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Background: Arsenic (As) metabolism by soil microorganisms has an impact on As geochemical cycling in paddy soils, which in turn affects As uptake in rice. However, little is known about the key microorganisms involved in this process in Japanese paddy soil., Results: Total RNA was extracted from Japanese paddy soils with different levels of dissolved As under flooded conditions, and the transcription of As metabolic genes (arrA, ttrA and arsM) was analyzed via a metatranscriptomic approach. The results showed that ttrA was the predominant respiratory arsenate reductase gene transcribed in these soils rather than arrA, suggesting that ttrA contributes to the reductive dissolution of As. The predominant taxa expressing ttrA differed among soils but were mostly associated with genera known for their iron- and/or sulfate-reduction activity. In addition, a wide variety of microorganisms expressed and upregulated arsM approximately 5.0- to 13.2-fold at 9 d compared with 3 d of incubation under flooded conditions in flasks., Conclusions: Our results support the involvement of microbial activity in the geochemical cycling of As in Japanese paddy soils and suggest that ttrA may be one of the key genes involved in the formation of arsenite, an inorganic species taken up by rice., (© 2024. The Author(s).)

Citrobacter sp. XT1-2-2, a functional microorganism with potential utilization, has the ability to immobilize soil cadmium. In this study, the regulatory gene cysH, as a rate-limiting enzyme in the sulfur metabolic pathway, was selected for functional analysis affecting cadmium immobilization in soil. To verify the effect of APS reductase on CdS formation, the ΔAPS and ΔAPS-com strains were constructed by conjugation transfer. Through TEM analysis, it was found that the adsorption of Cd 2+ was affected by the absence of APS reductase in XT1-2-2 strain. The difference analysis of biofilm formation indicated that APS reductase was necessary for cell aggregation and biofilm formation. The p-XRD, XPS and FT-IR analysis revealed that APS reductase played an important role in the cadmium immobilization process of XT1-2-2 strain and promoting the formation of CdS. According to the pot experiments, the cadmium concentration of roots, culms, leaves and grains inoculated with ΔAPS strain was significantly higher than that of wild-type and ΔAPS-com strains, and the cadmium removal ability of ΔAPS strain was significantly lower than that of wild-type strain. The study provided insights into the exploration of new bacterial assisted technique for the remediation and safe production of rice in cadmium-contaminated paddy soils., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351 T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni 2+ . As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin., (© 2024 John Wiley & Sons Ltd.)

As sessile organisms, plants encounter dynamic and challenging environments daily, including abiotic/biotic stresses. The regulation of carbon and nitrogen allocations for the synthesis of plant proteins, carbohydrates, and lipids is fundamental for plant growth and adaption to its surroundings. Light, one of the essential environmental signals, exerts a substantial impact on plant metabolism and resource partitioning (i.e., starch). However, it is not fully understood how light signaling affects carbohydrate production and allocation in plant growth and development. An orphan gene unique to Arabidopsis thaliana, named QUA-QUINE STARCH (QQS) is involved in the metabolic processes for partitioning of carbon and nitrogen among proteins and carbohydrates, thus influencing leaf, seed composition, and plant defense in Arabidopsis. In this study, we show that PHYTOCHROME-INTERACTING bHLH TRANSCRIPTION FACTORS (PIFs), including PIF4, are required to suppress QQS during the period at dawn, thus preventing overconsumption of starch reserves. QQS expression is significantly de-repressed in pif4 and pifQ, while repressed by overexpression of PIF4, suggesting that PIF4 and its close homologs (PIF1, PIF3, and PIF5) act as negative regulators of QQS expression. In addition, we show that the evening complex, including ELF3 is required for active expression of QQS, thus playing a positive role in starch catabolism during night-time. Furthermore, QQS is epigenetically suppressed by DNA methylation machinery, whereas histone H3 K4 methyltransferases (e.g., ATX1, ATX2, and ATXR7) and H3 acetyltransferases (e.g., HAC1 and HAC5) are involved in the expression of QQS. This study demonstrates that PIF light signaling factors help plants utilize optimal amounts of starch during the night and prevent overconsumption of starch before its biosynthesis during the upcoming day., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Arsenic (As) has become natural health hazard for millions of people across the world due to its distribution in the food chain. Naturally, it is present in different oxidative states of inorganic [As(V) and As(III)] and organic (DMA, MMA and TMA) forms. Among different mitigation approaches, microbe mediated mitigation of As toxicity is an effective and eco-friendly approach. The present study involves the characterization of bacterial strains containing arsenite methyltransferase (Pseudomonas oleovorans, B4.10); arsenate reductase (Sphingobacterium puteale, B4.22) and arsenite oxidase (Citrobacter sp., B5.12) activity with plant growth promoting (PGP) traits. Efficient reduction of grain As content by 61 % was observed due to inoculation of methyltransferase containing B4.10 as compared to B4.22 (47 %) and B5.12 (49 %). Reduced bioaccumulation of As in root (0.339) and shoot (0.166) in presence of B4.10 was found to be inversely related with translocation factor for Mn (3.28), Fe (0.073), and Se (1.82). Bioaccumulation of these micro elements was found to be associated with the modulated expression of different mineral transporters (OsIRT2, OsFRO2, OsTOM1, OsSultr4;1, and OsZIP2) in rice shoot. Improved dehydrogenase (407 %), and β-glucosidase (97 %) activity in presence of P. oleovorans (B4.10) as compared to arsenate reductase (198 and 50 %), and arsenite oxidase (134 and 69 %) containing bacteria was also observed. Our finding confers the potential of methyltransferase positive P. oleovorans (B4.10) for As stress amelioration. Reduced grain As uptake was found to be mediated by improved plant growth and nutrient uptake associated with enhanced soil microbial activity., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Selenate enhances arsenic (As) accumulation in As-hyperaccumulator Pteris vittata , but the associated molecular mechanisms are unclear. Here, we investigated the mechanisms of selenate-induced arsenic accumulation by exposing P. vittata to 50 μM arsenate (AsV 50 ) and 1.25 (Se 1.25 ) or 5 μM (Se 5 ) selenate in hydroponics. After 2 weeks, plant biomass, plant As and Se contents, As speciation in plant and growth media, and important genes related to As detoxification in P. vittata were determined. These genes included P transporters PvPht1;3 and PvPht1;4 (AsV uptake), arsenate reductases PvHAC1 and PvHAC2 (AsV reduction), and arsenite (AsIII) antiporters PvACR3 and PvACR3;2 (AsIII translocation) in the roots, and AsIII antiporters PvACR3;1 and PvACR3;3 (AsIII sequestration) in the fronds. The results show that Se 1.25 was more effective than Se 5 in increasing As accumulation in both P. vittata roots and fronds, which increased by 27 and 153% to 353 and 506 mg kg -1 . The As speciation analyses show that selenate increased the AsIII levels in P. vittata , with 124-282% more AsIII being translocated into the fronds. The qPCR analyses indicate that Se 1.25 upregulated the gene expression of PvHAC1 by 1.2-fold, and PvACR3 and PvACR3;2 by 1.0- to 2.5-fold in the roots, and PvACR3;1 and PvACR3;3 by 0.6- to 1.1-fold in the fronds under AsV 50 treatment. Though arsenate enhanced gene expression of P transporters PvPht1;3 and PvPht1;4 , selenate had little effect. Our results indicate that selenate effectively increased As accumulation in P. vittata , mostly by increasing reduction of AsV to AsIII in the roots, AsIII translocation from the roots to fronds, and AsIII sequestration into the vacuoles in the fronds. The results suggest that selenate may be used to enhance phytoremediation of As-contaminated soils using P. vittata .

