Arsenic stress triggers active exudation of arsenic-phytochelatin complexes from Lupinus albus roots.

Arsenic (As) contamination of soils threatens the health of millions globally through accumulation in crops. While plants detoxify As via phytochelatin (PC) complexation and efflux of arsenite from roots, arsenite efflux mechanisms are not fully understood. Here, white lupin (Lupinus albus) was grown in semi-hydroponics, and exudation of glutathione (GSH) derivatives and PCs in response to As was measured using LC-MS/MS. Inhibiting synthesis of the PC precursor GSH with l-buthionine sulfoximine (BSO) or ABC transporters with vanadate drastically reduced (>22%) GSH derivative and PC2 exudation, but not PC3 exudation. This was accompanied by As hypersensitivity in plants treated with BSO and moderate sensitivity with vanadate treatment. Investigating As-PC complexation revealed two distinct As-PC complexes, As bound to GSH and PC2 (GS-As-PC2) and As bound to PC3 (As-PC3), in exudates of As-treated lupin plants. Vanadate inhibited As-PC exudation, while BSO inhibited both the synthesis and exudation of As-PC complexes. These results demonstrate a role for GSH derivatives and PC exudation in lupin As tolerance and reveal As-PC exudation as a new potential mechanism contributing to active As efflux in plants. Overall, this study uncovers insights into rhizosphere As detoxification with potential to help mitigate pollution and reduce As accumulation in crops.

Genetic engineering low-arsenic and low-cadmium rice grain.

Rice is prone to take up the toxic elements arsenic (As) and cadmium (Cd) from paddy soil through the transporters for other essential elements. Disruption of these essential transporters usually adversely affects the normal growth of rice and the homeostasis of essential elements. Here we report on developing low-As and low-Cd rice grain through the co-overexpression of OsPCS1, OsABCC1, and OsHMA3 genes under the control of the rice OsActin1 promoter. Co-overexpression of OsPCS1 and OsABCC1 synergistically decreased As concentration in the grain. Overexpression of OsPCS1 also decreased Cd concentration in the grain by restricting the xylem-to-phloem Cd transport in node I, but paradoxically caused Cd hypersensitivity as the overproduced phytochelatins in OsPCS1-overexpressing plants suppressed OsHMA3-dependent Cd sequestration in vacuoles and promoted Cd transport from root to shoot. Co-overexpression of OsHAM3 and OsPCS1 overcame this suppression and complemented the Cd hypersensitivity. Compared with non-transgenic rice control, co-overexpression of OsABCC1, OsPCS1, and OsHMA3 in rice decreased As and Cd concentrations in grain by 92.1% and 98%, respectively, without causing any defect in plant growth and reproduction or of mineral nutrients in grain. Our research provides an effective approach and useful genetic materials for developing low-As and low-Cd rice grain.

The glutathione-dependent alarm triggers signalling responses involved in plant acclimation to cadmium.

Cadmium (Cd) uptake from polluted soils inhibits plant growth and disturbs physiological processes, at least partly due to disturbances in the cellular redox environment. Although the sulfur-containing antioxidant glutathione is important in maintaining redox homeostasis, its role as an antioxidant can be overruled by its involvement in Cd chelation as a phytochelatin precursor. Following Cd exposure, plants rapidly invest in phytochelatin production, thereby disturbing the redox environment by transiently depleting glutathione concentrations. Consequently, a network of signalling responses is initiated, in which the phytohormone ethylene is an important player involved in the recovery of glutathione levels. Furthermore, these responses are intricately connected to organellar stress signalling and autophagy, and contribute to cell fate determination. In general, this may pave the way for acclimation (e.g. restoration of glutathione levels and organellar homeostasis) and plant tolerance in the case of mild stress conditions. This review addresses connections between these players and discusses the possible involvement of the gasotransmitter hydrogen sulfide in plant acclimation to Cd exposure.

Degradation of glutathione and glutathione conjugates in plants.

