactions on three newly established tree stands (red maple (Acer rubrum), white birch (Betula papyrifera), and red oak (Quercus rubra)). 144 lysimeter pots were established in factorial design within a greenhouse canopy in Val Caron, Ontario. Each lysimeter had 6 trees of the same species per pot. Results from this study have shown that plants with simultaneous stressors have an additive effect. The plant leaves transpire less resulting in higher soil moisture levels, however leaves are experiencing symptoms of drought. One reason may be due to the low root density and possible inhibition of root aquaporins. P20. Effects of sand pH and nutrition on six species of boreal plant Feng XU, Janusz ZWIAZEK Department of Renewable Resources, University of Alberta, Alberta, Canada In the oil sands reclamation areas, soil pH (exceed 8.0) is higher compared with undisturbed boreal forests (below 6.0). High Soil pH affects nutrient availability, soil microbes, and root growth. The study examined growth and physiological responses of boreal plants to sand pH to understand their limits and mechanisms of pH tolerance or intolerance. In three series experiments, blueberry (Vaccinium myrtilloides), bearberry (Arctostaphyllos uva-ursi), aspen (Populus tremuloides), dogwood (Cornus sericea),jack pine (Pinus banksiana) and white spruce (Picea glauca) were grown in sand culture. Two nutrition levels (25% and 100% modified Hoagland’s solution) as well as seven pH levels (5, 6, 7, 7.5, 8, 8.5 and 9, which controlled with acids and bases) were applied to all the seedlings for eight weeks with automatic irrigation setup. Survival, growth, morphology of the root system, gas exchange, and leaf chlorophyll content were been measured. The results show that most parameters in the six species have the similar trend, which decreased as the pH increased. For the 2 nutrition level, there is not a big different effect in the same pH level. Both aspen and dogwood grow a lot in the low pH levels with 100% Hoagland solution. Blueberry rarely grows in alkaline sand. Bearberry grow badly when pH higher than 8 especially in 100% Hoagland. We recommend growing dogwood in oil sands reclamation. P21. Impact of cadmium-induced stress on phosphoenolpyruvate carboxylase isoenzyme concentrations in Arabidopsis thaliana Ian R. WILLICK, Sheila M. MACFIE Department of Biology, Western University, London, ON

Cadmium can negatively impact plant and human health. Understanding biochemical pathways that contribute to the uptake and sequestration of cadmium in plants would give insight into how toxic metal uptake can be minimized and ultimately ensure safer crops. Plants produce metal-chelating low molecular weight organic acids, which can be exuded into the rhizosphere resulting in increased solubility and uptake of metals and/or stored in the vacuole as non-toxic metal-ligand complexes. Phosphoenolpyruvate carboxylase (PEPC) catalyzes an anapleurotic pathway feeding into the tricarboxylic acid (TCA) cycle, increasing the production of malate and other metal-chelating organic acids. The purpose of this study is to compare responses to cadmium-stress in wildtype and three knockout mutants of Arabidopsis thaliana; each mutant lacks one of the PEPC isoenzymes (AtPPC1—AtPPC3). In wild type Arabidopsis thaliana (cv. Columbia), the genes encoding each of the PEPC isoenzymes were up-regulated in response to 1 and 5 µM CdCl2 relative to plants grown with no cadmium, and the plants did not show visible symptoms of cadmium toxicity. Transcription of AtPPC1—AtPPC3 in the

 

CSPB2012 p. 68

mutant plants will be evaluated, as will PEPC production, enzyme activity, cadmium uptake, organic acid content and cadmium-induced stress of all four plant lines. Measured changes in PEPC isoenzyme transcription and activity can then be compared to changes in physiology. This knowledge will be relevant to agricultural bioengineers involved in creating crops with an improved resistance to cadmium-induced stress as well as reduced uptake of cadmium.

P22. Overexpression of a poplar PIP2 aquaporin in poplar Kapilan RANGANATHAN1,2, Walid EL KAYAL2, Janice E.K. COOKE2 and Janusz J. ZWIAZEK1

1. Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada 2. Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada In plants, most of the symplastic water transport occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable and water stress conditions still remains unclear. This study was aimed at producing transgenic poplar over-expressing the poplar aquaporin PIP2:5 and to compare the physiological properties of the transgenic poplar with the wild type. PIP2:5 cloned from Populus trichocarpa × deltoides H11-11 was selected as it was previously confirmed in a previous study to function as a water-transporting aquaporin and also showed increased expression under water limitation. The coding sequence of PtdPIP2:5 was cloned into the expression vector pCambia1305.2, under the control of the Ubiquitin promoter. Agrobacterium mediated transformation was carried out to introduce the construct into the in vitro grown Populus tremula × alba INRA Clone 717 1B4. Rooted seedlings of transformed poplar were transferred to the growth chamber and transformed lines were confirmed by PCR using primers against the hygromycin resistance gene. qRT-PCR will be used to distinguish the expression pattern of the transformed lines. To help understand the role of PtdPIP2:5, the physiological and biochemical properties of the PtdPIP2:5 over expressing lines will be compared with the wild type in different drought and water recovery conditions.    

P23. Photoperiodic competency for dormancy induction in Populus balsamifera (balsam poplar) Li ZHANG, Robert D. GUY

Faculty of Forestry, University of British Columbia, Vancouver, Canada

Bud dormancy is an important overwintering mechanism for woody perennials and is induced in most species during late summer by an increase in night length and/or low temperature. Adaptively, however, it would be important for newly flushed growth to avoid responding prematurely to similar conditions that occur in early spring. A growth chamber experiment was conducted on four genotypes of balsam poplar, originating from two latitudes, by moving rooted cuttings into an inductive short-photoperiod at weekly intervals after flushing at either 15 or 20C. Plant height was measured every other day. Plants were harvested weekly for RNA extraction and transcriptome sequencing to propose candidate genes for qRT-PCR (pending). Analysis of height growth cessation data showed that before a certain age, no matter how soon plants were transferred to short day conditions, they continued to grow without interruption until they became competent to respond to photoperiod. Once competency was attained, all previously transferred cuttings of any given genotype ceased growth simultaneously and proceeded to set terminal buds, while plants not transferred into short-day conditions kept growing. The different genotypes became competent at different times, indicating possible genetic variation in this trait. Temperature did not seem to influence competency development. This research is meaningful in terms of offering insight into the risks of global warming to temperate and boreal trees. By advancing dates of bud burst, earlier springs may result in plants becoming competent to respond to photoperiod too early, causing premature height growth cessation and loss of productivity.

 

CSPB2012 p. 69

P24. Plant morphological and physiological responses to toxic metal stress when grown in the presence of different plant growth-promoting bacteria Melanie P. COLUMBUS, Sheila M. MACFIE Department of Biology, Western University, London, ON Accumulation of toxic metals in soil is an increasing concern for human health and food safety. Plants and soil microbes can remove soil contaminants efficiently, especially when they are able to grow under stress. Some plant growth-promoting bacteria (PGPB) are able to produce the ethylene-metabolizing enzyme 1-aminocyclopropane -1-carboylate deaminase (ACD) and beneficial plant hormones such as indole-3-acetic acid (IAA), which may increase plant survival and contaminant removal. The objectives of this project are to determine which aspects of plant growth and health can be altered by PGPB containing different combinations of these pathways, and to determine the effectiveness of each pathway under varying conditions of metal stress. This was accomplished by growing Arabidopsis thaliana in the presence of PGPB containing ACD and IAA pathways, only the IAA pathway, or no PGPB at cadmium concentrations ranging from 0 to 10.0 µM CdCl2. Many aspects of plant health believed to be affected by ethylene and IAA were measured, including root morphology, root and shoot biomass, leaf area, chlorophyll content, and photosystem II efficiency. The concentration of Cd in both root and shoot tissue was measured using ICP-MS. Finally, RT-PCR was used to determine the up- or down-regulation of important ethylene and auxin genes. Preliminary results suggest that not all morphological and physiological measures are affected by PGPB. Also, at a low stress level the IAA producing PGPB is more effective at maintaining plant growth than the ethylene metabolizing PGPB but the opposite is true at high stress.

