Phytochelatin Synthase Research Articles

Auxins and Cytokinins Regulate Phytohormone Homeostasis and Thiol-Mediated Detoxification in the Green Alga Acutodesmus obliquus Exposed to Lead Stress

Phytohormones, such as auxins and cytokinins, take part in the integration of growth control and stress response, but their role in algal adaptation to heavy metal remains to be elucidated. The current research indicated that lead (Pb), one of the most toxic metals in nature, causes severe depletion of endogenous cytokinins, auxins, and gibberellin and an increase in abscisic acid content in the green alga Acutodesmus obliquus. Exogenous auxins and cytokinins alleviate Pb toxicity through the regulation of the endogenous phytohormones’ levels. Exogenously applied auxins provoked the coordinated activation metal tolerance mechanisms leading to the increase in phytochelatin synthase activity and accumulation of phytochelatins and their precursors, which are essential for Pb sequestration. On the other hand, phytochelatin synthesis decreased in algal cells treated with cytokinins. Significant changes in the levels of low molecular weight metabolites, mainly involved in metal chelation and glutathione synthesis pathway under the influence of phytohormones in algal cells growing in the presence of Pb stress, were observed. This is the first report showing that auxins and cytokinins are important regulatory factors in algal adaptation strategies to heavy metal stress based on thiol-mediated compounds and the maintenance of phytohormone homeostasis.

MYB4 transcription factor, a member of R2R3-subfamily of MYB domain protein, regulates cadmium tolerance via enhanced protection against oxidative damage and increases expression of PCS1 and MT1C in Arabidopsis

Here, we describe functional characterization of Arabidopsis thaliana MYB4 transcription factor, a member of R2R3-subfamily of MYB domain protein, in the regulation of Cd-stress tolerance in Arabidopsis. Transgenic Arabidopsis plants overexpressing MYB4 showed appreciable Cd tolerance than wild-type plants, while MYB4 loss of function mutant lines (atmyb4) showed increased sensitivity to Cd-stress. MYB4 overexpression lines showed strong activation of anti-oxidant defense components and increased Cd accumulation than wild-type and atmyb4 mutant lines under Cd-stress. MYB4 overexpression resulted in the coordinated activation of the expression of phytochelatin (PC) synthesis related genes and specifically enhanced the transcript abundance of phytochelatin synthase 1 (PCS1) and metallothionein 1C (MT1C) genes under Cd-stress. In contrast, atmyb4 mutant lines showed reduced Cd accumulation and compromised expression of PC-synthesis related genes. Electrophoretic gel mobility shift assays have demonstrated specific binding activity of recombinant AtMYB4 to the putative MYB4-binding motifs ACCAACCAA and GGTAGGT identified in the promoters of PCS1 and MT1C genes, respectively. Further analyses have revealed that MYB4 binds directly to PCS1 and MT1C promoters in vivo and positively regulates their transcriptional expression, suggesting that PCS1 and MT1C are the key targets of MYB4. Overall, our results have provided evidence that MYB4 regulates Cd-tolerance via the coordinated activity of improved anti-oxidant defense system and through the enhanced expression of PCS1 and MT1C under Cd-stress in Arabidopsis.

Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase

Phytochelatins are small enzyme-synthesized peptides that mediate tolerance to several heavy metals. To gain insight into the unusual metal catalytic site of the Euglena gracilis phytochelatin synthase (EgPCS), site-directed mutagenesis was performed on the C-terminal Cys/His (C/H) rich region 421-C-C-X-H-X-H-X-H-H-H-430. EgPCS mutants were analyzed at the cellular, kinetic and structural levels. Cd2+-resistance was conferred to Cd2+-sensitive yeast cells by EgPCS_wt (476aa) and truncated EgPCS_435 (435aa), but not by EgPCS_415 (415aa), suggesting that the resistance phenotype was linked to the enzyme C/H rich region. Moreover, full-length mutants (in which C and H residues in the C/H rich region were replaced by Ala) EgPCS_C2 (C421A/C422A) and EgPCS_C2H5 (C421A/C422A/H424A/H426A/H428A/H429A/H430A) did not provide Cd2+-resistance; in contrast, EgPCS_H2 (H424A/H426A), EgPCS_H3 (H428A/H429A/H430A), and EgPCS_H5 (H424A/H426A/H428A/H429A/H430A) mutants conferred similar Cd2+-tolerance like EgPCS_wt. Kinetic analysis showed that maximal rate (Vmax) for PC2 synthesis, affinity constants (Km Zn-GS2 or Km Cd-GS2) and catalytic efficiencies (Vmax/Km) were differentially impaired in the mutants, as compared to EgPCS_wt, with EgPCS_C2 being the most perturbed enzyme; however, the K0.5 values for GSH were not affected. All EgPCS mutants were predominantly monomeric. Far UV circular dichroism spectra and differential scanning calorimetry endotherms, indicated that alterations of the catalytic properties of EgPCS_C2 were not due to partial unfolding or destabilization of the native state of this mutant. The results indicated that C421/C422 and H424A/H426A/H428/H429/H430 are essential components of EgPCS for catalysis and activation by metal-substrate complexes.

Morphological Responses and Gene Expression of Grain Amaranth (Amaranthus spp.) Growing under Cd.

Phytoremediation efficiency depends on the ability of plants to accumulate, translocate and resist high levels of metals without symptoms of toxicity. This study was conducted to evaluate the potential of grain amaranth for remediation of soils contaminated with Cd. Three grain amaranth varieties, “Pribina” (A. cruentus), “Zobor” (A. hypochondriacus x A. hybridus) and Plainsman (A. hypochondriacus x A. hybridus) were tested under different level of Cd (0, 5, 10 and 15 mg/L) in a hydroponic experimental treatment. All could be classified as Cd excluders or Cd-hypertolerant varieties able to grow and accumulate significant amounts of Cd from the hydroponic solution, preferentially in the roots. Under the highest level of Cd exposure, qRT-PCR expression analysis of five stress-related genes was examined in above- and below-ground biomass. The results show that the Cd concentration significantly increased the mRNA level of chitinase 5 (Chit 5) in amaranth roots as the primary site of metal stress. The involvement of phytochelatin synthase (PCS1) in Cd detoxification is suggested. Based on our findings, we can conclude that variety “Pribina” is the most Cd-tolerant among three tested and can be expected to be used in the phytomanagement of Cd loaded soils as an effective phytostabiliser.

Expression of Genes Involved in Heavy Metal Trafficking in Plants Exposed to Salinity Stress and Elevated Cd Concentrations.

Many areas intended for crop production suffer from the concomitant occurrence of heavy metal pollution and elevated salinity; therefore, halophytes seem to represent a promising perspective for the bioremediation of contaminated soils. In this study, the influence of Cd treatment (0.01–10.0 mM) and salinity stress (0.4 M NaCl) on the expression of genes involved in heavy metal uptake (irt2–iron-regulated protein 2, zip4–zinc-induced protein 4), vacuolar sequestration (abcc2–ATP-binding cassette 2, cax4–cation exchanger 2 pcs1–phytochelatin synthase 1) and translocation into aerial organs (hma4–heavy metal ATPase 4) were analyzed in a soil-grown semi-halophyte Mesembryanthemum crystallinum. The upregulation of irt2 expression induced by salinity was additionally enhanced by Cd treatment. Such changes were not observed for zip4. Stressor-induced alterations in abcc2, cax4, hma4 and pcs1 expression were most pronounced in the root tissue, and the expression of cax4, hma4 and pcs1 was upregulated in response to salinity and Cd. However, the cumulative effect of both stressors, similar to the one described for irt2, was observed only in the case of pcs1. The importance of salt stress in the irt2 expression regulation mechanism is proposed. To the best of our knowledge, this study is the first to report the combined effect of salinity and heavy metal stress on genes involved in heavy metal trafficking.

