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Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica juncea L.

Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica... Abstract In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was employed to identify genes that exhibited a modulated expression following cadmium (Cd) treatment in Brassica juncea grown in hydroponic culture. Plants were treated for 6 h, 24 h, and 6 weeks with 10 μM Cd(NO3)2 and untreated 6-week-old plants were used as controls. Cd content was measured at these four time points. Long exposure to Cd affected root morphology: roots appeared thinner and sent out side roots. Seventy-three transcript-derived fragments were identified as Cd responsive. Fifty-two of them showed significant homology to genes with known or putative function, 10 transcript-derived fragments were homologous to uncharacterized genes, while 11 transcript-derived fragments did not show significant matches. The expression pattern of several of these genes was confirmed by northern blot analysis. Fifty-two genes of known or putative function were transcriptional factors, expression regulators, and stress responding and transport facilitation genes, as well as genes involved in cellular metabolism and organization and the photosynthetic process, suggesting that a multitude of processes are implicated in Cd stress response. The transcription of drought- and abscisic acid-responsive genes observed in this study also suggested that Cd imposes water stress and that abscisic acid may be involved in the Cd plant response. Brassica juncea, cadmium, cDNA-AFLP, gene expression, heavy metals Introduction Environmental pollution by toxic metals has accelerated dramatically since the beginning of the industrial revolution (Nriagu, 1979). Cadmium, a non-essential heavy metal, is considered one of the major pollutants (Alloway and Steinnes, 1999). It is widespread in soil containing waste materials from zinc mines, in sewage water used for irrigation, in sludge-amended soils, and in soil fertilized with cadmium-rich phosphate fertilizers (Hüttermann et al., 1999). Cadmium is also released into the environment by urban traffic, heating systems, and waste incinerators (Sanità di Toppi and Gabbrielli, 1999). Toxicity to living cells is caused by very low cadmium concentrations and its presence in the food chain can be highly dangerous as it can cause significant damage to the human body (Buchet et al., 1990) and is a suspected carcinogen (Vido, 2001). Although Cd is a non-essential element for plant mineral nutrition it is taken up easily by roots and transported through the xylem to the vegetative and reproductive organs, thus affecting nutrient uptake and homeostasis and inhibiting root and shoot growth and yield production (Sanità di Toppi and Gabbrielli, 1999; Metwally et al., 2005). However, certain plant species have the ability to survive and reproduce on soils containing high concentrations of metals in forms that are toxic or inimical to other plants (Macnair and Baker, 1994). The ability of these plants to survive on metal-polluted soils is not only due to their capacity to take up, translocate, and sequester metals, but is also based on mechanisms that allow them to tolerate high levels of the element in root and shoot cells by alleviating their toxic effects (Salt et al., 1998). Tolerance is achieved by internal detoxification and probably involves cell compartmentation and metal complexation. For several metals and species, genetic analysis has demonstrated that tolerance is controlled by a small number of major genes, with additional modifiers determining the level of tolerance (Smith and Macnair, 1998; van Hoof et al., 2001) Extensive studies have characterized the mechanisms underlying metal accumulation and tolerance in plants (for reviews, see Clemens, 2001; Schützendübel and Polle, 2002) and several plant genes that regulate the adaptive response to heavy metal-contaminated soil have been identified. For instance, it is known that phytochelatins (PCs) play a major role in heavy-metal detoxification. Indeed, metals bound to PCs are probably transported into the vacuoles (Cobbett, 2000). Studies of mutants and transgenic plants corroborated the importance of PCs for protection from heavy metals and lack of PC synthase activity led to increased sensitivity (Howden et al., 1995; Zhu et al., 1999). However, most molecular components of the signal transduction pathway involved in gene regulation are as yet unidentified. cDNA-amplified fragment length polymorphism (cDNA-AFLP) is an efficient, sensitive, and reproducible technology for the isolation of differentially expressed genes (Bachem et al., 1996); it does not require prior sequence information and is therefore a useful tool for the identification of novel genes (Ditt et al., 2001). In this study, cDNA-AFLP was employed to identify genes that exhibited a modulated expression following Cd-treatment in Brassica juncea. This species was chosen because it is characterized by rapid growth, high biomass, and an appreciable capacity to take up Cd as well as other toxic metals (Kumar et al., 1995; Rugh, 2004). In particular, Cd appeared to accumulate preferentially in roots, part of it is translocated from root to shoot, where it finally accumulates in the leaves (Salt et al., 1995a, b; Clemens et al., 2002). High accumulation was also demonstrated for the trichomes covering the leaf surface (Salt et al., 1995a), and it has been reported that B. juncea seedlings grown in aquaculture were able to accumulate and remove Cd from contaminated water (Salt et al., 1997). Further characterization of the Cd-responsive genes isolated in this work, may be helpful for a better understanding of the mechanisms of Cd accumulation and tolerance in plants. Materials and methods Plant material, culture conditions, and Cd treatment Brassica juncea (L.) Czern, cv. Aurea, was selected because previous work screening 10 cultivars (Bona et al., unpublished results) had demonstrated its enhanced ability to accumulate Cd from hydroponic solution into the above-ground (harvestable) parts. Seeds were rinsed with distilled water and incubated on soaked 3MM Whatman paper (Whatman, Maidstone, UK) in Petri dishes at 20 °C for 4 d. Germinated seeds were mixed with sand and vermiculite (1:1) and transferred to holes (1.5 cm diameter) on polyethylene discs used as floating supports. Plants were grown for 6 weeks in continuously aerated hydroponic nutrient solution. Each pot held three plants and 1.0 l of half-strength Hoagland solution (Hoagland and Arnon, 1938) with pH adjusted to 5.7. The plants were maintained in greenhouse conditions, and the nutrient solution changed every 5–7 d depending on evaporative demand. To identify the gene transcriptional changes modulated by Cd, 6-week-old plants were treated for 6 h and 24 h with 10 μM Cd(NO3)2 while other plants had received the same Cd-treatment from seed germination for 6 weeks. Untreated 6-week-old plants were used as controls. Plants were then harvested. Some of the samples were quickly frozen in liquid nitrogen and stored at −80 °C for RNA extraction, while others were analysed for Cd content. Determination of Cd content For the assay of Cd concentration, plants were harvested, rinsed in distilled water, weighed, and oven-dried at 85 °C for 36 h. Dried samples were homogenized before analysis using a Wiley mill. Cd analysis was performed after microwave-assisted acid digestion (EPA 3052, 1996) by means of ICP-MS analysis (EPA 200.8). mRNA isolation and cDNA synthesis Total RNA was extracted from plants and sampled after the indicated treatments, using Trizol reagent (Gibco, Germany). Total RNA concentration was determined spectrophotometrically and adjusted to a final concentration of 1 μg μl−1. Poly (A)+ RNA fractions were isolated from 1 mg total RNA with the Oligotex mRNA Minikit (QIAGEN, Germany). First and second cDNA strands were synthesized according to standard protocols (Sambrook et al., 1989). cDNA-AFLP analysis The cDNA-AFLP-based transcript profiling procedure was performed according to the method described in Breyne et al. (2002). Double-stranded cDNA (500 ng) was used for cDNA-AFLP analysis. The restriction enzymes used were BstYI and MseI (New England Biolabs, Beverly, MA, USA). For pre-amplification, an MseI-primer without a selective nucleotide was combined with a BstYI-primer containing a T or a C at the 3′ end. The amplification mixtures obtained were diluted 600-fold and 5 μl were used for final selective amplifications according to Breyne et al. (2002). BstT- and MseI-primers and BstC- and MseI-primers with one selective nucleotide, respectively, were used for the cDNA-AFLP analysis, and all 32 possible primer combinations were performed. Selective [33P]ATP-labelled amplification products were separated on a 6% polyacrylamide gel run at 1100 V for 3 h. Gels were dried onto 3MM Whatman paper, and positionally marked before being exposed to Kodak Biomax film (Amersham, Pharmacia, USA) for 2 d. Isolation and sequencing of fragments Films were aligned with markings on the gels. The bands of interest were marked, cut out with a razor blade, and incubated in 100 μl of water at 65 °C for 15 min and then left overnight at room temperature for DNA elution. The eluted DNA was re-amplified using the same PCR conditions and primer combination as for the selective amplification. The re-amplified products representing the Cd-regulated transcript-derived fragments (TDFs) were checked on 2% agarose gel and directly sequenced using the selective BstYI-primer as a sequencing primer. TDFs that failed to be sequenced were ligated to the pGEM-T EASY vector (Promega, Southampton, UK) and clones were then sequenced using the T7 or SP6 primer. Database searches were performed using the BLAST Network service [NCBI (National Center for Biotechnology Information); http://www.ncbi.nlm.nih.gov/BLAST]. The sequence of each TDF was searched against all sequences in the databases using the BLASTN and BLASTX programs (Altschul et al., 1997). Northern blot analysis Poly (A)+ RNA was prepared for Cd-treated as well as untreated plants by chromatography on oligo dT-cellulose (Bartels and Thompson, 1983). Three micrograms of mRNA per lane were fractionated on a 1% denaturing formaldehyde/agarose gel and transferred onto a positively charged nylon membrane. The procedures for radiolabelling and hybridization were performed at 42 °C as described previously (Sambrook et al., 1989). Filters were washed three times for 10 min at 65 °C with 2× SSC and 0.1% SDS. To check that the RNAs were equally loaded, the filters were re-probed with a barley ubiquitin cDNA clone (Gausing and Barkardottir, 1986). Results Plant growth and Cd accumulation Plants of B. juncea cv. Aurea, grown for 6 weeks with 10 μM Cd(NO3)2 present in the nutrient solution, exhibited growth inhibition of both roots and shoots, with roots being more affected by Cd than shoots. At the end of the experimental period leaves of these plants showed symptoms of chlorosis. The decrease in root elongation was also accompanied by changes in morphology. Roots became thinner and were putting out numerous side roots. Control plants maintained under greenhouse conditions and grown for 6 weeks in hydroponic solution, without Cd, showed a concentration of 15.29 μg kg−1 in their dry biomass. After exposure to 10 μM Cd(NO3)2, for either 6 h or 24 h, Cd concentration increased. Under these conditions, the amount of Cd in the dry biomass was 70.59 μg kg−1 DW and 123.53 μg kg−1 DW at 6 h and 24 h, respectively, confirming the adsorption even after a short time of Cd exposure. Plants exposed for 6 weeks to 10 μM Cd(NO3)2 showed an accumulation of 805.88 μg kg−1 DW (Fig. 1). It is worth noting the accumulation capacity of B. juncea in hydroponic culture (0.08% of dry weight), although the severe growth impairment and stressed condition of these plants make a meaningful comparison between Cd content and gene expression difficult. Fig. 1. View largeDownload slide Cd contents of plants maintained under greenhouse conditions, grown for 6 weeks in hydroponic solution and exposed to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Fig. 1. View largeDownload slide Cd contents of plants maintained under greenhouse conditions, grown for 6 weeks in hydroponic solution and exposed to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Analysis of cDNA-AFLP cDNA-AFLP analysis was performed to identify the genes responsive to Cd in B. juncea (Fig. 2). Different exposure times to Cd (6 h, 24 h, and 6 weeks) were chosen to detect genes rapidly responding to Cd shock, and genes whose expression is modulated by the continuous presence of Cd in the culture medium. By using 32 primer combinations, about 3000 cDNA fragments were counted and all bands longer than 80 bp in length were compared in the four tested Cd conditions. About 100 up-regulated or down-regulated gene fragments were identified as Cd modulated. These Cd-regulated TDFs, varying in length from 80 to 500 bp, were excised from the gels, re-amplified by PCR, and sequenced. Only in a few cases did different cDNA fragments belong to the same transcript; in addition, sequencing failed for several TDFs even after cloning. These fragments were not characterized further. Fig. 2. View largeDownload slide cDNA-AFLP autoradiography showing the TDFs induced or repressed by Cd treatment. