Arabidopsis Enzyme Research Articles

Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

Guanidine production by plant homoarginine-6-hydroxylases

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

Droplet microfluidics for time-resolved serial crystallography.

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.

Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana.

The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering.

Suboptimal zinc supply affects the S-nitrosoglutathione reductase enzyme and nitric oxide signaling in Arabidopsis

The S-nitrosoglutathione reductase (GSNOR), a key regulator of nitric oxide (NO) signaling, requires zinc (Zn) cofactors for its catalytic activity. Possible changes in GSNOR function and NO signaling due to limited Zn supply and participation of GSNOR in response to Zn deficiency have not yet been examined. The wild-type Arabidopsis thaliana seedlings (Col-0) and the overproducer of GSNOR (35S::FLAG-GSNOR1) were grown in agar medium supplemented with 15 µM (full Zn), 1.5 µM (Zn/10) and 0 µM (Zn0) zinc sulfate. Limited availability of Zn in the nutrient medium during the experimental period resulted in a significantly reduced Zn content in the roots and shoots of Arabidopsis and altered the expression of Zn deficiency marker genes; however, the Zn content did not reflect Zn deficiency in the seedlings. GSNOR has no effect on the Zn limitation-induced reduction in the in planta Zn content. Suboptimal Zn supply results in a smaller but more active GSNOR protein pool in Col-0 A. thaliana. In 35S::FLAG-GSNOR1, overproduction possibly compensates for the negative effects of reduced Zn supply, resulting in unchanged GSNOR expression, protein abundance, and activity. Suboptimal Zn supply alters NO but did not modify peroxynitrite and hydrogen peroxide levels and exerts a pronitrant effect in the plant proteome. Overall, this is the first report to show that limited Zn supply affects the GSNOR enzyme at the gene and protein levels but not at the activity level and modifies reactive species levels and physiological nitroproteome in Arabidopsis.

The Function of Deubiquitinating Enzymes in Arabidopsis: Recent Progress of Ubiquitin-Specific Proteases (UBPs)

The Function of Deubiquitinating Enzymes in Arabidopsis: Recent Progress of Ubiquitin-Specific Proteases (UBPs)

The Functional Relationship between NADPH Thioredoxin Reductase C, 2-Cys Peroxiredoxins, and m-Type Thioredoxins in the Regulation of Calvin-Benson Cycle and Malate-Valve Enzymes in Arabidopsis.

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m are also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, growth retardation being noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin-Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system.

Distinctly localized lipid phosphate phosphatases mediate endoplasmic reticulum glycerolipid metabolism in Arabidopsis.

Inter-organelle communication is an integral subcellular process in cellular homeostasis. In plants, cellular membrane lipids are synthesized in the plastids and endoplasmic reticulum (ER). However, the crosstalk between these organelles in lipid biosynthesis remains largely unknown. Here, we show that a pair of lipid phosphate phosphatases (LPPs) with differential subcellular localizations is required for ER glycerolipid metabolism in Arabidopsis (Arabidopsis thaliana). LPPα2 and LPPε1, which function as phosphatidic acid phosphatases and thus catalyze the core reaction in glycerolipid metabolism, were differentially localized at ER and chloroplast outer envelopes despite their similar tissue expression pattern. No mutant phenotype was observed in single knockout mutants; however, genetic suppression of these LPPs affected pollen growth and ER phospholipid biosynthesis in mature siliques and seeds with compromised triacylglycerol biosynthesis. Although chloroplast-localized, LPPε1 was localized close to the ER and ER-localized LPPα2. This proximal localization is functionally relevant, because overexpression of chloroplastic LPPε1 enhanced ER phospholipid and triacylglycerol biosynthesis similar to the effect of LPPα2 overexpression in mature siliques and seeds. Thus, ER glycerolipid metabolism requires a chloroplast-localized enzyme in Arabidopsis, representing the importance of inter-organelle communication in membrane lipid homeostasis.

Plant Glutathione Peroxidases: Non-Heme Peroxidases with Large Functional Flexibility as a Core Component of ROS-Processing Mechanisms and Signalling.