P25. Impact of Solution Culture pH on Selected Boreal Tree Species for Oil Sands Reclamation Wenqing ZHANG, Janusz ZWIAZEK Department of Renewable Resources, University of Alberta, Edmonton, AB In some of the oil sands reclamation areas, soil pH is higher than in comparable soil horizons found in undisturbed boreal forests in the region. Potential differences in pH between reclamation soils and that of undisturbed forest soils can profoundly affect forest productivity. In the present project, we conducted experiments to investigate the mechanisms of reported differences in high pH tolerance in four dominant tree species of the northeastern boreal forest in Alberta. In the controlled-environment experiments, we compared growth, physiological parameters and micronutrient tissue concentrations in white spruce (Picea glauca), tamarack (Larix laricina), aspen (Populus tremuloides), and jack pine (Pinus banksiana). The plants were grown in solution culture with a continuously adjusted pH ranging from 5.0 to 9.0 for eight weeks. Our results demonstrated that only white spruce can grow relatively well in all pH levels, while for aspen, jack pine and tamarack both their growth and physiological parameters significantly decreased at pH higher than 6.0. We also found that the chlorisis of aspen and tamarack leaves at high pH are probably because of Fe or Mn deficiency especially in new leaves. An inhibition of cell elongation in roots was found at high pH in jack pine. Results in this investigation indicate that monitoring soil pH in oil sands reclamation areas is needed and pH tolerant species such as white spruce may be considered for planting where soil pH is high and vegetation performance is poor. We recommend that findings in this investigation be further tested in soil environments.

P26. Engineering nitrogen use efficiency in rice using a novel rice promoter to drive over-expression of barley AlaAT Yee Ying LOCK, Perrin H. BEATTY, Allen G. GOOD Department of Biological Sciences, University of Alberta, Edmonton, AB Nitrogen (N) is the primary rate limiting factor to plant growth and development and is an essential component to agriculture and food production. With the global population projected at 8.9 billion people by 2050, the production of sufficient food to feed the growing population has become a major economical and environmental concern. Cereal crops tend to be poor at N use efficiency (NUE), taking up approximately 50% of the N fertilizer that is applied. The remaining N fertilizer is lost to the plant by leaching, microbial use, and volatization, causing severe environmental impacts. The

 

CSPB2012 p. 70

development of NUE cereal crops may reduce both the detrimental environmental effects and food production costs. Many research efforts have been put into developing NUE cereal crops using transgenic. However, these efforts were met with limited success because most previous transgenic studies focus on the use of highly regulated primary N metabolism genes driven by constitutive promoters. The use of constitutive promoters can be energetically unfavorable because it expresses the transgene over all developmental stages and organs. Using previous microarray experiments and bioinformatics data mining, the novel PBpr1 rice promoter was selected, cloned and coupled to drive barley AlaAT for over-expression in rice. PBpr1::AlaAT plants exhibit higher seed yield, biomass and tillering compared to untransformed rice plants. Molecular studies showed elevated levels of barley AlaAT mRNA and protein. In addition, transgenic shoots exhibited high levels of AlaAT activity at all sampled times while root AlaAT activity increased after active tillering indicating developmental regulation.

P27. Gene expression in water-deficient finger millet (Eleusine corcana) Anupreeti RAMADOSS, Laki GOONEWARDENE, Hari D. UPADHYAYA, C.L. LAXMIPATHI GOWDA, Nat N.V. KAV, Michael K. DEYHOLOS 1. Department of Agricultural, Life, and Environmental Sciences, University of Alberta, Edmonton, AB 2. Department of Biological Sciences, University of Alberta, Edmonton, AB 3. International Crops Research Institute for the Semi Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh, India

Finger millet (Eleusine corocana) is an important cereal crop in many parts of the developing world. A preliminary survey of the physiological responses of finger millet to water limitation under laboratory conditions was conducted using 84 diverse accessions from the ICRISAT collection. Water was withheld from soil-grown plants 26 days after seedling (DAS). On the basis of this screen, eight accessions were selected for further characterization. These included accessions which, under our conditions, appeared highly susceptible (S1, S2, S3, S4) moderately susceptible (M1, M2), or relatively tolerant (C1, C2) to water withholding. We measured proline content, relative water content (RWC), anthocyanin content, chlorophyll content, ion leakage, and net photosynthetic rate. These experiments have shown that finger millet genotypes vary in their responses to water deficit stress. Characterizing the genes expressed under water deficit stress will give a better understanding of the drought inducible genes related to various signaling components and gene sets involved in the protecting cellular function.

P28. Abiotic and biotic factors affecting field performance of flax (Linum usitatissimum L.) under conditions of the Northern Prairies Jan J. SLASKI1, Cecil L. VERA2 1. Bioresource Technologies, Alberta Innovates - Technology Futures, Vegreville, AB 2. Agriculture and AgriFood Canada, Melfort, SK Flax is adapted to and primarily grown in the southern portion of the Canadian Prairies. Soil and climatic conditions prevalent in the Northern Prairies, particularly cooler temperatures and shorter growing season, could affect plant performance including emergence and seedling vigor, nutrient requirements and uptake or susceptibility to soil pathogens that will ultimately lead to reduction of grain yield. Three varieties: CDC Bethune, Prairie Grande and Nulin 50 were tested at Vegreville, AB and Melfort, SK. Biotic and abiotic stresses adversely affected crop performance. In plants seeded in early May, killing frost and severe spring drought resulted in poor plant establishment and grain yield 25% lower than crop seeded in early June. Flax responded well to increased rates of nitrogen supplied as urea; however, no yield advantages could be identified when using slow release fertilizer (ESN) as the only source of N. The application of Headline EC, a fungicide registered to control Septoria linicola causing pasmo, increased grain yield by up to 30% of control, despite the fact that only mild symptoms of disease were observed. Elucidation of physiological mechanisms contributing to this phenomenon should be warranted. When seedling establishment was moderately compromised by adverse biotic and abiotic factors (i.e. frost or herbicide injury, fungal disease, low seeding rate, deep seeding), flax plants were able to compensate for

 

CSPB2012 p. 71

lost grain yield by extensive development of secondary boll-bearing tillers. Our data indicate that better adapted, very early maturing cultivars would allow wider adoption of flax in the Northern Prairies.

P29. Comparison of aluminum tolerance mechanisms in two Madeira wheat cultivars using 2D electrophoresis and molecular markers José Filipe T. GANANÇA1, Teresa M. M. DOS SANTOS1, Miguel Ângelo A. PINHEIRO DE CARVALHO1, Janusz ZIMNY2, Neil S. HARRIS3, Gregory J. TAYLOR3, Jan J. SLASKI1,4 1. ISOPlexis Germplasm Bank / Germobanco, University of Madeira, Portugal 2. Plant Breeding and Acclimatization Institute, Radzikow, Poland 3. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada 4. Bioresource Technologies, Alberta Innovates - Technology Futures, Vegreville, Alberta, Canada The Portuguese Archipelago of Madeira, located 630 km west of the coast of North Africa, has soils of volcanic origin and low pH. These soil conditions make local wheat germplasm a potential source of genetic material for study and improvement of aluminum (Al) resistance in this crop. Over 50 wheat populations of local landraces were screened under Al stress, and several have shown high tolerance. Populations of two wheat cultivars showing different Al sensitivity have been chosen to study the mechanistic basis of Al tolerance. Two lines, one for each cultivar, were selected until the F3 generation. Double haploid lines were also obtained for the two cultivars. All generations of selected and double haploid lines of these cultivars were screened for Al tolerance in hydroponic conditions using erichrome cyanine root staining, root elongation, and callose production tests. We recorded differences in the Al tolerance response in these two cultivars, indicating that different Al tolerance mechanisms may be involved. Plant material from F0, F3 and DH lines from these cultivars will be used to study the physiological and molecular responses to Al stress. Efforts are being made to identify changes in root protein expression patterns under Al stress, assessed by 2D electrophoresis. Simultaneously, a preliminary molecular screening in the two selected cultivar lines for alleles of the wheat Al tolerance gene TaALMT1 was initiated, using the SPF and CAPS markers. Preliminary data obtained from these analyses will be presented.