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Fig. 2. View largeDownload slide cDNA-AFLP autoradiography showing the TDFs induced or repressed by Cd treatment. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. TDF sequences were compared with those present in the GenBank database (Table 1). The clones corresponding to different TDFs were renamed as BjCdR (B. juncea Cd-regulated). Of the total 73 TDFs sequenced (Table 1), 52 (71.2%) showed significant homology to genes with known or putative function, while 10 TDFs (13.7%) were homologous to uncharacterized genes (EST and unknown proteins). Of the remaining 11 TDFs, eight (11.0%) did not show significant matches; they may represent yet uncharacterized genes or the TDFs sequenced represent the 3′ end region of the transcripts, which is usually less conserved. For three (4.1%) TDFs, sequences match only genomic clones without allocated function. Table 1. Homologies of sequences of cDNA-AFLP fragments to sequences in the databases TDF   Length (bp)   Accession number   Homologya   BLAST scoreb,c   Expression patternd   BjCdR1  110  DT317657  Expressed protein from A. thaliana (NP_196620.1)  2e-12  0  BjCdR2  120  DT317658  Expressed protein from A. thaliana (NP_181834.1)  8e-09  6 h, 24 h, 6 weeks  BjCdR3  102  DT317659  Expressed protein from A. thaliana (NP_850785.1)  2e-07  6 h, 24 h, 6 weeks  BjCdR4  355  DT317660  Expressed protein from A. thaliana (NP_565726.1)  2e-24  0  BjCdR5  161  DT317661  Unknown protein from A. thaliana (AAM63312.1)  3e-04  6 h, 24 h, 6 weeks  BjCdR6  205  DT317662  Expressed protein from A. thaliana (NP_566625.1)  1e-14  24 h  BjCdR7  247  DT317663  Expressed protein from A. thaliana (NP_565922.1)  3e-29  6 h  BjCdR8  231  DT317664  Expressed protein from A. thaliana (NP_566847.1)  2e-15  6 h  BjCdR9  113  DT317665  Putative protein mRNA from A. thaliana (AY128790.1)  2e-10*  6 h  BjCdR10  379  DT317666  Expressed protein from A. thaliana (NP_174692.1)  3e-47  6 h, 24 h, 6 weeks  BjCdR11  130  DT317667  ABC1 family protein mRNA from A. thaliana (NM_180205)  4e-12*  24 h  BjCdR12  86  DT317668  MYB family transcription factor (MYB59) from A. thaliana (NP_851226.1)  3e-07  6 h  BjCdR13  176  DT317669  Putative initiation factor 5A mRNA A. thaliana (AF492850.1)  9e-08*  0  BjCdR14  151  DT317670  Transcription factor GBF5 from A. thaliana (AAG17474.1)  3e-08  6 h  BjCdR15  288  DT317671  bZIP family transcription factor (TGA3) from A. thaliana (NP_564156.1)  1e-11  6 h  BjCdR16  426  DT317672  Zinc-finger (B-box type) family protein/salt tolerance protein (STO) from A. thaliana (NP_172094.1)  2e-53  6 h, 24 h  BjCdR17  302  DT317673  CP12 protein, chloroplast precursor from P. sativum (T06562)  7e-21  6 h  BjCdR18  306  DT317674  Photosystem II family protein from A. thaliana (NP_563687.1)  6e-36  0  BjCdR19  331  DT317675  Photosystem I reaction centre subunit VI, chloroplast precursor (PSI-H) (light harvesting complex I 11 kDa protein) from B. rapa (O04006)  3e-25  6 h  BjCdR20  381  DT317676  Chorismate mutase, chloroplast precursor (CM1) from A. thaliana (P42738)  2e-65  24 h, 6 weeks  BjCdR21  392  DT317677  Chlorophyll a-b binding protein 1, chloroplast precursor (LCHII type I CAB-1) (LCHP) from S. alba (P13851)  3e-68  0  BjCdR22  168  DT317729  Putative cytochrome P450 protein mRNA from A. thaliana (AY050890)  7.2e-10*  24 h  BjCdR23  150  DT317678  LHCI type II from L. temulentum (CAA55864)  8e-13  0  BjCdR24  310  DT317679  ELIP from B. rapa subsp. pekinensis (AAR11456)  5e-10  24 h, 6 weeks  BjCdR25  287  DT317680  PSI-H subunit mRNA from B. rapa (U92504)  2e-06*  24 h  BjCdR26  86  DT317681  Glutamine synthetase, chloroplast precursor (glutamate–ammonia ligase) (GS2) from B. napus (Q42624)  2e-07  0  BjCdR27  135  DT317682  Glutamine-synthetase mRNA from A. thaliana (AY059932.1)  1e-15*  24 h  BjCdR28  127  DT317683  Cysteine synthase (O-acetylserine sulfhydrylase) (O-acetylserine (thiol)-lyase) from B. juncea (O23735)  6e-07  6 h, 24 h, 6 weeks  BjCdR29  321  DT317684  β-Hydroxyacyl-ACP dehydratase, putative from A. thaliana (NP_196578.1)  3e-29  24 h, 6 weeks  BjCdR30  386  DT317685  Putative histone deacetylase from A. thaliana (AAG28473.1)  6e-05  6 h  BjCdR31  287  DT317686  Ubiquitin-conjugating enzyme E2-17 kDa (ubiquitin-protein ligase) (ubiquitin carrier protein) from L. esculentum (P35135)  4e-51  24 h, 6 weeks  BjCdR32  236  DT317687  Starch excess protein (SEX1) from A. thaliana (NP_563877.1)  2e-35  6 h  BjCdR33  321  DT317688  AAA-type ATPase family protein from A. thaliana (NP_182074.2)  1e-48  6 h  BjCdR34  192  DT317689  NADH-ubiquinone oxireductase-related from A. thaliana (NP_566608.1)  3e-51  6 h  BjCdR35  174  DT317690  PBS lyase HEAT-like repeat-containing protein from A. thaliana (NP_197483.1)  3e-25  6 h  BjCdR36  485  DT317691  Phenylalanine ammonia lyase from A. thaliana (AAC18871)  1e-21  6 weeks  BjCdR37  355  DT317692  Putative serine-type carboxypeptidase II from A. thaliana (AAL33815)  2e-06  6 weeks  BjCdR38  123  DT317693  Ribulose-5-phosphate-3-epimerase from O. sativa (XP_470294)  5e-10  0  BjCdR39  202  DT317694  Aldehyde dehydrogenase mRNA from A. thaliana (NM_179476)  2e-06*  24 h  BjCdR40  141  DT317695  Pathogenesis-related protein, putative from A. thaliana (NP_195098.1)  2e-15  6 h  BjCdR41  309  DT317696  Pathogenesis-related family protein from A. thaliana (NP_849901.1)  2e-53  6 h  BjCdR42  133  DT317697  Ribosomal protein L35 from A. thaliana (CAA60774.1)  6e-07  0  BjCdR43  123  DT317698  60S ribosomal protein-like mRNA from A. thaliana (AY093180.1)  7e-11*  24 h  BjCdR44  225  DT317699  Putative ribosomal protein S3a homologue from A. thaliana (AAL15196)  9e-05  6 h  BjCdR45  240  DT317700  Putative 60S ribosomal protein from A. thaliana (AAN31827)  8e-17  0  BjCdR46  153  DT317701  Putative 60S ribosomal protein L6 from A. thaliana (AAO00948)  8e-12  0  BjCdR47  115  DT317702  Putative ribosomal protein S9 from A. thaliana (AAG51916)  2e-08  6 h, 24 h, 6 weeks  BjCdR48  152  DT317703  40S ribosomal protein S3 mRNA from A. thaliana (NM_115247)  4e-06*  0  BjCdR49  149  DT317704  Plasma membrane intrinsic protein 2 (PIP2) from B. napus (AAD39374.1)  3e-23  24 h  BjCdR50  262  DT317705  SNF7 family protein mRNA from A. thaliana (NM_127541)  2e-16*  6 h  BjCdR51  512  DT317706  Plasma membrane intrinsic protein 1 (PIP1) from B. napus (AAD39373.1)  5e-93  24 h  BjCdR52  148  DT317707  Sec61beta family protein from A. thaliana (NP_182033.1)  9e-08  6 h  BjCdR53  386  DT317708  Glutathione S-transferase, putative from A. thaliana (NP_177957.1)  2e-55  6 h  BjCdR54  134  DT317709  ERD9 mRNA for glutathione S-transferase from A. thaliana (AB039930.1)  7e-05*  6 weeks  BjCdR55  147  DT317710  RNA-binding protein homologue (grp2A) mRNA from S. alba (SALGRP2A)  3e-16*  6 h  BjCdR56  404  DT317711  Zinc finger (C3HC4-type RING finger) family protein from A. thaliana (NP_194556.1)  2e-56  0, 6 h  BjCdR57  396  DT317712  DNAJ heat-shock N-terminal domain-containing protein from A. thaliana (NP_178207.1)  8e-55  6 h, 24 h  BjCdR58  150  DT317713  Fasciclin-like arabinogalactan-protein, putative mRNA from A. thaliana (NM_123780.2)  1e-06*  6 h  BjCdR59  149  DT317714  Tetratricopeptide repeat (TRP)-containing protein from A. thaliana (NP_190782.3)  9e-08  6 h  BjCdR60  96  DT317715  Radical SAM domain-containing protein/ TRAM domain-containing protein from A. thaliana (NP_565035.1)  7e-11  0  BjCdR61  128  DT317716  Probable protein kinase mRNA from A. thaliana (NM_104060)  8e-36*  6 h, 24 h  BjCdR62  250  DT317728  Putative auxin-responsive protein mRNA from A. thaliana (AK117242)  3.3e-17*  6 h  BjCdR63  280  DT317717  DNA chromosome 4, BAC clone F25I24 from A. thaliana (ATF25I24)  0.00054*  6 h  BjCdR64  260  DT317718  Chromosome 2 clone from A. thaliana (AC004697)  9e-06*  6 weeks  BjCdR65  171  DT317719  Chromosome 2 clone F26B6 map CIC06C07 A. thaliana (AC003040)  1.1e-09*  24 h  BjCdR66  170  DT317720  No homologye    6 h  BjCdR67  222  DT317721  No homology    6 h, 24 h, 6 weeks  BjCdR68  203  DT317722  No homology    24 h, 6 weeks  BjCdR69  138  DT317723  No homology    24 h, 6 weeks  BjCdR70  103  DT317724  No homology    6 weeks  BjCdR71  188  DT317725  No homology    6 h  BjCdR72  256  DT317726  No homology    6 h  BjCdR73   339   DT317727   No homology     0   TDF   Length (bp)   Accession number   Homologya   BLAST scoreb,c   Expression patternd   BjCdR1  110  DT317657  Expressed protein from A. thaliana (NP_196620.1)  2e-12  0  BjCdR2  120  DT317658  Expressed protein from A. thaliana (NP_181834.1)  8e-09  6 h, 24 h, 6 weeks  BjCdR3  102  DT317659  Expressed protein from A. thaliana (NP_850785.1)  2e-07  6 h, 24 h, 6 weeks  BjCdR4  355  DT317660  Expressed protein from A. thaliana (NP_565726.1)  2e-24  0  BjCdR5  161  DT317661  Unknown protein from A. thaliana (AAM63312.1)  3e-04  6 h, 24 h, 6 weeks  BjCdR6  205  DT317662  Expressed protein from A. thaliana (NP_566625.1)  1e-14  24 h  BjCdR7  247  DT317663  Expressed protein from A. thaliana (NP_565922.1)  3e-29  6 h  BjCdR8  231  DT317664  Expressed protein from A. thaliana (NP_566847.1)  2e-15  6 h  BjCdR9  113  DT317665  Putative protein mRNA from A. thaliana (AY128790.1)  2e-10*  6 h  BjCdR10  379  DT317666  Expressed protein from A. thaliana (NP_174692.1)  3e-47  6 h, 24 h, 6 weeks  BjCdR11  130  DT317667  ABC1 family protein mRNA from A. thaliana (NM_180205)  4e-12*  24 h  BjCdR12  86  DT317668  MYB family transcription factor (MYB59) from A. thaliana (NP_851226.1)  3e-07  6 h  BjCdR13  176  DT317669  Putative initiation factor 5A mRNA A. thaliana (AF492850.1)  9e-08*  0  BjCdR14  151  DT317670  Transcription factor GBF5 from A. thaliana (AAG17474.1)  3e-08  6 h  BjCdR15  288  DT317671  bZIP family transcription factor (TGA3) from A. thaliana (NP_564156.1)  1e-11  6 h  BjCdR16  426  DT317672  Zinc-finger (B-box type) family protein/salt tolerance protein (STO) from A. thaliana (NP_172094.1)  2e-53  6 h, 24 h  BjCdR17  302  DT317673  CP12 protein, chloroplast precursor from P. sativum (T06562)  7e-21  6 h  BjCdR18  306  DT317674  Photosystem II family protein from A. thaliana (NP_563687.1)  6e-36  0  BjCdR19  331  DT317675  Photosystem I reaction centre subunit VI, chloroplast precursor (PSI-H) (light harvesting complex I 11 kDa protein) from B. rapa (O04006)  3e-25  6 h  BjCdR20  381  DT317676  Chorismate mutase, chloroplast precursor (CM1) from A. thaliana (P42738)  2e-65  24 h, 6 weeks  BjCdR21  392  DT317677  Chlorophyll a-b binding protein 1, chloroplast precursor (LCHII type I CAB-1) (LCHP) from S. alba (P13851)  3e-68  0  BjCdR22  168  DT317729  Putative cytochrome P450 protein mRNA from A. thaliana (AY050890)  7.2e-10*  24 h  BjCdR23  150  DT317678  LHCI type II from L. temulentum (CAA55864)  8e-13  0  BjCdR24  310  DT317679  ELIP from B. rapa subsp. pekinensis (AAR11456)  5e-10  24 h, 6 weeks  BjCdR25  287  DT317680  PSI-H subunit mRNA from B. rapa (U92504)  2e-06*  24 h  BjCdR26  86  DT317681  Glutamine synthetase, chloroplast precursor (glutamate–ammonia ligase) (GS2) from B. napus (Q42624)  2e-07  0  BjCdR27  135  DT317682  Glutamine-synthetase mRNA from A. thaliana (AY059932.1)  1e-15*  24 h  BjCdR28  127  DT317683  Cysteine synthase (O-acetylserine sulfhydrylase) (O-acetylserine (thiol)-lyase) from B. juncea (O23735)  6e-07  6 h, 24 h, 6 weeks  BjCdR29  321  DT317684  β-Hydroxyacyl-ACP dehydratase, putative from A. thaliana (NP_196578.1)  3e-29  24 h, 6 weeks  BjCdR30  386  DT317685  Putative histone deacetylase from A. thaliana (AAG28473.1)  6e-05  6 h  BjCdR31  287  DT317686  Ubiquitin-conjugating enzyme E2-17 kDa (ubiquitin-protein ligase) (ubiquitin carrier protein) from L. esculentum (P35135)  4e-51  24 h, 6 weeks  BjCdR32  236  DT317687  Starch excess protein (SEX1) from A. thaliana (NP_563877.1)  2e-35  6 h  BjCdR33  321  DT317688  AAA-type ATPase family protein from A. thaliana (NP_182074.2)  1e-48  6 h  BjCdR34  192  DT317689  NADH-ubiquinone oxireductase-related from A. thaliana (NP_566608.1)  3e-51  6 h  BjCdR35  174  DT317690  PBS lyase HEAT-like repeat-containing protein from A. thaliana (NP_197483.1)  3e-25  6 h  BjCdR36  485  DT317691  Phenylalanine ammonia lyase from A. thaliana (AAC18871)  1e-21  6 weeks  BjCdR37  355  DT317692  Putative serine-type carboxypeptidase II from A. thaliana (AAL33815)  2e-06  6 weeks  BjCdR38  123  DT317693  Ribulose-5-phosphate-3-epimerase from O. sativa (XP_470294)  5e-10  0  BjCdR39  202  DT317694  Aldehyde dehydrogenase mRNA from A. thaliana (NM_179476)  2e-06*  24 h  BjCdR40  141  DT317695  Pathogenesis-related protein, putative from A. thaliana (NP_195098.1)  2e-15  6 h  BjCdR41  309  DT317696  Pathogenesis-related family protein from A. thaliana (NP_849901.1)  2e-53  6 h  BjCdR42  133  DT317697  Ribosomal protein L35 from A. thaliana (CAA60774.1)  6e-07  0  BjCdR43  123  DT317698  60S ribosomal protein-like mRNA from A. thaliana (AY093180.1)  7e-11*  24 h  BjCdR44  225  DT317699  Putative ribosomal protein S3a homologue from A. thaliana (AAL15196)  9e-05  6 h  BjCdR45  240  DT317700  Putative 60S ribosomal protein from A. thaliana (AAN31827)  8e-17  0  BjCdR46  153  DT317701  Putative 60S ribosomal protein L6 from A. thaliana (AAO00948)  8e-12  0  BjCdR47  115  DT317702  Putative ribosomal protein S9 from A. thaliana (AAG51916)  2e-08  6 h, 24 h, 6 weeks  BjCdR48  152  DT317703  40S ribosomal protein S3 mRNA from A. thaliana (NM_115247)  4e-06*  0  BjCdR49  149  DT317704  Plasma membrane intrinsic protein 2 (PIP2) from B. napus (AAD39374.1)  3e-23  24 h  