Phytochelatin accumulation and stress tolerance in tomato cells exposed to cadmium

Cell suspension cultures of tomato (Lycopersicon esculentum) adapted to growing continuously in the presence of 0.1 mM CdCl₂ and accumulated phytochelatins (PCs, poly(-Glu-Cys)n-Gly). The highest level of PCs was measured 4 days after inoculation and coincided with the peak of cellular cadmium concentration. At this time there was an 8-fold molar excess of PC (-Glu-Cys) over Cd. PCs could not be detected after 12 days when the cellular concentration of cadmium was 0.2 mM. These results indicate that PCs are produced in excess of that required to bind the cellular cadmium in the early stage of the culture period followed by degradation of PCs during the stationary phase. Adaptation to 0.1 mM CdCl₂ did not increase tolerance to higher concentrations of cadmium when compared with control cells, but did significantly enhance tolerance to both anaerobiosis and heat shock. Exposure of tomato cells to 0.1 mM CdCl₂ resulted in several changes in proteins synthesized.     

Metal accumulation and damage in rice (cv. Vialone nano) seedlings exposed to cadmium

The effect of exposure to increasing cadmium concentrations was analyzed in rice seedlings (cv. Vialone nano). The highest Cd accumulation was found in roots, mostly in the apoplastic environment. Cd taken up in cells led to an increase in sulfhydryl groups, the appearance of phytochelatins, and formation of electron-dense vacuolar inclusions. The metal-exposure inhibited root growth and also interfered with correct root morphogenesis, causing disordered division and abnormal and forward enlargement of epidermal and cortical cell layers in the apical region. Cd accumulation in shoots was lower than in roots. In leaf cells, there was neither a substantial increase in sulfhydryl groups nor the appearance of phytochelatins. Shoot growth was reduced and, differently from in roots, leaf cell enlargement was inhibited. Chloroplasts had lowered contents of chlorophyll and a reduced number of thylakoids, but underwent structural alterations only at the highest Cd concentration tested (250 μM). Photosynthetic activity was limited due to the curtailment of CO₂ availability caused by the greater resistance of Cd-exposed leaves. The damage suffered by seedlings worsened with the increase in Cd concentration, but was already evident at the lowest concentration examined (50 μM), showing that the cv. Vialone nano has a Cd-sensitivity higher than other rice cultivars.     

Short term signaling responses in roots of young soybean seedlings exposed to cadmium stress

In the present study, the expression of fourteen genes involved in various signal transduction pathways was examined in young soybean (Glycine max) seedlings exposed to cadmium at two concentrations (10 mg L⁻¹ and 25 mg L⁻¹) for short time periods (3, 6 and 24 h). The results show that cadmium causes induction of genes encoding proteins involved in ethylene and polyamines metabolism, nitric oxide generation, MAPK cascades and regulation of other genes’ expression. The bioinformatic analysis of promoter sequences of Cd-inducible genes revealed that their promoters possess several regulative motifs associated with the plant response to stress factors and abscisic acid and ethylene signaling. The involvement of ethylene in the response of soybean seedlings to cadmium stress was further confirmed by the real-time analysis of ethylene production during 24 h of CdCl₂ treatment. The role of the described signaling elements in transduction of the cadmium signal in young soybean seedlings is discussed.     

Cadmium stress in wheat seedlings: growth,cadmium accumulation and photosynthesis

