Genomic overview of INA-induced NPR1 targeting and transcriptional cascades in Arabidopsis.

The phytohormone salicylic acid (SA) triggers transcriptional reprogramming that leads to SA-induced immunity in plants. NPR1 is an SA receptor and master transcriptional regulator in SA-triggered transcriptional reprogramming. Despite the indispensable role of NPR1, genome-wide direct targets of NPR1 specific to SA signaling have not been identified. Here, we report INA (functional SA analog)-specific genome-wide targets of Arabidopsis NPR1 in plants expressing GFP-fused NPR1 under its native promoter. Analyses of NPR1-dependently expressed direct NPR1 targets revealed that NPR1 primarily activates genes encoding transcription factors upon INA treatment, triggering transcriptional cascades required for INA-induced transcriptional reprogramming and immunity. We identified genome-wide targets of a histone acetyltransferase, HAC1, including hundreds of co-targets shared with NPR1, and showed that NPR1 and HAC1 regulate INA-induced histone acetylation and expression of a subset of the co-targets. Genomic NPR1 targeting was principally mediated by TGACG-motif binding protein (TGA) transcription factors. Furthermore, a group of NPR1 targets mostly encoding transcriptional regulators was already bound to NPR1 in the basal state and showed more rapid and robust induction than other NPR1 targets upon SA signaling. Thus, our study unveils genome-wide NPR1 targeting, its role in transcriptional reprogramming, and the cooperativity between NPR1, HAC1, and TGAs in INA-induced immunity.

Arsenate reductase of Rufibacter tibetensis is a metallophosphoesterase evolved to catalyze redox reactions.

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T 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 Ni2+. 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.

PHYTOCHROME-INTERACTING FACTORS are involved in starch degradation adjustment via inhibition of the carbon metabolic regulator QUA-QUINE STARCH in Arabidopsis.

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.

Sustained defense response via volatile signaling and its epigenetic transcriptional regulation.

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 ß-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to ß-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 ß-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.

LEUNIG_HOMOLOG Mediates MYC2-Dependent Transcriptional Activation in Cooperation with the Coactivators HAC1 and MED25.

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.

Dissimilatory and Cytoplasmic Antimonate Reductions in a Hydrogen-Based Membrane Biofilm Reactor.

A hydrogen-based membrane biofilm reactor (H2-MBfR) was operated to investigate the bioreduction of antimonate [Sb(V)] in terms of Sb(V) removal, the fate of Sb, and the pathways of reduction metabolism. The MBfR achieved up to 80% Sb(V) removal and an Sb(V) removal flux of 0.55 g/m2·day. Sb(V) was reduced to Sb(III), which mainly formed Sb2O3 precipitates in the biofilm matrix, although some Sb(III) was retained intracellularly. High Sb(V) loading caused stress that deteriorated performance that was not recovered when the high Sb(V) loading was removed. The biofilm community consisted of DSbRB (dissimilatory Sb-reduction bacteria), SbRB (Sb-resistant bacteria), and DIRB (dissimilatory iron-reducing bacteria). Dissimilatory antimonate reduction, mediated by the respiratory arsenate reductase ArrAB, was the main reduction route, but respiratory reduction coexisted with cytoplasmic Sb(V)-reduction mediated by arsenate reductase ArsC. Increasing Sb(V) loading caused stress that led to increases in the expression of arsC gene and intracellular accumulation of Sb(III). By illuminating the roles of the dissimilatory and cytoplasmic Sb(V) reduction mechanism in the biofilms of the H2-MBfR, this study reveals that the Sb(V) loading should be controlled to avoid stress that deteriorates Sb(V) reduction.

Selenium Increased Arsenic Accumulation by Upregulating the Expression of Genes Responsible for Arsenic Reduction, Translocation, and Sequestration in Arsenic Hyperaccumulator Pteris vittata.

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 (AsV50) and 1.25 (Se1.25) or 5 µM (Se5) 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 Se1.25 was more effective than Se5 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 Se1.25 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 AsV50 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.

A New Strategy for As(V) Biosensing Based on the Inhibition of the Phosphatase Activity of the Arsenate Reductase from Thermus thermophilus.

Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are non-competitive inhibitors affecting the Vmax but not the KM of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 µM for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a sensitivity of 0.53 ± 0.03 mU/mg/µM and a limit of detection (LOD) of 0.28 ± 0.02 µM. The good sensitivity and specificity for As(V) point to consider inhibition of TtArsC phosphatase activity for the setup of a novel biosensor for the detection of As(V).

New Inhibitors of the Human p300/CBP Acetyltransferase Are Selectively Active against the Arabidopsis HAC Proteins.

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.

Structural and Functional Investigation of the Periplasmic Arsenate-Binding Protein ArrX from Chrysiogenes arsenatis.

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) 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.

Targeted expression of the arsenate reductase HAC1 identifies cell type specificity of arsenic metabolism and transport in plant roots.