Sulfur application reduces cadmium uptake in edible parts of pakchoi (Brassica chinensis L.) by cadmium chelation and vacuolar sequestration.

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50μM, in comparison to the control condition, 0μM) and three S levels (0, 2, 4mM). The results showed that excessive S application (4mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Silicon induces phytochelatin and ROS scavengers facilitating cadmium detoxification in rice.

Cadmium (Cd) is detrimental to crops and the environment. This work examines the natural mechanisms underlying silicon- (Si-)directed Cd detoxification in rice plants. The addition of Si to plants under Cd stress caused significant improvements in morphological parameters, chlorophyll score, Fv /Fm and total soluble protein concentration compared to controls, confirming that Si is able to ameliorate Cd-induced damage in rice plants. This morpho-physiological evidence was correlated with decreased cell death and electrolyte leakage after Si application. The results showed no critical changes in root Cd concentration, while shoot Cd decreased significantly after Si supplementation in comparison with Cd-stressed rice. Additionally, expression of Cd transporters (OsNRAMP5 and OsHMA2) was significantly down-regulated while the concentration of phytochelatin, cysteine and glutathione, together with expression of OsPCS1 (phytochelatin synthase) in roots of Cd-stressed rice was significantly induced when subjected to Si treatment. This confirms that the alleviation of Cd stress is not only limited to the down-regulation of Cd transporters but also closely related to the phytochelatin-driven vacuolar storage of Cd in rice roots. The enzymatic analysis further revealed the role of SOD and GR enzymes in protecting rice plants from Cd-induced oxidative harm. These findings suggest a mechanistic basis in rice plants for Si-mediated mitigation of Cd stress.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

Protein phosphatase 2A alleviates cadmium toxicity by modulating ethylene production in Arabidopsis thaliana.

Cadmium (Cd) is phytotoxic and detoxified primarily via phytochelatin (PC) complexation in Arabidopsis. Here, we explore Cd toxicity responses and defence mechanisms beyond the PC pathway using forward genetics approach. We isolated an Arabidopsis thaliana Cd-hypersensitive mutant, Cd-induced short root 1 (cdsr1) in the PC synthase mutant (cad1-3) background. Using genomic resequencing and complementation, we identified PP2A-4C as the causal gene for the mutant phenotype, which encodes a catalytic subunit of protein phosphatase 2A (PP2A). Root and shoot growth of cdsr1 cad1-3 and cdsr1 were more sensitive to Cd than their respective wild-type cad1-3 and Col-0. A mutant of the PP2A scaffolding subunit 1A was also more sensitive to Cd. PP2A-4C was localized in the cytoplasm and nucleus and PP2A-4C expression was downregulated by Cd in cad1-3. PP2A enzyme activity was decreased in cdsr1 and cdsr1 cad1-3 under Cd stress. The expression of 1-aminocyclopropane-1-carboxylic acid synthase genes ACS2 and ACS6 was upregulated by Cd more in cad1-3 and cdsr1 cad1-3 than in Col-0 and the double mutant had a higher ACS activity. cdsr1 cad1-3 and cdsr1 overproduced ethylene under Cd stress. The results suggest that PP2A containing 1A and 4C subunits alleviates Cd-induced growth inhibition by modulating ethylene production.

Glutathione and Its Biosynthetic Intermediates Alleviate Cesium Stress in Arabidopsis.