Seedlings of wheat (Triticum aestivum L.) cultivars Jing 411, Jinmai 30 and Yangmai 10 were exposed to 0, 10, 20, 30, 40 or 50 μM of CdCl₂ in a solution culture experiment. The effects of cadmium (Cd) stress on wheat growth, leaf photon energy conversion, gas exchange, and Cd accumulation in wheat seedlings were investigated. Gas exchange was monitored at 3, 9, 24 days after treatment (DAT). Growth parameters, chlorophyll content, leaf chlorophyll fluorescence, and Cd concentration in shoot and root were measured at 24 DAT. Seedling growth, gas exchange, chlorophyll content, chlorophyll fluorescence parameters were generally depressed by Cd stress, especially under the high Cd concentrations. Cd concentration and accumulation in both shoots and roots increased with increasing external Cd concentrations. Relationships between corrected parameters of growth, photosynthesis and fluorescence and corrected Cd concentrations in shoots and roots could be explained by the regression model Y = K/(1 + exp(a + bX)). Jing 411 was found to be Cd tolerant considering parameters of chlorophyll content, photosynthesis and chlorophyll fluorescence in which less Cd translocation was from roots into shoots. The high Cd concentrations were in shoots and roots in Yangmai 10 which has been found to be a relative Cd tolerant cultivar in terms of most growth parameters.     

Abscisic acid accumulation and cadmium tolerance in rice seedlings

Rice ( Oryza sativa L.) seeds were soaked for 18 h in distilled water in the absence (–PBZ) or presence (+PBZ, a triazole) of 100 mg l⁻¹ paclobutrazol and then air dried. These air-dried seeds were germinated in the dark and then cultivated in a Phytotron. Twelve-day-old –PBZ and +PBZ seedlings were treated or not with CdCl₂. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll and protein content, increase in NH₄⁺ content and induction of oxidative stress. The results indicated that PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. On treatment with CdCl₂, the abscisic acid (ABA) content increased in +PBZ leaves, but not in –PBZ leaves. The decrease in the transpiration rate of –PBZ seedlings by CdCl₂ was less than that of +PBZ seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone (Flu), reduced ABA accumulation, increased the transpiration rate and Cd content, and decreased the Cd tolerance of +PBZ seedlings. The effects of Flu on the Cd toxicity, transpiration rate and Cd content were reversed by the application of ABA. It seems that the PBZ-induced Cd tolerance of rice seedlings is mediated through an accumulation of ABA.     

Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium

The effects of excess Cd on the contents of free cysteine, total glutathione and phytochelatin (PC) were measured in roots of intact maize seedlings. Exposure to 3 /tmM Cd for 15 min caused PCs to appe substrates for formation of longer PCs. Total glutathione levels declined with PC synthesis, free cysteine contents changed little. The reactions to excess Cd differed along the length of roots. In the 1 cm apical region a high production of PCs occurred with a moderate loss of total glutathione. In the mature region, PC content was 2.5-fold less than in apices, several unidentified thiols accumulated, and total glutathione levels declined drastically. Exposure to 0.05 μM Cd for 24 h induced PCs, the contents rose as Cd concentrations were increased. The roots produced PCs in excess of that required to chelate the Cd present, as if some PCs were compartmentalized or had not yet formed Cd-PC complexes. Phytochelatin formation was stimulated most effectively by Cd, less by Zn and Cu and negligibly by Ni. Total glutathione declined with Cd and Zn exposure, however, with excess Cu the roots contained 45% more total glutathione than did the controls.     

镉胁迫下玉米幼苗生理生态的变化

用不同浓度Cd2 + 处理玉米种子 ,在室内常规培养 ,研究了种子萌发和幼苗生理生态的变化。结果表明 ,镉影响玉米种子的萌发和幼苗的生长。当Cd2 + 浓度高于 5 0mg·L-1时 ,显著抑制种子的发芽率 ;随Cd2 + 浓度的增加 ,根系长度和侧根数减小 ;在Cd2 + 浓度小于 5mg·L-1时 ,镉刺激苗高和根系及地上部干物质量增加 ,当Cd2 + 浓度超过相应浓度时 ,苗高和根系及地上部的生长量随Cd2 + 浓度提高而降低。镉胁迫下幼苗根系活力和叶绿素含量明显降低 ,根内丙二醛含量增加。镉影响玉米幼苗对矿质元素的吸收。根系和茎叶中Ca、Mg、Fe、Cu的吸收量随Cd2 + 浓度提高而增加 ,K、Zn的吸收量随Cd2 + 浓度提高而减少。     