Glutathione (GSH) is a ubiquitous, abundant, and indispensable thiol for plants that participates in various biological processes, such as scavenging reactive oxygen species, redox signaling, storage and transport of sulfur, detoxification of harmful substances, and metabolism of several compounds. Therefore knowledge of GSH metabolism is essential for plant science. Nevertheless, GSH degradation has been insufficiently elucidated, and this has hampered our understanding of plant life. Over the last five decades, the γ-glutamyl cycle has been dominant in GSH studies, and the exoenzyme γ-glutamyl transpeptidase has been regarded as the major GSH degradation enzyme. However, recent studies have shown that GSH is degraded in cells by cytosolic enzymes such as γ-glutamyl cyclotransferase or γ-glutamyl peptidase. Meanwhile, a portion of GSH is degraded after conjugation with other molecules, which has also been found to be carried out by vacuolar γ-glutamyl transpeptidase, γ-glutamyl peptidase, or phytochelatin synthase. These findings highlight the need to re-assess previous assumptions concerning the γ-glutamyl cycle, and a novel overview of the plant GSH degradation pathway is essential. This review aims to build a foundation for future studies by summarizing current understanding of GSH/glutathione conjugate degradation.

TGase-induced Cd tolerance by boosting polyamine, nitric oxide, cell wall composition and phytochelatin synthesis in tomato.

In highly intensive greenhouse vegetable production, soil acidification was caused by excessive fertilization, increasing cadmium (Cd) concentrations in the vegetables, which bears environmental hazards and is a negative influence on vegetables and humans. Transglutaminases (TGases), a central mediator for certain physiological effects of polyamines (PAs) in the plant kingdom, play important roles in plant development and stress response. Despite increased research on the crucial role of TGase in protecting against environmental stresses, relatively little is known about the mechanisms of Cd tolerance. In this study, we found, TGase activity and transcript level, which was upregulated by Cd, and TGase-induced Cd tolerance related to endogenous bound PAs increase and formation of nitric oxide (NO). Plant growth of tgase mutants was hypersensitive to Cd, chemical complementation by putrescine, sodium nitroprusside (SNP, nitric oxide donor) or gain of function TGase experiments restore Cd tolerance. α-diflouromethylornithine (DFMO, a selective ODC inhibitor) and 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, NO scavenger), were respectively found declined drastically endogenous bound PA and NO content in TGase overexpression plants. Likewise, we reported that TGase interacted with polyamine uptake protein 3 (Put3), and the silencing of Put3 largely reduced TGase-induced Cd tolerance and bound PAs formation. This salvage strategy depends on TGase-regulated synthesis of bound PAs and NO that is able to positively increase the concentration of thiol and phytochelatins, elevate Cd in the cell wall, as well as induce the levels of expression Cd uptake and transport genes. Collectively, these findings indicate that TGase-mediated enhanced levels of bound PA and NO acts as a vital mechanism to protect the plant from Cd-caused toxicity.

Hormones and the antioxidant transduction pathway and gene expression, mediated by Serratia marcescens DB1, lessen the lethality of heavy metals (As, Ni, and Cr) in Oryza sativa L.

Microorganisms have recently gained recognition as efficient biological tool for reducing heavy metal toxicity in crops. In this experiment, we isolated a potent heavy metal (As, Ni, and Cr) resistant rhizobacterium Serratia marcescens DB1 and detected its plant growth promoting traits such as phosphate solubilization, gibberellin synthesis, organic acid production and amino acid regulation. Based on these findings, DB1 was further investigated for application in a rice var. Hwayeongbyeo subjected to 1 mM As, 4 mM Ni, and 4 mM Cr stress. The rice plants treated with Cr and Ni appeared healthy but were lethal, indicating unfitness for consumption due to toxic metal deposition, whereas the plants treated with > 1 mM As instantaneously died. Our results showed that DB1 inoculation significantly decreased metal accumulation in the rice shoots. Particularly, Cr uptake dropped by 16.55% and 22.12% in (Cr + DB1) and (Cr + As + Ni + DB1), respectively, As dropped by 48.90% and 35.82% in (As + DB1) and (Cr + As + Ni + DB1), respectively, and Ni dropped by 7.95% and 19.56% in (Ni + DB1) and (Cr + As + Ni + DB1), respectively. These findings were further validated by gene expression analysis results, which showed that DB1 inoculation significantly decreased the expression of OsPCS1 (a phytochelatin synthase gene), OsMTP1 (a metal transporting gene), and OsMTP5 (a gene for the expulsion of excess metal). Moreover, DB1 inoculation considerably enhanced the morphological growth of rice through modulation of endogenous phytohormones (abscisic acid, salicylic acid, and jasmonic acid) and uptake of essential elements such as K and P. These findings indicate that DB1 is an effective biofertilizer that can mitigate heavy metal toxicity in rice crops.

Phytochelatins: Sulfur-Containing Metal(loid)-Chelating Ligands in Plants.