Phytoremediation is optimized when plants grow vigorously while accumulating the contaminant of interest. Here we show that sulphur supply alleviates aerial chlorosis and growth retardation caused by cesium stress without reducing cesium accumulation in Arabidopsis thaliana. This alleviation was not due to recovery of cesium-induced potassium decrease in plant tissues. Sulphur supply also alleviated sodium stress but not potassium deficiency stress. Cesium-induced root growth inhibition has previously been demonstrated as being mediated through jasmonate biosynthesis and signalling but it was found that sulphur supply did not decrease the levels of jasmonate accumulation or jasmonate-responsive transcripts. Instead, induction of a glutathione synthetase gene GSH2 and reduction of a phytochelatin synthase gene PCS1 as well as increased accumulation of glutathione and cysteine were observed in response to cesium. Exogenous application of glutathione or concomitant treatments of its biosynthetic intermediates indeed alleviated cesium stress. Interestingly, concomitant treatments of glutathione biosynthetic intermediates together with a glutathione biosynthesis inhibitor did not cancel the alleviatory effects against cesium suggesting the existence of a glutathione-independent pathway. Taken together, our findings demonstrate that plants exposed to cesium increase glutathione accumulation to alleviate the deleterious effects of cesium and that exogenous application of sulphur-containing compounds promotes this innate process.

Domain exchange between Oryza sativa phytochelatin synthases reveals a region that determines responsiveness to arsenic and heavy metals

Phytochelatin synthases (PCSs) are activated by toxic metals/metalloids such as cadmium and arsenic and synthesize phytochelatins for detoxification of toxic elements. Rice (Oryza sativa L.) has two PCSs (OsPCS1 and OsPCS2), and we previously revealed that OsPCS1 has a higher responsiveness to arsenic than to cadmium, while OsPCS2 has a higher responsiveness to cadmium than to arsenic. Moreover, we found that the specific responsiveness of OsPCS1 to arsenic at rice nodes is a key factor in reducing arsenic in rice grains. However, the molecular characteristics of two PCSs in rice that contribute to the responsiveness to arsenic or heavy metals, including Cd, remain unclear. Here, we experimentally demonstrate that the C-terminal region in PCSs determines the responsiveness to arsenic or cadmium. We constructed chimeric proteins between OsPCS1 and OsPCS2 and performed an in vitro phytochelatin synthesis assay. A chimeric protein in which the 183 C-terminal amino acids of OsPCS2 were replaced with the 185 C-terminal amino acids of OsPCS1 showed higher responsiveness to arsenite than to cadmium, similar to OsPCS1. Contrary to expectations, mutations of cysteine residues that are unique to OsPCS1 or OsPCS2 had little influence on the responsiveness, although cysteine residues are reported to be representative of sites that interact with metals/metalloids. These results would enable the development of a breeding technology for reducing arsenic in rice grains by improving the arsenic-dependent activation of PCSs.

Deciphering the molecular mechanism behind stimulated co-uptake of arsenic and fluoride from soil, associated toxicity, defence and glyoxalase machineries in arsenic-tolerant rice

The current study elucidates the uncharacterized biohazard associated with rice growth in arsenic and fluoride co-contaminated sites. Analysis of the arsenic-tolerant rice cultivar, Muktashri (known to restrict arsenic uptake) revealed that fluoride largely stimulated arsenic bioaccumulation in the stressed tissues and vice versa. Gene expression studies revealed that high arsenic uptake was facilitated by the fluoride-dependent up regulation of phosphate transporter2 (PT2), PT8 and low silicon rice1 (Lsi1), and elevated fluoride accumulation was stimulated by the arsenic-mediated induction of chloride channels (CLCs). The endogenous accumulation of fluoride and arsenic increased reactive oxygen species (ROS), O2−, membrane peroxidation and arsenic localization within tissues. This inhibited plant growth by triggering chlorosis, electrolyte leakage, malondialdehyde production (due to high lipoxygenase activity), protein carbonylation, protease activity and methylglyoxal accumulation due to inhibited glyoxylase activity. Metabolic analysis showed inhibited proline biosynthesis along with increased channelization of glutathione towards phytochelatin synthase and glutathione-S-tranferase-dependent pathways. Inhibition of the antioxidant enzymes like catalase, ascorbate peroxidase and guaiacol peroxidase validated the inefficient scavenging of H2O2 during combined stress. In silico analyses predicted the ecotoxicological risks of arsenic-fluoride complex formed during joint stress. Overall, our investigation illustrated the underlying mechanism of arsenic-fluoride co-uptake which resulted in complete suppression of the ‘tolerant’-phenotype in Muktashri seedlings.