Glutathione peroxidases (GPXs) are non-heme peroxidases catalyzing the reduction of H2O2 or organic hydroperoxides to water or corresponding alcohols using glutathione (GSH) or thioredoxin (TRX) as a reducing agent. In contrast to animal GPXs, the plant enzymes are non-seleno monomeric proteins that generally utilize TRX more effectively than GSH but can be a putative link between the two main redox systems. Because of the substantial differences compared to non-plant GPXs, use of the GPX-like (GPXL) name was suggested for Arabidopsis enzymes. GPX(L)s not only can protect cells from stress-induced oxidative damages but are crucial components of plant development and growth. Due to fine-tuning the H2O2 metabolism and redox homeostasis, they are involved in the whole life cycle even under normal growth conditions. Significantly new mechanisms were discovered related to their transcriptional, post-transcriptional and post-translational modifications by describing gene regulatory networks, interacting microRNA families, or identifying Lys decrotonylation in enzyme activation. Their involvement in epigenetic mechanisms was evidenced. Detailed genetic, evolutionary, and bio-chemical characterization, and comparison of the main functions of GPXs, demonstrated their species-specific roles. The multisided involvement of GPX(L)s in the regulation of the entire plant life ensure that their significance will be more widely recognized and applied in the future.

Expression of Physaria longchain acyl-CoA synthetases and hydroxy fatty acid accumulation in transgenic Arabidopsis

Hydroxy fatty acids (HFA) are industrially useful chemical feedstocks that accumulate in seed-storage triacylglycerols (TAG) of several plant species, including castor (Ricinus communis) and Physaria (Physaria fendleri). For researchers, HFA also offer a unique opportunity to trace fatty acid metabolism and modification. Past work producing HFA in Arabidopsis (Arabidopsis thaliana) has demonstrated the importance of isozymes of TAG synthesis from plants that evolved to store HFA and as a result have a high degree of specificity towards HFA substrates. Castor phospholipase A2α (RcPLA2) has specificity for HFA-containing phosphatidylcholine. However, expression of RcPLA2 in HFA-accumulating Arabidopsis line CL37-PLA2 reduced HFA content of TAG. This loss was interpreted as being due to poor ability of Arabidopsis longchain acyl-CoA synthetases (LACSs) to utilize HFAs substrates. LACS enzymes are essential to activate HFA to HFA-CoA for TAG synthesis. Physaria is a close relative of Arabidopsis in the Brassicaceae family. To test the hypothesis that this close relatedness would allow Physaria LACSs to interface successfully with Arabidopsis enzymes of seed lipid metabolism and thereby restore HFA accumulation, we transformed PfLACS4 and PfLACS8 constructs into the CL37-PLA2 line. However, HFA content was not recovered, and biochemical characterization of recombinant PfLACS4 and PfLACS8 indicated that these isozymes have substrate specificities and selectivities that are similar to their Arabidopsis orthologues. These and other results pose an important question about how HFA synthesized on phosphatidylcholine can be transferred into the acyl-CoA pool for TAG synthesis.

Antimony-oxidizing bacteria alleviate Sb stress in Arabidopsis by attenuating Sb toxicity and reducing Sb uptake

Antimony (Sb)-oxidizing bacteria play an important role in Sb biogeochemical cycle in soil, but the benefits of microbial oxidation for plants have not been well documented. The aim of this study was to explore the contribution of Sb(III)-oxidizing bacteria to alleviate the Sb toxicity in plants. The plant growth-promoting (PGP) characteristics of Sb(III)-oxidizing bacterium Bacillus sp. S3 and the effects of bacterial inoculation on Arabidopsis plants were evaluated under controlled and Sb stressed conditions. Indole acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate-deaminase (ACC-deaminase) activity were the only two PGP strategies that Bacillus sp. S3 possessed, despite the production level of IAA and the activity of ACC deaminase sharply decreased under Sb stress. Bacillus sp. S3 inoculation significantly increased the plant biomass and chlorophyll content, alleviated the peroxidation of membrane lipids, decreased the activities of the antioxidant enzymes, and reduced the transcription of Sb transporters and reactive oxygen species (ROS)-related enzymes in Arabidopsis. Noteworthily, inoculation of Bacillus sp. S3 not only significantly decreased the Sb accumulation but also reduced the percentage of Sb(III) of total Sb in Arabidopsis. This study indicates that the Sb(III)-oxidizing strain of Bacillus sp. S3 is a promising inoculant used for bacteria-assisted phytoremediation on Sb-contaminated sites.

