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
?
?