Histone acetyltransferases (HATs) are involved in the epigenetic positive control of gene expression in eukaryotes. CREB-binding proteins (CBP)/p300, a subfamily of highly conserved HATs, have been shown to function as acetylases on both histones and non-histone proteins. In the model plant Arabidopsis thaliana among the five CBP/p300 HATs, HAC1, HAC5 and HAC12 have been shown to be involved in the ethylene signaling pathway. In addition, HAC1 and HAC5 interact and cooperate with the Mediator complex, as in humans. Therefore, it is potentially difficult to discriminate the effect on plant development of the enzymatic activity with respect to their Mediator-related function. Taking advantage of the homology of the human HAC catalytic domain with that of the Arabidopsis, we set-up a phenotypic assay based on the hypocotyl length of Arabidopsis dark-grown seedlings to evaluate the effects of a compound previously described as human p300/CBP inhibitor, and to screen previously described cinnamoyl derivatives as well as newly synthesized analogues. We selected the most effective compounds, and we demonstrated their efficacy at phenotypic and molecular level. The in vitro inhibition of the enzymatic activity proved the specificity of the inhibitor on the catalytic domain of HAC1, thus substantiating this strategy as a useful tool in plant epigenetic studies.

Plants perceive volatiles emitted from herbivore-damaged neighboring plants to urgently adapt or prime their defense responses to prepare for forthcoming herbivores. Mechanistically, these volatiles can induce epigenetic regulation based on histone modifications that alter the transcriptional status of defense genes, but little is known about the underlying mechanisms. To understand the roles of such epigenetic regulation of plant volatile signaling, we explored the response of Arabidopsis (Arabidopsis thaliana) plants to the volatile β-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to β-ocimene, through enriched histone acetylation and elevated transcriptional levels of defense gene regulators, including ethylene response factor genes (ERF8 and ERF104) in leaves. The enhanced defense ability of the plants was maintained for 5 d but not over 10 d after exposure to β-ocimene, and this coincided with elevated expression of those ERFs in their leaves. An array of histone acetyltransferases, including HAC1, HAC5, and HAM1, were responsible for the induction and maintenance of the anti-herbivore property. HDA6, a histone deacetylase, played a role in the reverse histone remodeling. Collectively, our findings illuminate the role of epigenetic regulation in plant volatile signaling., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In order to understand the mechanisms of arsenic (As) accumulation and detoxification in aquatic plants exposed to different As species, a hydroponic experiment was conducted and the three aquatic plants (Hydrilla verticillata, Pistia stratiotes and Eichhornia crassipes) were exposed to different concentrations of As(III), As(V) and dimethylarsinate (DMA) for 10 days. The biomass, the surface As adsorption and total As adsorption of three plants were determined. Furthermore, As speciation in the culture solution and plant body, as well as the arsenate reductase (AR) activities of roots and shoots, were also analyzed. The results showed that the surface As adsorption of plants was far less than total As absorption. Compared to As(V), the plants showed a lower DMA accumulation. P. stratiotes showed the highest accumulation of inorganic arsenic but E. crassipes showed the lowest at the same As treatment. E. crassipes showed a strong ability to accumulate DMA. Results from As speciation analysis in culture solution showed that As(III) was transformed to As(V) in all As(III) treatments, and the oxidation rates followed as the sequence of H. verticillata>P. stratiotes>E. crassipes>no plant. As(III) was the predominant species in both roots (39.4-88.3%) and shoots (39-86%) of As(III)-exposed plants. As(V) and As(III) were the predominant species in roots (37-94%) and shoots (31.1-85.6%) in As(V)-exposed plants, respectively. DMA was the predominant species in both roots (23.46-100%) and shoots (72.6-100%) in DMA-exposed plants. The As(III) contents and AR activities in the roots of P. stratiotes and in the shoots of H. verticillata were significantly increased when exposed to 1 mg·L -1 or 3 mg·L -1 As(V). Therefore, As accumulation mainly occurred via biological uptake rather than physicochemical adsorption, and AR played an important role in As detoxification in aquatic plants. In the case of As(V)-exposed plants, their As tolerance was attributed to the increase of AR activities., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, MEDIATOR is a conserved multi-subunit complex that links transcription factors and RNA polymerase II and that thereby facilitates transcriptional initiation. Although the composition of MEDIATOR has been well studied in yeast and mammals, relatively little is known about the composition of MEDIATOR in plants. By affinity purification followed by mass spectrometry, we identified 28 conserved MEDIATOR subunits in Arabidopsis thaliana, including putative MEDIATOR subunits that were not previously validated. Our results indicated that MED34, MED35, MED36, and MED37 are not Arabidopsis MEDIATOR subunits, as previously proposed. Our results also revealed that two homologous CBP/p300 histone acetyltransferases, HAC1 and HAC5 (HAC1/5) are in fact plant-specific MEDIATOR subunits. The MEDIATOR subunits MED8 and MED25 (MED8/25) are partially responsible for the association of MEDIATOR with HAC1/5, MED8/25 and HAC1/5 co-regulate gene expression and thereby affect flowering time and floral development. Our in vitro observations indicated that MED8 and HAC1 form liquid-like droplets by phase separation, and our in vivo observations indicated that these droplets co-localize in the nuclear bodies at a subset of nuclei. The formation of liquid-like droplets is required for MED8 to interact with RNA polymerase II. In summary, we have identified all of the components of Arabidopsis MEDIATOR and revealed the mechanism underlying the link of histone acetylation and transcriptional regulation., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