P30. Functional analysis and phylogenic classification of aquaporins in the ectomycorrhizal fungus Laccaria bicolor Hao XU1, Janice COOKE2, Walid EL KAYAL2, Janusz ZWIAZEK1 1. Department of Renewable Resources, University of Alberta, Edmonton, AB 2. Department of Biological Sciences, University of Alberta, Edmonton, AB Although the contributions of ectomycorrhizal associations to tree water relations have been recognized, the processes of water uptake by ectomycorrhizal fungi and its transport to host plants are not well understood. The transmembrane water transport via aquaporins in both plant roots and fungal mycelia likely plays a crucial role in regulating water transport of mycorrhizal associations to balance the rates of plant water loss and water uptake. The present study aims to functionally characterize aquaporins in Laccaria bicolor, an ectomycorrhizal fungus that forms associations with a wide range of plant hosts. Six of the seven aquaporin genes identified in the L. bicolor genome were cloned from the native L. bicolor strain UAMH8232. In silico analysis showed that deduced amino acid sequences representing the six genes had secondary structure and signature motifs typical of canonical aquaporins. Phylogenetic analysis of L. bicolor aquaporins together with 221 aquaporins from a diversity of fungal species indicated that JQ585592 (identified by NCBI accession number) was categorized into Cluster I (orthodox aquaporins); JQ585593 was classified into Cluster II (aquaglyceroporins); and JQ585594, JQ585595, and the isoforms JQ585596 and JQ585597 fell into Cluster IV (facultative aquaporins). Facultative aquaporins presumably have diverse capacity to transport water and small neutral molecules to different extents. Water transport functional assays for these six aquaporins have been carried out using the Xenopus oocyte expression system. Fungal aquaporins demonstrating higher water permeability in expression and co-expression assays are being further investigated for possible roles in regulating water transport in mycorrhizal plants colonized by L. bicolor.

 

CSPB2012 p. 72

P31. Mitigation of drought stress in transplanted trees by mycorrhiza fungi inoculation Josh H KERVIN School of Environmental Science, Guelph University, Guelph Ontario Canada A common affliction faced by newly transplanted trees is water stress probably resulting from the substantial root loss when dug. Popular nursery tree species were transplanted into a field and half were inoculated with a proprietary blend of mycorrhizal fungi. The blend was comprised of 9 endomycorrhizal fungi and 11 ectomycorrhizal fungi. The water status of the transplanted trees was monitored using in situ stem psychrometers measuring total water potential throughout periods of naturally imposed drought stress. The water status of trees inoculated with the mycorrhizal fungi exhibited significantly less water stress during extended dry spells. The field trials were initiated as part of the requirements to achieve CFIA registration of the product in which efficacy claims related to mitigation of drought stress had to be demonstrated. The field trials were also used to test newly developed automated datalogger interfaces for the stem psychrometers. The automated system allowed minimum temporal resolution of stem water potential of 10 minutes and provided detailed assessments of environmental effects on plant water status.      P32. Identification of single nucleotide polymorphisms to distinguish willow cultivars (Salix spp.) used in bioenergy applications Patrick LO1, Derek SIDDERS2, Janice COOKE1 1. Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada 2. Northern Forestry Centre, Canadian Forestry Service, Natural Resources Canada, Edmonton, AB, Canada Shrub willows (Salix spp.) are increasingly being grown as a short-rotation woody crop for various applications, including bioenergy. A reliable, cost-effective method to identify and distinguish between different willow cultivars is useful for willow growers and others involved in agriculture and silviculture, for use in clone verification and certification. The objective of this project is to develop a set of single nucleotide polymorphism (SNP) markers that can be used to discriminate between a number of commonly-used willow cultivars representing several different species and hybrids. As a starting point, Illumina next generation sequencing was carried out on shoot tip cDNA from eight willow clones representing six distinct species. Using this sequencing data, candidate SNPs were identified using the reference mapping and SNP detection tools of the CLC Genomics Workbench bioinformatics software. Reciprocal reference mapping was also performed to ensure each candidate SNP was present in at least 3 reference mappings. 36 candidate SNPs meeting marker development criteria were identified in silico from this training set, and are currently being validated by sequencing of amplified flanking genomic regions from each of the 8 willow clones. Validated SNPs will also be tested in a panel of 17 additional willow cultivars, and further next generation sequencing is planned to expand the set of diagnostic SNP markers available to distinguish between a greater number of willow clones. P33. Differential expression of proteins during seed germination in the wild type and abiotic stress resistant 7B-1 mutant of tomato (Solanum lycopersicum): a proteomic analysis Alan J. HIEBERT, Vipen K. SAWHNEY Department of Biology, University of Saskatchewan, Saskatoon, SK The initiation of plant growth and emergence from the seed is in part dependent on protein reserves in both the embryo and endosperm, more so under stress conditions. By using the proteomic approach, proteins associated with abiotic stress resistance in seed germination were investigated in a stress tolerant 7B-1 mutant and wild type (WT) of tomato. Proteins were extracted from the mutant and WT seeds from one to 120 hours post-imbibition with or without exposure to mannitol, an osmotic stressor. Two-dimensional fluorescence differential in-gel electrophoresis (2-D DIGE) of proteins extracted by the phenol method was used to analyse protein expression and differentially expressed proteins. Over 1700 protein spots were detected for each gel. At one hour post-imbibition 7 spots and at 48 hours 9 spots in the control, and 13 spots in the presence of mannitol at 48 hours showed 2-fold or greater volume ratio between WT and mutant. Proteins identified by MS+MS/MS included a LEA-like protein, a cystatin-like protein, and a storage protein. LEA proteins are known for

 

CSPB2012 p. 73

acquisition of stress resistance and cystatins and storage proteins have both previously been implicated in stress tolerance. These findings provide the proteomic basis of stress resistance in seed germination of the 7B-1 mutant. In addition, differences in protein profiles were observed in DIGE gels at one hour, 48 hours, and 120 hours post-imbibition which was in part related to protein mobilization during the germination period.

P34. Remap, Reuse, Remask: Annotating a loblolly pine cDNA microarray to extend its use to lodgepole pine and jack pine Dominik ROYKO, Janice COOKE

Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada Microarrays are useful tools for analysis of relative gene expression. Typically, a microarray will be designed for samples from a single species. Arrays can also be used for closely related species, provided that the length and similarity of the probe and target sequences is sufficiently high. Use of a pre-existing heterologous microarray can lead to significant  cost and time savings. An existing cDNA microarray, custom designed and validated for 25,000 loblolly pine (Pinus taeda) sequences, was used for lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana). To assess the suitability of the existing design, the loblolly pine cDNA sequences were compared with transcriptome assemblies for lodgepole and jack pine. Based on the similarity and length of matches between probe and target sequences, masks were created to exclude from analysis any spots where poor or ambiguous hybridisation was predicted. New annotations were then generated for the microarray for each target species, based on the matched target sequences from the transcriptome assemblies. Given that mismatches are inevitable with heterologous microarrays, we expect that in some cases more than one target cDNA will hybridize to a probe. Thus, rather than providing a single “best single match” between species, we allowed for more than one highly related lodgepole or jack pine sequence to assign to a single loblolly pine cDNA. Rather than matching gene-to-gene between species, groups of related genes were identified. This approach extends the use of the existing microarray design, while offering a novel approach to comparative genomics studies.

