Novel in silico screening system for plant defense activators using deep learning-based prediction of reactive oxygen species accumulation.

BACKGROUND: Plant defense activators offer advantages over pesticides by avoiding the emergence of drug-resistant pathogens. However, only a limited number of compounds have been reported. Reactive oxygen species (ROS) act as not only antimicrobial agents but also signaling molecules that trigger immune responses. They also affect various cellular processes, highlighting the potential ROS modulators as plant defense activators. Establishing a high-throughput screening system for ROS modulators holds great promise for identifying lead chemical compounds with novel modes of action (MoAs). RESULTS: We established a novel in silico screening system for plant defense activators using deep learning-based predictions of ROS accumulation combined with the chemical properties of the compounds as explanatory variables. Our screening strategy comprised four phases: (1) development of a ROS inference system based on a deep neural network that combines ROS production data in plant cells and multidimensional chemical features of chemical compounds; (2) in silico extensive-scale screening of seven million commercially available compounds using the ROS inference model; (3) secondary screening by visualization of the chemical space of compounds using the generative topographic mapping; and (4) confirmation and validation of the identified compounds as potential ROS modulators within plant cells. We further characterized the effects of selected chemical compounds on plant cells using molecular biology methods, including pathogenic signal-triggered enzymatic ROS induction and programmed cell death as immune responses. Our results indicate that deep learning-based screening systems can rapidly and effectively identify potential immune signal-inducible ROS modulators with distinct chemical characteristics compared with the actual ROS measurement system in plant cells. CONCLUSIONS: We developed a model system capable of inferring a diverse range of ROS activity control agents that activate immune responses through the assimilation of chemical features of candidate pesticide compounds. By employing this system in the prescreening phase of actual ROS measurement in plant cells, we anticipate enhanced efficiency and reduced pesticide discovery costs. The in-silico screening methods for identifying plant ROS modulators hold the potential to facilitate the development of diverse plant defense activators with novel MoAs.

Regulation of PaRBOH1-mediated ROS production in Norway spruce by Ca2+ binding and phosphorylation.

Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H2O2, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca2+ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.

Cell Cycle-Dependence of Autophagic Activity and Inhibition of Autophagosome Formation at M Phase in Tobacco BY-2 Cells.

Autophagy is ubiquitous in eukaryotic cells and plays an essential role in stress adaptation and development by recycling nutrients and maintaining cellular homeostasis. However, the dynamics and regulatory mechanisms of autophagosome formation during the cell cycle in plant cells remain poorly elucidated. We here analyzed the number of autophagosomes during cell cycle progression in synchronized tobacco BY-2 cells expressing YFP-NtATG8a as a marker for the autophagosomes. Autophagosomes were abundant in the G2 and G1 phases of interphase, though they were much less abundant in the M and S phases. Autophagosomes drastically decreased during the G2/M transition, and the CDK inhibitor roscovitine inhibited the G2/M transition and the decrease in autophagosomes. Autophagosomes were rapidly increased by a proteasome inhibitor, MG-132. MG-132-induced autophagosome formation was also markedly lower in the M phases than during interphase. These results indicate that the activity of autophagosome formation is differently regulated at each cell cycle stage, which is strongly suppressed during mitosis.

Impact of Autophagy on Gene Expression and Tapetal Programmed Cell Death During Pollen Development in Rice.

Autophagy has recently been shown to be required for tapetal programmed cell death (PCD) and pollen maturation in rice. A transcriptional regulatory network is also known to play a key role in the progression of tapetal PCD. However, the relationship between the gene regulatory network and autophagy in rice anther development is mostly unknown. Here, we comprehensively analyzed the effect of autophagy disruption on gene expression profile during the tapetal PCD in rice anther development using high-throughput RNA sequencing. Expression of thousands of genes, including specific transcription factors and several proteases required for tapetal degradation, fluctuated synchronously at specific stages during tapetal PCD progression in the wild-type anthers, while this fluctuation showed significant delay in the autophagy-deficient mutant Osatg7-1. Moreover, gene ontology enrichment analysis in combination with self-organizing map clustering as well as pathway analysis revealed that the expression patterns of a variety of organelle-related genes as well as genes involved in carbohydrate/lipid metabolism were affected in the Osatg7-1 mutant during pollen maturation. These results suggest that autophagy is required for proper regulation of gene expression and quality control of organelles and timely progression of tapetal PCD during rice pollen development.

Essential roles of autophagy in metabolic regulation in endosperm development during rice seed maturation.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.

Monitoring autophagy in rice tapetal cells during pollen maturation.

We have previously shown that autophagy is required for post meiotic anther development including programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. However, the spatiotemporal dynamics of autophagy in the tapetum remain poorly understood. We here established an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum cells by expressing green fluorescent protein-tagged AtATG8, a marker for autophagosomes. 3D-imaging analysis revealed that the number of autophagosomes/autophagy-related structures is extremely low at the tetrad stage (stage 8), and autophagy is dramatically induced at the uninucleate stages (stage 9-10) throughout the tapetal cells during anther development. The present monitoring system for autophagy offers a powerful tool to analyze the regulation of autophagy in rice tapetal cells during pollen maturation.

