Cadmium and oxidative stress influence on phytochelatin synthase activity in potato tuber

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ induced biosynthesis of phytochelatin synthase (PCS). The intensity of this process depended on the concentration of cadmium ions (0.01 – 1 mmol·dm⁻³), time and cadmium resistance of tissues. In more resistant tissues, PCS activity was much higher and PCS was more resistant to oxidative stress. It seems that these tissues possessed more efficient cadmium detoxification system.     

Effects of Chlorocholine Chloride on Phytohormones and Photosynthetic Characteristics in Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

Effects of chlorocholine chloride (CCC) on phytohormones and photosynthetic characteristics of Zhongshu 3, a potato (Solanum tuberosum L.) variety widely cultivated in south China, were studied by foliar CCC application on 24 and 28 days after emergence, that is, at the tuber initiation stage. It was found that on 42 days after emergence, that is, at the tuber bulking stage, spraying CCC increased indolacetic-3-acid (IAA) and zeatin (Z) contents but decreased abscisic acid (ABA) content in leaves. The content ratios of IAA/Z, IAA/ABA, Z/ABA, and (IAA + Z)/ABA in leaves treated with CCC were higher than those of the control. CCC plays a prominent regulating role in the photosynthesis of Zhongshu 3. The net photosynthetic rate (P _n), stomatal conductance (G _s), intercellular CO₂ concentration (C _i), and transpiration rate (T _r) of treated leaves were superior to those of controls at the tuber bulking stage. CCC markedly increased tuber yield and quality. The contents of sucrose and starch in tubers treated with CCC increased at the end of the vegetation period, whereas the contents of reducing sugars and solanine decreased. CCC at 2.0 g L⁻¹ was found to be the most effective concentration. Collectively, the results of this research identify phytohomonal metabolism and photosynthetic physiology of potato leaves as processes affected early after application of CCC resulting in significantly improved increases in tuber yield and quality.     

Cadmium effect on hydrogen peroxide, gluthatione and phytochelatins levels in potato tuber

Short-term treatment of potato tuber (Solanum tuberosum L.) dises with CdCl₂ (1mM) induced an oxidative stress, manifested by higher levels of H₂O₂, and activated the synthesis of phytochclatins ((γ-Glu-Cys)_n-Gly): PC₂, PC₃ and PC₄. If in the tissues with a lower GSH level, the oxidative stress was induced by treatment with 3-aminotriazol (AT), or with AT and H₂O₂, the elevation of H₂O₂ and GSH levels and then some accumulation of thiols, including PC₂, PC₃ and PC₄, were observed. However, this increase of PC concentration was considerably lower when compared with the effects brought about by Cd⁺² treatment. If such a procedure of evoking subsequent moderate oxidative stress in tissues preceded Cd-treatment, a marked limitation of PC synthesis was observed. The presented results support the role of H₂O₂ as the second messenger in activating GSH synthesis and thus suggest a possibility of redox type regulation mechanism of PCs synthesis.     

High concentration of cadmium induces AtPCS2 gene expression in Arabidopsis thaliana (L.) Heynh ecotype Wassilewskija seedlings

In Arabidopsis thaliana, two genes encoding phytochelatin synthase (PCS; EC 2.3.2.15), AtPCS1 and AtPCS2, have been identified. Until now, only AtPCS1 was shown to play a role in response to Cd. To gain insight into the putative role of AtPCS2, three Cd concentrations (50, 100 and 200 μM) and long-term exposure (7 days) were tested on 1-week-old A. thaliana ecotype Wassilewskija (Ws) seedlings. Since 100 μM Cd did not alter seedling metabolism, as shown by unchanged total soluble protein and free proline contents, we investigated plantlet response to this concentration in addition to Cd accumulation. Seedlings accumulated Cd in roots and shoots. As phytochelatins and glutathione (GSH) contents increased in treated seedlings, we suggested that Cd might be translocated via the phytochelatin pathway. Specific enzymatic activities of γ-glutamylcysteine synthetase (GCS; EC 6.3.2.2), glutathione synthetase (GS; EC 6.3.2.3) and PCS were twice much more stimulated in shoots and roots after Cd exposure except GS that remained constant in shoots. As expression of genes encoding GCS and GS was unchanged in response to Cd, we suggested a regulation at translational or post-translational level. Surprisingly, AtPCS1 and AtPCS2 were differentially up-regulated after Cd treatment: AtPCS1 in shoots and AtPCS2 in whole plantlets. This last result suggests that PCS2 could be involved in plant response to high concentration of Cd in Ws ecotype and supports a putative role of PCS2, not redundant with PCS1, in a long-term response to Cd.     