This study evaluated the phytoextraction capacity of the fern Pteris vittata grown on a natural arsenic-rich soil of volcanic-origin from the Viterbo area in central Italy. This calcareous soil is characterized by an average arsenic concentration of 750 mg kg -1 , of which 28% is bioavailable. By means of micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) we detected As in P. vittata fronds after just 10 days of growth, while a high As concentrations in fronds (5,000 mg kg -1 ), determined by Inductively coupled plasma-optical emission spectrometry (ICP-OES), was reached after 5.5 months. Sixteen arsenate-tolerant bacterial strains were isolated from the P. vittata rhizosphere, a majority of which belong to the Bacillus genus, and of this majority only two have been previously associated with As. Six bacterial isolates were highly As-resistant (> 100 mM) two of which, homologous to Paenarthrobacter ureafaciens and Beijerinckia fluminensis, produced a high amount of IAA and siderophores and have never been isolated from P. vittata roots. Furthermore, five isolates contained the arsenate reductase gene (arsC). We conclude that P. vittata can efficiently phytoextract As when grown on this natural As-rich soil and a consortium of bacteria, largely different from that usually found in As-polluted soils, has been found in P. vittata rhizosphere.

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO 4 3- ) as the terminal electron acceptor, where it is reduced to arsenite (AsO 3 3- ) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus , both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus , recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site., (Copyright © 2020 American Society for Microbiology.)

The three presently known enzymes responsible for arsenic-using bioenergetic processes are arsenite oxidase (Aio), arsenate reductase (Arr) and alternative arsenite oxidase (Arx), all of which are molybdoenzymes from the vast group referred to as the Mo/W-bisPGD enzyme superfamily. Since arsenite is present in substantial amounts in hydrothermal environments, frequently considered as vestiges of primordial biochemistry, arsenite-based bioenergetics has long been predicted to be ancient. Conflicting scenarios, however, have been put forward proposing either Arr/Arx or Aio as operating in the ancestral metabolism. Phylogenetic data argue in favor of Aio whereas biochemical and physiological data led several authors to propose Arx/Arr as the most ancient anaerobic arsenite metabolizing enzymes. Here we combine phylogenetic approaches with physiological and biochemical experiments to demonstrate that the Arx/Arr enzymes could not have been functional in the Archaean geological eon. We propose that Arr reacts with menaquinones to reduce arsenate whereas Arx reacts with ubiquinone to oxidize arsenite, in line with thermodynamic considerations. The distribution of the quinone biosynthesis pathways, however, clearly indicates that the ubiquinone pathway is recent. An updated phylogeny of Arx furthermore reinforces the hypothesis of a recent emergence of this enzyme. We therefore conclude that anaerobic arsenite redox conversion in the Archaean must have been performed in a metabolism involving Aio., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Arsenic (As) is a serious threat for environment and human health. Rice, the main staple crop is more prone to As uptake. Bioremediation strategies with heavy metal tolerant rhizobacteria are well known. The main objective of the study was to characterize arsenic-resistant yeast strains, capable of mitigating arsenic stress in rice. Three yeast strains identified as Debaryomyces hansenii (NBRI-Sh2.11), Candida tropicalis (NBRI-B3.4) and Candida dubliniensis (NBRI-3.5) were found to have As reductase activity. D. hansenii with higher As tolerance has As expulsion ability as compared to other two strains. Inoculation of D. hansenii showed improved detoxification through scavenging of reactive oxygen species (ROS) by the modulation of SOD and APX activity under As stress condition in rice. Modulation of defense responsive gene (NADPH, GST, GR) along with arsR and metal cation transporter are the probable mechanism of As detoxification as evident with improved membrane (electrolyte leakage) stability. Reduced grain As (~40% reduction) due to interaction with D. hansenii (NBRI-Sh2.11) further validated it's As mitigation property in rice. To the best of our knowledge D. hansenii has been reported for the first time for arsenic stress mitigation in rice with improved growth and nutrient status of the plant., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The yeast stress-activated protein kinase Hog1 is best known for its role in mediating the response to osmotic stress, but it is also activated by various mechanistically distinct environmental stressors, including heat shock, endoplasmic reticulum stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 through a kinase cascade. Here, we identified a mode of Hog1 function whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop. These residues were essential for Hog1-mediated protection against arsenic, were dispensable for the response to osmotic stress, and promoted the nuclear localization of Hog1 upon exposure of cells to arsenic. Hog1 promoted arsenic detoxification by stimulating phosphorylation of the transcription factor Yap8, promoting Yap8 nuclear localization, and stimulating the transcription of the only known Yap8 targets, ARR2 and ARR3 , both of which encode proteins that promote arsenic efflux. The related human kinases ERK1 and ERK2 also bound to arsenic in vitro, suggesting that this may be a conserved feature of some members of the mitogen-activated protein kinase (MAPK) family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Groucho/Thymidine uptake 1 (Gro/Tup1) family proteins are evolutionarily conserved transcriptional coregulators in eukaryotic cells. Despite their prominent function in transcriptional repression, little is known about their role in transcriptional activation and the underlying mechanism. Here, we report that the plant Gro/Tup1 family protein LEUNIG_HOMOLOG (LUH) activates MYELOCYTOMATOSIS2 (MYC2)-directed transcription of JAZ2 and LOX2 via the Mediator complex coactivator and the histone acetyltransferase HAC1. We show that the Mediator subunit MED25 physically recruits LUH to MYC2 target promoters that then links MYC2 with HAC1-dependent acetylation of Lys-9 of histone H3 (H3K9ac) to activate JAZ2 and LOX2 Moreover, LUH promotes hormone-dependent enhancement of protein interactions between MYC2 and its coactivators MED25 and HAC1. Our results demonstrate that LUH interacts with MED25 and HAC1 through its distinct domains, thus imposing a selective advantage by acting as a scaffold for MYC2 activation. Therefore, the function of LUH in regulating jasmonate signaling is distinct from the function of TOPLESS, another member of the Gro/Tup1 family that represses MYC2-dependent gene expression in the resting stage., (© 2019 American Society of Plant Biologists. All rights reserved.)