High Arsenic Concentration 1 (HAC1), an Arabidopsis thaliana arsenate reductase, plays a key role in arsenate [As(V)] tolerance. Through conversion of As(V) to arsenite [As(III)], HAC1 enables As(III) export from roots, and restricts translocation of As(V) to shoots. To probe the ability of different root tissues to detoxify As(III) produced by HAC1, we generated A. thaliana lines expressing HAC1 in different cell types. We investigated the As(V) tolerance phenotypes: root growth, As(III) efflux, As translocation, and As chemical speciation. We showed that HAC1 can function in the outer tissues of the root (epidermis, cortex, and endodermis) to confer As(V) tolerance, As(III) efflux, and limit As accumulation in shoots. HAC1 is less effective in the stele at conferring As(V) tolerance phenotypes. The exception is HAC1 activity in the protoxylem, which we found to be sufficient to restrict As translocation, but not to confer As(V) tolerance. In conclusion, we describe cell type-specific functions of HAC1 that spatially separate the control of As(V) tolerance and As translocation. Further, we identify a key function of protoxylem cells in As(V) translocation, consistent with the model where endodermal passage cells, above protoxylem pericycle cells, form a 'funnel' loading nutrients and potentially toxic elements into the vasculature.

Biochemical, molecular and in silico characterization of arsenate reductase from Bacillus thuringiensis KPWP1 tolerant to salt, arsenic and a wide range of pH.

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the KM and Kcat values as 25 µM and 0.00119 s-1, respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism.

Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations.

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 (Km = 44.6 ± 1.6 µM, kcat = 9,810 ± 220 seconds-1), and phosphate is a competitive inhibitor of arsenate reduction (Ki = 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.

Variations of arsenic forms and the role of arsenate reductase in three hydrophytes exposed to different arsenic species.

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.

The CBP/p300 histone acetyltransferases function as plant-specific MEDIATOR subunits in Arabidopsis.

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.

Isofunctional Clustering and Conformational Analysis of the Arsenate Reductase Superfamily Reveals Nine Distinct Clusters.

Arsenate reductase (ArsC) is a superfamily of enzymes that reduce arsenate. Due to active site similarities, some ArsC can function as low-molecular weight protein tyrosine phosphatases (LMW-PTPs). Broad superfamily classifications align with redox partners (Trx- or Grx-linked). To understand this superfamily's mechanistic diversity, the ArsC superfamily is classified on the basis of active site features utilizing the tools TuLIP (two-level iterative clustering process) and autoMISST (automated multilevel iterative sequence searching technique). This approach identified nine functionally relevant (perhaps isofunctional) protein groups. Five groups exhibit distinct ArsC mechanisms. Three are Grx-linked: group 4AA (classical ArsC), group 3AAA (YffB-like), and group 5BAA. Two are Trx-linked: groups 6AAAAA and 7AAAAAAAA. One is an Spx-like transcriptional regulatory group, group 5AAA. Three are potential LMW-PTP groups: groups 7BAAAA, and 7AAAABAA, which have not been previously identified, and the well-studied LMW-PTP family group 8AAA. Molecular dynamics simulations were utilized to explore functional site details. In several families, we confirm and add detail to literature-based mechanistic information. Mechanistic roles are hypothesized for conserved active site residues in several families. In three families, simulations of the unliganded structure sample specific conformational ensembles, which are proposed to represent either a more ligand-binding-competent conformation or a pathway toward a more binding-competent state; these active sites may be designed to traverse high-energy barriers to the lower-energy conformations necessary to more readily bind ligands. This more detailed biochemical understanding of ArsC and ArsC-like PTP mechanisms opens possibilities for further understanding of arsenate bioremediation and the LMW-PTP mechanism.

Improving Arsenic Tolerance of Pyrococcus furiosus by Heterologous Expression of a Respiratory Arsenate Reductase.

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.

Genetic Identification of Antimonate Respiratory Reductase in Shewanella sp. ANA-3.

Microbial antimonate [Sb(V)] respiratory reduction is an important process regulating Sb redox transformation in the environment. However, little is known about the microbial respiratory reductase for Sb(V). Herein, we report Sb(V)-respiring reduction by Shewanella sp. ANA-3 through an arsenate respiratory reductase encoded by arrAB. Incubation experiments showed that Shewanella sp. ANA-3 mediated Sb(V)-respiring reduction, which was dependent on the cell concentration. Both protein analysis and reverse transcriptase-polymerase chain reaction results revealed that arrAB was highly expressed in Sb(V)-respiring reduction. In vivo evidence with mutants indicated that neither ANA-3-ΔarrA nor ANA-3-ΔarrB was capable of reducing Sb(V) as efficiently as the wild type, whereas complementation by the wild-type sequences of arrA and arrB rescued the mutants' ability. Our in vitro results showed that ArrAB purified by His-Tag was able to mediate Sb(V) reduction, though with much suppressed catalytic kinetics compared with As(V) reduction. The cell-concentration-dependent reduction of Sb(V) was regulated by quorum sensing via the luxS gene. This study opens a new chapter in the mechanistic understanding of microbial Sb(V) respiratory reduction.

Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin.

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.

Yeast strain Debaryomyces hansenii for amelioration of arsenic stress in rice.

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.