CAX3 (cation/proton exchanger) mediates a Cd tolerance by decreasing ROS through Ca elevation in Arabidopsis.

KEY MESSAGE: Over-expression of CAX3 encoding a cation/proton exchanger enhances Cd tolerance by decreasing ROS (Reactive Oxygen Species) through activating anti-oxidative enzymes via elevation of Ca level in Arabidopsis CAXs (cation/proton exchangers) are involved in the sequestration of cations such as Mn, Li, and Cd, as well as Ca, from cytosol into the vacuole using proton gradients. In addition, it has been reported that CAX1, 2 and 4 are involved in Cd tolerance. Interestingly, it has been reported that CAX3 expressions were enhanced by Cd in Cd-tolerant transgenic plants expressing Hb1 (hemoglobin 1) or UBC1 (Ub-conjugating enzyme 1). Therefore, to investigate whether CAX3 plays a role in increasing Cd tolerance, CAX3 of Arabidopsis and tobacco were over-expressed in Arabidopsis thaliana. Compared to control plants, both transgenic plants displayed an increase in Cd tolerance, no change in Cd accumulation, and enhanced Ca levels. In support of these, AtCAX3-Arabidopsis showed no change in expressions of Cd transporters, but reduced expressions of Ca exporters and lower rate of Ca efflux. By contrast, atcax3 knockout Arabidopsis exhibited a reduced Cd tolerance, while the Cd level was not altered. The expression of Δ90-AtCAX3 (deletion of autoinhibitory domain) increased Cd and Ca tolerance in yeast, while AtCAX3 expression did not. Interestingly, less accumulation of ROS (H2O2 and O2-) was observed in CAX3-expressing transgenic plants and was accompanied with higher antioxidant enzyme activities (SOD, CAT, GR). Taken together, CAX3 over-expression may enhance Cd tolerance by decreasing Cd-induced ROS production by activating antioxidant enzymes and by intervening the positive feedback circuit between ROS generation and Cd-induced spikes of cytoplasmic Ca.

Development of bisphenol A (BPA)-sensing indicator Arabidopsis thaliana which synthesizes anthocyanin in response to BPA in leaves.

Bisphenol A (BPA) is an estrogenic endocrine disruptor which disturbs a normal animal development. We generated an indicator plant that senses and provides a clear visual indicator of an estrogen-like compound BPA in the environment. We developed transgenic Arabidopsis lines expressing a construct designed to synthesize anthocyanin (thus showing a red color) in response to BPA. We transformed Arabidopsis with a recombinant vector containing the chimeric estrogen receptor (XVE region), LAP and coding region of PtrMYB119 (transcription factor involved in anthocyanin biosynthesis in poplar and Arabidopsis). Upon binding of the estrogen compound to the ligand-binding domain of E (estrogen receptor) in XVE, the XV domain binds to LAP promoter and triggering the transcription of PtrMYB119 with a subsequent enhancement of anthocyanin biosynthetic gene expression, resulting in anthocyanin synthesis. The leaves of the transgenic Arabidopsis line XVE-PtrMYB119 turned red in the presence of 10 ppm BPA. The transcript level of PtrMYB119 peaked at day 3 of BPA exposure, then decreased to its minimal level at day 5. Similar expression patterns to that of PtrMYB119 were detected for genes encoding the anthocyanin biosynthetic enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and UFGT (UGT78D2). The leaves of transgenic plants did not turn red in response to BPA at concentrations below 10 ppm, but PtrMYB119 expression was induced by BPA at concentrations as low as 1 ppt BPA. Since this transgenic plant turns red in the presence of BPA without any experimental procedures, this line can be easily used by non-scientists.

Role of the INDETERMINATE DOMAIN Genes in Plants.

The INDETERMINATE DOMAIN (IDD) genes comprise a conserved transcription factor family that regulates a variety of developmental and physiological processes in plants. Many recent studies have focused on the genetic characterization of IDD family members and revealed various biological functions, including modulation of sugar metabolism and floral transition, cold stress response, seed development, plant architecture, regulation of hormone signaling, and ammonium metabolism. In this review, we summarize the functions and working mechanisms of the IDD gene family in the regulatory network of metabolism and developmental processes.

Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (Nicotiana tabacum).

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca2+/H+ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.

Transcriptome analysis and identification of genes associated with ω-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens.