四种园林植物对土壤镉污染的耐受性

采用模拟Cd污染土壤培养法,测定了Cd胁迫下山矾、桑树、绣线菊、山茶4种园林植物幼苗的生长、生物量变化,根茎叶的Cd含量,光合色素含量与MDA含量,对耐性指数(Ti)、转移系数(TF)、生物富集系数(BCF)进行了评价。结果表明:(1)山矾、桑树、绣线菊和山茶的平均耐性指数分别为93.99、82.33、82.10和87.25;(2)4个树种幼苗根茎叶的Cd含量都随着Cd处理浓度的增加而增加,转移系数值(TF)都小于1,转移能力为山矾山茶绣线菊桑树。对Cd累积能力为山矾山茶桑树绣线菊;山矾和山茶生物量吸收的Cd总量显著高于绣线菊和桑树。(3)Cd处理浓度的不断增加,叶绿素a/叶绿素b比值与对照相比变化不显著,类胡萝卜素的含量持续增加;桑树、山茶、山矾和绣线菊MDA含量分别平均上升为15%、10.17%、9.69%、12.86%。不同Cd浓度下,MDA上升幅度顺序为桑树绣线菊山茶山矾。研究表明山矾具有很高的Cd耐性、转移能力、以及地上部分积累镉的能力,是一种抗Cd污染较好的园林绿化树种。     

Identification of phytochelatin-related peptides in maize seedlings exposed to cadmium and obtained enzymatically in vitro

An analytical strategy based on the sensitivity of electrospray tandem mass spectrometry following a simplified and reproducible sample preparation procedure was evaluated for the determination of Cd-induced phytochelatins (PC) and related peptides in four maize varieties. In addition to the three known families of PC (PC, desGly-PC and iso-PC(Glu)) that were observed, novel PC and desGly-PC homologues lacking the N-terminal gamma-linked Glu were isolated from maize root extracts for the first time. Additionally the complete sequence of iso-PC3(Glu) was determined by tandem mass spectrometry. Peptides obtained in vivo and in vitro as the result of the reaction of glutathione with the enzyme phytochelatin synthase were compared. Minor forms detected from in vitro reactions include compounds with intramolecular or intermolecular disulfide bonds resulting from the oxidation of SH groups, phytochelatin homologues lacking the N-terminal gamma-linked Glu, and new PC-related peptides with a Cys-Cys motif. Since peptides lacking a gammaGlu residue could be generated as artifacts in electrospray mass spectrometry, the application of capillary electrophoresis with online electrospray mass spectrometry allowed the separation and detection of such peptides as endogenous molecules present in planta and as products of in vitro reactions.     

Antioxidant responses and water status in Brassica seedlings subjected to boron stress

The effect of boron (B) on growth, water status and oxidative damage was investigated in the leaves and roots of 7-day-old seedlings of Brassica juncea var. Varuna. For this seedlings of Brassica were grown in solution culture with variable boron supply (0.033, 0.33, 3.3 and 33 mg B L^?1) under controlled conditions in green house. Photosynthetic pigments were found to be decreased more under excess (3.3 and 33 mg B L^–1) than deficient boron supply (0.033 mg B L^–1) when compared to control (0.33 mg B L^–1). Accumulation of hydrogen peroxide and thiobarbituric acid reactive substances content in both leaves and roots under deficient and excess boron supply suggested oxidative damage due to excessive production of reactive oxygen species. Increased activity of antioxidative enzymes: superoxide dismutase, catalase and peroxidase along with polyphenol oxidase was observed in leaves and roots under boron deficiency and excess than in control. Increased proline concentration, decreased total water content and water saturation deficit also indicated the water deficit condition in leaves and roots of boron-stressed Brassica seedlings.     

