The Relationship between Cadmium Toxicity and the Modulation of Epigenetic Traits in Plants.

Elevated concentrations of heavy metals such as cadmium (Cd) have a negative impact on staple crop production due to their ability to elicit cytotoxic and genotoxic effects on plants. In order to understand the relationship between Cd stress and plants in an effort to improve Cd tolerance, studies have identified genetic mechanisms which could be important for conferring stress tolerance. In recent years epigenetic studies have garnered much attention and hold great potential in both improving the understanding of Cd stress in plants as well as revealing candidate mechanisms for future work. This review describes some of the main epigenetic mechanisms involved in Cd stress responses. We summarize recent literature and data pertaining to chromatin remodeling, DNA methylation, histone acetylation and miRNAs in order to understand the role these epigenetic traits play in cadmium tolerance. The review aims to provide the framework for future studies where these epigenetic traits may be used in plant breeding and molecular studies in order to improve Cd tolerance.

Identification of tRFs and phasiRNAs in tomato (Solanum lycopersicum) and their responses to exogenous abscisic acid.

BACKGROUND: The non-coding small RNA tRFs (tRNA-derived fragments) and phasiRNAs (plant-specific) exert important roles in plant growth, development and stress resistances. However, whether the tRFs and phasiRNAs respond to the plant important stress hormone abscisic acid (ABA) remain enigma. RESULTS: Here, the RNA-sequencing was implemented to decipher the landscape of tRFs and phasiRNAs in tomato (Solanum lycopersicum) leaves and their responses when foliar spraying exogenous ABA after 24 h. In total, 733 tRFs and 137 phasiRNAs were detected. The tRFs were mainly derived from the tRNAAla transporting alanine, which tended to be cleaved at the 5'terminal guanine site and D loop uracil site to produce tRFAla with length of 20 nt. Most of phasiRNAs originated from NBS-LRR resistance genes. Expression analysis revealed that 156 tRFs and 68 phasiRNAs expressed differentially, respectively. Generally, exogenous ABA mainly inhibited the expression of tRFs and phasiRNAs. Furthermore, integrating analysis of target gene prediction and transcriptome data presented that ABA significantly downregulated the abundance of phsaiRNAs associated with biological and abiotic resistances. Correspondingly, their target genes such as AP2/ERF, WRKY and NBS-LRR, STK and RLK, were mainly up-regulated. CONCLUSIONS: Combined with the previous analysis of ABA-response miRNAs, it was speculated that ABA can improve the plant resistances to various stresses by regulating the expression and interaction of small RNAs (such as miRNAs, tRFs, phasiRNAs) and their target genes. This study enriches the plant tRFs and phasiRNAs, providing a vital basis for further investigating ABA response-tRFs and phasiRNAs and their functions in biotic and abiotic stresses.

Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium.

MicroRNAs (miRNAs) are a distinct class of small RNAs in plants that not only regulate biological processes but also regulate response to environmental stresses. The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus), but it is not known on a genome-wide scale how the expression of miRNAs and their target genes, is regulated by Cd. In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Some of the miRNAs were validated by RNA gel blotting. Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure. The study simultaneously identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd.

The Arabidopsis abscisic acid catabolic gene CYP707A2 plays a key role in nitrate control of seed dormancy.

Nitrate releases seed dormancy in Arabidopsis (Arabidopsis thaliana) Columbia accession seeds in part by reducing abscisic acid (ABA) levels. Nitrate led to lower levels of ABA in imbibed seeds when included in the germination medium (exogenous nitrate). Nitrate also reduced ABA levels in dry seeds when provided to the mother plant during seed development (endogenous nitrate). Transcript profiling of imbibed seeds treated with or without nitrate revealed that exogenous nitrate led to a higher expression of nitrate-responsive genes, whereas endogenous nitrate led to a profile similar to that of stratified or after-ripened seeds. Profiling experiments indicated that the expression of the ABA catabolic gene CYP707A2 was regulated by exogenous nitrate. The cyp707a2-1 mutant failed to reduce seed ABA levels in response to both endogenous and exogenous nitrate. In contrast, both endogenous and exogenous nitrate reduced ABA levels of the wild-type and cyp707a1-1 mutant seeds. The CYP707A2 mRNA levels in developing siliques were positively correlated with different nitrate doses applied to the mother plants. This was consistent with a role of the CYP707A2 gene in controlling seed ABA levels in response to endogenous nitrate. The cyp707a2-1 mutant was less sensitive to exogenous nitrate for breaking seed dormancy. Altogether, our data underline the central role of the CYP707A2 gene in the nitrate-mediated control of ABA levels during seed development and germination.