P35. Detached leaf common bacterial blight susceptibility assay for common bean and Arabidopsis Denise M. COOPER1, Greg PERRY1, Weilong XIE2, K. Peter PAULS1

1. Department of Plant Agriculture, University of Guelph, Guelph ON 2. Agriculture and Agri-Food Canada, Guelph ON Common bacterial blight is a serious disease of common bean (Phaseolus vulgaris L.) caused by the bacterial pathogen Xanthomonas axonopodis pv. phaseoli (Xap). As part of efforts to isolate and characterize major resistance genes from the resistant common bean variety OAC-Rex, candidate genes will be cloned into Arabidopsis thaliana, which is susceptible to Xap, to test for the ability to confer resistance to the pathogen. Common bean leaves are generally inoculated while on the plant to test for resistance. However, inoculation of detached leaves maintained in tissue culture would provide a more controlled environment for the determination of disease susceptibility. The objective of this study was to test if a detached leaf assay would provide sufficient indication of susceptibility to Xap. Leaves from Arabidopsis, susceptible common bean cultivar Nordica and resistant common bean cultivar OAC-Rex were detached from the plants and inoculated. Inoculums consisted of two controls (water and 10 mM MgCl2) and four different Xap isolates (12, 18, 98, and 118). The response was shown by determining colony forming units from inoculated tissue samples that grew on XCP media plates at different times post inoculation (0, 48, 96, 144 and 192 hours). PCR, using Xap-specific primers, was performed to confirm the identity of colonies grown from inoculated tissues. Image analysis was used to determine the extent of symptomatic lesions on the leaves. It is hoped that this assay will allow for the rapid assaying of resistance to Xap and will provide a more controlled alternative to plant inoculation.

P36. Cellular and Molecular Basis of Plant-Plasmodiophora Interaction

 

CSPB2012 p. 74

Jiangying TU, Peta BONHAM-SMITH & Yangdou WEI Department of Biology, University of Saskatchewan, Saskatoon, SK Plasmodiophora brassicae (Woron.) is the causal agent of clubroot in the agriculturally important Brassicaceae family, and is an emerging threat to Canadian canola (Brassica napus) production. Because of the longevity of resting spores in soil and the obligate nature of this pathogen, a satisfactory control measure has yet to be established. Furthermore, little is known about P. brassicae at the molecular level as its obligate biotrophic nature also hampers the application of typical experimental systems for the study of the molecular mechanism of pathogenesis. In this project, I aim to 1) exploit the model Arabidopsis-P. brassicae pathosystem to investigate the cellular events during infection development, and 2) isolate, identify and characterize P. brassicae effector genes, from a cDNA library, to investigate the molecular mechanisms of obligate intracellular parasitism. The project is based on establishing a pure culture system for clubroot. Expected data analysis will enhance our understanding of the biology of the pathogen and the mechanism of pathogenesis. The ultimate goal of the project is to develop novel sources of resistance and other disease control measures. P37. Characterization of agglutinin domain-containing proteins in flax Kashfia FARUQUE, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, Edmonton, Alberta. The recent sequencing of flax genome has provided us with tools to gain new insights into this economically important crop grown mainly for its seed oil and stem fibers. The sequence analysis resulted in a prediction of the presence of 19 agglutinin domain (PFAM PF07468) containing proteins in flax. Agglutinins are carbohydrate binding lectin proteins that are well known defense proteins of plants against insects and pathogens. Interesting fact is that plants like cassava, poplar which are close relatives of flax are not enriched for these proteins. This study was conducted to characterize these proteins of flax and determine their potential function. The phylogenetic analysis of these proteins revealed that flax has a unique group of agglutinin domain containing proteins that does not fit with the already classified lectin domain families. These proteins in flax can be separated into 4 groups based on the sequence similarity of their agglutinin domains. Analysis with ‘SignalP 4.0 server’ found no evidence of signal peptides in any of these proteins. Thus they are probably cytoplasmic proteins. The expression patterns of these genes were determined from EST libraries and by QRT-PCR. They were found to be expressed in stem, leaves, cotyledons, roots, floral buds and embryos. So the potential functions of these flax agglutinins include defense against predators and pathogens, seed storage, symbiotic recognition, vegetative and reproductive growth and embryogenesis. Further studies will be conducted in order to determine the function of these proteins in flax. P38. Multiple effects of simulated mountain pine beetle disturbance on seedling growth of lodgepole pine and white spruce Paul W. CIGAN1, Gregory PEC2, Justine KARST2, Nadir ERBILGIN1, Janice COOKE2, JC CAHILL2 1. Department of Renewable Resources, University of Alberta, Edmonton, AB 2. Department of Biological Sciences, University of Alberta, Edmonton, AB Mountain pine beetle (Dendroctonus ponderosae; MPB) outbreaks result in mortality of mature pines, leading to increased levels of understory light and litter input, altered soil nutrition, and a potential influence on the diversity and function of mycorrhizal fungal communities. Because ectomycorrhizal communities are critical for establishment and growth of conifer seedlings, we hypothesize that MPB-associated changes to ectomycorrhizal community composition impact seedling regeneration, with implications for ecological resilience in post-outbreak forests. The goal of this study is to test the effects and interactions among light, litter, and inoculation of ecotomycorrhizas on the growth of seedlings of lodgepole pine (Pinus contorta Douglas ex Louden) and white spruce (Picea glauca (Moench) Voss). We sowed seeds from each species separately into 1 L pots, and thinned to one individual per pot after one month. We added and mixed into each pot 20 ml of field-collected soil from two lodgepole pine-dominated stands that lacked MPB infestation and two that displayed severe MPB infestation. Using the same collection scheme, we added to each pot 12 grams of surface collected litter. Light treatments comprise two light levels: ambient light and shade-cloth modified light (i.e., 65% shade). Results from a measurement trial performed 4 months post-germination show that seedling height, canopy diameter, and bud development

 

CSPB2012 p. 75

stage increased for both species when grown with fungal inoculum. Future measurements will include these as well as measures of photosynthesis, duration of active growth and harvested biomass. Characterization of the ectomycorrhizal community using morphological and molecular assessments will also be performed. P39. Localization of proanthocyanidis and flavonols in developing highbush blueberry (Vaccinium corymbosum L.) fruit Alena JIN, Jocelyn A. OZGA Plant BioSystems Group, Department of Agriculture, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada Blueberry fruit have been the focus of much attention due to numerous reports of their positive effects on human health. These benefits are generally attributed to high level of polyphenolics, in particular the flavonoids. Three common types of flavonoids that accumulate in blueberry fruit are the flavonols, anthocyanins and proanthocyanidins (PAs). Anthocyanins are pigments that can easily be localized in fruit tissues by visual assessment. However, to date, there are few reports of localization of PAs and flavonols in the fruit as histochemical methods and specific staining reagents are required. Using these methods, we observed that in very young fruit PAs accumulated in all the ovary tissues as well as in developing seed coats, but that as the fruit mature, PAs in the ovary primarily were concentrated in placentae and the exocarp (skin). Flavonol localization and accumulation during fruit development in general paralleled that of PAs. We also detected flavonols in the seed coat, and in the epidermal layer of the seed coat there appeared to be redistribution of flavonols toward the cell periphery in early seed development. The specificity of staining to specific cell types emphasizes that these flavonoid pathways are controlled in both time and space in the fruit and seeds, and suggests important functions for these compounds during fruit development. P40. Functional analysis of a putative regulator of proanthocyanidin synthesis in poplar, MYB115 Amy M. FRANKLIN, Kazuko YOSHIDA, C. Peter CONSTABEL Centre for Forest Biology, University of Victoria, Victoria, BC Proanthocyanidins, also known as condensed tannins, are wide-spread plant polymers synthesized via the flavonoid pathway. Proanthocyanidins influence the quality and flavor of fruits and plant-derived products such as tea and wine, and have many positive effects on human health. As they are often induced by biotic and abiotic stresses, in plants proanthocyanidins may have functions in defense and stress responses. Transcriptional regulators of proanthocyanidin synthesis have been identified from a number of plants including grape, persimmon and poplar. An R2R3 MYB transcription factor, MYB134, was recently shown to be a stress-responsive regulator of proanthocyanidin synthesis in poplar (Mellway et al., 2009, Plant Physiol 150: 924-941). Through microarray analysis of MYB134 overexpressing plants, a second putative regulator of proanthocyanidin synthesis was identified: the R2R3 MYB transcription factor, MYB115. In this study, MYB115 is being characterized using a functional approach. The dual-luciferase transient assay was employed to test direct regulation of flavonoid biosynthetic gene promoters by MYB115. Preliminary assay results indicate that MYB115 transcriptionally activates the promoter for a proanthocyanidin-specific biosynthetic enzyme, anthocyanidin reductase, similarly to MYB134. This indicates that MYB115 is likely a direct regulator of proanthocyanidin synthesis. MYB115 overexpressing plants have been generated and show increased accumulation of proanthocyanins in leaves as shown by DMACA staining, suggesting that MYB115 overexpression results in the increased expression of the major flavonoid biosynthetic genes leading to the synthesis of proanthocyanidins. Our results suggest a role of MYB115 in the regulation of the proanthocyanidin pathway. P41. The effect of flavonoids on cuticular wax loads in Arabidopsis thaliana Brittany A. WILLIAMSON1, David A. BIRD1,2 1. Department of Chemical and Biological Sciences, Mount Royal University, Calgary, AB