Autophagic flux analysis of Arabidopsis seedlings exposed to salt stress

In plant cells, autophagy is required for efficient recycling of cytoplasmic macromolecules in vacuoles. It was previously shown that autophagy-deficient mutants also exhibited hypersensitivity to various abiotic stresses, such as salt, osmotic changes, heat, drought, and oxidative damage. However, it has not been clearly determined whether autophagy is induced or inhibited by these environmental stressors. Using the GFP-ATG8 (green fluorescent protein fused to AUTOPHAGY-RELATED PROTEIN 8) processing assay and confocal microscopy, we assessed autophagic flux of Arabidopsis seedlings exposed to salt stress. Treatment with 150 mM NaCl resulted in an increase in the processing of GFP-ATG8. Notably, the effects of concanamycin A, an inhibitor of vacuolar proton pumps, on GFP-ATG8 processing indicated that the apparent increase in GFP-ATG8 processing by salt-induced stress was due to inefficient vacuolar degradation of the GFP moiety processed from GFP-ATG8. Salt and osmotic stresses did not increase the abundance of autophagic vesicles in the root cells. Although NaCl, KCl, and mannitol did not greatly inhibit the vacuolar trafficking of GFP-ATG8, LiCl partially inhibited autophagy. These data indicated that NaCl stress neither increases nor substantially inhibits autophagic flux. Our work illustrates the importance of autophagic flux analysis to assess the effect of abiotic stresses on plant autophagy.     

Toxicity in leaves of rice exposed to cadmium is due to hydrogen peroxide accumulation

The production of H₂O₂ in detached rice leaves of Taichung Native 1 (TN1) caused by CdCl₂ was investigated. CdCl₂ treatment resulted in H₂O₂ production in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase (NOX), prevented CdCl₂-induced H₂O₂ production, suggesting that NOX is a H₂O₂-genearating enzyme in CdCl₂-treated detached rice leaves. Phosphatidylinositol 3-kinase inhibitors wortmanin (WM) or LY294002 (LY) inhibited CdCl₂-inducted H₂O₂ production in detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY, suggesting that phosphatidylinositol 3-phosphate is required for Cd-induced H₂O₂ production in detached rice leaves. Nitric oxide donor sodium nitroprusside (SNP) was also effective in reducing CdCl₂-inducing accumulation of H₂O₂ in detached rice leaves. Cd toxicity was judged by the decrease in chlorophyll content. The results indicated that DPI, IMD, WM, LY, and SNP were able to reduce Cd-induced toxicity of detached rice leaves. Twelve-day-old TN1 and Tainung 67 (TNG67) rice seedlings were treated with or without CdCl₂. In terms of Cd toxicity (leaf chlorosis), it was observed that rice seedlings of cultivar TN1 are Cd-sensitive and those of cultivar TNG67 are Cd-tolerant. On treatment with CdCl₂, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Prior exposure of TN1 seedlings to 45^oC for 3 h resulted in a reduction of H₂O₂ accumulation, as well as Cd tolerance of TN1 seedlings treated with CdCl₂. The results strongly suggest that Cd toxicity of detached leaves and leaves attached to rice seedlings are due to H₂O₂ accumulation.     

Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity

Seed germination is a complex physiological process in plants that can be affected severely by heavy metals. The interference of germination by cadmium stress has not been well documented at the proteomic level. In the present study, in order to investigate the protein profile alternations during the germination stage following exposure to cadmium, a proteomic approach has been adopted in combination with morphological and physiological parameters. Seeds were exposed with a wide range of cadmium between 0.2 and 1.0 mM. Increases of cadmium concentration in the medium resulted in increased cadmium accumulation in seeds and TBARS content, whereas germination rate, shoot elongation, biomass, and water content were decreased significantly. Temporal changes of the total proteins were investigated by two-dimensional electrophoresis (2-DE). Twenty-one proteins were identified using MALDI-TOF mass spectrometry, which were upregulated at least 1.5-fold in response to cadmium stress. The identified proteins are involved in several processes, including defense and detoxification, antioxidant, protein biosynthesis, and germination processes. The identification of these proteins in the cadmium stress response provides new insight that can lead to a better understanding of the molecular basis of heavy metal responses of seeds at the germination stage.     