Endogenous hormonal and enzymatic responses of Catharanthus roseus with triadimefon application under water deficits.

The effect of triadimefon was investigated in a medicinal plant, Catharanthus roseus subjected to water deficit stress. The abscisic acid (ABA) level, DNA and RNA contents and activities of ATPase and protease were found varying in different parts of the plants under treatment. Drought treatment increased the ABA level more than twofold in all parts of the plants. TDM treatment to the drought stressed plants showed highest contents. In roots, stem and leaves, drought stress caused a decrease in the DNA and RNA contents when compared with control and other treatments. TDM treatment with drought increased the nucleic acid contents to the level of the control roots. The activity of ATPase and protease were increased under drought treatment and lowered due to TDM applications. This information could be useful in the field of soil water deficits reclamation efforts by using plant growth regulators.

Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis.

Delta-1-pyrroline-5-carboxylate synthetase enzymes, which catalyse the rate-limiting step of proline biosynthesis, are encoded by two closely related P5CS genes in Arabidopsis. Transcription of the P5CS genes is differentially regulated by drought, salinity and abscisic acid, suggesting that these genes play specific roles in the control of proline biosynthesis. Here we describe the genetic characterization of p5cs insertion mutants, which indicates that P5CS1 is required for proline accumulation under osmotic stress. Knockout mutations of P5CS1 result in the reduction of stress-induced proline synthesis, hypersensitivity to salt stress, and accumulation of reactive oxygen species. By contrast, p5cs2 mutations cause embryo abortion during late stages of seed development. The desiccation sensitivity of p5cs2 embryos does not reflect differential control of transcription, as both P5CS mRNAs are detectable throughout embryonic development. Cellular localization studies with P5CS-GFP gene fusions indicate that P5CS1 is sequestered into subcellular bodies in embryonic cells, where P5CS2 is dominantly cytoplasmic. Although proline feeding rescues the viability of mutant embryos, p5cs2 seedlings undergo aberrant development and fail to produce fertile plants even when grown on proline. In seedlings, specific expression of P5CS2-GFP is seen in leaf primordia where P5CS1-GFP levels are very low, and P5CS2-GFP also shows a distinct cell-type-specific and subcellular localization pattern compared to P5CS1-GFP in root tips, leaves and flower organs. These data demonstrate that the Arabidopsis P5CS enzymes perform non-redundant functions, and that P5CS1 is insufficient for compensation of developmental defects caused by inactivation of P5CS2.

Role of Arabidopsis RAP2.4 in regulating light- and ethylene-mediated developmental processes and drought stress tolerance.

Light and the plant hormone ethylene regulate many aspects of plant growth and development in an overlapping and interdependent fashion. Little is known regarding how their signal transduction pathways cross-talk to regulate plant development in a coordinated manner. Here, we report functional characterization of an AP2/DREB-type transcription factor, Arabidopsis RAP2.4, in mediating light and ethylene signaling. Expression of the RAP2.4 gene is down-regulated by light but up-regulated by salt and drought stresses. RAP2.4 protein is constitutively targeted to the nucleus and it can bind to both the ethylene-responsive GCC-box and the dehydration-responsive element (DRE). We show that RAP2.4 protein possesses an intrinsic transcriptional activation activity in yeast cells and that it can activate a reporter gene driven by the DRE cis-element in Arabidopsis protoplasts. Overexpression of RAP2.4 or mutation in RAP2.4 cause altered expression of representative light-, ethylene-, and drought-responsive genes. Although no salient phenotype was observed with a rap2.4 loss-of-function mutant, constitutive overexpression of RAP2.4 results in defects in multiple developmental processes regulated by light and ethylene, including hypocotyl elongation and gravitropism, apical hook formation and cotyledon expansion, flowering time, root elongation, root hair formation, and drought tolerance. Based on these observations, we propose that RAP2.4 acts at or downstream of a converging point of light and ethylene signaling pathways to coordinately regulate multiple developmental processes and stress responses.

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

The peptaibol alamethicin induces an rRNA-cleavage-associated death in Arabidopsis thaliana.

The plant-metabolic response to amphipathic peptides produced by the soil fungi of the genus Trichoderma remains largely unknown. The present investigation was undertaken to examine the death process in alamethicin-treated Arabidopsis thaliana plantlets. The rapid death triggered by alamethicin (at 50 microM) was shown to be associated with protein-synthesis arrest and with specific cleavage of 18S and 25S ribosomal RNA. The use of an inhibitor of nitric oxide (NO) synthases and of an NO scavenger suggested that rRNA cleavage was suppressed by NO. Experiments conducted with a synthetic alamethicin analogue, in which all alpha-aminoisobutyric acid (Aib) residues have been replaced by leucine moieties, showed that the non-coded residues are essential for the ability of the peptaibol to induce rRNA cleavage in Arabidopsis. Our data indicate that further investigations on the mode of action of alamethicin in planta could be of great interest to study the death-signaling pathway associated with rRNA degradation in plants.