BACKGROUND: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of α-linolenic acid (ALA), a ω-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cDNAs from developing seeds and leaves for understanding the mechanism underlying ALA and seed TAG biosynthesis. RESULTS: Perilla cultivar Dayudeulkkae contains 66.0 and 56.2 % ALA in seeds and leaves, respectively. Using Illumina HiSeq 2000, we have generated a total of 392 megabases of raw sequences from four mRNA samples of seeds at different developmental stages and one mature leaf sample of Dayudeulkkae. De novo assembly of these sequences revealed 54,079 unique transcripts, of which 32,237 belong to previously annotated genes. Among the annotated genes, 66.5 % (21,429 out of 32,237) showed highest sequences homology with the genes from Mimulus guttatus, a species placed under the same Lamiales order as perilla. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome and identified 540 unique perilla genes involved in all known pathways of acyl-lipid metabolism. We characterized the expression profiles of 43 genes involved in FA and TAG synthesis using quantitative PCR. Key genes were identified through sequence and gene expression analyses. CONCLUSIONS: This work is the first report on building transcriptomes from perilla seeds. The work also provides the first comprehensive expression profiles for genes involved in seed oil biosynthesis. Bioinformatic analysis indicated that our sequence collection represented a major transcriptomic resource for perilla that added valuable genetic information in order Lamiales. Our results provide critical information not only for studies of the mechanisms involved in ALA synthesis, but also for biotechnological production of ALA in other oilseeds.

Tobacco NtUBC1 and NtUBQ2 enhance salt tolerance by reducing sodium accumulation and oxidative stress through proteasome activation in Arabidopsis.

The ubiquitin/proteasome system plays a crucial role in the regulation of plant responses to environmental stress. Here, we studied the involvement of the UBC1 and UBQ2 genes encoding a ubiquitin conjugating enzyme (E2) and ubiquitin extension protein, respectively, in the response to salt stress. Our results showed that the constitutive expression of tobacco NtUBC1 and NtUBQ2 in Arabidopsis thaliana improved salt tolerance, along with the lower Na+ level and higher K+/Na+ ratio compared to control plants. Moreover, the expression levels of sodium transporters, including AtHKT1 (High-Affinity K+ Transporter1) and AtSOS1 (Salt Overly Sensitive 1), were higher in NtUBC1- and NtUBQ2-Arabidopsis. However, the transcript level of AtNHX1 (Na+/H+ Exchanger 1) was similar between control and transgenic plants. After salt exposure, the activity of the 26S proteasome markedly increased in NtUBC1- and NtUBQ2-expressing plants; however, ubiquitinated protein levels decreased compared to control plants. Furthermore, higher activity of antioxidant enzymes and lower ROS production were observed in UBC1- and UBQ2-expressing plants. We further challenged atubc1, atubc2, and atubq2 single mutants and atubc1ubc2 double mutant lines with salt stress; interestingly, the salt sensitivity and sodium levels of the studied mutants were enhanced, while the potassium levels were reduced. However, the atubc1ubc2 double mutant illustrated a more severe phenotype than the single mutants, probably due to the redundant function of UBC1 and UBC2 in Arabidopsis. Taken together, NtUBC1 and NtUBQ2 enhance salt tolerance by enhancing 26S proteasome activity and reducing Na+ accumulation, ROS, and ubiquitinated/salt-denatured proteins.

Ethylene and ROS mediate root growth inhibition induced by the endocrine disruptor bisphenol A (BPA).

Bisphenol A (BPA) functions as a detrimental substance that disrupts the endocrine system in animals while also impeding the growth and development of plants. In our previous study, we demonstrated that BPA hinders the growth of roots in Arabidopsis by diminishing cell division and elongation, which is ascribed to the increased accumulation and redistribution of auxin. Here, we examined the mediation of ROS and ethylene in BPA-induced auxin accumulation and root growth inhibition. BPA enhanced ROS levels, and ROS increased auxin contents but reduced cell division activity and the expression of EXPA8 involved in root elongation. ROS scavenger treatment reversed BPA-triggered root growth retardation, auxin accumulation, and cell division inhibition. In addition, BPA induced ethylene, and ethylene synthesis inhibitor treatment reversed BPA-triggered root growth retardation and auxin accumulation. Taken together, ROS and ethylene are involved in BPA-inhibited cell elongation and cell division by mediating auxin accumulation and redistribution.

Decreases in arsenic accumulation by the plasma membrane intrinsic protein PIP2;2 in Arabidopsis and yeast.