 

CSPB2012 p. 76

2. University of Calgary, Calgary, AB The plant cuticle is composed of cutin, a polyester matrix made up of C16 and C18 fatty acid derivatives, and waxes, very long chain (>C20) fatty acid derivatives. It functions to protect plants from water loss, pathogens, and UV radiation. However the principle UV protectant in plants is anthocyanin. In Arabidopsis thaliana, the biosynthesis of flavonoids, including anthocyanin, proceeds from the condensation of coumaroyl-CoA and mevalonate to produce naringenin chalcone. This reaction is catalyzed by chalcone synthase, encoded by TRANSPARENT TESTA4 (TT4). Naringenin chalcone is subsequently converted to chalcone by chalcone isomerase, encoded by TT5. In addition to loss of flavonoids, tt5 mutants show a ~25% reduction in cuticular wax, as measured by gas chromatography. Surprisingly, tt4 mutants do not any significant change in wax load compared to wild type. Similarly, the double mutant tt4 tt5 also demonstrates a normal wax load. These results imply that the accumulation of the flavonoid intermediate, naringenin chalcone, is causing a reduction in wax biosynthesis and/or transport. This effect could be due to enzymatic inhibition, trafficking of enzymes, or transcriptional regulation. Because flavonoids have been shown to be involved in gene regulation, we wanted to determine if the loss of TT5 causes a down-regulation of wax-biosynthetic genes. We are analyzing the expression of wax biosynthetic genes in tt4, tt5, and tt4 tt5 mutant lines using RT-qPCR. Until now, flavonoids have not been recognized for involvement in wax biosynthesis or transport. The results of this study imply that such a connection exists. P42. Quantification and localization of proanthocyanidins in pea (Pisum sativum L.) seed coats Alena (Lihua) JIN, Jocelyn A. OZGA, and Dennis M. REINECKE Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada Proanthocyanidins (PAs), also known as condensed tannins, mainly accumulate in seed coats of grain legume seeds. Historically, PAs were considered as anti-nutritional compounds in pulse nutritional studies because they can precipitate proteins and reduce bioavailability of some minerals. However, recent research on the roles of PAs as plant-based health-beneficial components in the human diet has lead to renewed interest in this class of flavonoids in food crops. The objective of this study was to identify, quantify (using high performance liquid chromatography-photodiode array detector and liquid chromatography-mass spectrometry techniques), and localize (using histological techniques) proanthocyanidins in pea seed coats over development to aid in understanding the spatial and temporal regulation of PA biosynthesis and accumulation in this tissue. Pea seed coats of ‘Courier’ contained proanthocyanidins (435.9 mg/100 g dry seed weight at maturity) almost exclusively consisting of prodelphinidin-type subunits. PA content increased during the early stages of pea seed development and peaked at 20 days after anthesis (DAA). After 20 DAA, the PA content steadily decreased until seed maturation. Seed coat proanthocyanidins were mainly localized in the ground parenchyma layer and epidermal layer of this tissue. These data show PA biosynthesis and accumulation is regulated temporally and spatially during seed coat development and that this tissue can serve as a good dietary source of proanthocyanidins.      P43. Investigating methionine aminotransferases to identify the first enzyme of the algal dimethylsulfoniopropionate biosynthesis pathway Monica R. JEPSON1, Caitlin A. GELDART, Michelle L. McLAUCHLAN, Jeffrey C. WALLER Department of Chemistry and Biochemistry, Mount Allison University, Barclay Building, 63C York Street, Sackville, New Brunswick, Canada, E4L 1G8 It is not without irony that tiny oceanic microalgae have a huge global impact. Many taxa of micro- and macro- algae enzymatically synthesize dimethylsulfoniopropionate (DMSP), an important osmoregulatory compound, with multiple proposed functions. DMSP is broken down to the volatile compound dimethylsulfide (DMS), primarily by marine bacteria. DMS accounts for about half the global atmospheric sulfur budget, and influences global climate. As DMSP is the source of DMS, it is surprising that almost no research on DMSP synthesis has been done - not one enzyme is known! What are the molecular identities and structures of the enzymes catalyzing this major biogeochemical flux? Are they conserved across phylogenetically diverse algal species? Will gene identification predict an alga's capacity to make DMSP? Recent advances in sequencing diverse algal genomes, creation of algal cDNA libraries, and development of genetic tools for algae can facilitate algal gene discovery. We have been applying comparative genomic approaches and developing bacterial

 

CSPB2012 p. 77

functional complementation assays to help identify methionine aminotransferase, the first enzyme of the algal DMSP biosynthesis pathway. Progress in developing this bacterial functional complementation assay and an enzyme assay sensitive enough to detect the native algal enzyme in protein purification efforts will be presented.    P44. Studies on the regulation of resistant starch biosynthesis in barley Zahir AHMED Starch is an important carbohydrate with wide spread distribution among plant species and of great economic value. Besides providing many commercial uses, starch is an important part of the human diet, accounting for 70-80% daily caloric intake. An important form of starch is high amylose or resistant starch (RS) which has associated human health benefits including lowering glycaemic index and reduced risk of colon cancer. The present study was conducted to understand the mechanisms underpinning the regulation of RS biosynthesis in barley. 33 genotypes of barley were studied which varied considerably in their total starch and RS contents. It was found that smaller B-type granules were positively correlated with the amount of RS, while large A-type granules were negatively correlated with the RS contents. Scanning electron microscopy revealed that genotypes with higher RS contents have altered granule morphology, size and number. The amount of total granule-bound protein and individual granule-bound proteins (principally isoforms of starch synthases and branching enzymes) involved in the pathway were measured and exhibited a positive correlation with RS content. Future studies aim to understand the biochemical pathway of starch biosynthesis in more detail by investigating role of protein-protein interactions in the formation of RS. P45. Regulation of maize (Zea mays L.) starch synthase IIa by protein phosphorylation. Usha P. RAYIRATH, Ian J. TETLOW and Michael J. EMES Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada Starch is a significant carbohydrate reserve in plants and forms the major dietary energy source for humans. Biosynthesis of storage starch in maize (Zea mays L.) occurs in the amyloplasts of the developing endosperm, through the co-ordinated actions of several enzymes including ADP-glucose pyrophosphorylase (AGPase), five isoforms of starch synthases (SS), three isoforms of branching enzymes (SBE) and two isoforms of debranching enzymes (DBE). Previous studies indicate that in cereal endosperm, protein phosphorylation regulates the formation of functional multi-enzyme complexes between SSs and SBEs during starch biosynthesis and that SSIIa forms the core of a functional protein complex with SSI and SBEIIb. The present study investigated whether SSIIa is itself regulated by protein phosphorylation. Evidence indicating the regulation of SSIIa in maize amyloplasts by protein phosphorylation comes from studies of migration of the protein following electrophoresis, autoradiography following labelling with γ-32P-ATP and the use of reagents which interact with phospho-peptides. The effect of physiological concentrations of ATP and potent protein kinase inhibitors on the post translational modification of SSIIa will also be discussed. Future investigations aim to identify the phosphorylation sites within SSIIa and identify and characterize the protein phosphatases and kinases that modulate this post translational modification. P46. Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation Dylan M. SILVER1, Leslie P. SILVA2, Emmanuelle ISSAKIDIS-BOURGUET3, Mikkel A. GLARING4, David C. SCHRIEMER2 and Greg B. G. MOORHEAD1