Phytohormone Profiling During Tuber Development of Chinese Yam by Ultra-high performance Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

Changes in agronomic characters and the profile of various endogenous phytohormones during tuber development were studied in Dioscorea opposite (Chinese yam) cv. Guihuai 16. Tuber development exhibited a sigmoidal growth pattern according to the changes in tuber agronomic characters. The growth cycle of yam tuber could be divided into three stages: initiation stage, enlargement stage, and maturation stage. Moreover, the enlargement stage could be separated into three phases—slow growth phase, rapid growth phase, and late growth phase. Endogenous changes in phytohormones were associated with developmental changes in the tubers. The pulses of bioactive gibberellins (such as GA₃ and GA₄) were measured in tubers. The highest contents of GA₃ and GA₄ were reached 90 days after field planting, corresponding to the beginning of the rapid growth phase of tuber enlargement. Changes in trans-zeatin (tZ), jasmonic acid (JA), indole-3-acetic acid (IAA), and abscisic acid (ABA) levels were also observed, and seemed to be related to tuber enlargement at different phases. Continuous increases in JA and tZ contents accompanied tuber enlargement. Transient pulses of both IAA and ABA contents were also observed at the start of tuber rapid growth. Additionally, a second peak level of IAA was detected at the tuber maturation stage. These results suggest GAs play a key role at the beginning of the tuber rapid growth stage, and there is a close relationship between whole tuber enlargement and the contents of JA and tZ. Moreover, it is suggested that IAA and ABA also may be linked to the beginning of tuber rapid growth, and IAA also seems to be correlated to late tuber maturation.     

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8′-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Mechanism of cadmium tolerance in Salicornia europaea at optimum levels of NaCl

• Many saline-alkali soils around the world are polluted by the heavy metal Cd, restricting the development of agriculture and ecology in those regions. The halophyte Salicornia europaea L. is capable of growing healthily in Cd-contaminated saline-alkali soil, suggesting that the species is tolerant to stress caused by both salt and heavy metals. In this study, the mechanism of Cd tolerance in this species was explored under 200 mM NaCl.
• Flame spectrophotometric assays for ions content and spectrophotometric for organic soluble substances, antioxidant enzyme activity, phytochelatins (PCs) content and phytochelatin synthase (PCS) activity, the photosynthetic parameters by portable photosynthesis measurement system, genes expression by qRT-PCR analysis were carried out.
• Cd treatment significantly decreased the dry weight, photosynthetic rate, K⁺, Zn²⁺, and Fe^2+/3+ content, while significantly increasing Na⁺ and Cd⁺, soluble organic matter, and reactive oxygen species (ROS) levels. Compared with Cd treatment at 0 mM NaCl, Cd treatment at 200 mM NaCl significantly increased dry weight and photosynthetic rate while significantly decreasing ROS content through increased antioxidant enzyme activity. When exposed to Cd stress, treatment with 200 mM NaCl significantly increased PCs content and PCS activity and up-regulated the expression of the phytochelatin synthase genes CDA1 and PCS1 were, thereby increasing resistance to Cd.
• NaCl treatment increases the tolerance of S. europaea to the heavy metal Cd by growing rapidly, reducing the quantity of Cd²⁺ from entering the plant shoots, increasing the levels of PCs that chelate Cd²⁺, thereby reducing its toxicity.