To study systems-level properties of the cell, it is necessary to go beyond individual regulators and target genes to study the regulatory network among transcription factors (TFs). However, it is difficult to directly dissect the TFs mediated genome-wide gene regulatory network (GRN) by experiment. Here, we proposed a hierarchical graphical model to estimate TF activity from mRNA expression by building TF complexes with protein cofactors and inferring TF's downstream regulatory network simultaneously. Then we applied our model on flower development and circadian rhythm processes in Arabidopsis thaliana . The computational results show that the sequence specific bHLH family TF HFR1 recruits the chromatin regulator HAC1 to flower development master regulator TF AG and further activates AG's expression by histone acetylation. Both independent data and experimental results supported this discovery. We also found a flower tissue specific H3K27ac ChIP-seq peak at AG gene body and a HFR1 motif in the center of this H3K27ac peak. Furthermore, we verified that HFR1 physically interacts with HAC1 by yeast two-hybrid experiment. This HFR1-HAC1-AG triplet relationship may imply that flower development and circadian rhythm are bridged by epigenetic regulation and enrich the classical ABC model in flower development. In addition, our TF activity network can serve as a general method to elucidate molecular mechanisms on other complex biological regulatory processes., Competing Interests: Competing interestsThe authors declare no competing interests.

The lipid-derived hormone jasmonate (JA) regulates plant immunity and adaptive growth by triggering a genome-wide transcriptional programme. In Arabidopsis thaliana, JA-triggered transcriptional programming is largely orchestrated by the master transcription factor MYC2. The function of MYC2 is dependent on its physical interaction with the MED25 subunit of the Mediator transcriptional co-activator complex. Here we report the identification of JA enhancers (JAEs) through profiling the occupancy pattern of MYC2 and MED25. JA regulates the dynamic chromatin looping between JAEs and their promoters in a MED25-dependent manner, while MYC2 auto-regulates itself through JAEs. Interestingly, the JAE of the MYC2 locus (named ME2) positively regulates MYC2 expression during short-term JA responses but negatively regulates it during constant JA responses. We demonstrate that new gene editing tools open up new avenues to elucidate the in vivo function of enhancers. Our work provides a paradigm for functional study of plant enhancers in the regulation of specific physiological processes.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Arsenic is a carcinogenic metalloid, exists in two important oxidation states-arsenate (As-V) and arsenite (As-III). The influence of arsenate with or without silicate on the growth and thiol metabolism in rice (Oryza sativa L. cv. MTU-1010) seedlings were investigated. Arsenate was more toxic for root growth than shoot growth where the root lengths were short, characteristically fragile and root tips turned brown. The multiple comparison analysis using Tukey's HSD (honest significant difference) tests indicated that the rate of arsenate accumulation and its conversion to arsenite by arsenate reductase were significantly increased in all arsenate treated seedlings while in seedlings treated jointly with arsenate and silicate, arsenate accumulation and its conversion to arsenite decreased. Silicate content was detected in the seedlings treated with silicate alone and under co-application of arsenate with silicate. In the test seedlings arsenic toxicity increased ascorbate and glutathione contents along with the activities of their regulatory enzymes, viz., ascorbate peroxidase, glutathione reductase, glutathione peroxidase and glutathione-s-transferase to reduce the toxicity level induced by arsenic whereas ascorbate oxidase activity was decreased to maintain sufficient ascorbate pool under arsenate treatment. Phytochelatins production were increased in both root and shoot of the test seedlings under arsenate exposure to alter the detrimental effects of arsenic by chelation with arsenite and their subsequent sequestration into vacuole. Thus, joint application of silicate along with arsenate showed significant alterations on all the parameters tested compared to arsenate treatment alone due to less availability of arsenic in the tissue leading to better growth and metabolism in rice seedlings. Thus use of silicon in arsenic contaminated medium may help to grow rice with improved vigour.

The major hazard of arsenic in living organisms is increasingly being recognized. Marine mollusks are apt to accumulate high levels of arsenic, but knowledge related to arsenic detoxification in marine mollusks is still less than sufficient. In this study, arsenic bioaccumulation as well as the role of glutathione S-transferase omega (GSTΩ) in the process of detoxification were investigated in the Ruditapes philippinarum clam after waterborne exposure to As(III) or As(V) for 30 days. The results showed that the gills accumulated significantly higher arsenic levels than the digestive glands. Arsenobetaine (AsB) and dimethylarsenate (DMA) accounted for most of the arsenic found, and monomethylarsonate (MMA) can be quickly metabolized. A subcellular distribution analysis showed that most arsenic was in biologically detoxified metal fractions (including metal-rich granules and metallothionein-like proteins), indicating their important roles in protecting cells from arsenic toxicity. The relative mRNA expressions of two genes encoding GSTΩ were up-regulated after arsenic exposure, and the transcriptional responses were more sensitive to As(III) than As(V). The recombinant GSTΩs exhibited high activities at optimal conditions, especially at 37 °C and pH 4-5, with an As(V) concentration of 60 mM. Furthermore, the genes encoding GSTΩ significantly enhance the arsenite tolerance but not the arsenate tolerance of E. coli AW3110 (DE3) (ΔarsRBC). It can be deduced from these results that GSTΩs play an important role in arsenic detoxification in R. philippinarum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The paddy soils in some areas in Jianghan Plain were severely contaminated by arsenic. However, little is known about the activity and diversity of the dissimilatory arsenate-respiring prokaryotes (DARPs) in the paddy soils, and the effects of sulfate on the microbial mobilization and release of arsenic from soils into solution. To address this issue, we collected arsenic-rich soils from the depths of 1.6 and 4.6 m in a paddy region in the Xiantao city, Hubei Province, China. Microcosm assays indicated that all of the soils have significant arsenate-respiring activities using lactate, pyruvate or acetate as the sole electron donor. Functional gene cloning and analysis suggest that there are diverse DARPs in the indigenous microbial communities of the soils. They efficiently promoted the mobilization, reduction and release of arsenic and iron from soils under anaerobic conditions. Remarkably, when sulfate was amended into the microcosms, the microorganisms-catalyzed reduction and release of arsenic and iron were significantly increased. We further found that sulfate significantly enhanced the arsenate-respiring reductase gene abundances in the soils. Taken together, a diversity of DARPs in the paddy soils significantly catalyzed the dissolution, reduction and release of arsenic and iron from insoluble phase into solution, and the presence of sulfate significantly increased the microbial reactions.