1. Department of Biological Sciences, University of Calgary, Calgary, AB, CAN 2. Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, CAN 3. Institut de Biologie des Plantes, UMR 8618, CNRS ⁄ Universite ́ Paris-Sud 11, Orsay Cedex, FRA 4. Department of Biology, ETH Zürich, Zürich, CHE

 

CSPB2012 p. 78

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and Cys130 within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

P47. Characterization of phloem fiber related NAC domain protein genes in flax Ningyu ZHANG, Michael K. DEYHOLOS Department of Biological Sciences, University of Alberta, Canada Fiber harvested from flax phloem tissue is a renewable resource with promising uses in eco-friendly composites. Most molecular and cellular research to date as focused on later stages of fiber differentiation including the development of the fiber cell wall. On the other hand, the molecular mechanisms that govern specification of fibers are largely unknown. All phloem fibers in flax are formed during primary growth. Therefore gene expression patterns in the shoot apex are likely to govern fiber identity, and therefore fiber yield. In this study, I will focus on flax NAC domain protein genes whose homologs have been indicated to be involved in regulating the differentiation of xylem vessels and xylem fiber cells in other species. A total of 187 predicted NAC domain genes were identified within the whole-genome sequence of flax. To select potential NAC domain protein genes related to the flax phloem fiber development, transcription profile analysis will be conducted through RNA-seq. Furthermore, the function of candidate NAC domain protein genes will be studied by VIGS or other reverse-genetics technologies. P48. Proteomic analysis on the effects of salicylic acid-induced priming in flax and its relationship to cadmium pollution response Aïcha BELKADHI1,2, Leonardo GALINDO1, Wahbi DJEBALI2, Wided CHAÏBI2 and Michael K. DEYHOLOS1

1. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 2. Unité de Recherche de Physiologie et Biochimie de la tolérance des plantes aux contraintes abiotiques, Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia Flax, one of the most important fiber crops in the world, is generally exposed to cadmium (Cd) pollution, notably affecting its growth performance. Towards a better understanding of flax response to Cd stress, we have used proteomics to characterize protein changes occurring in Cd-stressed plants that have been pre-treated with salicylic acid (SA) which is known to alleviate the negative effects of the pollutant. The aim of the present study is to analyze the effect of SA induction on growth-related processes and to characterize the proteins involved in such response, including the potential defense mechanisms enabling primed seedlings to overcome environmental stresses. This work provides novel avenues toward understanding the mechanisms of plant enhancement of tolerance to Cd pollution by priming treatments to be used both in commercial applications and in contaminated soils (on farm seed priming) to improve crop yields.

 

CSPB2012 p. 79

 

CSPB2012 p. 80

   2D electrophoresis, 71 ABA, 56, 62, 63, 64 ABC transporter, 25 abscisic acid, 42, 56, 62, 63, 64 Acer rubrum, 67 affinity chromatography, 27 agglutinin, 74 AHMED, 77 AKHTER, 30 AL-FORKAN, 34 algae, 54, 76 alkaloid, 25 allotetraploid, 24 ALMEIDA-RODRIGUEZ, 45 alternative oxidase, 55, 56 aluminum, 47, 71 AMBROSE, 23 amino acids, 27, 47, 48, 50, 56, 61 aminotransferase, 47, 48, 61, 77 amylopectin, 51 amylose, 77 AMYOT, 40 Anabaena flos-aquae, 30 aquaporin, 16, 42, 45, 67, 68, 71 Arabidopsis, 24 Arabidopsis thaliana, 14, 16, 19, 24, 28, 31, 33, 34, 35,

37, 38, 39, 40, 45, 48, 50, 52, 53, 54, 55, 56, 60, 61, 62, 64, 67, 69, 73, 74, 75, 76

arabinogalactan, 41 ARANGO, 46 ARANGO-VELEZ, 46 ARIF, 41 ARNOLD, 14 ARSHAD, 43 Ascophyllum nodosum, 30 Asteraceae, 26 ATTARAN, 32 auxin, 16, 32, 63, 69 AVERINA, 65 AYELE, 50, 64 AYTON, 43 Bacillus subtilis, 51 BAYLIS, 32 BEATTY, 47, 69 BEGAM, 48 BELKADHI, 78 BENDER, 55 BENLLOCH, 19 Beta vulgaris, 19 beta-galactosidase, 41 BEYZAEI, 65 bHLH, 64

bimolecular fluorescence complementation, 62 BIN YAMEEN, 31 bioactive, 24 bioenergy, 72 biofuel, 38 bioinformatics, 20, 54, 56, 60, 70, 72 biomass, 35, 44, 45, 47, 69, 70, 75 BIRD, 25, 75 BISGROVE, 43, 53 BISWAS, 43 blight, 36, 73 BOMBLIES, 14 BONHAM-SMITH, 33, 74 BONNET, 19 BOYCHUCK, 46 BOYCHUK, 37 Brassica, 19, 24, 29, 34, 37, 38, 52, 74 Brassica juncea, 29 Brassica napus, 24, 29, 37, 38, 52, 74 Brassica oleracea, 24, 29 Brassica rapa, 19, 24, 29 breeding, 17, 38, 43, 65 BRULE-BABEL, 50 bud, 34, 65, 68, 74 bud set, 65 BUSOV, 33 BYKOVA, 49 cadmium, 30, 31, 67, 69, 78 CAHILL, 74 calcium, 50, 55, 60 calmodulin, 55, 60 cambium, 40 Cannabis sativa, 23 CAO, 36, 52 CAPO-CHICHI, 29 carbohydrate, 17, 18, 24, 74, 77, 78 carbon, 18, 19, 26, 31, 32, 33, 37, 43, 46, 48, 50, 55, 65 carbon assimilation, 19, 31, 65 CARROLL, 47 cavitation, 43, 44, 45 cDNA, 27, 39, 62, 72, 73, 74, 76 cDNA library, 62, 74 cell wall, 9, 40, 41, 52, 55, 60, 61, 64, 78 cellulose, 40, 41, 52, 64 CesA, 40 CHAÏBI, 78 CHAKRABARTY, 25 chalcone synthase, 76 CHAMOT, 28 CHANG, 31, 65 CHEBLI, 41 CHEN, 24, 32, 54 CHENG, 61 chlorophyll, 29, 31, 34, 65, 66, 67, 69, 70

 