     

不同成花量金花茶花果期果枝叶内源激素的变化

该研究采用ISSR分子标记,对黄枝油杉7个自然种群的遗传多样性进行了分析。结果表明：用12条ISSR引物对218个黄枝油杉个体进行扩增,共扩增出125个位点。在物种水平上,多态性位点百分数( PPL)为100.00％,Shannon信息多样性指数( I)为0.4177,Nei’ s基因多样性指数( H)为0.2666;在种群水平上,多态性位点百分数(PPL)在71.20％~92.00％之间,平均值为80.69％,Shannon信息多样性指数(I)在0.3273~0.3886之间,平均值为0.3548,Nei’ s基因多样性指数( H)在0.2139~0.2478之间,平均值为0.2291。这说明黄枝油杉在物种水平和种群水平上均显示出较高的遗传多样性。 Nei’ s遗传多样性分析( Gst＝0.1433)和AMOVA分析(Φst＝17.91％)表明,黄枝油杉的遗传变异主要存在于种群内,种群间的遗传分化程度较低,种群间保持一定的基因交流( Nm＝2.9890>1)。 Mantel分析显示,黄枝油杉种群间的遗传距离和地理距离之间不存在显著的相关关系( r＝0.4567, P＝0.0610>0.05)。     

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga <Emphasis Type="Italic">Trebouxia erici</Emphasis>: metal accumulation,toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC₂–PC₅), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC₅. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC₂ were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.     

Endogenous inhibitors and seasonal changes in abscisic acid in Dioscorea floribunda Mart. & Gal.

In the tubers of medicinal yam (Dioscorea floribunda Mart. & Gal.) abscisic acid (ABA) content was high in all the parts during the dormant condition in the winter, but it decreased to a very low level in the actively growing plants. ABA content of the entire tuber was negatively correlated with temperature and photoperiod. Growth inhibitors including batatasin-I and phthalic acid were identified in the dormant tuber.CIMAP Communication No. 673.     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

梾木种子低温层积过程中内源激素含量的动态变化特征

应用酶联免疫吸附测定法(ELISA)研究了梾木种子低温层积过程中内源激素含量的动态变化,分析了内源激素与种子休眠与发芽的关系。结果表明:(1)梾木种子中IAA含量在层积处理初期剧烈降低,持续一段时间后又显著升高,但后期下降,且IAA/ABA也出现同样的变化;种子中ABA含量在层积处理前期较高,但随着处理时间的延长趋于下降;种子内GA1/3含量以及GA1/3/ABA均随层积处理时间的延长逐渐增大;种子内ZRs和iPAs含量的变化相对较为平稳,尽管有一定的波动,但整体呈渐趋增高趋势。(2)梾木种子发芽率及发芽势在未经层积处理以及处理时间少于90d的条件下均为0,但随着层积处理时间的延长二者明显上升,层积处理的时间长短对梾木种子萌发有显著影响。(3)相关分析表明,梾木种子内GA1/3含量与种子的发芽率、发芽势均呈显著正相关关系,相关系数分别为0.688、0.662;种子内GA1/3/ABA增大有利于种子休眠解除和萌发。     

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Localization and functional characterization of metal-binding sites in phytochelatin synthases

Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd²⁺ and other toxic metal and metalloid ions. In order to localize Cd²⁺-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with ¹⁰⁹Cd²⁺. Distinct binding sites and binding motifs could be localized based on the patterns of Cd²⁺-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd²⁺-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity.Abbreviations EMM Edinburgh's minimal medium - GSH glutathione - HA hemagglutinin - PC phytochelatin - PCS phytochelatin synthase     

中国沙棘克隆生长对灌水强度的响应规律及其激素调控机制

关于中国沙棘克隆生长调节研究目前局限于外在机制,旨在探讨其克隆生长对灌水强度的响应规律及其激素调控的内在机制。结果表明:随着灌水强度的增大,分株生长、克隆繁殖、克隆扩散能力先升后降,IAA(吲哚-3-乙酸)、ZR(玉米素核苷)、GA_3(赤霉酸)含量及其与ABA(脱落酸)的比值先升后降而ABA含量先降后升。同时,分株生长、克隆繁殖、克隆扩散能力与IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA呈极显著或显著正相关,与ABA含量呈极显著负相关。灌水强度过小或过大,IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA低而ABA含量高,克隆生长潜力受到抑制,种群以分株小、数量少(分布稀疏)、扩散(水平根延伸和分枝)能力弱为特征,克隆生长格局倾向于"游击型"、种群早衰概率高;灌水强度适宜,IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA高而ABA含量低,克隆生长潜力得以充分发挥,种群以分株大、数量多(分布密集)、扩散能力强为特征,克隆生长格局倾向于"聚集型"、种群稳定性高。随着灌水强度过小-适宜-过大的连续变化,中国沙棘通过改变激素状况调控克隆生长,从而形成与灌水强度相适应的克隆生长格局连续体"游击型-聚集型-游击型",种群稳定性呈"低-高-低"的连续变化过程。由此可见:灌水强度诱导内源激素发生改变,激素特征调控克隆生长格局,克隆生长格局决定种群稳定性。     