[The effect of Furolan on the physiological and biochemical characteristics of ripening winter wheat grain].

The effects of the preparation Furolan, (2-furyl-2)-1,3-dioxolane, on the degree of mRNA polyadenylation and the pattern of protein synthesis in the ripening grain of several soft winter wheat (Triticum aestivum L.) cultivars were studied. It was demonstrated that Furolan stabilized mRNA in a cultivar-specific manner, thereby accelerating to various degrees the biochemical processes taking place in the ripening grain. Of the wheat cultivars studied, Krasnodarskaya 99 was the most responsive cultivar with respect to a set of changes in nucleic-protein metabolism; the cultivar Deya was next followed by the cultivar Bat'ko. The cultivar Kroshka did not respond to the treatment with Furolan. The cultivar specificity of this preparation allows its practical application to be optimized.

Changes in PEP Carboxylase, Rubisco and Rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites.

Methyl jasmonate (MeJA) treatment increases the levels of plant secondary metabolites, including ginsenosides, which are considered to be the main active compounds in ginseng (Panax ginseng C.A. Meyer). To create a ginseng gene resource that contains the genes involved in the biosynthesis of secondary metabolites, including ginsenosides, we generated 3,134 expression sequence tags (ESTs) from MeJA-treated ginseng hairy roots. These ESTs assembled into 370 clusters and 1,680 singletons. Genes yielding highly abundant transcripts were those encoding proteins involved in fatty acid desaturation, the defense response, and the biosynthesis of secondary metabolites. Analysis of the latter group revealed a number of genes that may be involved in the biosynthesis of ginsenosides, namely, oxidosqualene cyclase (OSC), cytochrome P450, and glycosyltransferase. A novel OSC gene was also identified by this analysis. RNA gel blot analysis confirmed that transcription of this OSC gene, along with squalene synthase (SS) and squalene epoxidase (SE) gene transcription, is increased by MeJA treatment. This ginseng EST data set will also provide important information on the genes that are involved in the biosynthesis of other secondary metabolites and the genes that are responsive to MeJA treatment.

cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit.

The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin (PIP2a), 9-cis-epoxycarotenoid dioxygenase (NCED1) and a negative regulator of gibberellin signal transduction (SPY); eight other genes participated in cell wall loosening or vesicle traffic.

GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency.

Purple acid phosphatases (PAPs) are commonly found in plants, but the physiological functions of different classes of PAPs are not thoroughly understood. In the present study, we identified a novel gene, GmPAP3, from salt-stressed soybean using suppression subtractive hybridization (SSH) techniques. Protein sequence alignment studies and phylogenetic analysis strongly suggested that GmPAP3 belongs to the group of plant PAPs and PAP-like proteins that are distinct from those of fungi and animals. In addition, the invariable consensus metal binding residues of PAPs were all conserved in GmPAP3. Surprisingly, analysis of protein sorting signals showed that a putative mitochondrion targeting transit peptide is present on GmPAP3. Northern blot analysis revealed that NaCl stress causes a general induction of GmPAP3 expression in both roots and leaves of various cultivated (Glycine max) and wild (Glycine soja) soybean varieties. Further test using two genetically unrelated cultivated soybean varieties showed that the expression pattern of GmPAP3 is distinct from other PAP genes in soybeans. NaCl stress and oxidative stress but not phosphorus (P) starvation induces the expression of GmPAP3. These results suggest that the physiological role of GmPAP3 might be related to the adaptation of soybean to NaCl stress, possibly through its involvement in reactive oxygen species (ROS) forming and/or scavenging or stress-responding signal transduction pathways.

Temporal progression of gene expression responses to salt shock in maize roots.

Using a cDNA microarray containing 7943 ESTs, the behavior of the maize root transcriptome has been monitored in a time course for 72 h after imposition of salinity stress (150 mM NaCI). Under these conditions, root sodium amounts increased faster than in leaves, and root potassium decreased significantly. Although the overall free amino acid concentration was not affected, amino acid composition was changed with proline and asparagine increasing. Microarray analysis identified 916 ESTs representing genes whose steady-state RNA levels were significantly altered at various time points, corresponding to 11% of the ESTs printed. The response of the transcriptome to sub-lethal salt stress was rapid and transient, leading to a burst of changes at the three-hour time point. The salt-regulated ESTs represented 472 tentatively unique genes (TUGs), which, based on functional category analysis, are involved in a broad range of cellular and biochemical activities, prominent amongst which were transport and signal transduction pathways. Clustering of regulated transcripts based on the timing and duration of changes suggests a structured succession of induction and repression for salt responsive genes in multiple signal and response cascades. Within this framework, 16 signaling molecules, including six protein kinases, two protein phosphatases and eight transcription factors, were regulated with distinct expression patterns by high salinity.