Functional Features of TREHALOSE-6-PHOSPHATE SYNTHASE1, an Essential Enzyme in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4.

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5′ end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5′ ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3′ fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.

The identification of small molecule inhibitors of the plant inositol phosphorylceramide synthase which demonstrate herbicidal activity

Resistance to 157 different herbicides and 88% of known sites of action has been observed, with many weeds resistant to two or more modes. Coupled with tighter environmental regulation, this demonstrates the need to identify new modes of action and novel herbicides. The plant sphingolipid biosynthetic enzyme, inositol phosphorylceramide synthase (IPCS), has been identified as a novel, putative herbicide target. The non-mammalian nature of this enzyme offers the potential of discovering plant specific inhibitory compounds with minimal impact on animals and humans, perhaps leading to the development of new non-toxic herbicides. The best characterised and most highly expressed isoform of the enzyme in the model-dicot Arabidopsis, AtIPCS2, was formatted into a yeast-based assay which was then utilized to screen a proprietary library of over 11,000 compounds provided by Bayer AG. Hits from this screen were validated in a secondary in vitro enzyme assay. These studies led to the identification of a potent inhibitor that showed selectivity for AtIPCS2 over the yeast orthologue, and activity against Arabidopsis seedlings. This work highlighted the use of a yeast-based screening assay to discover herbicidal compounds and the status of the plant IPCS as a novel herbicidal target.

The metabolite repair enzyme Nit1 is a dual-targeted amidase that disposes of damaged glutathione in Arabidopsis.

The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 mM range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant Arabidopsis Nit1 (At4g08790) has high and specific amidase activity towards dGSH. Ablating the Arabidopsis Nit1 gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays show that both potential translation start codons in Arabidopsis Nit1 were used and confocal microscopy of Nit1-GFP fusions in plant cells confirmed both cytoplasmic and plastidial localization. Furthermore, we show that Arabidopsis enzymes present in leaf extracts convert GSH to dGSH at a rate of 2.8 pmol min-1 mg-1 in the presence of glyoxalate as an amino acceptor. Our data demonstrate that plants have a dGSH repair system that is directed to at least two cellular compartments via the use of alternative translation start sites.

CYSTM3 negatively regulates salt stress tolerance in Arabidopsis.

CYSTM3, a small mitochondrial protein, acts as a negative regulator in salt stress response by preventing Na+ efflux and disturbing reactive oxygen species (ROS) homeostasis in Arabidopsis. Cysteine-rich transmembrane module (CYSTM) is a not well characterized small peptide family in plants. In this study, we identified a novel mitochondrion-localized CYSTM member CYSTM3 from Arabidopsis, which was ubiquitously expressed in different tissues and dramatically induced by salt stress. Transgenic plants overexpressing CYSTM3 (OE) displayed hypersensitivity to salt stress compared with wild type (WT) plants, whereas a knockout mutant cystm3 was more tolerant to high salinity than WT. Moreover, OE lines accumulated higher contents of Na+ and ROS than WT and cystm3 upon exposure to high salinity. Further analysis revealed that CYSTM3 could deter root Na+ efflux and inhibit the activities of a range of ROS scavenging enzymes in Arabidopsis. In addition, the transcripts of nuclear salt stress-responsive genes were over-activated in cystm3 than those in WT and OE lines. Taken together, Arabidopsis CYSTM3 acts as a negative regulator in salt stress tolerance.

Four Arabidopsis berberine bridge enzyme-like proteins are specific oxidases that inactivate the elicitor-active oligogalacturonides.

Recognition of endogenous molecules acting as 'damage-associated molecular patterns' (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper-accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth-defence trade-off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper-accumulation. By combining affinity chromatography on acrylamide-trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo-protein, OGOX1 is a sulphite-sensitive H2 O2 -producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD-binding proteins [Berberine Bridge Enzyme-like (BBE-like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE-like enzymes in Arabidopsis are OG oxidases (OGOX1-4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.

Changes by cadmium stress in lipid peroxidation and activities of lipoxygenase and antioxidant enzymes in Arabidopsis are associated with extracellular ATP

AbstractExposure of Arabidopsis leaves to cadmium (Cd) stress led to an increase in the level of extracellular ATP (eATP). And, Cd stress also caused significant increases in lipid peroxidation and the activities of lipoxygenase (LOX) and antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT). Treatment of Arabidopsis leaves with