24-Epibrassinolide modulates primary metabolites, antioxidants, and phytochelatins in Acutodesmus obliquus exposed to lead stress

Aquatic organisms are exposed to many stressors, e.g., heavy metals. Brassinosteroids, a plant hormone group, can effectively stimulate plants to defend against the negative impact of a heavy metal. The present study was conducted with an aim to find out the influence of 24-epibrassinolide (EBL) on Acutodesmus obliquus treated with 0.01 and 500 μM of lead (Pb) ions during 7 days of cultivation. Pb has a toxic effect on algal cultures because it limits both the growth and development and induces oxidative stress. Simultaneously, 1 μM EBL was involved in protecting algal cells against the toxic effect of Pb. Despite the presence of Pb, EBL significantly increased the number of algal cells and their metabolite content (e.g., proteins, monosaccharides, chlorophylls, carotenes, and xanthophylls). Cultures treated concurrently with EBL and Pb were characterized by a reduction in their content of endogenous Pb, H2O2, and malondialdehyde. Also, EBL increased the activity of catalase, ascorbate peroxidase, superoxide dismutase, glutathione reductase, and the content of ascorbate and glutathione. EBL increased the phytochelatin synthase activity, thereby enhancing the production of phytochelatins accountable for both binding and detoxification of Pb. These results indicate the influence of EBL on the inhibitory effect of Pb in A. obliquus. These findings help to clarify the role of BRs in the algal adaptation to the prevailing stressful conditions.

Cultivar-specific cadmium uptake strategies in flax – a hyperaccumulating plant

‘Lvbao-701’ is a cultivar of Chinese flowering cabbage (Brassica parachinensis) that exhibits low cadmium (Cd) accumulation and high Cd tolerance. In this study, this cultivar was compared with a high-Cd accumulating cultivar, ‘Chixin-4’, to characterize the mechanisms influencing Cd accumulation in B. parachinensis. Root cell walls were isolated by dissolving the cytoplasm with an organic solvent, and root Cd and phytochelatin (PC) contents were analyzed. In addition, a PC synthase gene fragment was cloned and expressed under Cd stress conditions. The proportions of Cd bound to root cell walls were higher in the ‘Lvbao-701’ plants (68.32%–76.80%) than in the ‘Chixin-4’ plants (35.36%–54.18%) after exposure to Cd stress. The proportions of Cd bound to root cell walls measured using cell walls isolated with a non-destructive method were higher than those obtained using a conventional method that required grinding and centrifugation. Exposure to Cd stress induced the PC production and resulted in higher PC contents in the ‘Lvbao-701’ plants than in the ‘Chixin-4’ plants. Cloning and expression analysis of a B. parachinensis PC synthase cDNA fragment indicated that PC synthase gene expression was induced by Cd and occurred mainly in the roots of both ‘Lvbao-701’ and ‘Chixin-4’ plants. However, the PC synthase gene expression level was higher in the ‘Lvbao-701’ roots than in the ‘Chixin-4’ roots. Therefore, a higher abundance of Cd in the root cell walls of ‘Lvbao-701’ and up-regulated PC production in the roots are probably the main reasons why ‘Lvbao-701’ exhibits lower Cd translocation to the shoots and higher tolerance to Cd than ‘Chixin-4’.

Transcriptome analysis of genes expressed in the earthworm Eisenia fetida in response to cadmium exposure

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.

Enhanced Arsenic Tolerance in Triticum aestivum Inoculated with Arsenic-Resistant and Plant Growth Promoter Microorganisms from a Heavy Metal-Polluted Soil.