Salt-dependent expression of a nitrate transporter and two amino acid transporter genes in Mesembryanthemum crystallinum.

Uptake and transport of inorganic nitrogen and allocation of amino acids are essential for plant growth and development. To study the effects of salinity on the regulation of transporters for nitrogenous compounds, we characterized the putative nitrate transporter McNRT1 and the amino acid transporters McAAT1 and McAAT2 from Mesembryanthemum crystallinum. By transcript analyses, McAAT1 was found in leaves, McAAT2 in roots, and McNRT1 in both tissues. By in situ PCR McNRT1 was localized, for example, to epidermal and vascular cells whereas McAAT2 was abundant in most cell types in mature roots and McAAT1 in the mesophyll and cells neighbouring xylem vessels in leaves. In response to salt stress, expression of McAAT2 and McNRT1 was stimulated in the root vasculature. In addition, McNRT1 and McAAT1 signals increased in the leaf phloem. Growth of yeast mutants deficient in histidine uptake was restored by McAAT2 whereas both McAAT1 and McAAT2 complemented a yeast mutant carrying a defect in proline uptake. The differential and cell-specific transcriptional activation of genes encoding nitrogen and amino acid transporters under salt stress suggest complex coordinated regulation of these transporter families to maintain uptake and distribution of nitrogenous compounds and amino acids under conditions of high salinity in plants.

Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: implications of the signal divergence by cytokinin-inducible response regulators in the cytosol and the nuclei.

Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L. to characterize their function in cytokinin signaling. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4-ZmRR7), 3 type-B RRs (ZmRR8-ZmRR10), and 3 HPs (ZmHP1-ZmHP3) were found in leaves. All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin. Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus. Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6). In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs. In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4. These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status.

Expression of an evolutionarily distinct novel BiP gene during the unfolded protein response in Arabidopsis thaliana.

Compared to mammals, little is known about the unfolded protein response (UPR) in plants. Using an oligonucleotide array comprising approximately 8200 Arabidopsis genes we investigated the effect of endoplasmic reticulum (ER) stress on gene expression. Expression of 26 genes increased, including at least nine whose products act in the ER, while their transcriptional activations were confirmed by promoter analyses. Among them, BiP-L, a novel BiP, whose expression appeared to be regulated by two promoter sequences perfectly matching mammalian ERSE. Cloning and sequencing of full-length BiP-L cDNA showed it contained a signal peptide sequence and the ER retention signal (HDEL). Interestingly, BiP-L was substantially different from the other two Arabidopsis BiP genes in genomic organization and sequence homology. Furthermore, phylogenetic analysis showed that the BiP-L protein is the most distal form among the reported plant BiP proteins. RNA levels of BiP-L were very low in various mature Arabidopsis plant organs, while significant levels of BiP-L only observed in stressed seedlings. Transcription of BiP-L during ER stress was shown to be regulated by a feedback loop.

Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana.

Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene beta-glucuronidase (GUS) fused with DNA sequences flanking the DGAT1 coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1 gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.

The serine acetyltransferase gene family in Arabidopsis thaliana and the regulation of its expression by cadmium.

Expression of the serine acetyltransferase (SAT) gene family from Arabidopsis thaliana was investigated in response to treatment with the heavy metal cadmium (Cd). A fourth member of the SAT gene family, Sat-106, was also cloned and the complete SAT gene family from A. thaliana is discussed. Northern analysis of the gene family revealed tissue-specific expression patterns for each isogene. A. thaliana plants grown under 50 microM CdCl2 for a 24 h time course were also used for northern analysis. Expression of all SAT genes was increased to some extent by Cd treatment. Sat-5 expression showed particularly high levels of induction in the leaves of treated plants and was chosen for study by in situ hybridisation. Sat-5 expression was induced in the root and stem cortex and the leaf lamella and trichomes in response to heavy metal stress. SAT and its product O-acetylserine have previously been shown to be implicated in the control of sulphate reduction and cysteine biosynthesis in plants. These results suggest that specific SAT isoforms have a role in increasing cysteine production under conditions of heavy-metal stress when increased biosynthesis of glutathione and phytochelatins is required for detoxification purposes.