Arsenic (As) is a toxic pollutant that mainly enters the human body via plants. Therefore, understanding the strategy for reducing arsenic accumulation in plants is important to human health and the environment. Aquaporins are ubiquitous water channel proteins that bidirectionally transport water across cell membranes and play a role in the transportation of other molecules, such as glycerol, ammonia, boric acid, and arsenic acid. Previously, we observed that Arabidopsis PIP2;2, encoding a plasma membrane intrinsic protein, is highly expressed in NtCyc07-expressing Arabidopsis, which shows a higher tolerance to arsenite (As(III)). In this study, we report that the overexpression of AtPIP2;2 enhanced As(III) tolerance and reduced As(III) levels in yeast. Likewise, AtPIP2;2-overexpressing Arabidopsis exhibited improved As(III) tolerance and lower accumulation of As(III). In contrast, atpip2;2 knockout Arabidopsis showed reduced As(III) tolerance but no significant change in As(III) levels. Interestingly, the AtPIP2;2 transcript and protein levels were increased in roots and shoots of Arabidopsis in response to As(III). Furthermore, As(III) efflux was enhanced and As(III) influx/accumulation was reduced in AtPIP2;2-expressing plants. The expression of AtPIP2;2 rescued the As(III)-sensitive phenotype of acr3 mutant yeast by reducing As levels and slightly reduced the As(III)-tolerant phenotype of fps1 mutant yeast by enhancing As content, suggesting that AtPIP2; 2 functions as a bidirectional channel of As(III), while the As(III) exporter activity is higher than the As(III) importer activity. All these results indicate that AtPIP2;2 expression promotes As(III) tolerance by decreasing As(III) accumulation through enhancing As(III) efflux in Arabidopsis. This finding can be applied to the generation of low arsenic crops for human health.

Wood transcriptome analysis of Pinus densiflora identifies genes critical for secondary cell wall formation and NAC transcription factors involved in tracheid formation.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

Mechanism for Higher Tolerance to and Lower Accumulation of Arsenite in NtCyc07-Overexpressing Tobacco.

Arsenite [As(III)] is a highly toxic chemical to all organisms. Previously, we reported that the overexpression of NtCyc07 enhanced As(III) tolerance and reduced As(III) accumulation in yeast (Saccharomyces cerevisiae) and tobacco (Nicotiana tabacum). To understand a mechanism for higher As(III) tolerance and lower As(III) accumulation in NtCyc07-overexpressing tobacco, we examined the expression levels of various putative As(III) transporters (aquaporin). The expressions of putative As(III) exporter NIP1;1, PIP1;1, 1;5, 2;1, 2;2, and 2;7 were enhanced, while the expressions of putative As(III) importer NIP3;1, 4;1, and XIP2;1 were decreased, contributing to the reduced accumulation of As(III) in NtCyc07-overexpressing tobacco. In addition, the levels of oxidative stress indicators (H2O2, superoxide and malondialdehyde) were lower, and the activities of antioxidant enzymes (catalase, superoxide dismutase and glutathione reductase) were higher in NtCyc07-tobacco than in the control tobacco. This suggests that the lower oxidative stress in transgenic tobacco may be attributed to the higher activities of antioxidant enzymes and lower As(III) levels. Taken together, the overexpression of NtCyc07 enhances As(III) tolerance by reducing As(III) accumulation through modulation of expressions of putative As(III) transporters in tobacco.

The mechanism of root growth inhibition by the endocrine disruptor bisphenol A (BPA).

Bisphenol A (BPA) is a harmful environmental contaminant acting as an endocrine disruptor in animals, but it also affects growth and development in plants. Here, we have elucidated the functional mechanism of root growth inhibition by BPA in Arabidopsis thaliana using mutants, reporter lines and a pharmacological approach. In response to 10 ppm BPA, fresh weight and main root length were reduced, while auxin levels increased. BPA inhibited root growth by reducing root cell length in the elongation zone by suppressing expansin expression and by decreasing the length of the meristem zone by repressing cell division. The inhibition of cell elongation and cell division was attributed to the enhanced accumulation/redistribution of auxin in the elongation zone and meristem zone in response to BPA. Correspondingly, the expressions of most auxin biosynthesis and transporter genes were enhanced in roots by BPA. Taken together, it is assumed that the endocrine disruptor BPA inhibits primary root growth by inhibiting cell elongation and division through auxin accumulation/redistribution in Arabidopsis. This study will contribute to understanding how BPA affects growth and development in plants.

A Convenient Plant-Based Detection System to Monitor Androgenic Compound in the Environment.