Biofilms, microbial mats, and microbialites dwell under highly limiting conditions (high salinity, extreme aridity, pH, and elevated arsenic concentration) in the Andean Puna. Only recent pioneering studies have described the microbial diversity of different Altiplano lakes and revealed their unexpectedly diverse microbial communities. Arsenic metabolism is proposed to be an ancient mechanism to obtain energy by microorganisms. Members of Bacteria and Archaea are able to exploit arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. Only six aioAB sequences coding for arsenite oxidase and three arrA sequences coding for arsenate reductase from haloarchaea were previously deposited in the NCBI database. However, no experimental data on their expression and function has been reported. Recently, our working group revealed the prevalence of haloarchaea in a red biofilm from Diamante Lake and microbial mat from Tebenquiche Lake using a metagenomics approach. Also, a surprisingly high abundance of genes used for anaerobic arsenate respiration (arr) and arsenite oxidation (aio) was detected in the Diamante's metagenome. In order to study in depth the role of arsenic in these haloarchaeal communities, in this work, we obtained 18 haloarchaea belonging to the Halorubrum genus, tolerant to arsenic. Furthermore, the identification and expression analysis of genes involved in obtaining energy from arsenic compounds (aio and arr) showed that aio and arr partial genes were detected in 11 isolates, and their expression was verified in two selected strains. Better growth of two isolates was obtained in presence of arsenic compared to control. Moreover, one of the isolates was able to oxidize As[III]. The confirmation of the oxidation of arsenic and the transcriptional expression of these genes by RT-PCR strongly support the hypothesis that the arsenic can be used in bioenergetics processes by the microorganisms flourishing in these environments.

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit ( K m = 44.6 ± 1.6 μM, k cat = 9,810 ± 220 seconds -1 ), and phosphate is a competitive inhibitor of arsenate reduction ( K i = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that ( i ) arsenate desorption limits microbiologically induced arsenate reductive mobilization and ( ii ) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level., Competing Interests: The authors declare no conflict of interest.

Background: Microorganisms specifically bacteria play a crucial role in arsenic mobilization and its distribution in aquatic systems. Although bacteria are well known for their active participation in the different biogeochemical cycles, the role of these bacteria in regulating the concentration of arsenic in Brahmaputra valley has not been investigated in detail., Results: In this paper, we report the isolation of an arsenic resistant bacterium TA6 which can efficiently reduce arsenate. The isolate identified as Staphylococcus sp. TA6 based on the molecular and chemotaxonomic identification (FAME) showed resistance to the high concentration of both arsenate and arsenite (As(III) = 30 mM; As(V) = 250 mM), along with cross-tolerance to other heavy metals viz., Hg 2+ , Cd 2+ , Co 2+ , Ni 2+ , Cr 2+ . The bacterium also had a high siderophore activity (78.7 ± 0.004 μmol) that positively correlated with its ability to resist arsenic. The isolate, Staphylococcus sp. TA6 displayed high bio-transformation ability and reduced 2 mM As(V) initially added into As(III) in a period of 72 h with 88.2% efficiency. The characterization of arsenate reductase enzyme with NADPH coupled assay showed the highest activity at pH 5.5 and temperature of 50 °C., Conclusions: This study demonstrates the role of an isolate, Staphylococcus sp. TA6, in the biotransformation of arsenate to arsenite. The presence of ars operon along with the high activity of the arsenate reductase and siderophore production in this isolate may have played an important role in mobilizing arsenate to arsenite and thus increasing the toxicity of arsenic in the aquatic systems of the Brahmaputra valley.

Citrobacter sp. strain TSA-1 is an enteric bacterium isolated from the hindgut of the termite. Strain TSA-1 displays anaerobic growth with selenite, fumarate, tetrathionate, nitrate, or arsenate serving as electron acceptors, and it also grows aerobically. In regards to arsenate, genome sequencing revealed that strain TSA-1 lacks a homolog for respiratory arsenate reductase, arrAB, and we were unable to obtain amplicons of arrA. This raises the question as to how strain TSA-1 achieves As(V)-dependent growth. We show that growth of strain TSA-1 on glycerol, which it cannot ferment, is linked to the electron acceptor arsenate. A series of transcriptomic experiments were conducted to discern which genes were upregulated during growth on arsenate, as opposed to those on fumarate or oxygen. For As(V), upregulation was noted for 1 of the 2 annotated arsC genes, while there was no clear upregulation for tetrathionate reductase (ttr), suggesting that this enzyme is not an alternative to arrAB as occurs in certain hyperthermophilic archaea. A gene-deletion mutant strain of TSA-1 deficient in arsC could not achieve anaerobic respiratory growth on As(V). Our results suggest that Citrobacter sp. strain TSA-1 has an unusual and as yet undefined means of achieving arsenate respiration, perhaps involving its ArsC as a respiratory reductase as well as a detoxifying agent.