In soils multi-contaminated with heavy metal and metalloids, the establishment of plant species is often hampered due to toxicity. This may be overcome through the inoculation of beneficial soil microorganisms. In this study, two arsenic-resistant bacterial isolates, classified as Pseudomonas gessardii and Brevundimonas intermedia, and two arsenic-resistant fungi, classified as Fimetariella rabenhortii and Hormonema viticola, were isolated from contaminated soil from the Puchuncaví valley (Chile). Their ability to produce indoleacetic acid and siderophores and mediate phosphate solubilization as plant growth-promoting properties were evaluated, as well as levels of arsenic resistance. A real time PCR applied to Triticum aestivum that grew in soil inoculated with the bacterial and fungal isolates was performed to observe differences in the relative expression of heavy metal stress defense genes. The minimum inhibitory concentration of the bacterial strains to arsenate was up to 7000 mg·L−1 and that of the fungal strains was up to 2500 mg·L−1. P. gessardi was able to produce siderophores and solubilize phosphate; meanwhile, B. intermedia and both fungi produced indoleacetic acid. Plant dry biomass was increased and the relative expression of plant metallothionein, superoxide dismutase, ascorbate peroxidase and phytochelatin synthase genes were overexpressed when P. gessardii plus B. intermedia were inoculated.

Improving biosynthesis of Au[sbnd]Pd core-shell nanoparticles through Escherichia coli with the assistance of phytochelatin for catalytic enhanced chemiluminescence and benzyl alcohol oxidation

In this work, AuPd core-shell nanoparticles (NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (Au-Pd/AtPCS1-E. coli) with catalytic enhanced chemiluminescence (CL) and benzyl alcohol oxidation (BAO) was investigated. Such biosynthesis of AuPd core-shell NPs was obviously enhanced due to insertion of the gene sequence of Arabidopsis thaliana phytochelatin synthase (AtPCS1) to a plasmid vector (pET-28b) of Escherichia coli (E. coli). The obtained Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (AtPCS1-E. coli) could generate phytochelatins (PCs, (γ-Glu-Cys)n-Gly, n > 1) for efficient capture and enrichment of Au3+. The component and morphology of AuPd core-shell NPs were checked through X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). Catalytic CL (in H2O2-luminol system) and BAO (in H2O2-benzyl alcohol system) effect with different experimental conditions were examined, respectively. These results revealed that multifunctional PCs could effectively facilitate biosynthetic process of AuPd core-shell NPs with better distribution, higher yield and lower cost while stronger CL intensity and higher conversion could be obtained for further quantitative analysis and application.

Overexpression of phytochelatin synthase AtPCS2 enhances salt tolerance in Arabidopsis thaliana

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.

Safe Cultivation of Medicago sativa in Metal-Polluted Soils from Semi-Arid Regions Assisted by Heat- and Metallo-Resistant PGPR.

Soil contamination with heavy metals is a constraint for plant establishment and development for which phytoremediation may be a solution, since rhizobacteria may alleviate plant stress under these conditions. A greenhouse experiment was conducted to elucidate the effect of toxic metals on growth, the activities of ROS (reactive oxygen species)-scavenging enzymes, and gene expression of Medicago sativa grown under different metal and/or inoculation treatments. The results showed that, besides reducing biomass, heavy metals negatively affected physiological parameters such as chlorophyll fluorescence and gas exchange, while increasing ROS-scavenging enzyme activities. Inoculation of M. sativa with a bacterial consortium of heat- and metallo-resistant bacteria alleviated metal stress, as deduced from the improvement of growth, lower levels of antioxidant enzymes, and increased physiological parameters. The bacteria were able to effectively colonize and form biofilms onto the roots of plants cultivated in the presence of metals, as observed by scanning electron microscopy. Results also evidenced the important role of glutathione reductase (GR), phytochelatin synthase (PCS), and metal transporter NRAMP1 genes as pathways for metal stress management, whereas the gene coding for cytochrome P450 (CP450) seemed to be regulated by the presence of the bacteria. These outcomes showed that the interaction of metal-resistant rhizobacteria/legumes can be used as an instrument to remediate metal-contaminated soils, while cultivation of inoculated legumes on these soils is still safe for animal grazing, since inoculation with bacteria diminished the concentrations of heavy metals accumulated in the aboveground parts of the plants to below toxic levels.