Phytochelatins (PCs) are small cysteine-rich peptides capable of binding metal(loid)s via SH-groups. Although the biosynthesis of PCs can be induced in vivo by various metal(loid)s, PCs are mainly involved in the detoxification of cadmium and arsenic (III), as well as mercury, zinc, lead, and copper ions, which have high affinities for S-containing ligands. The present review provides a comprehensive account of the recent data on PC biosynthesis, structure, and role in metal(loid) transport and sequestration in the vacuoles of plant cells. A comparative analysis of PC accumulation in hyperaccumulator plants, which accumulate metal(loid)s in their shoots, and in the excluders, which accumulate metal(loid)s in their roots, investigates the question of whether the endogenous PC concentration determines a plant's tolerance to metal(loid)s. Summarizing the available data, it can be concluded that PCs are not involved in metal(loid) hyperaccumulation machinery, though they play a key role in metal(loid) homeostasis. Unraveling the physiological role of metal(loid)-binding ligands is a fundamental problem of modern molecular biology, plant physiology, ionomics, and toxicology, and is important for the development of technologies used in phytoremediation, biofortification, and phytomining.

Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

Sec24C mediates a Golgi-independent trafficking pathway that is required for tonoplast localisation of ABCC1 and ABCC2.

Protein sorting is an essential biological process in all organisms. Trafficking membrane proteins generally relies on the sorting machinery of the Golgi apparatus. However, many proteins have been found to be delivered to target locations via Golgi-independent pathways, but the mechanisms underlying this delivery system remain unknown. Here, we report that Sec24C mediates the direct secretory trafficking of the phytochelatin transporters ABCC1 and ABCC2 from the endoplasmic reticulum (ER) to prevacuolar compartments (PVCs) in Arabidopsis thaliana. Genetic analysis showed that the sec24c mutants are hypersensitive to cadmium (Cd) and arsenic (As) treatments due to mislocalisation of ABCC1 and ABCC2, which results in defects in the vacuole compartmentalisation of the toxic metals. Furthermore, we found that Sec24C recognises ABCC1 and ABCC2 through direct interactions to mediate their exit from the ER to PVCs, which is independent of brefeldin A-sensitive post-Golgi trafficking pathway. These findings expand our understanding of Golgi-independent trafficking, which also provide key insights regarding the mechanism of tonoplast protein sorting and open a new perspective on the function of Sec24 proteins.

Phytochelatin and coumarin enrichment in root exudates of arsenic-treated white lupin.

Soil contamination with toxic metalloids, such as arsenic, can represent a substantial human health and environmental risk. Some plants are thought to tolerate soil toxicity using root exudation, however, the nature of this response to arsenic remains largely unknown. Here, white lupin plants were exposed to arsenic in a semi-hydroponic system and their exudates were profiled using untargeted liquid chromatography-tandem mass spectrometry. Arsenic concentrations up to 1 ppm were tolerated and led to the accumulation of 12.9 µg As g-1 dry weight (DW) and 411 µg As g-1 DW in above-ground and belowground tissues, respectively. From 193 exuded metabolites, 34 were significantly differentially abundant due to 1 ppm arsenic, including depletion of glutathione disulphide and enrichment of phytochelatins and coumarins. Significant enrichment of phytochelatins in exudates of arsenic-treated plants was further confirmed using exudate sampling with strict root exclusion. The chemical tolerance toolkit in white lupin included nutrient acquisition metabolites as well as phytochelatins, the major intracellular metal-binding detoxification oligopeptides which have not been previously reported as having an extracellular role. These findings highlight the value of untargeted metabolite profiling approaches to reveal the unexpected and inform strategies to mitigate anthropogenic pollution in soils around the world.

Influence of arsenate imposition on modulation of antioxidative defense network and its implication on thiol metabolism in some contrasting rice (Oryza sativa L.) cultivars.

Globally, many people have been suffering from arsenic poisoning. Arsenate (AsV) exposure to twelve rice cultivars caused growth retardation, triggered production of As-chelatin biopeptides and altered activities of antioxidants along with increase in ascorbate (AsA)-glutathione (GSH) contents as a protective measure. The effects were more conspicuous in cvs. Swarnadhan, Tulaipanji, Pusa basmati, Badshabhog, Tulsibhog and IR-20 to attenuate oxidative-overload mediated adversities. Contrastingly, in cvs. Bhutmuri, Kumargore, Binni, Vijaya, TN-1 and IR-64, effects were less conspicuous in terms of alterations in the said variables due to reduced generation of oxidative stress. Under As(V) imposition, the protective role of phytochelatins (PCs) were recorded where peaks height and levels of PCs (PC2, PC3 and PC4) were elevated significantly in the test seedlings with an endeavour to detoxify cells by sequestering arsenic-phytochelatin (As-PC) complex into vacuole that resulted in reprogramming of antioxidants network. Additionally, scatter plot correlation matrices, color-coded heat map analysis and regression slopes demonstrated varied adaptive responses of test cultivars, where cvs. Bhutmuri, Kumargore, Binni, Vijaya, TN-1 and IR-64 found tolerant against As(V) toxicity. Results were further justified by hierarchical clustering. These findings could help to grow identified tolerant rice cultivars in As-prone soil with sustainable growth and productivity after proper agricultural execution.