Arsenic (As) is an important environmental and food-chain toxin. We investigated the key components controlling As accumulation and tolerance in Arabidopsis thaliana. We tested the effects of different combinations of gene knockout, including arsenate reductase (HAC1), γ-glutamyl-cysteine synthetase (γ-ECS), phytochelatin synthase (PCS1) and phosphate effluxer (PHO1), and the heterologous expression of the As-hyperaccumulator Pteris vittata arsenite efflux (PvACR3), on As tolerance, accumulation, translocation and speciation in A. thaliana. Heterologous expression of PvACR3 markedly increased As tolerance and root-to-shoot As translocation in A. thaliana, with PvACR3 being localized to the plasma membrane. Combining PvACR3 expression with HAC1 mutation led to As hyperaccumulation in the shoots, whereas combining HAC1 and PHO1 mutation decreased As accumulation. Mutants of γ-ECS and PCS1 were hypersensitive to As and had higher root-to-shoot As translocation. Combining γ-ECS or PCS1 with HAC1 mutation did not alter As tolerance or accumulation beyond the levels observed in the single mutants. PvACR3 and HAC1 have large effects on root-to-shoot As translocation. Arsenic hyperaccumulation can be engineered in A. thaliana by knocking out the HAC1 gene and expressing PvACR3. PvACR3 and HAC1 also affect As tolerance, but not to the extent of γ-ECS and PCS1., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Jasmonoyl-isoleucine (JA-Ile), the active form of the plant hormone jasmonate (JA), is sensed by the F-box protein CORONATINE INSENSITIVE 1 (COI1), a component of a functional Skp-Cullin-F-box E3 ubiquitin ligase complex. Sensing of JA-Ile by COI1 rapidly triggers genome-wide transcriptional changes that are largely regulated by the basic helix-loop-helix transcription factor MYC2. However, it remains unclear how the JA-Ile receptor protein COI1 relays hormone-specific regulatory signals to the RNA polymerase II general transcriptional machinery. Here, we report that the plant transcriptional coactivator complex Mediator directly links COI1 to the promoters of MYC2 target genes. MED25, a subunit of the Mediator complex, brings COI1 to MYC2 target promoters and facilitates COI1-dependent degradation of jasmonate-ZIM domain (JAZ) transcriptional repressors. MED25 and COI1 influence each other's enrichment on MYC2 target promoters. Furthermore, MED25 physically and functionally interacts with HISTONE ACETYLTRANSFERASE1 (HAC1), which plays an important role in JA signaling by selectively regulating histone (H) 3 lysine (K) 9 (H3K9) acetylation of MYC2 target promoters. Moreover, the enrichment and function of HAC1 on MYC2 target promoters depend on COI1 and MED25. Therefore, the MED25 interface of Mediator links COI1 with HAC1-dependent H3K9 acetylation to activate MYC2-regulated transcription of JA-responsive genes. This study exemplifies how a single Mediator subunit integrates the actions of both genetic and epigenetic regulators into a concerted transcriptional program., Competing Interests: The authors declare no conflict of interest.

This study evaluates the transcriptionally active, dissimilatory sulfur- and arsenic-cycling components of the microbial community in alkaline, hypersaline Mono Lake, CA, USA. We sampled five depths spanning the redox gradient (10, 15, 18, 25 and 31 m) during maximum thermal stratification. We used custom databases to identify transcripts of genes encoding complex iron-sulfur molybdoenzyme (CISM) proteins, with a focus on arsenic (arrA, aioA and arxA) and sulfur cycling (dsrA, aprA and soxB), and assigned them to taxonomic bins. We also report on the distribution of transcripts related to the ars arsenic detoxification pathway. Transcripts from detoxification pathways were not abundant in oxic surface waters (10 m). Arsenic cycling in the suboxic and microaerophilic zones of the water column (15 and 18 m) was dominated by arsenite-oxidizing members of the Gammaproteobacteria most closely affiliated with Thioalkalivibrio and Halomonas, transcribing arxA. We observed a transition to arsenate-reducing bacteria belonging to the Deltaproteobacteria and Firmicutes transcribing arsenate reductase (arrA) in anoxic bottom waters of the lake (25 and 31 m). Sulfur cycling at 15 and 18 m was dominated by Gammaproteobacteria (Thioalkalivibrio and Thioalkalimicrobium) oxidizing reduced S species, with a transition to sulfate-reducing Deltaproteobacteria at 25 and 31 m. Genes related to arsenic and sulfur oxidation from Thioalkalivibrio were more highly transcribed at 15 m relative to other depths. Our data highlight the importance of Thioalkalivibrio to arsenic and sulfur biogeochemistry in Mono Lake and identify new taxa that appear capable of transforming arsenic.

Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As(V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes., (Copyright © 2017. Published by Elsevier B.V.)

This study aimed to identify arsenic resistant mechanisms in As-resistant purple nonsulfur bacteria (PNSB) by screening them for presence of As-resistance genes and related enzymes. Resistance to As(III) and As(V) of four As-resistant PNSB determined in terms of median inhibition concentration (IC 50 values) were in the order of strains Rhodopseudomonas palustris C1 > R. palustris AB3 > Rubrivivax benzoatilyticus C31 > R. palustris L28 which corresponded to the presence of As-resistance genes in these bacteria. The strain C1 showed all As-marker genes; arsC, arsM, aioA, and acr3, while aioA was not detected in strain AB3. Strains C31 and L28 had only Arsenite-transporter gene, acr3. Translation of all these detected gene sequences of strain C1 to amino acid sequences showed that these proteins have vicinal cysteine; Cys126, Cys105, and Cys178 of Acr3, ArsC, AioA, respectively. Tertiary structure of proteins Acr3, ArsC, AioA, and ArsM showed strain C1 exhibits the high activities of arsenite oxidase and arsenate reductase enzymes that are encoded by aioA and arsC genes, respectively. Moreover, strain C1 with arsM gene produced volatile-methylated As-compounds; monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), and arsenobetaine (AsB) in the presence of either As(III) or As(V). In conclusion, the strain C1 has great potential for its application in bioremediation of As-contaminated sites., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Arsenic (As) is a ubiquitous environmental toxin that has created catastrophic human health and environmental problems around world. Physcomitrella patens is a potential model plant for the study of environmental monitoring, which exists in all kinds of ecosystems. In this study, arsenic metabolism was investigated by this moss. When supplied with different levels of arsenate (50, 100, 200 µmol/L) for a 4-week period, the total arsenic concentrations were up to 231.4-565.4 mg/kg DW in this moss. Arsenite concentration increased with increasing external arsenate concentrations, the proportion was up to 25.1-36.8% of the total As. An arsenate reductase, PpACR2, was identified and functionally characterized. Heterologous expression of PpACR2 in an As(V)-sensitive strain WC3110 (ΔarsC) of Escherichia coli conferred As(V) resistance. Purified PpACR2 protein exhibited the arsenate reductase activity. Given its powerful As accumulation ability, the bryophyte could be exploited in bioremediation of As-contaminated environments.