CSPB2012 p. 81

CIERLIK, 32 CIGAN, 74 cis-prenyltransferase, 25 CLARKE, 31 clubroot, 74 cold, 20, 28, 31, 32, 34, 65, 66 COLTMAN, 37 COLUMBUS, 69 COLVILLE, 54 CONSTABEL, 75 CONTRERAS, 30 COOKE, 34, 37, 46, 68, 71, 72, 73, 74 COOPER, 36, 73 COPELAND, 46 Coptis japonica, 25 Cornus sericea, 67 CRANSTON, 62 CRITCHLEY, 30 CROSBY, 36 CULLINGHAM, 37 cutin, 76 CVETKOVSKA, 55 cyanobacteria, 28, 29, 30, 66 cyanogenesis, 15 cytokinin, 63 cytoskeleton, 53, 60 DAIGLE, 36 DANG, 37 DASTMALCHI, 23 DATLA, 52 DE CARVALHO, 71 defense, 26, 31, 32, 46, 74, 75, 78 defensive, 26 DEMISSIE, 27 Dendroctonus ponderosae, 74 DENG, 24 DEOL, 64 DEPAUW, 41 DESPRÉS, 20 DEYHOLOS, 36, 41, 50, 52, 54, 56, 70, 74, 78 DGAT, 37, 38, 39 DHAUBHADEL, 23 DHUGGA, 49 DIGE, 72 dimethylsulfide, 76 disease, 20, 36, 61, 63, 70, 73, 74 DJEBALI, 78 DNA repair, 61 DONNELLY, 60 dormancy, 34, 49, 64, 65, 68 DOS SANTOS, 71 DOUGLAS, 24, 64 DOYLE, 54 drought, 17, 19, 26, 43, 44, 45, 46, 67, 68, 70, 72

ectomycorrhizal, 71, 72, 74 EL KAYAL, 34, 46, 68, 71 Eleusine corocana, 70 ELIAS, 33 ELLENS, 51 ELLIS, 21, 60, 61 EL-MEZAWY, 34 embryo, 24, 33, 39, 40, 49, 63, 72 EMES, 51, 77 endoplasmic reticulum, 53, 54 endosperm, 72, 77 ENSMINGER, 31, 65 epibrassinolide, 40, 63 EQUIZA, 44 ERBILGIN, 74 Eschscholzia californica, 25 ESPIE, 66 EST, 36, 74 ESTs, 27 ETHERINGTON, 33 ethylene, 33, 35, 51, 63, 69 EWERS, 19 faba bean, 24 FACETTE, 48 FANG, 62 FARUQUE, 74 fatty acid, 38, 39, 76 FEDOSEJEVS, 50 fiber, 9, 11, 33, 41, 43, 50, 52, 54, 74, 78 fibre, 24 flavonoid, 24, 75, 76 flooding, 35, 45 flower, 23, 27, 63 FRANKLIN, 75 FRÉCHETTE, 31, 65 frost, 70 fruit, 34, 62, 75 FUJITA, 40 fungal, 26, 46, 70, 71, 74 fungi, 26, 46, 54, 71, 72 galactose, 51 GALINDO, 36, 46, 78 GALWAY, 40 GANANÇA, 71 GANDHI, 33 GARNETT, 49 GC-MS, 62 GEITMANN, 41 GELDART, 76 GELVIN, 62 gene expression, 14, 23, 32, 34, 45, 52, 56, 60, 66, 73, 78 genome-wide association (GWA), 35 genomics, 14, 17, 18, 26, 34, 36, 37, 49, 51, 72, 73 GEORG, 28

 

CSPB2012 p. 82

geranyl diphosphate, 27 germination, 29, 49, 63, 64, 72, 73, 74 gibberellin, 33, 62, 63, 64 GIELEN, 19 glandular trichomes, 23 GLARING, 77 global warming, 43, 68 glutathione, 49 GOOD, 47, 48, 69 GOONEWARDENE, 70 GORING, 52 GOWDA, 70 GREER, 37 GRIEGOSCHEWSKI, 30 GRIENENBERGER, 64 Grosmannia clavigera, 26 GRUBER, 40 GRZESIAK, 45 GUY, 35, 65, 68 HACKE, 32, 42, 44, 45 Hakea prostrata, 47 HAMANN, 32 HANNOUFA, 40 HANSON, 18, 51 HARRIS, 31, 71 HE, 32 heavy metal, 27, 30, 31, 67, 69, 71, 78 Helianthus, 14, 63 Helianthus tuberosus, 63 helicase, 28, 29, 66 hemicellulose, 41 HESS, 28 heterochromatin, 28 heterosis, 65 HIEBERT, 72 HIMMELSPACH, 40 His-tag, 61 HOBSON, 41 HOCART, 40 HOLLISTER, 14 HOLT, 48 HOLTHAM, 49 homogalacturonan, 55 Hordeum vulgare, 30, 49, 64 HOSSAIN, 40 HOWE, 32 HSU, 62 hydraulic, 42 hypocotyl, 35, 40, 55 IGAMBERDIEV, 49 Illumina, 23 imaging, 25 INDRIOLO, 21, 52 intellectual property, 62

intrinsically disordered proteins, 56 isoflavonoid, 23 isoprene, 25 isoprenoid, 26 ISSAKIDIS-BOURGUET, 77 JACOBSEN, 42, 43 JANG, 45 jasmonate, 32, 45 jasmonic acid, 60 JEPSON, 76 JIN, 75, 76 JU, 34, 40 KAISER, 49 KARST, 74 KAV, 70 KAZALA, 62 KERVIN, 72 KEURENTJES, 35 KHAN, 50 KHUU, 53 kinase, 47, 50, 54, 56, 57, 60, 61, 77 KIRKEY, 67 KLAPSTE, 35 KOOYERS, 15 KOSOLA, 33 KOZIEL, 34 KRAFT, 19 KULICHIKHIN, 50 KUREPIN, 62 Laccaria bicolor, 71 LACHENBRUCH, 17 Lactuca sativa, 25, 30 LAHARI, 61 LANGE, 29 LAUR, 42, 45 Lavandula, 27 LEA, 72 lectin, 74 LEE, 45, 62 legume, 24 lentil, 24 lettuce, 25 LIAO, 62 lignin, 17, 41 lily, 63 LIN, 62 LINSKY, 46 Linum usitatissimum, 8, 9, 11, 36, 39, 41, 52, 54, 55, 70,

71, 74, 78 LIU, 40, 51, 54, 56, 62, 64 LO, 72 LOCK, 69 LOPES, 17

 

CSPB2012 p. 83

low temperature, 65 LUKENS, 28 LUNG, 37 LUSEBRINK, 46 LYZENGA, 56 MA, 33, 49 MACFIE, 30, 67, 69 MACLACHLAN, 31 MAHMOUD, 26, 27 MAJ,, 60 MAJOR, 32 MANSFIELD, 40 MAP kinase, 36, 37, 60, 61 MARRSOLAIS, 36 MARSOLAIS, 61 MARTIN, 41 MATTON, 36 MATTSSON, 32, 43 MAUREL, 16 McALLISTER, 48 McCLUNG, 19 McDONALD, 56 McGARVEY, 40 McKOWN, 35 McLAUCHLAN, 76 MEENTS, 46 MEHROKE, 63 meristem, 63 microarray, 24, 29, 32, 46, 52, 60, 61, 70, 73, 75 micronutrient, 69 microtubule, 53, 60, 64 MIETKIEWSKA, 38 MILES, 38 miRNA, 20 mitochondria, 53, 55 MOORHEAD, 54, 77 mountain pine beetle, 26, 37, 46, 74 MUISE-HENNESSEY, 56 MUKHERJEE, 50 MULLEN, 51, 53 MYB, 75 NAC, 34, 78 NADEAU, 62 natural antisense transcripts, 28 natural variation, 35 NAVABI, 36 NEILY, 30 NEIMANIS, 56 NILSSON, 19 nitrate reductase, 66 nitrogen, 23, 44, 47, 48, 49, 55, 61, 65, 66, 69, 70 nucleotide excision repair, 61 nutraceutical, 38 nutrition, 24, 26, 47, 62, 67, 74, 76

oilseed, 38, 50, 62 OLSEN, 15 orchid, 62 organogenesis, 63 ornamental, 63 OWTTRIM, 28, 29, 66 OZGA, 24, 62, 75, 76 P. deltoides, 44 P450, 26 PAGE, 23 PAN, 39 PARK, 53 PARKIN, 29 pathogen, 32 PAULS, 36, 41, 73 pea, 24 PEARCE, 33 PEC, 74 pectinesterases, 11, 54, 55 PENG, 24 PEPC, 47, 53, 67 peroxisome, 23 PERRY, 36, 73 persimmon, 75 pH, 67, 69, 71 PHARIS, 62 Phaseolus acutifolius, 36 Phaseolus vulgaris, 36, 73 phenolic, 63, 72 phenylpropanoid, 23 phloem, 18 phosphatase, 47, 54, 57, 60, 77, 78 photoperiod, 19, 31, 34, 35, 68 photorespiratory, 61 photosynthesis, 18, 26, 28, 29, 31, 32, 34, 46, 65, 66, 70,