Gamma-Glutamylcysteine Production Using Phytochelatin Synthase-Like Enzyme Derived from Nostoc sp. Covalently Immobilized on a Cellulose Carrier.

Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer's disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications.

Biological responses to heavy metal stress in the moss Leptodictyum riparium (Hedw.) Warnst.

Leptodictyum riparium, a widely distributed aquatic moss, can both tolerate and accumulate very high concentrations of toxic heavy metals, with only slight apparent damage. Here we report the effects on photosynthetic yield, glutathione (GSH), phytochelatin (PCn) synthesis, nitrogen metabolism and cellular localization of molecules rich in SH groups in L. riparium exposed in vitro to heavy metals. We simulated the concentrations of Cu, Zn, Cd, Pb detected in Regi Lagni, Italy, one of the most contaminated freshwater sites in Southern Europe, in the laboratory to test how the moss responds to heavy metal contamination. There was a steady decrease of photosynthetic efficiency correlated with the heavy metal concentrations and ultrastructural organization. All PCn levels increased significantly as the concentration of heavy metals increased, while the GSH levels did not appear to be particularly affected. A significant increase of GDH and NADH-GOGAT activities increased with increasing heavy metal concentration. Immunoblotting analysis revealed an increase of the chl-GS2 while no significant increase was detected in the cyt-GS1. These results give insight into the molecular events underlying the metal-tolerance of the aquatic moss L. riparium exposed to environmental heavy metal concentrations.

Foliar spray with rutin improves cadmium remediation efficiency excellently by enhancing antioxidation and phytochelatin detoxification of Amaranthus hypochondriacus.

Rutin is a flavonoid with strong antioxidative effects on plant metabolism that facilitates resistance to environmental stress. The effect of foliar rutin on cadmium (Cd) uptake in Amaranthus hypochondriacus (K472) was studied. The results showed that a foliar spray of rutin alleviated Cd toxicity, promoted plant growth, improved Cd transfer to and storage in aerial plant parts and Cd accumulation with positive effects over time. A rutin concentration of 1.5 mg/mL showed the strongest promotion effect: the biomass and Cd content were increased at 13 days by 68.62% and 405.54% compared to 3 days, respectively, whereas a high concentration of rutin (5 mg/mL) inhibited plant growth and hindered Cd absorption. Two stages of Cd detoxification were identified in K472 after appropriate rutin application. First, an antioxidant system including an enzymatic antioxidant (superoxide dismutase [SOD]) and nonenzymatic antioxidants (glutathione [GSH] and flavonoids) was activated to enhance plant stress resistance. Quercetin and phytochelatin (PC) synthesis were then enhanced to perform detoxification synergistically with the antioxidant system to improve stress tolerance and achieve stable Cd detoxification. The results demonstrated that appropriately prolonging the application time of exogenous rutin to K472 is an effective way to improve the Cd remediation efficiency.

The application of exogenous rutin to regulate the growth and Cd absorption of grain amaranth is reported for the first time. A foliar spray of rutin enriches Cd by regulating the metabolism of flavonoids and enhancing antioxidation and phytochelatin detoxification under Cd stress. Properly prolonging the harvest time after rutin treatment can greatly improve the Cd remediation efficiency of soil. The findings of the present study would be helpful for the remediation of Cd-contaminated soils.

Effects of Phytochelatin-like Gene on the Resistance and Enrichment of Cd2+ in Tobacco.