BjCdR50  262  DT317705  SNF7 family protein mRNA from A. thaliana (NM_127541)  2e-16*  6 h  BjCdR51  512  DT317706  Plasma membrane intrinsic protein 1 (PIP1) from B. napus (AAD39373.1)  5e-93  24 h  BjCdR52  148  DT317707  Sec61beta family protein from A. thaliana (NP_182033.1)  9e-08  6 h  BjCdR53  386  DT317708  Glutathione S-transferase, putative from A. thaliana (NP_177957.1)  2e-55  6 h  BjCdR54  134  DT317709  ERD9 mRNA for glutathione S-transferase from A. thaliana (AB039930.1)  7e-05*  6 weeks  BjCdR55  147  DT317710  RNA-binding protein homologue (grp2A) mRNA from S. alba (SALGRP2A)  3e-16*  6 h  BjCdR56  404  DT317711  Zinc finger (C3HC4-type RING finger) family protein from A. thaliana (NP_194556.1)  2e-56  0, 6 h  BjCdR57  396  DT317712  DNAJ heat-shock N-terminal domain-containing protein from A. thaliana (NP_178207.1)  8e-55  6 h, 24 h  BjCdR58  150  DT317713  Fasciclin-like arabinogalactan-protein, putative mRNA from A. thaliana (NM_123780.2)  1e-06*  6 h  BjCdR59  149  DT317714  Tetratricopeptide repeat (TRP)-containing protein from A. thaliana (NP_190782.3)  9e-08  6 h  BjCdR60  96  DT317715  Radical SAM domain-containing protein/ TRAM domain-containing protein from A. thaliana (NP_565035.1)  7e-11  0  BjCdR61  128  DT317716  Probable protein kinase mRNA from A. thaliana (NM_104060)  8e-36*  6 h, 24 h  BjCdR62  250  DT317728  Putative auxin-responsive protein mRNA from A. thaliana (AK117242)  3.3e-17*  6 h  BjCdR63  280  DT317717  DNA chromosome 4, BAC clone F25I24 from A. thaliana (ATF25I24)  0.00054*  6 h  BjCdR64  260  DT317718  Chromosome 2 clone from A. thaliana (AC004697)  9e-06*  6 weeks  BjCdR65  171  DT317719  Chromosome 2 clone F26B6 map CIC06C07 A. thaliana (AC003040)  1.1e-09*  24 h  BjCdR66  170  DT317720  No homologye    6 h  BjCdR67  222  DT317721  No homology    6 h, 24 h, 6 weeks  BjCdR68  203  DT317722  No homology    24 h, 6 weeks  BjCdR69  138  DT317723  No homology    24 h, 6 weeks  BjCdR70  103  DT317724  No homology    6 weeks  BjCdR71  188  DT317725  No homology    6 h  BjCdR72  256  DT317726  No homology    6 h  BjCdR73   339   DT317727   No homology     0   a GenBank accession numbers of sequences homologous to AFLP fragments are in parentheses. b All are BLASTX scores except for those marked with * which are BLASTN scores. c e-value cut-off=1e−5. d Gene expression pattern after exposure to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 weeks (6 w) represent the Cd exposure time. e No significant sequence homology found in genome, EST, and protein database. View Large Considering the expression pattern of Cd-modulated genes, among the TDFs isolated and sequenced, Table 1 shows that genes were either up- or down-regulated by Cd treatment. It is possible to distinguish 14 genes that were expressed only in untreated control plants and down-regulated by Cd treatment, while seven genes were induced after Cd exposure and the transcription was sustained throughout the Cd treatment (6 h, 24 h, and 6 weeks). Table 1 also shows that 27 genes, the majority of the TDFs identified, are detected within 6 h of exposure to Cd and their expression is suppressed with longer Cd treatment times, suggesting that many of them may have roles in further signalling. Ten genes were up-regulated only 24 h after the addition of Cd and three genes were induced at 6 h and 24 h of Cd treatment. For six genes the transcription was observed at 24 h and 6 weeks of Cd exposure, and only one was up-regulated in untreated plants and after the addition of Cd for 6 h. In plants treated with Cd for 6 weeks the transcript accumulation of five genes was detected. The induction of the latter genes might probably be due to general stress conditions imposed on the plants by the continuous presence of Cd in the culture medium. Indeed, protracted exposure to Cd inhibited growth, and the generally stunted conditions and leaf chlorosis indicated that general metabolism such as photosynthesis and respiration were affected. Northern analysis of Cd-regulated genes To validate the cDNA-AFLP expression patterns, several TDFs were selected for RNA gel blot analysis. To minimize problems of cross-hybridization, full-length TDFs were used and their kinetics of transcript accumulation in response to the Cd presence in the culture medium are shown in Fig. 3. Moreover, to confirm the expression pattern observed, each hybridization was repeated at least twice (data not shown). For this expression analysis, TDFs from genes induced at 6 h of Cd treatment were preferentially chosen as probes to further characterize genes which may have roles in the early events of the signal transduction pathways leading to Cd translocation and sequestration in B. juncea. Fig. 3. View largeDownload slide Northern analysis showing the expression profiles of several genes induced by Cd treatment in B. juncea. These experiments were repeated at least twice for each TDF. Poly (A)+ RNA was isolated from whole plants after Cd exposure for 0, 6 h, 24 h, and 6 weeks. Fig. 3. View largeDownload slide Northern analysis showing the expression profiles of several genes induced by Cd treatment in B. juncea. These experiments were repeated at least twice for each TDF. Poly (A)+ RNA was isolated from whole plants after Cd exposure for 0, 6 h, 24 h, and 6 weeks. The induction pattern observed in northern analysis showed that six of the nine TDFs tested (BjCdR15, BjCdR7, BjCdR35, BjCdR14, BjCdR29, and BjCdR30) fully confirmed the expression profiles observed with the cDNA-AFLP analysis. This technique was thus validated in 67% of cases. It is noteworthy that there are slight differences in expression pattern for the three TDFs (BjCdR47, BjCdR43, and BjCdR51) for which the transcription profile seen with the cDNA-AFLP analysis was not confirmed by RNA gel blot analysis. Northern analysis showed that the ribosomal protein S9 (BjCdR47) is mainly induced at 6 h and 24 h of Cd exposure and that its transcription decreased with longer Cd treatment; whereas the 60S ribosomal protein (BjCdR43) showed the highest transcript level when Cd was added to the culture medium for 6 h, and the plasma membrane intrinsic protein 1 (BjCdR51) is mainly expressed at 24 h and 6 weeks of Cd treatment. These discrepancies between northern blotting and cDNA-AFLP analysis may either be due to changes in the intensity of individual bands in the cDNA-AFLP gels or to gene family complexity. Nevertheless, the cDNA-AFLP technique allowed the isolation of differentially expressed genes under the conditions tested. Functional classification and temporal expression pattern of Cd-responsive genes Of the 73 sequenced Cd-modulated genes, 52 were genes of known or putative function which could be grouped into several major functional categories (Table 2). Four were transcriptional factors possibly involved in the transcriptional control of plant stress response. BjCdR15 and BjCdR14 activated at 6 h of Cd treatment showed homology to Arabidopsis TGA3 and GBF5, respectively, which belong to the bZIP protein family of transcriptional factors that have been implicated in stress signalling (Jakoby et al., 2002). In particular, the gene corresponding to BjCdR15 is under investigation and preliminary data suggest that it is transcribed in leaves and roots after 0.5 h of Cd treatment and is also activated by other heavy metals, such as Pb and Zn (Micheletto et al., unpublished results). In addition, a cDNA fragment (BjCdR12) homologous to the Arabidopsis Myb59 was induced at 6 h of Cd treatment, while BjCdR16 expressed at 6 and 24 h of Cd exposure showed homology to zinc finger protein. Cd modulated the expression of several stress-responding proteins. Genes whose expression improves plant stress protection were considered to be in this functional category. At 24 h of Cd stress, BjCdR39 was isolated, which showed homology to an aldehyde dehydrogenase induced by dehydration, NaCl, heavy metal, and oxidative stress (Sunkar et al., 2003), whereas 6 h of Cd stress induced the transcription of a gene (BjCdR55) homologous to Sinapis alba mRNA cap-binding protein. It has been reported that the Arabidopsis homologue is implicated in abscisic acid (ABA) signalling (Hugouvieux et al., 2001). Two cDNA fragments (BjCdR40 and BjCdR41) induced early by Cd exhibited homology to pathogenesis-related protein. The stress-responding category also includes a gene (BjCdR24) transcribed at 24 h and 6 weeks of Cd treatment that showed homology to a gene encoding for an early light-induced protein (ELIP) from Brassica rapa, and a gene transcribed at 24 h of Cd stress (BjCdR22) homologous to the Arabidopsis cytochrome P450. Transcript induction was observed in all Cd time treatments for a gene encoding O-acetylserine (thiol) lyase (BjCdR28), an enzyme that plays a role in Cd tolerance (Domínguez-Solis et al., 2001). Furthermore, BjCdR53 and BjCdR54, detected, respectively, at 6 h and 6 weeks of Cd exposure, showed high homology to Arabidopsis genes encoding glutathione S-transferase directly involved in Cd stress tolerance. Not unexpectedly, at 6 h and 24 h of Cd exposure, a cDNA fragment (BjCdR57) was isolated homologous to a gene encoding for a DNAJ heat-shock protein probably participating in protein folding, and BjCdR36 detected after 6 weeks of Cd treatment showed homology to an Arabidopsis gene encoding for a phenylalanine ammonia lyase, a key enzyme leading to lignin synthesis (Mao et al., 2004). Table 2. Functional classification of Cd-responsive gene product in B. juncea Functiona   Identification of BjCdR clones   No. clones   %   Transcriptional factor  12, 14, 15, 16  4  5.5  Stress responding  40, 41, 53, 54, 57, 36, 22, 24, 39, 55, 38, 28  12  16.4  Cellular metabolism and organization  26, 27, 29, 20, 34, 37, 58, 59  8  11.0  Photosynthetic process  17, 18, 19, 32, 21, 23, 25  7  9.6  Transport facilitation  33, 52, 50, 51, 49, 11  6  8.2  Unclassified protein  1, 2, 3, 4, 5, 6, 7, 8, 9, 10  10  13.7  Expression regulator  42, 43, 44, 45, 46, 47, 48, 30, 31, 56, 13  11  15.0  Miscellaneous  35, 61, 62, 60  4  5.5  No hitb  63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73  11  15.1  Total     73   100   Functiona   Identification of BjCdR clones   No. clones   %   Transcriptional factor  12, 14, 15, 16  4  5.5  Stress responding  40, 41, 53, 54, 57, 36, 22, 24, 39, 55, 38, 28  12  16.4  Cellular metabolism and organization  26, 27, 29, 20, 34, 37, 58, 59  8  11.0  Photosynthetic process  17, 18, 19, 32, 21, 23, 25  7  9.6  Transport facilitation  33, 52, 50, 51, 49, 11  6  8.2  Unclassified protein  1, 2, 3, 4, 5, 6, 7, 8, 9, 10  10  13.7  Expression regulator  42, 43, 44, 45, 46, 47, 48, 30, 31, 56, 13  11  15.0  Miscellaneous  35, 61, 62, 60  4  5.5  No hitb  63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73  11  15.1  Total     73   100   a Unclassified protein indicates sequence that is homologous to unknown, putative, and expressed proteins without annotated function in other organisms. Other sequence homologies denoted in Table 1 with a putative or probable function are included in their probable function categories. b No hit indicates identity only to unannotated genomic sequences or low similarity to existing nucleotide sequences. View Large A group of eight genes were considered to be related to cellular metabolism and organization. Fragments homologous to Arabidopsis chorismate mutase (BjCdR20), and β-hydroxyacyl-ACP (BjCdR29) were observed at 24 h and 6 weeks of Cd treatment and are involved in amino acid and fatty acid biosynthesis, respectively, while BjCdR34 observed at 6 h of Cd exposure showed similarity to a gene encoding a NADH-ubiquinone oxidoreductase involved in cellular respiration. BjCdR26 and BjCdR27, which are down-regulated by Cd or induced at 24 h of Cd treatment, respectively, showed homology to genes encoding glutamine-synthetase, an enzyme involved in the synthesis of glutamate. In addition, cDNA fragments detected after long Cd exposure (BjCdR37) showed similarity to a serine carboxypeptidase probably involved in plant development, whereas BjCdR59 and BjCdR58 detected at 6 h of Cd treatment were similar to a gene encoding a tetratricopeptide repeat-containing protein implicated in protein–protein interaction (Das et al., 1998) and a fasciclin-like arabinogalactan-protein involved in cell adhesion (Johnson et al., 2003), respectively. The presence of Cd in plants causes an overall inhibition of photosynthesis. In this work, seven genes related to the photosynthetic process were found to be modulated by Cd. The expression of three of them (BjCdR18, BjCdR21, and BjCdR23) was inhibited by the presence of Cd in the culture medium, while three cDNA fragments (BjCdR17, BjCdR19, and BjCdR32) were detected only after 6 h of Cd exposure, and BjCdR25 was found instead at 24 h of Cd treatment. The presence of Cd in the culture medium rapidly induced the synthesis of six proteins that were included in the transport facilitation category. Following the expression pattern in Table 1, BjCdR49 and BjCdR51 were isolated at 24 h of Cd treatment, which showed sequence similarity to Brassica napus aquaporins PIP2 and PIP1, respectively, although northern analysis (Fig. 3) indicated that PIP1 is also transcribed at 6 weeks of Cd exposure. The expression of these genes suggests that Cd evoked water stress. After 24 h of Cd addition, BjCdR11 homologous to an Arabidopsis ABC1 transporter was also induced. Moreover, genes with similarity to an AAA-type ATPase (BjCdR33), Sec61 