The metalloid arsenic predominantly exists in the arsenite [As(III)] and arsenate [As(V)]. These two forms are respectively oxidized and reduced by microbial redox processes. This study was designed to bioprospect arsenic tolerating bacteria from Lonar lake and to characterize their arsenic redoxing ability. Screening of sixty-nine bacterial species isolated from Lonar lake led to identification of three arsenic-oxidizing and seven arsenic-reducing species. Arsenite oxidizing isolate Roseomonas sp. L-159a being closely related to Roseomonas cervicalis ATCC 49957 oxidized 2mM As(III) in 60h. Gene expression of large and small subunits of arsenite oxidase respectively showed 15- and 17-fold higher expression. Another isolate Nocardioides sp. L-37a formed a clade with Nocardioides ghangwensis JC2055, exhibited normal growth with different carbon sources and pH ranges. It reduced 2mM As(V) in 36h and showed constitutive expression of arsenate reductase which increased over 4-fold upon As(V) exposure. Genetic markers related to arsenic transformation were identified and characterized from the two isolates. Moderate resistance against the arsenicals was exhibited by the two isolates in the range of 1-5mM for As(III) and 1-200mM for As(V). Altogether we provide multiple evidences to indicate that Roseomonas sp. and Nocardioides sp. exhibited arsenic transformation ability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Arsenic (As) contamination in water is a cause of major concern to human population worldwide, especially in Bangladesh and West Bengal, India. Arsenite (As(III)) and arsenate (As(V)) are the two common forms in which arsenic exists in soil and groundwater, the former being more mobile and toxic. A large number of arsenic metabolising microorganisms play a crucial role in microbial transformation of arsenic between its different states, thus playing a key role in remediation of arsenic contaminated water. This review focuses on advances in biochemical, molecular and genomic developments in the field of arsenic metabolising bacteria - covering recent developments in the understanding of structure of arsenate reductase and arsenite oxidase enzymes, their gene and operon structures and their mechanism of action. The genetic and molecular studies of these microbes and their proteins may lead to evolution of successful strategies for effective implementation of bioremediation programs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rice is a major dietary source of the toxic metalloid arsenic (As). Reducing its accumulation in rice (Oryza sativa) grain is of critical importance to food safety. Rice roots take up arsenate and arsenite depending on the prevailing soil conditions. The first step of arsenate detoxification is its reduction to arsenite, but the enzyme(s) catalyzing this reaction in rice remains unknown. Here, we identify OsHAC1;1 and OsHAC1;2 as arsenate reductases in rice. OsHAC1;1 and OsHAC1;2 are able to complement an Escherichia coli mutant lacking the endogenous arsenate reductase and to reduce arsenate to arsenite. OsHAC1:1 and OsHAC1;2 are predominantly expressed in roots, with OsHAC1;1 being abundant in the epidermis, root hairs, and pericycle cells while OsHAC1;2 is abundant in the epidermis, outer layers of cortex, and endodermis cells. Expression of the two genes was induced by arsenate exposure. Knocking out OsHAC1;1 or OsHAC1;2 decreased the reduction of arsenate to arsenite in roots, reducing arsenite efflux to the external medium. Loss of arsenite efflux was also associated with increased As accumulation in shoots. Greater effects were observed in a double mutant of the two genes. In contrast, overexpression of either OsHAC1;1 or OsHAC1;2 increased arsenite efflux, reduced As accumulation, and enhanced arsenate tolerance. When grown under aerobic soil conditions, overexpression of either OsHAC1;1 or OsHAC1;2 also decreased As accumulation in rice grain, whereas grain As increased in the knockout mutants. We conclude that OsHAC1;1 and OsHAC1;2 are arsenate reductases that play an important role in restricting As accumulation in rice shoots and grain., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Arsenic (As) is an acute poison and class I carcinogen, can cause a serious health risk. Staple crops like rice are the primary source of As contamination in human food. Rice grown on As contaminated areas accumulates higher As in their edible parts. Based on our previous transcriptome data, two rice glutaredoxins (OsGrx_C7 and OsGrx_C2.1) were identified that showed up-regulated expression during As stress. Here, we report OsGrx_C7 and OsGrx_C2.1 from rice involved in the regulation of intracellular arsenite (AsIII). To elucidate the mechanism of OsGrx mediated As tolerance, both OsGrxs were cloned and expressed in Escherichia coli (Δars) and Saccharomyces cerevisiae mutant strains (Δycf1, Δacr3). The expression of OsGrxs increased As tolerance in E. coli (Δars) mutant strain (up to 4 mM AsV and up to 0.6 mM AsIII). During AsIII exposure, S. cerevisiae (Δacr3) harboring OsGrx_C7 and OsGrx_C2.1 have lower intracellular AsIII accumulation (up to 30.43% and 24.90%, respectively), compared to vector control. Arsenic accumulation in As-sensitive S. cerevisiae mutant (Δycf1) also reduced significantly on exposure to inorganic As. The expression of OsGrxs in yeast maintained intracellular GSH pool and increased extracellular GSH concentration. Purified OsGrxs displays in vitro GSH-disulfide oxidoreductase, glutathione reductase and arsenate reductase activities. Also, both OsGrxs are involved in AsIII extrusion by altering the Fps1 transcripts in yeast and protect the cell by maintaining cellular GSH pool. Thus, our results strongly suggest that OsGrxs play a crucial role in the maintenance of the intracellular GSH pool and redox status of the cell during both AsV and AsIII stress and might be involved in regulating intracellular AsIII levels by modulation of aquaporin expression and functions., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The arsenic (As) is a toxic element causing major health concern worldwide. Arsenate stress caused no significant reduction in growth parameters and shoot electrolyte leakage but showed increased root arsenate reductase activity along with relatively lower root As content and shoot translocation rate in As-tolerant BRRI 33 than in As-sensitive BRRI 51. It indicates that As inhibition and tolerance mechanisms are driven by root responses. Interestingly, As stress showed consistent decrease in phosphate content and expression of phosphate transporters (OsPT8, OsPT4, OsPHO1;2) under both high and low phosphate conditions in roots of BRRI 33, suggesting that limiting phosphate transport mainly mediated by OsPHO1;2 directs less As accumulation in BRRI 33. Further, BRRI 33 showed simultaneous increase in OsPCS1 (phytochelatin synthase) expression and phytochelatins (PCs) content in roots under As exposure supporting the hypothesis that root As sequestration acts as 'firewall system' in limiting As translocation in shoots. Furthermore, increased CAT, POD, SOD, GR, along with elevated glutathione, methionine, cysteine and proline suggests that strong antioxidant defense plays integral part to As tolerance in BRRI 33. Again, BRRI 33 self-grafts and plants having BRRI 33 rootstock combined with BRRI 51 scion had no adverse effect on morphological parameters but showed reduced As translocation rate, increased root arsenate reductase activity, shoot PC synthesis and root OsPHO1;2 expression due to As stress. It confirms that signal driving As tolerance mechanisms is generated in the roots. These findings can be implemented for As detoxification and As-free transgenic rice production for health safety., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC.