Phytochelatins (PCs) are class III metallothioneins in plants. They are low molecular-weight polypeptides rich in cysteine residues which can bind to metal ions and affect the physiological metabolism in plants. Unlike other types of metallothioneins, PCs are not the product of gene coding but are synthesized by phytochelatin synthase (PCS) based on glutathione (GSH). The chemical formula of phytochelatin is a mixture of (γ-Glu-Cys)n-Gly (n = 2-11) and is influenced by many factors during synthesis. Phytochelatin-like (PCL) is a gene-encoded peptide (Met-(α-Glu-Cys)11-Gly) designed by our laboratory whose amino acid sequence mimics that of a natural phytochelatin. This study investigated how PCL expression in transgenic plants affects resistance to Cd and Cd accumulation. Under Cd2+ stress, transgenic plants were proven to perform significantly better than the wild-type (WT), regarding morphological traits and antioxidant abilities, but accumulated Cd to higher levels, notably in the roots. Fluorescence microscopy showed that PCL localized in the cytoplasm and nucleus.

Significance of vacuolar proton pumps and metal/H+ antiporters in plant heavy metal tolerance.

Soil and water are among the most valuable resources on earth. Unfortunately, their contamination with heavy metals has become a global problem. Heavy metals are not biodegradable and cannot be chemically degraded; therefore, they tend to accumulate in soils or to be transported by streaming water and contaminate both surface and groundwater. Cadmium (Cd) has no known biological function but is one of the most toxic metals. It represents a serious environmental concern since its accumulation in soils is associated with health risks to plants, animals and humans. On the other hand, copper (Cu) and zinc (Zn) are heavy metals that are indispensable to plants but become toxic when their concentration in soils exceeds a certain optimal level. Plants have evolved many mechanisms to cope with heavy metal toxicity; vacuolar sequestration is one of them. Vacuolar sequestration can be achieved through either phytochelatin-dependent or phytochelatin-independent pathways. Most of the transgenic plants meant for phytoremediation described in the literature result from the manipulation of genes involved in the phytochelatin-dependent pathway. However, recent evidence has emerged to support the importance of the phytochelatin-independent pathway in heavy metal sequestration into the vacuole, with metal/H+ antiporters and proton pumps playing an important role. In this review, the importance of vacuolar proton pumps and metal/H+ antiporters transporting Cd, Cu, and Zn is discussed. In addition, the recent advances in the production of transgenic plants with potential application in phytoremediation and food safety through the manipulation of genes encoding V-PPase proton pumps is described.

Optimization of Reaction Conditions for γ-Glutamylcysteine Production from Glutathione Using a Phytochelatin Synthase-Like Enzyme from Nostoc sp. Pasteur Culture Collection 7120.

γ-Glutamylcysteine (γ-EC) has antioxidant properties similar to those of glutathione (GSH) and acts as its precursor in mammals. There are a few procedures for the production of γ-EC, such as chemical synthesis or enzymatic synthesis from glutamate and cysteine; however, they are very costly and not suitable for industrial production. A phytochelatin synthase-like enzyme derived from Nostoc sp. Pasteur Culture Collection 7120 (NsPCS) catalyzes the hydrolysis of GSH to γ-EC and glycine in the absence of ATP or other additives. Our research aims to establish an alternative γ-EC production procedure with low cost and high productivity. To this end, we optimized the reaction conditions of NsPCS and characterized its properties in this study. We found that 200 mM potassium phosphate buffer, pH 8.0, at 37 °C, had the highest NsPCS activity among the conditions we tested. Under these conditions, NsPCS had a Km of 385 µM and a Vmax of 26 mol/min/mg-protein. In addition, NsPCS converted 100 mM GSH into γ-EC with high yields. These results suggest that the NsPCS reaction has great potential for the low-cost, industrial-scale production of γ-EC.

Exogenous melatonin regulates endogenous phytohormone homeostasis and thiol-mediated detoxification in two indica rice cultivars under arsenic stress.