beta (BjCdR52) and SNF7 (BjCdR50), all involved in protein transport across membranes, were only induced at 6 h of Cd treatment. Genes encoding for ribosomal proteins (BjCdR42, BjCdR43, BjCdR44, BjCdR45, BjCdR46, BjCdR47, and BjCdR48) were classified as expression regulators. The transcription of four of them (BjCdR42, BjCdR45, BjCdR46, and BjCdR48) was only observed in untreated control plants. BjCdR47 was up-regulated by Cd and its transcription was maintained at all times of Cd treatment, although northern analysis (Fig. 3) showed the higher expression at 6 and 24 h of Cd treatment. The expressions of BjCdR44 and BjCdR43 were induced at 6 h and 24 h of Cd exposure, respectively. Also for the latter gene, an RNA gel blot (Fig. 3) revealed the highest expression after 6 h of Cd addition. BjCdR30, homologous to a histone deacetylase, up-regulated at 6 h of Cd treatment, BjCdR31 with similarity to a ubiquitin carrier protein, activated at 24 h and 6 weeks of Cd treatment and probably involved in protein degradation, were also included in this functional category. Furthermore, transcription was inhibited by Cd for a gene homologous to an initiation factor BjCdR13, whereas inhibition was seen after 24 h and 6 weeks of Cd exposure for a gene similar to a zinc finger RING (BjCdR56). The other four clones were clustered as miscellaneous. A PBS lyase HEAT-like repeat-containing protein, BjCdR35, was detected at 6 h of Cd treatment; a putative protein kinase, BjCdR61, probably involved in signalling is activated at 6 h and 24 h of Cd treatment and a putative auxin-responsive protein, BjCdR62, is transcribed at 6 h of Cd exposure. A gene encoding for a protein containing the SAM and TRAM domain, BjCdR60, repressed by Cd is also included in this functional category. Discussion Some plants species can grow on soil that naturally, or due to human activities, contains growth-prohibiting concentrations of metals (Clemens, 2001). For instance, Brassica juncea, a fast-growing plant with high biomass, has the ability to take up Cd, as other heavy metals, from the soil and to accumulate substantial amounts of it in the shoot (Salt et al., 1995a, 1997). Therefore, B. juncea is an interesting species to use in the study of Cd accumulation and tolerance mechanisms (Clemens et al., 2002; Rugh, 2004). In this study, cDNA-AFLP technology was employed to identify Cd-regulated gene expression in B. juncea to gain new insights into the molecular mechanisms governing Cd accumulation. Plants of B. juncea grown in hydroponic culture with Cd added for 6 weeks showed symptoms of toxicity. Long exposure to Cd induced leaf chlorosis. This observation is in agreement with other works describing a reduction of chlorophyll concentration in plants of B. juncea (Salt et al., 1995b) and B. napus (Larsson et al., 1998) exposed to Cd, indicating a general inhibition of the photosynthetic process. Cd also inhibited plant growth, affecting roots to a greater extent than leaves, while plants exposed to Cd for short times (6 h and 24 h) showed no symptoms. Similar root growth inhibition was described for seedlings of several genotypes of Pisum sativum grown in hydroponic culture for 10 d and exposed to 5 μM CdCl2 (Metwally et al., 2005). Long exposure to Cd affected root morphology; not only root elongation was inhibited, but roots appeared thinner and sent out side roots. A plant with numerous thin roots would accumulate more metals than one with a few thick roots (Schierup and Larsen, 1981), plus, to take up heavy metal ions, plants must be able to renew the active parts of their root biomass (Das et al., 1997). The development of thin and side roots in plants exposed to Cd during growth may thus represent an adaptive strategy to cope with Cd ions. As a first step in the identification of Cd-modulated genes in B. juncea, Cd contents of whole plants were measured and similar plant materials were used for the cDNA-AFLP analysis. The Cd content analysis showed that B. juncea plants grown in hydroponic solution take up Cd and the metal concentration in plants increased with the duration of Cd treatment. The cDNA-AFLP technique allowed transcription changes to be surveyed with no prior assumptions about which genes might be induced or repressed by Cd treatment. Northern blotting analysis of nine TDFs confirmed that expression of the identified genes is Cd modulated. With the enzyme combination chosen, 3000 cDNA fragments were visualized on the gels, and about 100 were found to be Cd regulated, of which 73 are reported in Table 1. They belong to different functional categories, which indicates that Cd affected different physiological and biochemical pathways. Sequencing analyses revealed that one gene identified is homologous to a protein kinase (BjCdR61) and four encode transcriptional factors (BjCdR12, BjCdR14, BjCdR15, and BjCdR16), indicating that signal transduction pathways are rapidly activated by the presence of Cd in the nutrient solution. Overall the transcriptional factors identified in this work have been linked to plant stress responses (Singh et al., 2002), particularly TGA and GBF; members of the bZIP protein family are involved in responses to ABA and ethylene as well as in pathogen attack (Jakoby et al., 2002). The induction by Cd of transcripts for bZIP, Myb, and zinc finger transcriptional factor observed in this study suggest, as previously reported by Knight and Knight (2001), that plant response to environmental stresses, including heavy metals, may be regulated by multiple signalling pathways. Among the Cd-regulated genes detected, 12 appear to encode proteins which protect against Cd stress. The transcription upon exposure to Cd of a DNAJ heat shock protein (BjCdR57), a chaperone involved in protein protection in times of cellular stress, confirmed that protein denaturation is one of the effects of Cd toxicity (Suzuki et al., 2001). Furthermore, the expression of two pathogenesis-related proteins (BjCdR40 and BjCdR41) at 6 h after Cd addition indicates that Cd induces defence reactions. Indeed, H2O2 accumulation was observed in Cd-exposed roots (Schützendübel et al., 2001), and it was suggested that H2O2 would act as a signalling molecule triggering secondary defences leading to cell wall rigidification and lignification in Cd-exposed cells (Schützendübel and Polle, 2002). In addition, the transcript of phenylalanine ammonia lyase (BjCdR36), an enzyme of the phenylpropanoid pathway leading to lignin synthesis, detected in 6-week-old Cd-treated plants, is in accordance with the finding that Cd induces lignin deposition in cell walls. One of the most sensitive responses of higher plants to Cd is stomata closure (Sanità di Toppi and Gabbrielli, 1999), which is a symptom of water stress mediated by ABA. Based on transcript accumulation, aldehyde dehydrogenase has been correlated with ABA treatment, drought stress, UV light, NaCl, and heavy metals (Chen et al., 2002; Sunkar et al., 2003). The expression of an aldehyde dehydrogenase (BjCdR39) and an RNA-binding protein (BjCdR55) involved in ABA signalling, induced by Cd in B. juncea, corroborates the idea of existing cross-talk between Cd-induced and water stress-induced signalling that may also employ ABA a as signal transduction compound (Polle and Schützendübel, 2004). Genes encoding for glutathione S-transferases (BjCdR53 and BjCdR54) and cytochrome P450 (BjCdR22), were previously reported as responsive to Cd and other stresses (Marrs and Walbot, 1997; Suzuki et al., 2001) and are probably functioning in cytotoxic product detoxification. In particular, glutathione S-transferases catalyse the synthesis of glutathione S-conjugates, allowing them to be recognized for transport into the vacuole (Marrs and Walbot, 1997). Chelation of metals by high-affinity ligands, such as PSs and thiolated peptides, is considered a principal mechanism of Cd detoxification in plants, and numerous physiological, biochemical, and genetic studies have confirmed that glutathione is the substrate for PS biosynthesis (Cobbett, 2000; Cobbett and Goldsbrough, 2002). Moreover, in B. juncea, changes of expression of a glutathione transporter in response to Cd exposure has been reported (Bogs et al., 2003) also indicating that glutathione plays a prominent role in Cd accumulation and/or detoxification. The amount of glutathione transporter protein decreased in older leaves 48 h after the onset of Cd exposure and reached a minimum at 96 h, while an increase in protein amount was detected at 120 h and 144 h of Cd treatment. It was suggested (Heiss et al., 2003) that, during Cd exposure, glutathione export to the various sink tissues is reduced to meet the high demand of glutathione for PS synthesis during Cd accumulation and, therefore, the observed decrease in glutathione transporter protein may reflect this adaptation. In this cDNA-AFLP analysis TDFs for glutathione transporters were not detected. This may simply be due to the technique employed that allows discrimination between the presence or absence of bands, or to the experimental conditions used. In this study, a gene (BjCdR28) encoding for O-acetylserine (thiol) lyase enzyme (OASTL) was up-regulated in all Cd exposure times. OASTL catalyses the last step of cysteine biosynthesis and Arabidopsis plants overexpressing OASTL showed high Cd resistance, suggesting that cysteine pool requirement for glutathione biosynthesis is a main factor for tolerance (Domínguez-Solis et al., 2001). However, previous studies in B. juncea, also indicated an enhanced OASTL expression, although glutathione biosynthesis, more than cysteine availability, was considered the limiting step (Schäfer et al., 1998). Cd toxicity is generally associated with inhibition of chlorophyll synthesis and damage to photosynthetic apparatus (Sanità di Toppi and Gabbrielli, 1999). In this work, a group of genes related to the photosynthetic process showed expression only in Cd-untreated control plants (BjCdR18, BjCdR21, and BjCdR23), while others (BjCdR19, BjCdR32, and BjCdR25) were induced by Cd treatments, indicating that the presence of Cd in plant tissues disturbs photosynthesis. Similarly, the effect of Cd was evident from the expression pattern of genes that were grouped under the function of cellular metabolism and organization and that may be involved in different cellular processes, such as a tetratricopeptide repeat-containing protein (BjCdR59) induced after 6 h of Cd addition, probably acting as a scaffold for the assembly of multi-protein complexes (Das et al., 1998), and a fasciclin-like arabinogalactan-protein (BjCdR58) putatively working in cell adhesion (Johnson et al., 2003). Furthermore, Cd affected the transcription of genes encoding for glutamine-synthetase (GS) (BjCdR26 and BjCdR27) involved in nitrogen metabolism. The expression of chloroplastic GS was inhibited by Cd treatment, while transcription of the cytosolic GS was detected 24 h after Cd addition. Protein and transcript analyses of Cd-treated tomato plants showed that cytosolic GS increased in leaves and chloroplastic GS decreased in parallel, suggesting that plants subjected to Cd stress induced cytosolic GS to compensate and continue glutamine biosynthesis when Cd affected chloroplastic GS activity (Chaffei et al., 2004). In addition, the induction of chorismate mutase (BjCdR20) observed in this work at 24 h and 6 weeks of Cd exposure also confirms an increased amino acid synthesis in Cd-treated plants (Chaffei et al., 2004). Sequence analysis of Cd-responsive genes also revealed the induction of genes whose gene products are involved in cellular transport. Aquaporins mediate the passive movement of water in cellular membranes and the expression of plasma membrane intrinsic proteins, PIP1 and PIP2, is regulated by ABA and water stress (Gao et al., 1999; Yang et al., 2003). The transcription of aquaporins PIP1 and PIP2 (BjCdR51 and BjCdR49) seen in B. juncea upon exposure to Cd for 24 h, together with the expression of other drought- and ABA-responsive genes (BjCdR39 and BjCdR55) strengthen the idea that Cd imposes water stress and that both ABA and Cd act synergistically (Polle and Schülzendübel, 2004). In addition, a gene encoding for an ABC-transporter protein (BjCdR11) was up-regulated by Cd treatment. ABC transporters directly involved in Cd transport have not been identified in plants, whereas it has been shown that in yeast and fission yeast they act directly in the final step of Cd detoxification by mediating the vacuolar transport of Cd complexes (Ortiz et al., 1995; Li et al., 1997). However, recent analyses of AtMRPs, a subfamily of Arabidopsis ABC transporters, showed that AtMRP3 was induced by Cd and not by oxidative stress (Bovet et al., 2003), suggesting that ABC transporters in plants, as in yeast, are involved in heavy metal fluxes, although a direct role of AtMRP3 in Cd transport has not been demonstrated (Bovet et al., 2003). In conclusion, the cDNA-AFLP technique allowed genes to be identified whose expression is modulated by Cd. This study reveals that a multitude of processes are implicated in determining response to metal in plants and these processes require the activation of different sets of genes. Identification of components of the signal transduction cascades suggests that Cd triggers stress signals and that ABA may be involved in plant response to Cd. 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Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica juncea L.