75 Physcomitrella patens, 56 phytochelatin, 30 Picea glauca, 34, 67, 69, 74 PIERIK, 35 PIN, 19 Pinus banksiana, 46, 67, 69, 73 Pinus contorta, 26, 46, 73, 74 Pinus strobus, 65 Pinus taeda, 73 PINZON, 52, 54 Pisum sativum, 62, 76 PLANT, 43 Plasmodiophora brassicae, 74 PLAVCOVÁ, 44 PLAXTON, 47, 50, 53 PLETT, 49 PLINER, 34, 63 pollen, 24, 37, 42, 52

 

CSPB2012 p. 84

polyketide, 23, 24 polyphenolics, 75 polyploidy, 24 polypoidy, 14 Populus balsamifera, 65, 68 Populus tremula, 33, 68 Populus tremuloides, 44, 67, 69 Populus trichocarpa, 17, 32, 33, 35, 37, 42, 43, 44, 68,

74, 75 post harvest, 63 POWER, 36 POZNIAK, 31 PRATT, 43 proanthocyanidin, 24, 75, 76 proline, 66, 70 promoter, 34, 40, 41, 57, 60, 64, 68, 69, 70, 75 propyzamide, 60 proteolysis, 29 protoplast, 16, 48, 62 PUJOL, 41 PURVES, 23 QTL, 17, 19, 41 QU, 25 QUANDT, 34 QUILICHINI, 24 RAFALSKI, 49 raffinose, 18 RAHMAN, 34 RAMADOSS, 70 RANGANATHAN, 68 RAYIRATH, 77 reclamation, 33, 67, 69 recombinant inbred, 19, 41 redox, 29, 66, 77, 78 REINECKE, 24, 62, 76 REINPRECHT, 41 RENAUT, 14 reverse genetic, 55, 60, 64, 78 REYNOLDS, 17 RHEAULT, 27 rhizosphere, 67 RIAZ, 41 ribosome, 33 RICHARDSON, 51 RIESEBERG, 14 RNA chaperone, 28 RNA helicase, 28 RNAi, 24, 25, 34, 41, 52 RO, 25, 26 ROACH, 41 ROESSNER, 49 ROOD, 33 root, 16, 17, 23, 30, 33, 40, 42, 45, 47, 49, 53, 54, 64, 67,

69, 70, 71, 72

ROS, 16 ROSA, 32 ROSANA, 28, 66 ROSENBAUM, 55 ROYKO, 73 rubber, 25 RYAN, 65 RYSER, 67 Saccharomyces cerevisiae, 37, 39 S-adenosylmethionine, 51 SAFAVIAN, 52 SAINT PIERRE, 17 salicylic acid, 20 salinity, 16, 30, 65, 66 Salix, 72 SAMUELS, 22, 24 SARHAN, 20 SARKER, 27 SASIDHARAN, 35 SAWHNEY, 72 SCHOFIELD, 56 SCHREIBER, 32 SCHRIEMER, 77 SCHROEDER, 61 seaweed, 30 seed, 19, 24, 29, 34, 35, 37, 38, 39, 40, 49, 50, 53, 62, 64,

70, 72, 74, 75, 76, 78 seed coat, 24 self-incompatibility, 52 self-incompatible, 52, 53 senescence, 32, 44, 63 Septoria linicola, 70 sesquiterpene lactone, 26 shade avoidance, 19, 35 SHAH, 34 SHAHIDI, 53 SHANE, 47 SHARMA, 63 SHEN, 62 SHERBAKOV, 65 SHEVCHENKO, 33 SHISHKOV, 30 SHRAWAT1, 47 SIDDERS, 72 siderophore, 30 SILOTO, 38, 39 SILVA, 77 SILVER, 77 SIMON, 41 SINGER, 62 SINGH, 21, 63, 64 single nucleotide polymorphism (SNP), 72 single nucleotide polymorphisms (SNP), 35 siRNA, 28 SLASKI, 29, 63, 70, 71

 

CSPB2012 p. 85

SMALL, 15 SNEDDEN, 55, 60 SNP, 35, 72 soil, 16, 29, 45, 47, 67, 69, 70, 71, 74 soil microbes, 67, 69 Solanum lycopersicum, 37, 72 SONIER, 30 soybean, 23, 56 sporopollenin, 24 St.ONGE, 35 stachyose, 18 starch, 24, 50, 51, 77, 78 STASOLLA, 50 statolith, 41 STEWART, 33 stomata, 42, 46 STONE, 56 STOUT, 23 STRAUSS, 33 stress, 16, 17, 23, 24, 28, 29, 30, 32, 33, 35, 37, 41, 42,

43, 44, 45, 46, 56, 60, 66, 67, 68, 69, 70, 71, 72, 73, 75, 78

sucrose, 18, 40, 45, 50, 63 SUNDBERG, 32 suspension cultures, 25 symbiont, 26 Synechocystis, 28, 29, 66 TAG, 37, 38, 39 TARASSOVA, 29 TAYLOR, 31, 71 temperature, 20, 28, 29, 31, 40, 41, 56, 65, 66, 68 terpene synthase, 27 terpene synthases, 27 terpenoid, 26 TESTER, 49 TETLOW, 51, 77 THARMAPALAN, 52 THOMAS, 32, 43 TITUS, 30 TO, 52 tobacco, 33, 34, 53, 55, 56, 61, 64 TOBIN, 43 tonoplast, 31 transcriptome, 23, 28, 32, 34, 35, 37, 47, 56, 68, 73 TRANSPARENT TESTA 16, 24 transport, 18 transposable elements, 36 TREBLE, 30 triacylglycerol, 37, 39 trichome, 27 trichomes, 27 Trifolium repens, 15 triticale, 45 Triticum turgidum, 31 TRUKSA, 24, 37

TRUONG, 40 TU, 74 tubulin, 60 TURGEON, 18 TYREE, 44 ubiquitin, 47, 52, 56 UHRIG, 54 UPADHYAYA, 70 UV, 61, 76 UZUEGBU, 25 Vaccinium corymbosum, 75 Vaccinium myrtilloides, 67 VANLERBERGHE, 55 variegation, 34 VERA, 70 vessel, 32, 42, 43, 44 VIDMAR, 34, 63 VIGS, 78 VOESENEK, 35 VREUGDENHIL, 35 WALLER, 76 WARD, 40 WASTENEYS, 40, 60 water, 16, 17, 19, 24, 26, 32, 34, 42, 43, 44, 45, 46, 52,

67, 68, 70, 71, 72, 73, 76 water deficit, 26 water transport, 16, 17, 42, 45, 68, 71 wax, 75, 76 WEERSINK, 36 WEGER, 30 WEI, 74 WEINIG, 19 WESELAKE, 24, 37, 38, 39, 62 wheat, 17, 31, 50, 66, 71 WHITFORD, 66 WHITTINGTON, 40 WICKRAMARATHNA, 39 WIEBE, 31 WILLIAMSON, 75 WILLICK, 67 WOJAS, 60 WREMERTH-WEICH, 19 WU, 29, 34, 62, 63 XAIOLU, 36 Xanthomonas axonopodis, 73 xanthophyll, 65 XIE, 36, 73 XU, 67, 71 XUE, 14 xylem, 16, 17, 32, 40, 42, 43, 44, 45, 64, 78 YANG, 24

 

CSPB2012 p. 86

YAO, 64 YEH, 62 YOSHIDA, 75 YU, 36 ZANGHELLINI, 49 Zea mays, 45, 49, 51, 77

ZHAN, 28 ZHANG, 61, 68, 69, 78 ZHENG, 54 ZHUANG, 56 ZIMNY, 71 ZWIAZEK, 43, 44, 45, 67, 68, 69, 71

 

CSPB2012 p. 87