Takashi Akihiro, Sayo Yoshikawa, Katsuhiko Sato, Tatsuhito FujimuraGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba City, Ibaraki, 305-8572 Japan

Transcriptomic responses to cadmium in Brassica juncea

0 hr

CdSO4

250 (M)

Plant growth and cadmium treatment Seeds of Brassica juncea (accesion no.173874) were germinated on vermiculite and grown in a controlled growth chamber with a day/night photoperiod of 12/12 h at 227 mol photons m–2s–1 and temperature regimes of 25 / 20ºC, respectively. After 10 days, plants were transferred to hydroponic system and cultured in a Kasugai hydroponic solution. After 38 days after germination, plant were treated with 25 M CdSO4 for 24 hours and then harvest.

24 hr

Total 10776 TDFsUp-regulation 52 TDFs (0.48%) Down-regulation 21 TDFs (0.19%) Total of 10,776 TDFs were observed on gels using 128 pairs of primer sets for the cDNA-AFLP analysis. Among them, 52 TDFs were Up-regulated, 21 TDFs were down-regulated by Cd treatment.

- - - - - -+ + + + + +

　　　　 AA　　　　　　AT AC AG GA GT GC

AvaIITaqICd

cDNA-AFLP autoradiography showing the transcripts derived fragments (TDFs) patterns. Primer combinations for restriction endonucleases (AvaII and TaqI ) with extensions are shown at the top of the cDNA-AFLP gel.

Cd

Oxygen reactive species ↑

2OG-Fe(II) oxygenase family protein

thioredoxin4 ×ubiquitinperoxidase

lipoxygenase 3 × heat shock protein

ferredoxin--nitrite reductase

MAP3K?

ABC transporter protein

Tonoplast ATPase

vacuole

Cd

Cys

Sulfate

APS

Glu

γ-glutamylcysteine synthetase

γGluCysGl ｙ

GSH

phytochelatin

ATP sulfurylase

NADP-specific glutatamate

dehydrogenase

2-oxoglutarate

s

Cds

s CdsCd

s s

?depletion

ACC oxidase

Met

SAM

ACC

Ethylene

nucleus zinc finger (C3HC4-type RING finger) family protein

WD repeat and FYVE domain containing 3 isoform 1

zinc finger (FYVE type) family protein

CONSTANS B-box zinc finger protein

4 × ribosomal protain

?

?

?

Transcriptional regulation in response to cadmium treatment in Cd highper-accumulator Brassica juncea was investigated with cDNA-amplified fragment length polymorphism technique. Total of 10,776 transcripts derived fragments (TDF) were observed on gels. Among them, 52 TDFs were Up-regulated and 21 TDFs were down-regulated by Cd treatment. The genes encoding for ATP-sulfurylase, gamma-glutamylcysteine synthase, ABC transporter, heat shock protein, and ubiquitin are identified. Based on our date, a simplified multilevel modulation scheme for cadmium tolerance mechanisms in the Brasicca juncea is depicted in figure 3

Fig1

Fig2

Fig3

ATP sulfurylasegamma-glutamylcysteine synthetaseNADP-specific glutatamate dehydrogenase

Tonoplast ATPase ABC transporter protein ABC transporter protein

2OG-Fe(II) oxygenase family proteinthioredoxinubiquitin 　ubiquitinubiquitin 　ubiquitinperoxidase lipoxygenase 71 kDa heat shock protein73 kDa heat shock protein70 kDa heat shock protein ferredoxin--nitrite reductase

1-aminocyclopropane-2-carboxylate oxidaseefflux carrier, pin2

zinc finger (C3HC4-type RING finger) family protein MAP3K epsilon 1 protein kinase proteinWD repeat and FYVE domain containing 3 isoform 1 zinc finger (FYVE type) family protein putative CONSTANS B-box zinc finger protein

dehydration-responsive protein-related /ankyrin-likepathogenesis-related protein 1 precursorWound induced protein nodulin MtN21

lectin-like proteinlectin-like proteinputative pectinesterase

16S ribosomal RNA16S ribosomal RNA23S ribosomal RNA60S ribosomal protein L34

DetoxificationUp UpDown

CompartmentalizationUpUpUp?

Oxygen and radical detoxificationUpUpUpUpUpUpUpUpUpUpUpDown?

Plant hormoneUpUp

Signal transduction & Transcriptional factorUp

UpUp

UpDown

Response to stressUpUpDownDown

Cell wall relatedUpDownDown

rRNA proteinUpUpUpUp

Unknown proteinUpUpUpUpUpUpUpUp

No hitUpUpDownDownDownDown

UnclasifiedUpUpUp

UpUpUpUpUpDownDown

DownDownDown

Unknown proteinUnknown proteinUnknown proteinUnknown proteinUnknown proteinUnknown proteinUnknown proteinUnknown protein

No HitNo HitNo HitNo HitNo HitNo Hit

zinc-binding family protein O-methyltransferase, putative leucine-rich repeat family protein / protein kinase family proteinpoly(A)-binding proteinU2 SnRNP-specific A'proteinRas-related GTP-binding proteinglutamyl-tRNA synthetase Putative glycine-rich protein 　putative pyruvate decarboxylase-1 Pdc1S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase-likeglutamyl-tRNA synthetase trans-cinnamate 4-hydroxylasebeta-1,3-glucanase; bgl gene.

Northern blot analysis

Cd - +

Northern blot analysis

Cd - +

Table 1 Table 2

Content of Cd

Roots 　 2205 ppm

Leaves 51 ppm

Stems 268 ppm

72 TDFs were recovered from the cDNA-AFLP gels and subcloned into plasmid vectors and then sequenced. These sequences can be grouped into 11 functional categories. Northern blot analysis was performed to confirm results with the cDNA-AFLP procedure

TATA

Up-regulationDown-regulation

?

?