The protective role of selenium in rape seedlings subjected to cadmium stress

The effect of selenium (Se) on rape (Brassica napus) seedlings subjected to cadmium (Cd) stress was studied in vitro by investigating plant growth and changes in fatty acid composition, activity of antioxidative enzymes and DNA methylation pattern. Physiological experiments were carried out on seedlings cultured for 2 weeks on Murashige-Scoog (MS) media with Cd concentrations of 0, 400 and 600 μM, and on corresponding media supplied with Se (2 μM). Exposure to increasing Cd concentrations reduced the fresh weight of the upper part (hypocotyls+cotyledons) of the seedlings more strongly than that of the root system, which was accompanied by higher Cd accumulation in these tissues. In the upper part, Cd exposure led to significant changes in the biochemical parameters: fatty acid unsaturation of plasmalemma decreased, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPOX) diminished and that of ascorbate peroxidase (APX) increased. In contrast, the roots showed an increase in fatty acid unsaturation and in the activity of antioxidative enzymes. In both parts of rape seedlings H₂O₂ level and lipid peroxidation increased. Se addition to medium considerably reversed the Cd-induced decrease in fresh mass as well as the changes in lipid unsaturation and peroxidation. Se applied separately or in combination with Cd did not significantly affect the activity of antioxidative enzymes in the roots, but diminished it in the upper part. Moreover, the presence of Se in medium prevented changes in the DNA methylation pattern triggered in rape seedlings by high Cd concentrations. Two possible mechanisms for the action of Se were considered: (1) removal of Cd from metabolically active cellular sites, and (2) reduction of oxygen radicals.     

Antioxidant capacity of Lemna gibba L. exposed to wastewater treatment

In this study, the amounts of antioxidant vitamins (A, E and C), selenium (Se), reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and malondialdehyde (MDA) that is the indicator of lipid peroxidation were determined in Lemna gibba L. plants placed in the secondary clarifier and grown in natural water. The amounts of antioxidant vitamins (A, E and C), GSH, GSSG and MDA were determined with HPLC (high performance liquid chromatography) and the amounts of Se were determined fluorimetrically. While significant decrease in amounts of antioxidant vitamins (A, E and C) and Se takes place between the first and the second or third day depending on species and insignificant decrease takes place after that day, the amount of GSH/GSSG ratio decreases until the second day (p < 0.05) and insignificantly increases after that day. Amounts of vitamins (A, E and C), Se and GSH/GSSG ratio for the plants placed in the secondary clarifier are much less than that for control group while an opposite trend was observed in MDA level. The MDA for the plants placed in secondary clarifier has maximum value at the second day, which can be considered as the maximum stress occurring at that day. Consequently, the first two days of treatment time can be taken as acclimation time and after the acclimation time the plant lives in the hostile environmental condition with the certain amount of oxidative stress. As a result, it is determined that wastewater decreases the lifetime of plant by causing metabolic stress on it and affects antioxidant capacity. The lifetime of the plants in the secondary clarifier was determined to be five days since fading toward yellow color (necrosis) in the plants was observed at the fifth day.     

镉胁迫下金银花的生长反应及积累特性

采用水培试验方法,研究了藤本植物金银花在不同浓度镉（Cd）胁迫下的生长反应和积累特 性,结果表明,Cd对金银花生长未造成显著影响,甚至低浓度(5和10 mg·L^-1) Cd具有一定的生长促进作用,表现为植物的株高和叶绿素含量有所增加。当Cd处理浓度达到50 mg·L^-1时,植物的株高和根长虽有所降低,但与对照相比并无显著差异,且耐性系数均＞0.8,表明金银花对Cd具有较强的耐性。当Cd处理浓度为25 mg·L^-1时,金银花地上部中Cd含量达到286.12 μg·g^-1,而富集系数均＞1,说明金银花具备Cd超富集植物的特征,对Cd污染土壤的修复具有很大的应用潜力。     

Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress

The effect of proline (Pro) accumulation on heat sensitivity was investigated using transgenic Arabidopsis (Arabidopsis thaliana) plants ectopically expressing the Δ(1)-pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of a heat shock protein 17.6II gene promoter. During heat stress, the heat-inducible expression of the AtP5CS1 transgene was capable of enhancing Pro biosynthesis. Twelve-day-old seedlings were first treated with heat at 37 °C for 24 h to induce Pro and then were stressed at 50 °C for 4 h. After recovery at 22 °C for 96 h, the growth of Pro-overproducing plants was significantly more inhibited than that of control plants that do not accumulate Pro, manifested by lower survival rate, higher ion leakage, higher reactive oxygen species (ROS) and malondialdehyde levels, and increased activity of the Pro/P5C cycle. The activities of antioxidant enzymes superoxide dismutase, guaiacol peroxidase, and catalase, but not those of glutathione reductase and ascorbate peroxidase, increased in all lines after heat treatment, but the increase was more significant in Pro-overproducing seedlings. Staining with MitoSox-Red, reported for being able to specifically detect superoxide formed in mitochondria, showed that Pro accumulation during heat stress resulted in elevated levels of ROS in mitochondria. Interestingly, exogenous abscisic acid (ABA) and ethylene were found to partially rescue the heat-sensitive phenotype of Pro-overproducing seedlings. Measurement of ethylene and ABA levels further confirmed that these two hormones are negatively affected in Pro-overproducing seedlings during heat stress. Our results indicated that Pro accumulation under heat stress decreases the thermotolerance, probably by increased ROS production via the Pro/P5C cycle and inhibition of ABA and ethylene biosynthesis.     

Fermentative capacity of baker's yeast exposed to hyperbaric stress

Baker's yeast suspensions were incubated at different pressures (from 1 bar to 6 bar) and different gases [air, O(2) and a mixture of 8% (v/v) CO(2), 21% O(2) and N(2)]. Raising the air pressure from 1 bar to 6 bar stimulated cell growth but had no effect on leavening ability or viability of the cells. A 50% reduction of the CO(2) produced in dough occurred with 6 bar O(2) which also stopped growth. The fermentative capacity of the cells was stimulated by the cells exposure to increased CO(2) partial pressure up to 0.48 bar.     

互花米草幼苗对重金属镉胁迫的生理响应

以互花米草为实验材料,通过模拟海水河沙培养实验,研究不同浓度镉胁迫对互花米草生理生化的影响。结果表明:随着镉浓度的增大,互花米草叶、根生物量逐渐降低,膜透性、丙二醛、SOD、POD酶活性随着镉浓度的增加而增加,其酶抗性也发挥到最大的程度。随着镉浓度的增加互花米草的光合特性发生较大变化,净光合速率、胞间CO₂和气孔导度都下降和减少。     

The response of maize seedlings to cadmium stress under hydroponic conditions

The effects of cadmium (Cd) supply level in nutrient solution (0, 12.5, 25, 50, 100, 200, 400, and 800 μM) on growth, Cd accumulation ability, and the related physiological indices of maize (Zea mays L.) seedlings were studied under hydroponic conditions. The results showed that the increments in the shoot height and biomass were stimulated at relatively low external Cd supply levels (<100 μM), while they were inhibited at Cd supply levels over 200 μM. Cd accumulation ability of the maize seedlings also showed the similar stimulation/inhibition pattern as shoot growth, and the Cd contents in the shoots and roots reached the peaks (389.5 and 505.5 mg/kg dry wt, respectively) at 50 μM Cd. The contents of chlorophyll a, chlorophyll b, and carotenoids in the maize leaf blades decreased with increasing external Cd supply level. At the highest Cd supply level (800 μM), the contents of chlorophyll a, chlorophyll b, and carotenoids in the leaf blade were only 38.9, 46.0, and 29.7% of the control plants, respectively. Moreover, chlorophyll b was more sensitive to the Cd stress than chlorophyll a. The increased proline content in the leaf blade of maize seedlings resulted from external Cd stress indicates that maize can adapt to the adversity menace via changing the content of proline.