KEY MESSAGE: Melatonin enhanced arsenic (As) tolerance by inhibiting As bioaccumulation, modulating the expression of As transporters and phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid. The present study was aimed at investigating the influence of exogenous melatonin on the regulation of endogenous plant growth regulators and their cumulative effects on metal(loid)-binding ligands in two contrasting indica rice cultivars, viz., Khitish (arsenic sensitive) and Muktashri (arsenic tolerant) under arsenic stress. Melatonin supplementation ameliorated arsenic-induced perturbations by triggering endogenous levels of gibberellic acid and melatonin, via up-regulating the expression of key biosynthetic genes like GA3ox, TDC, SNAT and ASMT. The endogenous abscisic acid content was also enhanced upon melatonin treatment by induced expression of the key anabolic gene, NCED3 and concomitant suppression of ABA8ox1. Enhanced melatonin content induced accumulation of higher polyamines (spermidine and spermine), together with up-regulation of SPDS and SPMS in Khitish, thereby modulating stress condition. On the contrary, melatonin escalated putrescine and spermidine levels in Muktashri, via enhanced expression of ADC and SAMDC. The role of melatonin appeared to be more prominent in Khitish, as evident from better utilization of thiol components like cysteine, GSH, non-protein thiols and phytochelatins, with higher GSH/GSSG ratio, despite down-regulated expression of corresponding thiol-metabolic genes (OsMT2 and OsPCS1) to deal with arsenic toxicity. The extent of arsenic bioaccumulation, which was magnified several folds, particularly in Khitish, was decreased upon melatonin application. Overall, our observation highlighted the fact that melatonin enhanced arsenic tolerance by inhibiting arsenic bioaccumulation, via modulating the expression levels of selected arsenic transporters (OsNramp1, OsPT2, OsPT8, OsLsi1) and controlling endogenous phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid.

Analyzing cadmium-phytochelatin2 complexes in plant using terahertz and circular dichroism information.

Phytochelatins are plants' small metal-binding peptides which chelate internal heavy metals to form nontoxic complexes. Detecting the complexes in plants would simplify identification of cultivars with both high tolerance and enrichment capabilities for heavy metals which represent phytoextraction performance. Thus, a terahertz spectroscopy combined with density functional theory, chemometrics and circular dichroism was used for characterization of phytochelatin2 (PC2), Cd-PC2 mixture standards, and pak choi (Brassica chinensis) leaves as a plant model. Results showed PC2 chelates Cd2+ in a 2:1 ratio to form Cd(PC2)2 complex; Cd connected to thoils of PC2 and changed ß-turn and random coil of PC2 peptide chain to ß-Sheet which presented as terahertz vibrations of PC2 around 1.03 and 1.71 THz being suppressed; the best models for detecting the complex in pak choi were obtained by partial least squares regression modeling combined with successive projections algorithm selection; the models used PC2 as a natural probe for visualizing and quantifying chelated Cd in pak choi leaf and achieved a limit of detection up to 1.151 ppm. This study suggested that terahertz information of the heavy metal-PCs complexes is qualified for representing a simpler alternative to classical index for evaluating phytoextraction performance of plant; it provided a general protocol for structure analysis and detection of heavy metal-PCs complexes in plant by terahertz absorbance.

Arsenic accumulation and speciation in two cultivars of Pteris cretica L. and characterization of arsenate reductase PcACR2 and arsenite transporter PcACR3 genes in the hyperaccumulating cv. Albo-lineata.

Pollution and poisoning with carcinogenic arsenic (As) is of major concern globally. Interestingly, there are ferns that can naturally tolerate remarkably high As concentrations in soils while hyperaccumulating this metalloid in their fronds. Besides Pteris vittata in which As-related traits and molecular determinants have been studied in detail, the As hyperaccumulation status has been attributed also to Pteris cretica. We thus inspected two P. cretica cultivars, Parkerii and Albo-lineata, for As hyperaccumulation traits. The cultivars were grown in soils supplemented with 20, 100, and 250 mg kg-1 of inorganic arsenate (iAsV). Unlike Parkerii, Albo-lineata was confirmed to be As tolerant and hyperaccumulating, with up to 1.3 and 6.4 g As kg-1 dry weight in roots and fronds, respectively, from soils amended with 250 mg iAsV kg-1. As speciation analyses rejected that organoarsenical species and binding with phytochelatins and other proteinaceous ligands would play any significant role in the biology of As in either cultivar. While in Parkerii, the dominating As species, particularly in roots, occurred as iAsV, in Albo-lineata the majority of the root and frond As was apparently converted to iAsIII. Parkerii markedly accumulated iAsIII in its fronds when grown on As spiked soils. Considering the roles iAsV reductase ACR2 and iAsIII transporter ACR3 may have in the handling of iAs, we isolated Albo-lineata PcACR2 and PcACR3 genes closely related to P. vittata PvACR2 and PvACR3. The gene expression analysis in Albo-lineata fronds revealed that the transcription of PcACR2 and PcACR3 was clearly As responsive (up to 6.5- and 45-times increase in transcript levels compared to control soil conditions, respectively). The tolerance and uptake assays in yeasts showed that PcACRs can complement corresponding As-sensitive mutations, indicating that PcACR2 and PcACR3 encode functional proteins that can perform, respectively, iAsV reduction and membrane iAsIII transport tasks in As-hyperaccumulating Albo-lineata.