Environmental androgen analogues act as endocrine disruptors, which inhibit the normal function of androgen in animals. In the present work, through the expression of a chimeric gene specified for the production of the anthocyanin in response to androgen DHT (dihydrotestosterone), we generated an indicator Arabidopsis that displays a red color in leaves in the presence of androgen compounds. This construct consists of a ligand-binding domain of the human androgen receptor gene and the poplar transcription factor gene PtrMYB119, which is involved in anthocyanin biosynthesis in poplar and Arabidopsis. The transgenic Arabidopsis XVA-PtrMYB119 displayed a red color in leaves in response to 10 ppm DHT, whereas it did not react in the presence of other androgenic compounds. The transcript level of PtrMYB119 peaked at day 13 of DHT exposure on agar media and then declined to its normal level at day 15. Expressions of anthocyanin biosynthesis genes including chalcone flavanone isomerase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, UFGT (UGT78D2), and anthocyanidin synthase were similar to that of PtrMYB119. It is assumed that this transgenic plant can be used by nonscientists for the detection of androgen DHT in the environment and samples such as food solution without any experimental procedures.

Expression of the Tobacco Non-symbiotic Class 1 Hemoglobin Gene Hb1 Reduces Cadmium Levels by Modulating Cd Transporter Expression Through Decreasing Nitric Oxide and ROS Level in Arabidopsis.

Hemoglobin (Hb) proteins are ubiquitous in plants, and non-symbiotic class 1 hemoglobin (Hb1) is involved in various biotic and abiotic stress responses. Here, the expression of the tobacco (Nicotiana tabacum) hemoglobin gene NtHb1 in Arabidopsis (Arabidopsis thaliana) showed higher cadmium (Cd) tolerance and lower accumulations of Cd, nitric oxide (NO), and reactive oxygen species (ROS) like hydrogen peroxide (H2O2). NtHb1-expressing Arabidopsis exhibited a reduced induction of NO levels in response to Cd, suggesting scavenging of NO by Hb1. In addition, transgenic plants had reduced accumulation of ROS and increased activities of antioxidative enzymes (catalase, superoxide dismutase, and glutathione reductase) in response to Cd. While the expression of the Cd exporters ABC transporter (PDR8) and Ca2+/H+ exchangers (CAXs) was increased, that of the Cd importers iron responsive transporter 1 (IRT1) and P-type 2B Ca2+ ATPase (ACA10) was reduced in response to Cd. When Col-0 plants were treated with the NO donor sodium nitroprusside (SNP) and H2O2, the expression pattern of Cd transporters (PDR8, CAX3, IRT1, and ACA10) was reversed, suggesting that NtHb1 expression decreased the Cd level by regulating the expression of Cd transporters via decreased NO and ROS. Correspondingly, NtHb1-expressing Arabidopsis showed increased Cd export. In summary, the expression of NtHb1 reduces Cd levels by regulating Cd transporter expression via decreased NO and ROS levels in Arabidopsis.

The tobacco gene Ntcyc07 confers arsenite tolerance in Saccharomyces cerevisiae by reducing the steady state levels of intracellular arsenic.

We cloned a plant gene, Ntcyc07, conferring arsenite tolerance by expressing a tobacco expression library in WT yeast (Y800). Expression of Ntcyc07 increased the tolerance to As(III) and decreased its accumulation, suggesting that the enhanced As(III) tolerance resulted from a reduction of the intracellular arsenic level. Interestingly, expression of Ntcyc07 increased the expression of the As(III) export carrier ACR3, but repressed that of As(III) uptake channel FPS1. Ntcyc07p interacted with Acr1p, which is the transcriptional activator of ACR3, but not with the ACR3 promoter. Taken together, the data indicated that Ntcyc07p promoted As(III) tolerance by decreasing the intracellular level of As(III) via increasing the expression of ACR3 and reducing that of FPS1.

Expression of yeast transcriptional activator MSN1 promotes accumulation of chromium and sulfur by enhancing sulfate transporter level in plants.

MSN1 is a putative yeast transcriptional activator involved in chromium (Cr) accumulation. Here we show that overexpression of MSN1 enhances Cr and sulfur accumulation and Cr tolerance in transgenic tobacco. In addition, we found that expression of NtST1 (Nicotiana tabacum sulfate transporter 1) was elevated in MSN1- expressing transgenic tobacco, suggesting that chromate and sulfate are taken up via the sulfate transporter in plants. Supporting this, expression of NtST1 increased levels of Cr and S in Saccharomyces cerevisiae. Our findings suggest that yeast transcriptional activators can be used for developing effective metal remediators, and for improving the nutritional status of plants.