Effect of abscisic acid on heat stress tolerance in the calli from two ecotypes of <Emphasis Type="Italic">Phragmites communis</Emphasis>

Dune reed (DR) and swamp reed (SR) are two ecotypes of reed (Phragmites communis Trin.) that displayed differences in stress tolerance. To uncover the molecular basis for such difference, the effects of heat stress were studied using the calli derived from the two ecotypes. Heat stress caused increased ion leakage, inhibited growth, decreased cell viability, and elevated hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents in the calli of both ecotypes, but DR callus showed better heat tolerance than SR callus. In DR callus, heat stress caused significant increase in the endogenous ABA content but not in SR callus. Application of fluridone (an ABA synthesis inhibitor) aggravated the heat stress damages on the DR callus whereas it had only minimal impact on the SR callus. Exogenous application of ABA alleviated the heat stress symptoms in the calli of both ecotypes. ABA treatment increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase, and also decreased H₂O₂ and MDA contents. These results indicate that the ability of ABA synthesis under heat stress is a key factor attributing to the higher heat tolerance of DR than SR.     

Multi-tasking phytochelatin synthases

Phytochelatins are essential for cadmium and arsenic detoxification in plants, some fungi, and animals. It is mysterious, that the responsible enzymes, phytochelatin synthases (PCS), are constitutively expressed and so widespread in nature. Phylogenetic analysis indicates multiple horizontal transfers of PCS genes, but a bacterial origin appears unlikely. Differences between bacterial and eukaryotic PCS proteins in structure and activity had indicated bi-functionality of phytochelatin synthases as peptidases and transpeptidases. Recent observations indicate that PCS indeed serve physiological functions that most likely are much more prevalent than cadmium or arsenic detoxification. First, PCS-deficient Arabidopsis thaliana mutants are hypersensitive to zinc suggesting a role of phytochelatin synthesis, i.e. the formation of metal-binding peptides from glutathione in a transpeptidase reaction, in Zn homeostasis. Second, these mutants are also impaired in defense responses conferring resistance to incompatible pathogens (= nonhost resistance). The latter is hypothesized to be attributable to an involvement of PCS as a peptidase in indole glucosinolate metabolism. Possibly, micronutrient homeostasis and nonhost resistance are closely connected as PCS are not the only proteins involved in both processes.     

Leaf-targeted phytochelatin synthase in Arabidopsis thaliana

One of the key steps in developing transgenic plants for the phytoremediation of metal containing soils is to develop plants that accumulate metals in the aerial tissues. With the goal of changing the distribution of phytochelatin (PC)-dependent cadmium accumulation from roots to the leaves, the phytochelatin synthase (PCS) deficient cad1-3 mutant and wild type (Col-0) Arabidopsis plants were transformed with an Arabidopsis phytochelatin synthase (AtPCS1) under the control of a leaf-specific promoter. Three independent transformant lines from each genetic background were chosen for further analysis and designated cad-PCS and WT-PCS. PCS activity in the cadPCS lines was restored in the leaves, but not in the roots. Additionally, when whole plants were treated with cadmium, PCs were found only in the leaves of cad-PCS plants. Although the inserted AtPCS1 gene was leaf-specific, cad-PCS lines showed an overall decrease in cadmium toxicity evidenced by a partial amelioration of the "brown-root" phenotype and root growth was restored to wild type levels when treated with cadmium and arsenate. WT-PCS lines showed an increase in leaf PCS activity but had only wild type PC levels. In addition, cadmium uptake studies indicated that there was no difference in cadmium accumulation among all types tested. So, while we were able to protect the plants against cadmium by expressing PC synthase only in the leaves, we were not able to limit cadmium accumulation to aerial tissues.