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Oxford University Press
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
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0022-0957
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10.1093/jxb/eri299
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Abstract

Abstract In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was employed to identify genes that exhibited a modulated expression following cadmium (Cd) treatment in Brassica juncea grown in hydroponic culture. Plants were treated for 6 h, 24 h, and 6 weeks with 10 μM Cd(NO3)2 and untreated 6-week-old plants were used as controls. Cd content was measured at these four time points. Long exposure to Cd affected root morphology: roots appeared thinner and sent out side roots. Seventy-three transcript-derived fragments were identified as Cd responsive. Fifty-two of them showed significant homology to genes with known or putative function, 10 transcript-derived fragments were homologous to uncharacterized genes, while 11 transcript-derived fragments did not show significant matches. The expression pattern of several of these genes was confirmed by northern blot analysis. Fifty-two genes of known or putative function were transcriptional factors, expression regulators, and stress responding and transport facilitation genes, as well as genes involved in cellular metabolism and organization and the photosynthetic process, suggesting that a multitude of processes are implicated in Cd stress response. The transcription of drought- and abscisic acid-responsive genes observed in this study also suggested that Cd imposes water stress and that abscisic acid may be involved in the Cd plant response. Brassica juncea, cadmium, cDNA-AFLP, gene expression, heavy metals Introduction Environmental pollution by toxic metals has accelerated dramatically since the beginning of the industrial revolution (Nriagu, 1979). Cadmium, a non-essential heavy metal, is considered one of the major pollutants (Alloway and Steinnes, 1999). It is widespread in soil containing waste materials from zinc mines, in sewage water used for irrigation, in sludge-amended soils, and in soil fertilized with cadmium-rich phosphate fertilizers (Hüttermann et al., 1999). Cadmium is also released into the environment by urban traffic, heating systems, and waste incinerators (Sanità di Toppi and Gabbrielli, 1999). Toxicity to living cells is caused by very low cadmium concentrations and its presence in the food chain can be highly dangerous as it can cause significant damage to the human body (Buchet et al., 1990) and is a suspected carcinogen (Vido, 2001). Although Cd is a non-essential element for plant mineral nutrition it is taken up easily by roots and transported through the xylem to the vegetative and reproductive organs, thus affecting nutrient uptake and homeostasis and inhibiting root and shoot growth and yield production (Sanità di Toppi and Gabbrielli, 1999; Metwally et al., 2005). However, certain plant species have the ability to survive and reproduce on soils containing high concentrations of metals in forms that are toxic or inimical to other plants (Macnair and Baker, 1994). The ability of these plants to survive on metal-polluted soils is not only due to their capacity to take up, translocate, and sequester metals, but is also based on mechanisms that allow them to tolerate high levels of the element in root and shoot cells by alleviating their toxic effects (Salt et al., 1998). Tolerance is achieved by internal detoxification and probably involves cell compartmentation and metal complexation. For several metals and species, genetic analysis has demonstrated that tolerance is controlled by a small number of major genes, with additional modifiers determining the level of tolerance (Smith and Macnair, 1998; van Hoof et al., 2001) Extensive studies have characterized the mechanisms underlying metal accumulation and tolerance in plants (for reviews, see Clemens, 2001; Schützendübel and Polle, 2002) and several plant genes that regulate the adaptive response to heavy metal-contaminated soil have been identified. For instance, it is known that phytochelatins (PCs) play a major role in heavy-metal detoxification. Indeed, metals bound to PCs are probably transported into the vacuoles (Cobbett, 2000). Studies of mutants and transgenic plants corroborated the importance of PCs for protection from heavy metals and lack of PC synthase activity led to increased sensitivity (Howden et al., 1995; Zhu et al., 1999). However, most molecular components of the signal transduction pathway involved in gene regulation are as yet unidentified. cDNA-amplified fragment length polymorphism (cDNA-AFLP) is an efficient, sensitive, and reproducible technology for the isolation of differentially expressed genes (Bachem et al., 1996); it does not require prior sequence information and is therefore a useful tool for the identification of novel genes (Ditt et al., 2001). In this study, cDNA-AFLP was employed to identify genes that exhibited a modulated expression following Cd-treatment in Brassica juncea. This species was chosen because it is characterized by rapid growth, high biomass, and an appreciable capacity to take up Cd as well as other toxic metals (Kumar et al., 1995; Rugh, 2004). In particular, Cd appeared to accumulate preferentially in roots, part of it is translocated from root to shoot, where it finally accumulates in the leaves (Salt et al., 1995a, b; Clemens et al., 2002). High accumulation was also demonstrated for the trichomes covering the leaf surface (Salt et al., 1995a), and it has been reported that B. juncea seedlings grown in aquaculture were able to accumulate and remove Cd from contaminated water (Salt et al., 1997). Further characterization of the Cd-responsive genes isolated in this work, may be helpful for a better understanding of the mechanisms of Cd accumulation and tolerance in plants. Materials and methods Plant material, culture conditions, and Cd treatment Brassica juncea (L.) Czern, cv. Aurea, was selected because previous work screening 10 cultivars (Bona et al., unpublished results) had demonstrated its enhanced ability to accumulate Cd from hydroponic solution into the above-ground (harvestable) parts. Seeds were rinsed with distilled water and incubated on soaked 3MM Whatman paper (Whatman, Maidstone, UK) in Petri dishes at 20 °C for 4 d. Germinated seeds were mixed with sand and vermiculite (1:1) and transferred to holes (1.5 cm diameter) on polyethylene discs used as floating supports. Plants were grown for 6 weeks in continuously aerated hydroponic nutrient solution. Each pot held three plants and 1.0 l of half-strength Hoagland solution (Hoagland and Arnon, 1938) with pH adjusted to 5.7. The plants were maintained in greenhouse conditions, and the nutrient solution changed every 5–7 d depending on evaporative demand. To identify the gene transcriptional changes modulated by Cd, 6-week-old plants were treated for 6 h and 24 h with 10 μM Cd(NO3)2 while other plants had received the same Cd-treatment from seed germination for 6 weeks. Untreated 6-week-old plants were used as controls. Plants were then harvested. Some of the samples were quickly frozen in liquid nitrogen and stored at −80 °C for RNA extraction, while others were analysed for Cd content. Determination of Cd content For the assay of Cd concentration, plants were harvested, rinsed in distilled water, weighed, and oven-dried at 85 °C for 36 h. Dried samples were homogenized before analysis using a Wiley mill. Cd analysis was performed after microwave-assisted acid digestion (EPA 3052, 1996) by means of ICP-MS analysis (EPA 200.8). mRNA isolation and cDNA synthesis Total RNA was extracted from plants and sampled after the indicated treatments, using Trizol reagent (Gibco, Germany). Total RNA concentration was determined spectrophotometrically and adjusted to a final concentration of 1 μg μl−1. Poly (A)+ RNA fractions were isolated from 1 mg total RNA with the Oligotex mRNA Minikit (QIAGEN, Germany). First and second cDNA strands were synthesized according to standard protocols (Sambrook et al., 1989). cDNA-AFLP analysis The cDNA-AFLP-based transcript profiling procedure was performed according to the method described in Breyne et al. (2002). Double-stranded cDNA (500 ng) was used for cDNA-AFLP analysis. The restriction enzymes used were BstYI and MseI (New England Biolabs, Beverly, MA, USA). For pre-amplification, an MseI-primer without a selective nucleotide was combined with a BstYI-primer containing a T or a C at the 3′ end. The amplification mixtures obtained were diluted 600-fold and 5 μl were used for final selective amplifications according to Breyne et al. (2002). BstT- and MseI-primers and BstC- and MseI-primers with one selective nucleotide, respectively, were used for the cDNA-AFLP analysis, and all 32 possible primer combinations were performed. Selective [33P]ATP-labelled amplification products were separated on a 6% polyacrylamide gel run at 1100 V for 3 h. Gels were dried onto 3MM Whatman paper, and positionally marked before being exposed to Kodak Biomax film (Amersham, Pharmacia, USA) for 2 d. Isolation and sequencing of fragments Films were aligned with markings on the gels. The bands of interest were marked, cut out with a razor blade, and incubated in 100 μl of water at 65 °C for 15 min and then left overnight at room temperature for DNA elution. The eluted DNA was re-amplified using the same PCR conditions and primer combination as for the selective amplification. The re-amplified products representing the Cd-regulated transcript-derived fragments (TDFs) were checked on 2% agarose gel and directly sequenced using the selective BstYI-primer as a sequencing primer. TDFs that failed to be sequenced were ligated to the pGEM-T EASY vector (Promega, Southampton, UK) and clones were then sequenced using the T7 or SP6 primer. Database searches were performed using the BLAST Network service [NCBI (National Center for Biotechnology Information); http://www.ncbi.nlm.nih.gov/BLAST]. The sequence of each TDF was searched against all sequences in the databases using the BLASTN and BLASTX programs (Altschul et al., 1997). Northern blot analysis Poly (A)+ RNA was prepared for Cd-treated as well as untreated plants by chromatography on oligo dT-cellulose (Bartels and Thompson, 1983). Three micrograms of mRNA per lane were fractionated on a 1% denaturing formaldehyde/agarose gel and transferred onto a positively charged nylon membrane. The procedures for radiolabelling and hybridization were performed at 42 °C as described previously (Sambrook et al., 1989). Filters were washed three times for 10 min at 65 °C with 2× SSC and 0.1% SDS. To check that the RNAs were equally loaded, the filters were re-probed with a barley ubiquitin cDNA clone (Gausing and Barkardottir, 1986). Results Plant growth and Cd accumulation Plants of B. juncea cv. Aurea, grown for 6 weeks with 10 μM Cd(NO3)2 present in the nutrient solution, exhibited growth inhibition of both roots and shoots, with roots being more affected by Cd than shoots. At the end of the experimental period leaves of these plants showed symptoms of chlorosis. The decrease in root elongation was also accompanied by changes in morphology. Roots became thinner and were putting out numerous side roots. Control plants maintained under greenhouse conditions and grown for 6 weeks in hydroponic solution, without Cd, showed a concentration of 15.29 μg kg−1 in their dry biomass. After exposure to 10 μM Cd(NO3)2, for either 6 h or 24 h, Cd concentration increased. Under these conditions, the amount of Cd in the dry biomass was 70.59 μg kg−1 DW and 123.53 μg kg−1 DW at 6 h and 24 h, respectively, confirming the adsorption even after a short time of Cd exposure. Plants exposed for 6 weeks to 10 μM Cd(NO3)2 showed an accumulation of 805.88 μg kg−1 DW (Fig. 1). It is worth noting the accumulation capacity of B. juncea in hydroponic culture (0.08% of dry weight), although the severe growth impairment and stressed condition of these plants make a meaningful comparison between Cd content and gene expression difficult. Fig. 1. View largeDownload slide Cd contents of plants maintained under greenhouse conditions, grown for 6 weeks in hydroponic solution and exposed to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Fig. 1. View largeDownload slide Cd contents of plants maintained under greenhouse conditions, grown for 6 weeks in hydroponic solution and exposed to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Analysis of cDNA-AFLP cDNA-AFLP analysis was performed to identify the genes responsive to Cd in B. juncea (Fig. 2). Different exposure times to Cd (6 h, 24 h, and 6 weeks) were chosen to detect genes rapidly responding to Cd shock, and genes whose expression is modulated by the continuous presence of Cd in the culture medium. By using 32 primer combinations, about 3000 cDNA fragments were counted and all bands longer than 80 bp in length were compared in the four tested Cd conditions. About 100 up-regulated or down-regulated gene fragments were identified as Cd modulated. These Cd-regulated TDFs, varying in length from 80 to 500 bp, were excised from the gels, re-amplified by PCR, and sequenced. Only in a few cases did different cDNA fragments belong to the same transcript; in addition, sequencing failed for several TDFs even after cloning. These fragments were not characterized further. Fig. 2. View largeDownload slide cDNA-AFLP autoradiography showing the TDFs induced or repressed by Cd treatment. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. Fig. 2. View largeDownload slide cDNA-AFLP autoradiography showing the TDFs induced or repressed by Cd treatment. 0, 6 h, 24 h, and 6 w (weeks) represent the exposure times to Cd. TDF sequences were compared with those present in the GenBank database (Table 1). The clones corresponding to different TDFs were renamed as BjCdR (B. juncea Cd-regulated). Of the total 73 TDFs sequenced (Table 1), 52 (71.2%) showed significant homology to genes with known or putative function, while 10 TDFs (13.7%) were homologous to uncharacterized genes (EST and unknown proteins). Of the remaining 11 TDFs, eight (11.0%) did not show significant matches; they may represent yet uncharacterized genes or the TDFs sequenced represent the 3′ end region of the transcripts, which is usually less conserved. For three (4.1%) TDFs, sequences match only genomic clones without allocated function. Table 1. Homologies of sequences of cDNA-AFLP fragments to sequences in the databases TDF   Length (bp)   Accession number   Homologya   BLAST scoreb,c   Expression patternd   BjCdR1  110  DT317657  Expressed protein from A. thaliana (NP_196620.1)  2e-12  0  BjCdR2  120  DT317658  Expressed protein from A. thaliana (NP_181834.1)  8e-09  6 h, 24 h, 6 weeks  BjCdR3  102  DT317659  Expressed protein from A. thaliana (NP_850785.1)  2e-07  6 h, 24 h, 6 weeks  BjCdR4  355  DT317660  Expressed protein from A. thaliana (NP_565726.1)  2e-24  0  BjCdR5  161  DT317661  Unknown protein from A. thaliana (AAM63312.1)  3e-04  6 h, 24 h, 6 weeks  BjCdR6  205  DT317662  Expressed protein from A. thaliana (NP_566625.1)  1e-14  24 h  BjCdR7  247  DT317663  Expressed protein from A. thaliana (NP_565922.1)  3e-29  6 h  BjCdR8  231  DT317664  Expressed protein from A. thaliana (NP_566847.1)  2e-15  6 h  BjCdR9  113  DT317665  Putative protein mRNA from A. thaliana (AY128790.1)  2e-10*  6 h  BjCdR10  379  DT317666  Expressed protein from A. thaliana (NP_174692.1)  3e-47  6 h, 24 h, 6 weeks  BjCdR11  130  DT317667  ABC1 family protein mRNA from A. thaliana (NM_180205)  4e-12*  24 h  BjCdR12  86  DT317668  MYB family transcription factor (MYB59) from A. thaliana (NP_851226.1)  3e-07  6 h  BjCdR13  176  DT317669  Putative initiation factor 5A mRNA A. thaliana (AF492850.1)  9e-08*  0  BjCdR14  151  DT317670  Transcription factor GBF5 from A. thaliana (AAG17474.1)  3e-08  6 h  BjCdR15  288  DT317671  bZIP family transcription factor (TGA3) from A. thaliana (NP_564156.1)  1e-11  6 h  BjCdR16  426  DT317672  Zinc-finger (B-box type) family protein/salt tolerance protein (STO) from A. thaliana (NP_172094.1)  2e-53  6 h, 24 h  BjCdR17  302  DT317673  CP12 protein, chloroplast precursor from P. sativum (T06562)  7e-21  6 h  BjCdR18  306  DT317674  Photosystem II family protein from A. thaliana (NP_563687.1)  6e-36  0  BjCdR19  331  DT317675  Photosystem I reaction centre subunit VI, chloroplast precursor (PSI-H) (light harvesting complex I 11 kDa protein) from B. rapa (O04006)  3e-25  6 h  BjCdR20  381  DT317676  Chorismate mutase, chloroplast precursor (CM1) from A. thaliana (P42738)  2e-65  24 h, 6 weeks  BjCdR21  392  DT317677  Chlorophyll a-b binding protein 1, chloroplast precursor (LCHII type I CAB-1) (LCHP) from S. alba (P13851)  3e-68  0  BjCdR22  168  DT317729  Putative cytochrome P450 protein mRNA from A. thaliana (AY050890)  7.2e-10*  24 h  BjCdR23  150  DT317678  LHCI type II from L. temulentum (CAA55864)  8e-13  0  BjCdR24  310  DT317679  ELIP from B. rapa subsp. pekinensis (AAR11456)  5e-10  24 h, 6 weeks  BjCdR25  287  DT317680  PSI-H subunit mRNA from B. rapa (U92504)  2e-06*  24 h  BjCdR26  86  DT317681  Glutamine synthetase, chloroplast precursor (glutamate–ammonia ligase) (GS2) from B. napus (Q42624)  2e-07  0  BjCdR27  135  DT317682  Glutamine-synthetase mRNA from A. thaliana (AY059932.1)  1e-15*  24 h  BjCdR28  127  DT317683  Cysteine synthase (O-acetylserine sulfhydrylase) (O-acetylserine (thiol)-lyase) from B. juncea (O23735)  6e-07  6 h, 24 h, 6 weeks  BjCdR29  321  DT317684  β-Hydroxyacyl-ACP dehydratase, putative from A. thaliana (NP_196578.1)  3e-29  24 h, 6 weeks  BjCdR30  386  DT317685  Putative histone deacetylase from A. thaliana (AAG28473.1)  6e-05  6 h  BjCdR31  287  DT317686  Ubiquitin-conjugating enzyme E2-17 kDa (ubiquitin-protein ligase) (ubiquitin carrier protein) from L. esculentum (P35135)  4e-51  24 h, 6 weeks  BjCdR32  236  DT317687  Starch excess protein (SEX1) from A. thaliana (NP_563877.1)  2e-35  6 h  BjCdR33  321  DT317688  AAA-type ATPase family protein from A. thaliana (NP_182074.2)  1e-48  6 h  BjCdR34  192  DT317689  NADH-ubiquinone oxireductase-related from A. thaliana (NP_566608.1)  3e-51  6 h  BjCdR35  174  DT317690  PBS lyase HEAT-like repeat-containing protein from A. thaliana (NP_197483.1)  3e-25  6 h  BjCdR36  485  DT317691  Phenylalanine ammonia lyase from A. thaliana (AAC18871)  1e-21  6 weeks  BjCdR37  355  DT317692  Putative serine-type carboxypeptidase II from A. thaliana (AAL33815)  2e-06  6 weeks  BjCdR38  123  DT317693  Ribulose-5-phosphate-3-epimerase from O. sativa (XP_470294)  5e-10  0  BjCdR39  202  DT317694  Aldehyde dehydrogenase mRNA from A. thaliana (NM_179476)  2e-06*  24 h  BjCdR40  141  DT317695  Pathogenesis-related protein, putative from A. thaliana (NP_195098.1)  2e-15  6 h  BjCdR41  309  DT317696  Pathogenesis-related family protein from A. thaliana (NP_849901.1)  2e-53  6 h  BjCdR42  133  DT317697  Ribosomal protein L35 from A. thaliana (CAA60774.1)  6e-07  0  BjCdR43  123  DT317698  60S ribosomal protein-like mRNA from A. thaliana (AY093180.1)  7e-11*  24 h  BjCdR44  225  DT317699  Putative ribosomal protein S3a homologue from A. thaliana (AAL15196)  9e-05  6 h  BjCdR45  240  DT317700  Putative 60S ribosomal protein from A. thaliana (AAN31827)  8e-17  0  BjCdR46  153  DT317701  Putative 60S ribosomal protein L6 from A. thaliana (AAO00948)  8e-12  0  BjCdR47  115  DT317702  Putative ribosomal protein S9 from A. thaliana (AAG51916)  2e-08  6 h, 24 h, 6 weeks  BjCdR48  152  DT317703  40S ribosomal protein S3 mRNA from A. thaliana (NM_115247)  4e-06*  0  BjCdR49  149  DT317704  Plasma membrane intrinsic protein 2 (PIP2) from B. napus (AAD39374.1)  3e-23  24 h  BjCdR50  262  DT317705  SNF7 family protein mRNA from A. thaliana (NM_127541)  2e-16*  6 h  BjCdR51  512  DT317706  Plasma membrane intrinsic protein 1 (PIP1) from B. napus (AAD39373.1)  5e-93  24 h  BjCdR52  148  DT317707  Sec61beta family protein from A. thaliana (NP_182033.1)  9e-08  6 h  BjCdR53  386  DT317708  Glutathione S-transferase, putative from A. thaliana (NP_177957.1)  2e-55  6 h  BjCdR54  134  DT317709  ERD9 mRNA for glutathione S-transferase from A. thaliana (AB039930.1)  7e-05*  6 weeks  BjCdR55  147  DT317710  RNA-binding protein homologue (grp2A) mRNA from S. alba (SALGRP2A)  3e-16*  6 h  BjCdR56  404  DT317711  Zinc finger (C3HC4-type RING finger) family protein from A. thaliana (NP_194556.1)  2e-56  0, 6 h  BjCdR57  396  DT317712  DNAJ heat-shock N-terminal domain-containing protein from A. thaliana (NP_178207.1)  8e-55  6 h, 24 h  BjCdR58  150  DT317713  Fasciclin-like arabinogalactan-protein, putative mRNA from A. thaliana (NM_123780.2)  1e-06*  6 h  BjCdR59  149  DT317714  Tetratricopeptide repeat (TRP)-containing protein from A. thaliana (NP_190782.3)  9e-08  6 h  BjCdR60  96  DT317715  Radical SAM domain-containing protein/ TRAM domain-containing protein from A. thaliana (NP_565035.1)  7e-11  0  BjCdR61  128  DT317716  Probable protein kinase mRNA from A. thaliana (NM_104060)  8e-36*  6 h, 24 h  BjCdR62  250  DT317728  Putative auxin-responsive protein mRNA from A. thaliana (AK117242)  3.3e-17*  6 h  BjCdR63  280  DT317717  DNA chromosome 4, BAC clone F25I24 from A. thaliana (ATF25I24)  0.00054*  6 h  BjCdR64  260  DT317718  Chromosome 2 clone from A. thaliana (AC004697)  9e-06*  6 weeks  BjCdR65  171  DT317719  Chromosome 2 clone F26B6 map CIC06C07 A. thaliana (AC003040)  1.1e-09*  24 h  BjCdR66  170  DT317720  No homologye    6 h  BjCdR67  222  DT317721  No homology    6 h, 24 h, 6 weeks  BjCdR68  203  DT317722  No homology    24 h, 6 weeks  BjCdR69  138  DT317723  No homology    24 h, 6 weeks  BjCdR70  103  DT317724  No homology    6 weeks  BjCdR71  188  DT317725  No homology    6 h  BjCdR72  256  DT317726  No homology    6 h  BjCdR73   339   DT317727   No homology     0   TDF   Length (bp)   Accession number   Homologya   BLAST scoreb,c   Expression patternd   BjCdR1  110  DT317657  Expressed protein from A. thaliana (NP_196620.1)  2e-12  0  BjCdR2  120  DT317658  Expressed protein from A. thaliana (NP_181834.1)  8e-09  6 h, 24 h, 6 weeks  BjCdR3  102  DT317659  Expressed protein from A. thaliana (NP_850785.1)  2e-07  6 h, 24 h, 6 weeks  BjCdR4  355  DT317660  Expressed protein from A. thaliana (NP_565726.1)  2e-24  0  BjCdR5  161  DT317661  Unknown protein from A. thaliana (AAM63312.1)  3e-04  6 h, 24 h, 6 weeks  BjCdR6  205  DT317662  Expressed protein from A. thaliana (NP_566625.1)  1e-14  24 h  BjCdR7  247  DT317663  Expressed protein from A. thaliana (NP_565922.1)  3e-29  6 h  BjCdR8  231  DT317664  Expressed protein from A. thaliana (NP_566847.1)  2e-15  6 h  BjCdR9  113  DT317665  Putative protein mRNA from A. thaliana (AY128790.1)  2e-10*  6 h  BjCdR10  379  DT317666  Expressed protein from A. thaliana (NP_174692.1)  3e-47  6 h, 24 h, 6 weeks  BjCdR11  130  DT317667  ABC1 family protein mRNA from A. thaliana (NM_180205)  4e-12*  24 h  BjCdR12  86  DT317668  MYB family transcription factor (MYB59) from A. thaliana (NP_851226.1)  3e-07  6 h  BjCdR13  176  DT317669  Putative initiation factor 5A mRNA A. thaliana (AF492850.1)  9e-08*  0  BjCdR14  151  DT317670  Transcription factor GBF5 from A. thaliana (AAG17474.1)  3e-08  6 h  BjCdR15  288  DT317671  bZIP family transcription factor (TGA3) from A. thaliana (NP_564156.1)  1e-11  6 h  BjCdR16  426  DT317672  Zinc-finger (B-box type) family protein/salt tolerance protein (STO) from A. thaliana (NP_172094.1)  2e-53  6 h, 24 h  BjCdR17  302  DT317673  CP12 protein, chloroplast precursor from P. sativum (T06562)  7e-21  6 h  BjCdR18  306  DT317674  Photosystem II family protein from A. thaliana (NP_563687.1)  6e-36  0  BjCdR19  331  DT317675  Photosystem I reaction centre subunit VI, chloroplast precursor (PSI-H) (light harvesting complex I 11 kDa protein) from B. rapa (O04006)  3e-25  6 h  BjCdR20  381  DT317676  Chorismate mutase, chloroplast precursor (CM1) from A. thaliana (P42738)  2e-65  24 h, 6 weeks  BjCdR21  392  DT317677  Chlorophyll a-b binding protein 1, chloroplast precursor (LCHII type I CAB-1) (LCHP) from S. alba (P13851)  3e-68  0  BjCdR22  168  DT317729  Putative cytochrome P450 protein mRNA from A. thaliana (AY050890)  7.2e-10*  24 h  BjCdR23  150  DT317678  LHCI type II from L. temulentum (CAA55864)  8e-13  0  BjCdR24  310  DT317679  ELIP from B. rapa subsp. pekinensis (AAR11456)  5e-10  24 h, 6 weeks  BjCdR25  287  DT317680  PSI-H subunit mRNA from B. rapa (U92504)  2e-06*  24 h  BjCdR26  86  DT317681  Glutamine synthetase, chloroplast precursor (glutamate–ammonia ligase) (GS2) from B. napus (Q42624)  2e-07  0  BjCdR27  135  DT317682  Glutamine-synthetase mRNA from A. thaliana (AY059932.1)  1e-15*  24 h  BjCdR28  127  DT317683  Cysteine synthase (O-acetylserine sulfhydrylase) (O-acetylserine (thiol)-lyase) from B. juncea (O23735)  6e-07  6 h, 24 h, 6 weeks  BjCdR29  321  DT317684  β-Hydroxyacyl-ACP dehydratase, putative from A. thaliana (NP_196578.1)  3e-29  24 h, 6 weeks  BjCdR30  386  DT317685  Putative histone deacetylase from A. thaliana (AAG28473.1)  6e-05  6 h  BjCdR31  287  DT317686  Ubiquitin-conjugating enzyme E2-17 kDa (ubiquitin-protein ligase) (ubiquitin carrier protein) from L. esculentum (P35135)  4e-51  24 h, 6 weeks  BjCdR32  236  DT317687  Starch excess protein (SEX1) from A. thaliana (NP_563877.1)  2e-35  6 h  BjCdR33  321  DT317688  AAA-type ATPase family protein from A. thaliana (NP_182074.2)  1e-48  6 h  BjCdR34  192  DT317689  NADH-ubiquinone oxireductase-related from A. thaliana (NP_566608.1)  3e-51  6 h  BjCdR35  174  DT317690  PBS lyase HEAT-like repeat-containing protein from A. thaliana (NP_197483.1)  3e-25  6 h  BjCdR36  485  DT317691  Phenylalanine ammonia lyase from A. thaliana (AAC18871)  1e-21  6 weeks  BjCdR37  355  DT317692  Putative serine-type carboxypeptidase II from A. thaliana (AAL33815)  2e-06  6 weeks  BjCdR38  123  DT317693  Ribulose-5-phosphate-3-epimerase from O. sativa (XP_470294)  5e-10  0  BjCdR39  202  DT317694  Aldehyde dehydrogenase mRNA from A. thaliana (NM_179476)  2e-06*  24 h  BjCdR40  141  DT317695  Pathogenesis-related protein, putative from A. thaliana (NP_195098.1)  2e-15  6 h  BjCdR41  309  DT317696  Pathogenesis-related family protein from A. thaliana (NP_849901.1)  2e-53  6 h  BjCdR42  133  DT317697  Ribosomal protein L35 from A. thaliana (CAA60774.1)  6e-07  0  BjCdR43  123  DT317698  60S ribosomal protein-like mRNA from A. thaliana (AY093180.1)  7e-11*  24 h  BjCdR44  225  DT317699  Putative ribosomal protein S3a homologue from A. thaliana (AAL15196)  9e-05  6 h  BjCdR45  240  DT317700  Putative 60S ribosomal protein from A. thaliana (AAN31827)  8e-17  0  BjCdR46  153  DT317701  Putative 60S ribosomal protein L6 from A. thaliana (AAO00948)  8e-12  0  BjCdR47  115  DT317702  Putative ribosomal protein S9 from A. thaliana (AAG51916)  2e-08  6 h, 24 h, 6 weeks  BjCdR48  152  DT317703  40S ribosomal protein S3 mRNA from A. thaliana (NM_115247)  4e-06*  0  BjCdR49  149  DT317704  Plasma membrane intrinsic protein 2 (PIP2) from B. napus (AAD39374.1)  3e-23  24 h  BjCdR50  262  DT317705  SNF7 family protein mRNA from A. thaliana (NM_127541)  2e-16*  6 h  BjCdR51  512  DT317706  Plasma membrane intrinsic protein 1 (PIP1) from B. napus (AAD39373.1)  5e-93  24 h  BjCdR52  148  DT317707  Sec61beta family protein from A. thaliana (NP_182033.1)  9e-08  6 h  BjCdR53  386  DT317708  Glutathione S-transferase, putative from A. thaliana (NP_177957.1)  2e-55  6 h  BjCdR54  134  DT317709  ERD9 mRNA for glutathione S-transferase from A. thaliana (AB039930.1)  7e-05*  6 weeks  BjCdR55  147  DT317710  RNA-binding protein homologue (grp2A) mRNA from S. alba (SALGRP2A)  3e-16*  6 h  BjCdR56  404  DT317711  Zinc finger (C3HC4-type RING finger) family protein from A. thaliana (NP_194556.1)  2e-56  0, 6 h  BjCdR57  396  DT317712  DNAJ heat-shock N-terminal domain-containing protein from A. thaliana (NP_178207.1)  8e-55  6 h, 24 h  BjCdR58  150  DT317713  Fasciclin-like arabinogalactan-protein, putative mRNA from A. thaliana (NM_123780.2)  1e-06*  6 h  BjCdR59  149  DT317714  Tetratricopeptide repeat (TRP)-containing protein from A. thaliana (NP_190782.3)  9e-08  6 h  BjCdR60  96  DT317715  Radical SAM domain-containing protein/ TRAM domain-containing protein from A. thaliana (NP_565035.1)  7e-11  0  BjCdR61  128  DT317716  Probable protein kinase mRNA from A. thaliana (NM_104060)  8e-36*  6 h, 24 h  BjCdR62  250  DT317728  Putative auxin-responsive protein mRNA from A. thaliana (AK117242)  3.3e-17*  6 h  BjCdR63  280  DT317717  DNA chromosome 4, BAC clone F25I24 from A. thaliana (ATF25I24)  0.00054*  6 h  BjCdR64  260  DT317718  Chromosome 2 clone from A. thaliana (AC004697)  9e-06*  6 weeks  BjCdR65  171  DT317719  Chromosome 2 clone F26B6 map CIC06C07 A. thaliana (AC003040)  1.1e-09*  24 h  BjCdR66  170  DT317720  No homologye    6 h  BjCdR67  222  DT317721  No homology    6 h, 24 h, 6 weeks  BjCdR68  203  DT317722  No homology    24 h, 6 weeks  BjCdR69  138  DT317723  No homology    24 h, 6 weeks  BjCdR70  103  DT317724  No homology    6 weeks  BjCdR71  188  DT317725  No homology    6 h  BjCdR72  256  DT317726  No homology    6 h  BjCdR73   339   DT317727   No homology     0   a GenBank accession numbers of sequences homologous to AFLP fragments are in parentheses. b All are BLASTX scores except for those marked with * which are BLASTN scores. c e-value cut-off=1e−5. d Gene expression pattern after exposure to 10 μM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 weeks (6 w) represent the Cd exposure time. e No significant sequence homology found in genome, EST, and protein database. View Large Considering the expression pattern of Cd-modulated genes, among the TDFs isolated and sequenced, Table 1 shows that genes were either up- or down-regulated by Cd treatment. It is possible to distinguish 14 genes that were expressed only in untreated control plants and down-regulated by Cd treatment, while seven genes were induced after Cd exposure and the transcription was sustained throughout the Cd treatment (6 h, 24 h, and 6 weeks). Table 1 also shows that 27 genes, the majority of the TDFs identified, are detected within 6 h of exposure to Cd and their expression is suppressed with longer Cd treatment times, suggesting that many of them may have roles in further signalling. Ten genes were up-regulated only 24 h after the addition of Cd and three genes were induced at 6 h and 24 h of Cd treatment. For six genes the transcription was observed at 24 h and 6 weeks of Cd exposure, and only one was up-regulated in untreated plants and after the addition of Cd for 6 h. In plants treated with Cd for 6 weeks the transcript accumulation of five genes was detected. The induction of the latter genes might probably be due to general stress conditions imposed on the plants by the continuous presence of Cd in the culture medium. Indeed, protracted exposure to Cd inhibited growth, and the generally stunted conditions and leaf chlorosis indicated that general metabolism such as photosynthesis and respiration were affected. Northern analysis of Cd-regulated genes To validate the cDNA-AFLP expression patterns, several TDFs were selected for RNA gel blot analysis. To minimize problems of cross-hybridization, full-length TDFs were used and their kinetics of transcript accumulation in response to the Cd presence in the culture medium are shown in Fig. 3. Moreover, to confirm the expression pattern observed, each hybridization was repeated at least twice (data not shown). For this expression analysis, TDFs from genes induced at 6 h of Cd treatment were preferentially chosen as probes to further characterize genes which may have roles in the early events of the signal transduction pathways leading to Cd translocation and sequestration in B. juncea. Fig. 3. View largeDownload slide Northern analysis showing the expression profiles of several genes induced by Cd treatment in B. juncea. These experiments were repeated at least twice for each TDF. Poly (A)+ RNA was isolated from whole plants after Cd exposure for 0, 6 h, 24 h, and 6 weeks. Fig. 3. View largeDownload slide Northern analysis showing the expression profiles of several genes induced by Cd treatment in B. juncea. These experiments were repeated at least twice for each TDF. Poly (A)+ RNA was isolated from whole plants after Cd exposure for 0, 6 h, 24 h, and 6 weeks. The induction pattern observed in northern analysis showed that six of the nine TDFs tested (BjCdR15, BjCdR7, BjCdR35, BjCdR14, BjCdR29, and BjCdR30) fully confirmed the expression profiles observed with the cDNA-AFLP analysis. This technique was thus validated in 67% of cases. It is noteworthy that there are slight differences in expression pattern for the three TDFs (BjCdR47, BjCdR43, and BjCdR51) for which the transcription profile seen with the cDNA-AFLP analysis was not confirmed by RNA gel blot analysis. Northern analysis showed that the ribosomal protein S9 (BjCdR47) is mainly induced at 6 h and 24 h of Cd exposure and that its transcription decreased with longer Cd treatment; whereas the 60S ribosomal protein (BjCdR43) showed the highest transcript level when Cd was added to the culture medium for 6 h, and the plasma membrane intrinsic protein 1 (BjCdR51) is mainly expressed at 24 h and 6 weeks of Cd treatment. These discrepancies between northern blotting and cDNA-AFLP analysis may either be due to changes in the intensity of individual bands in the cDNA-AFLP gels or to gene family complexity. Nevertheless, the cDNA-AFLP technique allowed the isolation of differentially expressed genes under the conditions tested. Functional classification and temporal expression pattern of Cd-responsive genes Of the 73 sequenced Cd-modulated genes, 52 were genes of known or putative function which could be grouped into several major functional categories (Table 2). Four were transcriptional factors possibly involved in the transcriptional control of plant stress response. BjCdR15 and BjCdR14 activated at 6 h of Cd treatment showed homology to Arabidopsis TGA3 and GBF5, respectively, which belong to the bZIP protein family of transcriptional factors that have been implicated in stress signalling (Jakoby et al., 2002). In particular, the gene corresponding to BjCdR15 is under investigation and preliminary data suggest that it is transcribed in leaves and roots after 0.5 h of Cd treatment and is also activated by other heavy metals, such as Pb and Zn (Micheletto et al., unpublished results). In addition, a cDNA fragment (BjCdR12) homologous to the Arabidopsis Myb59 was induced at 6 h of Cd treatment, while BjCdR16 expressed at 6 and 24 h of Cd exposure showed homology to zinc finger protein. Cd modulated the expression of several stress-responding proteins. Genes whose expression improves plant stress protection were considered to be in this functional category. At 24 h of Cd stress, BjCdR39 was isolated, which showed homology to an aldehyde dehydrogenase induced by dehydration, NaCl, heavy metal, and oxidative stress (Sunkar et al., 2003), whereas 6 h of Cd stress induced the transcription of a gene (BjCdR55) homologous to Sinapis alba mRNA cap-binding protein. It has been reported that the Arabidopsis homologue is implicated in abscisic acid (ABA) signalling (Hugouvieux et al., 2001). Two cDNA fragments (BjCdR40 and BjCdR41) induced early by Cd exhibited homology to pathogenesis-related protein. The stress-responding category also includes a gene (BjCdR24) transcribed at 24 h and 6 weeks of Cd treatment that showed homology to a gene encoding for an early light-induced protein (ELIP) from Brassica rapa, and a gene transcribed at 24 h of Cd stress (BjCdR22) homologous to the Arabidopsis cytochrome P450. Transcript induction was observed in all Cd time treatments for a gene encoding O-acetylserine (thiol) lyase (BjCdR28), an enzyme that plays a role in Cd tolerance (Domínguez-Solis et al., 2001). Furthermore, BjCdR53 and BjCdR54, detected, respectively, at 6 h and 6 weeks of Cd exposure, showed high homology to Arabidopsis genes encoding glutathione S-transferase directly involved in Cd stress tolerance. Not unexpectedly, at 6 h and 24 h of Cd exposure, a cDNA fragment (BjCdR57) was isolated homologous to a gene encoding for a DNAJ heat-shock protein probably participating in protein folding, and BjCdR36 detected after 6 weeks of Cd treatment showed homology to an Arabidopsis gene encoding for a phenylalanine ammonia lyase, a key enzyme leading to lignin synthesis (Mao et al., 2004). Table 2. Functional classification of Cd-responsive gene product in B. juncea Functiona   Identification of BjCdR clones   No. clones   %   Transcriptional factor  12, 14, 15, 16  4  5.5  Stress responding  40, 41, 53, 54, 57, 36, 22, 24, 39, 55, 38, 28  12  16.4  Cellular metabolism and organization  26, 27, 29, 20, 34, 37, 58, 59  8  11.0  Photosynthetic process  17, 18, 19, 32, 21, 23, 25  7  9.6  Transport facilitation  33, 52, 50, 51, 49, 11  6  8.2  Unclassified protein  1, 2, 3, 4, 5, 6, 7, 8, 9, 10  10  13.7  Expression regulator  42, 43, 44, 45, 46, 47, 48, 30, 31, 56, 13  11  15.0  Miscellaneous  35, 61, 62, 60  4  5.5  No hitb  63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73  11  15.1  Total     73   100   Functiona   Identification of BjCdR clones   No. clones   %   Transcriptional factor  12, 14, 15, 16  4  5.5  Stress responding  40, 41, 53, 54, 57, 36, 22, 24, 39, 55, 38, 28  12  16.4  Cellular metabolism and organization  26, 27, 29, 20, 34, 37, 58, 59  8  11.0  Photosynthetic process  17, 18, 19, 32, 21, 23, 25  7  9.6  Transport facilitation  33, 52, 50, 51, 49, 11  6  8.2  Unclassified protein  1, 2, 3, 4, 5, 6, 7, 8, 9, 10  10  13.7  Expression regulator  42, 43, 44, 45, 46, 47, 48, 30, 31, 56, 13  11  15.0  Miscellaneous  35, 61, 62, 60  4  5.5  No hitb  63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73  11  15.1  Total     73   100   a Unclassified protein indicates sequence that is homologous to unknown, putative, and expressed proteins without annotated function in other organisms. Other sequence homologies denoted in Table 1 with a putative or probable function are included in their probable function categories. b No hit indicates identity only to unannotated genomic sequences or low similarity to existing nucleotide sequences. View Large A group of eight genes were considered to be related to cellular metabolism and organization. Fragments homologous to Arabidopsis chorismate mutase (BjCdR20), and β-hydroxyacyl-ACP (BjCdR29) were observed at 24 h and 6 weeks of Cd treatment and are involved in amino acid and fatty acid biosynthesis, respectively, while BjCdR34 observed at 6 h of Cd exposure showed similarity to a gene encoding a NADH-ubiquinone oxidoreductase involved in cellular respiration. BjCdR26 and BjCdR27, which are down-regulated by Cd or induced at 24 h of Cd treatment, respectively, showed homology to genes encoding glutamine-synthetase, an enzyme involved in the synthesis of glutamate. In addition, cDNA fragments detected after long Cd exposure (BjCdR37) showed similarity to a serine carboxypeptidase probably involved in plant development, whereas BjCdR59 and BjCdR58 detected at 6 h of Cd treatment were similar to a gene encoding a tetratricopeptide repeat-containing protein implicated in protein–protein interaction (Das et al., 1998) and a fasciclin-like arabinogalactan-protein involved in cell adhesion (Johnson et al., 2003), respectively. The presence of Cd in plants causes an overall inhibition of photosynthesis. In this work, seven genes related to the photosynthetic process were found to be modulated by Cd. The expression of three of them (BjCdR18, BjCdR21, and BjCdR23) was inhibited by the presence of Cd in the culture medium, while three cDNA fragments (BjCdR17, BjCdR19, and BjCdR32) were detected only after 6 h of Cd exposure, and BjCdR25 was found instead at 24 h of Cd treatment. The presence of Cd in the culture medium rapidly induced the synthesis of six proteins that were included in the transport facilitation category. Following the expression pattern in Table 1, BjCdR49 and BjCdR51 were isolated at 24 h of Cd treatment, which showed sequence similarity to Brassica napus aquaporins PIP2 and PIP1, respectively, although northern analysis (Fig. 3) indicated that PIP1 is also transcribed at 6 weeks of Cd exposure. The expression of these genes suggests that Cd evoked water stress. After 24 h of Cd addition, BjCdR11 homologous to an Arabidopsis ABC1 transporter was also induced. Moreover, genes with similarity to an AAA-type ATPase (BjCdR33), Sec61 beta (BjCdR52) and SNF7 (BjCdR50), all involved in protein transport across membranes, were only induced at 6 h of Cd treatment. Genes encoding for ribosomal proteins (BjCdR42, BjCdR43, BjCdR44, BjCdR45, BjCdR46, BjCdR47, and BjCdR48) were classified as expression regulators. The transcription of four of them (BjCdR42, BjCdR45, BjCdR46, and BjCdR48) was only observed in untreated control plants. BjCdR47 was up-regulated by Cd and its transcription was maintained at all times of Cd treatment, although northern analysis (Fig. 3) showed the higher expression at 6 and 24 h of Cd treatment. The expressions of BjCdR44 and BjCdR43 were induced at 6 h and 24 h of Cd exposure, respectively. Also for the latter gene, an RNA gel blot (Fig. 3) revealed the highest expression after 6 h of Cd addition. BjCdR30, homologous to a histone deacetylase, up-regulated at 6 h of Cd treatment, BjCdR31 with similarity to a ubiquitin carrier protein, activated at 24 h and 6 weeks of Cd treatment and probably involved in protein degradation, were also included in this functional category. Furthermore, transcription was inhibited by Cd for a gene homologous to an initiation factor BjCdR13, whereas inhibition was seen after 24 h and 6 weeks of Cd exposure for a gene similar to a zinc finger RING (BjCdR56). The other four clones were clustered as miscellaneous. A PBS lyase HEAT-like repeat-containing protein, BjCdR35, was detected at 6 h of Cd treatment; a putative protein kinase, BjCdR61, probably involved in signalling is activated at 6 h and 24 h of Cd treatment and a putative auxin-responsive protein, BjCdR62, is transcribed at 6 h of Cd exposure. A gene encoding for a protein containing the SAM and TRAM domain, BjCdR60, repressed by Cd is also included in this functional category. Discussion Some plants species can grow on soil that naturally, or due to human activities, contains growth-prohibiting concentrations of metals (Clemens, 2001). For instance, Brassica juncea, a fast-growing plant with high biomass, has the ability to take up Cd, as other heavy metals, from the soil and to accumulate substantial amounts of it in the shoot (Salt et al., 1995a, 1997). Therefore, B. juncea is an interesting species to use in the study of Cd accumulation and tolerance mechanisms (Clemens et al., 2002; Rugh, 2004). In this study, cDNA-AFLP technology was employed to identify Cd-regulated gene expression in B. juncea to gain new insights into the molecular mechanisms governing Cd accumulation. Plants of B. juncea grown in hydroponic culture with Cd added for 6 weeks showed symptoms of toxicity. Long exposure to Cd induced leaf chlorosis. This observation is in agreement with other works describing a reduction of chlorophyll concentration in plants of B. juncea (Salt et al., 1995b) and B. napus (Larsson et al., 1998) exposed to Cd, indicating a general inhibition of the photosynthetic process. Cd also inhibited plant growth, affecting roots to a greater extent than leaves, while plants exposed to Cd for short times (6 h and 24 h) showed no symptoms. Similar root growth inhibition was described for seedlings of several genotypes of Pisum sativum grown in hydroponic culture for 10 d and exposed to 5 μM CdCl2 (Metwally et al., 2005). Long exposure to Cd affected root morphology; not only root elongation was inhibited, but roots appeared thinner and sent out side roots. A plant with numerous thin roots would accumulate more metals than one with a few thick roots (Schierup and Larsen, 1981), plus, to take up heavy metal ions, plants must be able to renew the active parts of their root biomass (Das et al., 1997). The development of thin and side roots in plants exposed to Cd during growth may thus represent an adaptive strategy to cope with Cd ions. As a first step in the identification of Cd-modulated genes in B. juncea, Cd contents of whole plants were measured and similar plant materials were used for the cDNA-AFLP analysis. The Cd content analysis showed that B. juncea plants grown in hydroponic solution take up Cd and the metal concentration in plants increased with the duration of Cd treatment. The cDNA-AFLP technique allowed transcription changes to be surveyed with no prior assumptions about which genes might be induced or repressed by Cd treatment. Northern blotting analysis of nine TDFs confirmed that expression of the identified genes is Cd modulated. With the enzyme combination chosen, 3000 cDNA fragments were visualized on the gels, and about 100 were found to be Cd regulated, of which 73 are reported in Table 1. They belong to different functional categories, which indicates that Cd affected different physiological and biochemical pathways. Sequencing analyses revealed that one gene identified is homologous to a protein kinase (BjCdR61) and four encode transcriptional factors (BjCdR12, BjCdR14, BjCdR15, and BjCdR16), indicating that signal transduction pathways are rapidly activated by the presence of Cd in the nutrient solution. Overall the transcriptional factors identified in this work have been linked to plant stress responses (Singh et al., 2002), particularly TGA and GBF; members of the bZIP protein family are involved in responses to ABA and ethylene as well as in pathogen attack (Jakoby et al., 2002). The induction by Cd of transcripts for bZIP, Myb, and zinc finger transcriptional factor observed in this study suggest, as previously reported by Knight and Knight (2001), that plant response to environmental stresses, including heavy metals, may be regulated by multiple signalling pathways. Among the Cd-regulated genes detected, 12 appear to encode proteins which protect against Cd stress. The transcription upon exposure to Cd of a DNAJ heat shock protein (BjCdR57), a chaperone involved in protein protection in times of cellular stress, confirmed that protein denaturation is one of the effects of Cd toxicity (Suzuki et al., 2001). Furthermore, the expression of two pathogenesis-related proteins (BjCdR40 and BjCdR41) at 6 h after Cd addition indicates that Cd induces defence reactions. Indeed, H2O2 accumulation was observed in Cd-exposed roots (Schützendübel et al., 2001), and it was suggested that H2O2 would act as a signalling molecule triggering secondary defences leading to cell wall rigidification and lignification in Cd-exposed cells (Schützendübel and Polle, 2002). In addition, the transcript of phenylalanine ammonia lyase (BjCdR36), an enzyme of the phenylpropanoid pathway leading to lignin synthesis, detected in 6-week-old Cd-treated plants, is in accordance with the finding that Cd induces lignin deposition in cell walls. One of the most sensitive responses of higher plants to Cd is stomata closure (Sanità di Toppi and Gabbrielli, 1999), which is a symptom of water stress mediated by ABA. Based on transcript accumulation, aldehyde dehydrogenase has been correlated with ABA treatment, drought stress, UV light, NaCl, and heavy metals (Chen et al., 2002; Sunkar et al., 2003). The expression of an aldehyde dehydrogenase (BjCdR39) and an RNA-binding protein (BjCdR55) involved in ABA signalling, induced by Cd in B. juncea, corroborates the idea of existing cross-talk between Cd-induced and water stress-induced signalling that may also employ ABA a as signal transduction compound (Polle and Schützendübel, 2004). Genes encoding for glutathione S-transferases (BjCdR53 and BjCdR54) and cytochrome P450 (BjCdR22), were previously reported as responsive to Cd and other stresses (Marrs and Walbot, 1997; Suzuki et al., 2001) and are probably functioning in cytotoxic product detoxification. In particular, glutathione S-transferases catalyse the synthesis of glutathione S-conjugates, allowing them to be recognized for transport into the vacuole (Marrs and Walbot, 1997). Chelation of metals by high-affinity ligands, such as PSs and thiolated peptides, is considered a principal mechanism of Cd detoxification in plants, and numerous physiological, biochemical, and genetic studies have confirmed that glutathione is the substrate for PS biosynthesis (Cobbett, 2000; Cobbett and Goldsbrough, 2002). Moreover, in B. juncea, changes of expression of a glutathione transporter in response to Cd exposure has been reported (Bogs et al., 2003) also indicating that glutathione plays a prominent role in Cd accumulation and/or detoxification. The amount of glutathione transporter protein decreased in older leaves 48 h after the onset of Cd exposure and reached a minimum at 96 h, while an increase in protein amount was detected at 120 h and 144 h of Cd treatment. It was suggested (Heiss et al., 2003) that, during Cd exposure, glutathione export to the various sink tissues is reduced to meet the high demand of glutathione for PS synthesis during Cd accumulation and, therefore, the observed decrease in glutathione transporter protein may reflect this adaptation. In this cDNA-AFLP analysis TDFs for glutathione transporters were not detected. This may simply be due to the technique employed that allows discrimination between the presence or absence of bands, or to the experimental conditions used. In this study, a gene (BjCdR28) encoding for O-acetylserine (thiol) lyase enzyme (OASTL) was up-regulated in all Cd exposure times. OASTL catalyses the last step of cysteine biosynthesis and Arabidopsis plants overexpressing OASTL showed high Cd resistance, suggesting that cysteine pool requirement for glutathione biosynthesis is a main factor for tolerance (Domínguez-Solis et al., 2001). However, previous studies in B. juncea, also indicated an enhanced OASTL expression, although glutathione biosynthesis, more than cysteine availability, was considered the limiting step (Schäfer et al., 1998). Cd toxicity is generally associated with inhibition of chlorophyll synthesis and damage to photosynthetic apparatus (Sanità di Toppi and Gabbrielli, 1999). In this work, a group of genes related to the photosynthetic process showed expression only in Cd-untreated control plants (BjCdR18, BjCdR21, and BjCdR23), while others (BjCdR19, BjCdR32, and BjCdR25) were induced by Cd treatments, indicating that the presence of Cd in plant tissues disturbs photosynthesis. Similarly, the effect of Cd was evident from the expression pattern of genes that were grouped under the function of cellular metabolism and organization and that may be involved in different cellular processes, such as a tetratricopeptide repeat-containing protein (BjCdR59) induced after 6 h of Cd addition, probably acting as a scaffold for the assembly of multi-protein complexes (Das et al., 1998), and a fasciclin-like arabinogalactan-protein (BjCdR58) putatively working in cell adhesion (Johnson et al., 2003). Furthermore, Cd affected the transcription of genes encoding for glutamine-synthetase (GS) (BjCdR26 and BjCdR27) involved in nitrogen metabolism. The expression of chloroplastic GS was inhibited by Cd treatment, while transcription of the cytosolic GS was detected 24 h after Cd addition. Protein and transcript analyses of Cd-treated tomato plants showed that cytosolic GS increased in leaves and chloroplastic GS decreased in parallel, suggesting that plants subjected to Cd stress induced cytosolic GS to compensate and continue glutamine biosynthesis when Cd affected chloroplastic GS activity (Chaffei et al., 2004). In addition, the induction of chorismate mutase (BjCdR20) observed in this work at 24 h and 6 weeks of Cd exposure also confirms an increased amino acid synthesis in Cd-treated plants (Chaffei et al., 2004). Sequence analysis of Cd-responsive genes also revealed the induction of genes whose gene products are involved in cellular transport. Aquaporins mediate the passive movement of water in cellular membranes and the expression of plasma membrane intrinsic proteins, PIP1 and PIP2, is regulated by ABA and water stress (Gao et al., 1999; Yang et al., 2003). The transcription of aquaporins PIP1 and PIP2 (BjCdR51 and BjCdR49) seen in B. juncea upon exposure to Cd for 24 h, together with the expression of other drought- and ABA-responsive genes (BjCdR39 and BjCdR55) strengthen the idea that Cd imposes water stress and that both ABA and Cd act synergistically (Polle and Schülzendübel, 2004). In addition, a gene encoding for an ABC-transporter protein (BjCdR11) was up-regulated by Cd treatment. ABC transporters directly involved in Cd transport have not been identified in plants, whereas it has been shown that in yeast and fission yeast they act directly in the final step of Cd detoxification by mediating the vacuolar transport of Cd complexes (Ortiz et al., 1995; Li et al., 1997). However, recent analyses of AtMRPs, a subfamily of Arabidopsis ABC transporters, showed that AtMRP3 was induced by Cd and not by oxidative stress (Bovet et al., 2003), suggesting that ABC transporters in plants, as in yeast, are involved in heavy metal fluxes, although a direct role of AtMRP3 in Cd transport has not been demonstrated (Bovet et al., 2003). In conclusion, the cDNA-AFLP technique allowed genes to be identified whose expression is modulated by Cd. This study reveals that a multitude of processes are implicated in determining response to metal in plants and these processes require the activation of different sets of genes. Identification of components of the signal transduction cascades suggests that Cd triggers stress signals and that ABA may be involved in plant response to Cd. 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Journal of Experimental Botany – Oxford University Press

Published: Oct 10, 2005

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