Constitutive expression of a high-affinity sulfate transporter in Indian mustard affects metal tolerance and accumulation.

The Stylosanthes hamata SHST1 gene encodes a high-affinity sulfate transporter located in the plasma membrane. In this study the S. hamata SHST1 gene was constitutively expressed in Indian mustard [Brassica juncea (L.) Czern.] to investigate its importance for tolerance and accumulation of various oxyanions that may be transported by SHST1 and for cadmium, which is detoxified by sulfur-rich compounds. The transgenic SHST1 lines SHST1-12C and SHST1-4C were compared with wild-type Indian mustard for tolerance and accumulation of arsenate, chromate, tungstate, vanadate, and cadmium. As seedlings the SHST1 plants accumulated significantly more Cd and W, and somewhat more Cr and V. The SHST1 seedlings were less tolerant to Cd, Mo, and V compared to wild-type plants. Mature SHST1 plants were less tolerant than wild-type plants to Cd and Cr. SHST1 plants accumulated significantly more Cd, Cr, and W in their roots than wild-type plants. In their shoots they accumulated significantly more Cr and somewhat more V and W. Shoot Cd accumulation was significantly lower than in wild-type, and As levels were somewhat reduced. Compared to wild-type plants, sulfur accumulation was enhanced in roots of SHST1 plants but not in shoots. Together these results suggest that SHST1 can facilitate uptake of other oxyanions in addition to sulfate and that SHST1 mediates uptake in roots rather than root-to-shoot translocation. Since SHST1 overexpression led to enhanced accumulation of Cr, Cd, V, and W, this approach shows some potential for phytoremediation, especially if it could be combined with the expression of a gene that confers enhanced metal translocation or tolerance.

The Density and Length of Root Hairs Are Enhanced in Response to Cadmium and Arsenic by Modulating Gene Expressions Involved in Fate Determination and Morphogenesis of Root Hairs in Arabidopsis.

Root hairs are tubular outgrowths that originate from epidermal cells. Exposure of Arabidopsis to cadmium (Cd) and arsenic [arsenite, As(III)] increases root hair density and length. To examine the underlying mechanism, we measured the expression of genes involved in fate determination and morphogenesis of root hairs. Cd and As(III) downregulated TTG1 and GL2 (negative regulators of fate determination) and upregulated GEM (positive regulator), suggesting that root hair fate determination is stimulated by Cd and As(III). Cd and As(III) increased the transcript levels of genes involved in root hair initiation (RHD6 and AXR2) and root hair elongation (AUX1, AXR1, ETR1, and EIN2) except CTR1. DR5::GUS transgenic Arabidopsis showed a higher DR5 expression in the root tip, suggesting that Cd and As(III) increased the auxin content in the root tip. Knockdown of TTG1 in Arabidopsis resulted in increased root hair density and decreased root hair length compared with the control (Col-0) on 1/2 MS media. This phenotype may be attributed to the downregulation of GL2 and CTR1 and upregulation of RHD6. By contrast, gem mutant plants displayed a decrease in root hair density and length with reduced expression of RHD6, AXR2, AUX1, AXR1, ETR1, CTR1, and EIN2. Taken together, our results indicate that fate determination, initiation, and elongation of root hairs are stimulated in response to Cd and As(III) through the modulation of the expression of genes involved in these processes in Arabidopsis.

The putative transcriptional activator MSN1 promotes chromium accumulation in Saccharomyces cerevisiae.

Yeast is a good system for studying molecular mechanisms of metal tolerance. Using a mini-Tn mutagenized yeast pool, we isolated a chromate-tolerant mutant, CrT9, that displayed metal-specific tolerance since it was only tolerant to Cr(VI), not to Cr(III), Cd, As, or Fe. The Cr-tolerance of CrT9 appeared to be due to reduced Cr accumulation as it accumulated only 56% as much as WT (Y800). Using IPCR (inverse PCR), we found that the mini-Tn had been inserted at nt 741 of the transcriptional activator, MSN1. MSN1 is a multifunctional protein involved in invertase activity, iron uptake, starch degradation, pseudohyphal growth, and osmotic gene expression. We found that there was only one mini-Tn insertion in CrT9 since MSN1 and mini-Tn probes hybridized to the same DNA fragment, and the MSN1 probe detected an enlarged MSN1 mRNA. When we over-expressed MSN1 in CrT9 and WT, both accumulated larger amounts of Cr. We conclude that Cr accumulation in S. cerevisiae is promoted by the transcriptional activator MSN1.