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.

Arsenite [As(III)]-oxidizing bacteria were isolated from heavy metal contaminated shore of Gulf of Cambay at Alang, India. The most efficient bacterial strain Alang-4 could tolerate up to 15 mM arsenite [As(III)] and 200 mM of arsenate [As(V)]. Its 16S rRNA gene sequence was 99% identical to the 16S rRNA genes of genus Halomonas (Accession no. HQ659187). Arsenite oxidase enzyme localized on membrane helped in conversion of As(III) to As(V). Arsenite transporter genes (arsB, acr3(1) and acr3(2)) assisted in extrusion of arsenite from Halomonas sp. Alang-4. Generation of ROS in response to arsenite stress was alleviated by higher activities of catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase enzymes. Down-regulation in the specific activities of nearly all dehydrogenases of carbon assimilatory pathway viz., glucose-6-phosphate, pyruvate, α-ketoglutarate, isocitrate and malate dehydrogenases, was observed in presence of As(III), whereas, the specific activities of phosphoenol pyruvate carboxylase, pyruvate carboxylase and isocitrate lyase enzymes were found to increase two times in As(III) treated cells. The results suggest that in addition to efficient ars operon, alternative pathways of carbon utilization exist in the marine bacterium Halomonas sp. Alang-4 to overcome the toxic effects of arsenite on its dehydrogenase enzymes.

The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological-metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV-vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles' aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 μM concentration. Moreover, the assay shows partial selectivity against other heavy metals.

Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Contamination of arsenic (As) in rice (Oryza sativa L.) paddies and subsequent uptake by rice plants is a serious concern, because rice is a staple crop for millions of people. Identification of As toxicity and detoxification mechanisms in paddy rice cultivars would help to reduce As-associated risk. Arsenic tolerance and susceptibility mechanisms were investigated in 2 differential As-accumulating rice genotypes, Triguna and IET-4786, selected from initial screening of 52 rice cultivars as an As-tolerant and an As-sensitive cultivar, respectively, on the basis of root and shoot length during various arsenite (AsIII) exposures (0-50 μM). Indicators of oxidative stress, such as pro-oxidant enzymes (reduced nicotinamide adenine dinucleotide phosphate [NADPH] oxidase and ascorbate oxidase) and nitric oxide, were more numerous in the sensitive cultivar than in the tolerant cultivar. Arsenic-induced anatomical deformities were frequent in the sensitive cultivar, showing more distorted and flaccid root cells than the tolerant cultivar. Chlorophyll and carotenoid synthesis were inhibited in both cultivars, although the decline was more prominent in the sensitive cultivar at higher doses of As. Furthermore, the tolerant cultivar tolerated As stress by producing more antioxidants, such as proline, sustaining the ratio of ascorbate, dehydroascorbate, and glutathione peroxidase (GPX) activity as well as As detoxifying enzymes arsenate reductase, whereas these respective metabolic activities declined in sensitive cultivar, resulting in greater susceptibility to As toxicity., (© 2015 SETAC.)

The unfolded protein response (UPR) signaling network encompasses two pathways in plants, one mediated by inositol-requiring protein-1 (IRE1)-bZIP60 mRNA and the other by site-1/site-2 proteases (S1P/S2P)-bZIP17/bZIP28. As the major sensor of UPR in eukaryotes, IRE1, in response to endoplasmic reticulum (ER) stress, catalyzes the unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans. Recent studies suggest that IRE1p and HAC1 mRNA, the only UPR pathway found in yeast, evolves as a cognate system responsible for the robust UPR induction. However, the functional connectivity of IRE1 and its splicing target in multicellular eukaryotes as well as the degree of conservation of IRE1 downstream signaling effectors across eukaryotes remains to be established. Here, we report that IRE1 and its substrate bZIP60 function as a strictly cognate enzyme-substrate pair to control viral pathogenesis in plants. Moreover, we show that the S1P/S2P-bZIP17/bZIP28 pathway, the other known branch of UPR in plants, does not play a detectable role in virus infection, demonstrating the distinct function of the IRE1-bZIP60 pathway in plants. Furthermore, we provide evidence that bZIP60 and HAC1, products of the enzyme-substrate duet, rather than IRE1, are functionally replaceable to cope with ER stress in yeast. Taken together, we conclude that the downstream signaling of the IRE1-mediated splicing is evolutionarily conserved in yeast and plants, and that the IRE1-bZIP60 UPR pathway not only confers overlapping functions with the other UPR branch in fundamental biology but also may exert a unique role in certain biological processes such as virus-plant interactions.

The present study deals with the enzyme mediated biotransformation of arsenic in five arsenic tolerant strains (Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Paenibacillus macerans and Escherichia coli). Biotransformation ability of these isolates was evaluated by monitoring arsenite oxidase and arsenate reductase activity. Results showed that arsenic oxidase activity was exclusively present in P. macerans and B. pumilus while B. subtilis, B. megaterium and E. coli strains showed presence of Arsenic oxido-reductase enzyme. The reversible nature of arsenic oxido- reductase suggested that same enzyme can carry out oxidation and reduction of arsenic depending upon the relative concentration of arsenic species. Lineweaver-Burk plot of the arsenite oxidase activity in P. macerans showed highest Km value (Km- 200 μM) and lower Vmax (0.012 μmol mg-1 protein min-1) indicating lowest affinity of the enzyme for arsenite. On the contrary, E. coli showed the lower Km value ( Km- 38.46 μM) and higher Vmax (0.044 μmol mg-1 protein min-1) suggesting for higher affinity for the arsenite. Lineweaver-Burk plot of arsenate reductase activity showed the presence of this enzyme in B. subtilis, B. megaterium and E. coli which were in the range of 200-360 μM Km and Vmax value between 0.256- 0.129 mmol mg-1 protein min-1. These results suggested that affinity of the as reductase enzyme is lowest for arsenate than that for the arsenite. Thus, arsenite oxidase system appears to be a predominant mechanism of cellular defense in these bacterial strains.

Competing Interests: The author has declared that no competing interests exist.

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice., Competing Interests: The authors have declared that no competing interests exist.