1. Takashi Akihiro, Sayo Yoshikawa, Katsuhiko Sato, Tatsuhito Fujimura Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba City, Ibaraki, 305-8572 Japan Transcriptomic responses to cadmium in Brassica juncea 0 hr CdSO 4 25 0 (  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 –2 s –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 TDFs Up-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 AvaII TaqI Cd 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 thioredoxin 4 × ubiquitin peroxidase lipoxygenase 3 × heat shock protein ferredoxin--nitrite reductase MAP3K ? ABC transporter protein Tonoplast ATPase vacuole Cd Cys Sulfate APS Glu γ-glutamylcysteine synthetase γGluCys Gl ｙ GSH phytochelatin ATP sulfurylase NADP-specific glutatamate dehydrogenase 2-oxoglutarate s Cd s s s 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 sulfurylase gamma-glutamylcysteine synthetase NADP-specific glutatamate dehydrogenase Tonoplast ATPase ABC transporter protein 2OG-Fe(II) oxygenase family protein thioredoxin ubiquitin peroxidase lipoxygenase 71 kDa heat shock protein 73 kDa heat shock protein 70 kDa heat shock protein ferredoxin--nitrite reductase 1-aminocyclopropane-2-carboxylate oxidase efflux carrier, pin2 zinc finger (C3HC4-type RING finger) family protein MAP3K epsilon 1 protein kinase protein WD 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-like pathogenesis-related protein 1 precursor Wound induced protein nodulin MtN21 lectin-like protein putative pectinesterase 16S ribosomal RNA 23S ribosomal RNA 60S ribosomal protein L34 Detoxification Up Down Compartmentalization Up Up? Oxygen and radical detoxification Up Down? Plant hormone Up Signal transduction & Transcriptional factor Up Down Response to stress Up Down Cell wall related Up Down rRNA protein Up Unknown protein Up No hit Up Down Unclasified Up Down Unknown protein No Hit zinc-binding family protein O-methyltransferase, putative leucine-rich repeat family protein / protein kinase family protein poly(A)-binding protein U2 SnRNP-specific A'protein Ras-related GTP-binding protein glutamyl-tRNA synthetase Putative glycine-rich protein putative pyruvate decarboxylase-1 Pdc1 S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase-like glutamyl-tRNA synthetase trans-cinnamate 4-hydroxylase beta-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-regulation Down-regulation ? ?