Functional screening of salt tolerance genes from a halophyte Sporobolus virginicus and transcriptomic and metabolomic analysis of salt tolerant plants expressing glycine-rich RNA-binding protein.

Sporobolus virginicus is a halophytic C4 grass found worldwide, from tropical to warm temperate regions. One Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a three-fold higher concentration than the salinity of sea water. To identify the key genes involved in the regulation of salt tolerance in S. virginicus, we produced 3500 independent transgenic Arabidopsis lines expressing random cDNA from S. virginicus and screened 10 lines which showed enhanced salt tolerance compared with the wild type in a medium containing 150 mM NaCl. Among the selected lines, two contained cDNA coding glycine-rich RNA-binding proteins (SvGRP1 and SvGRP2). This is the first reports on the function of GRPs from halophytes in salt tolerance though reports have shown GRPs are involved in diverse biological and biochemical processes including salt tolerance in Arabidopsis and some other glycophytes. Transcriptomic analysis and GO enrichment analysis of SvGRP1-expressing Arabidopsis under salt stress revealed upregulation of polyol and downregulation of glucosinolate and indole acetic acid biosynthesis/metabolic pathways. Metabolomic analysis of the SvGRP1-transformant suggested that the increase in 3-aminoppropanoic acid, citramalic acid, and isocitric acid content was associated with enhanced salt tolerance. These findings could provide novel insight into the roles of GRPs in plant salt tolerance.

High-Affinity K+ Transporters from a Halophyte, Sporobolus virginicus, Mediate Both K+ and Na+ Transport in Transgenic Arabidopsis, X. laevis Oocytes and Yeast.

Class II high-affinity potassium transporters (HKTs) have been proposed to mediate Na+-K+ co-transport in plants, as well as Na+ and K+ homeostasis under K+-starved and saline environments. We identified class II HKTs, namely SvHKT2;1 and SvHKT2;2 (SvHKTs), from the halophytic turf grass, Sporobolus virginicus. SvHKT2;2 expression in S. virginicus was up-regulated by NaCl treatment, while SvHKT2;1 expression was assumed to be up-regulated by K+ starvation and down-regulated by NaCl treatment. Localization analysis revealed SvHKTs predominantly targeted the plasma membrane. SvHKTs complemented K+ uptake deficiency in mutant yeast, and showed both inward and outward K+ and Na+ transport activity in Xenopus laevis oocytes. When constitutively expressed in Arabidopsis, SvHKTs mediated K+ and Na+ accumulation in shoots under K+-starved conditions, and the K+ concentration in xylem saps of transformants was also higher than in those of wild-type plants. These results suggest transporter-enhanced K+ and Na+ uploading to the xylem from xylem parenchyma cells. Together, our data demonstrate that SvHKTs mediate both outward and inward K+ and Na+ transport in X. laevis oocytes, and possibly in plant and yeast cells, depending on the ionic conditions.

Involvement of S-type anion channels in disease resistance against an oomycete pathogen in Arabidopsis seedlings.

Pharmacological indications suggest that anion channel-mediated plasma membrane (PM) anion efflux is crucial in early defense signaling to induce immune responses and programmed cell death in plants. Arabidopsis SLAC1, an S-type anion channel required for stomatal closure, is involved in cryptogein-induced PM Cl- efflux to positively modulate the activation of other ion fluxes, production of reactive oxygen species and a wide range of defense responses including hypersensitive cell death in tobacco BY-2 cells. We here analyzed disease resistance against several pathogens in multiple mutants of the SLAC/SLAH channels of Arabidopsis. Resistance against a biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 was significantly enhanced in the SLAC1-overexpressing plants than in the wild-type, while that against a bacteria Pseudomonas syringae was not affected significantly. Possible regulatory roles of S-type anion channels in plant immunity and disease resistance against bacterial and oomycete pathogens is discussed.

Autophagy-mediated regulation of phytohormone metabolism during rice anther development.

Autophagy has recently been shown to be required for postmeiotic anther development including anther dehiscence, programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. Several phytohormones are known to play essential roles during male reproductive development including pollen maturation. However, the relationship between phytohormone metabolism and autophagy in plant reproductive development is unknown. We here comprehensively analyzed the effect of autophagy disruption on phytohormone contents in rice anthers at the flowering stage, and found that endogenous levels of active-forms of gibberellins (GAs) and cytokinin, trans-zeatin, were significantly lower in the autophagy-defective mutant, Osatg7-1, than in the wild type. Treatment with GA4 partially recovered maturation of the mutant pollens, but did not recover the limited anther dehiscence as well as sterility phenotype. These results suggest that autophagy affects metabolism and endogenous levels of GAs and cytokinin in rice anthers. Reduction in bioactive GAs in the autophagy-deficient mutant may partially explain the defects in pollen maturation of the autophagy-deficient mutant, but tapetal autophagy also plays other specific roles in fertilization.

Autophagy, programmed cell death and reactive oxygen species in sexual reproduction in plants.

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.

Plant signaling networks involving Ca(2+) and Rboh/Nox-mediated ROS production under salinity stress.

Salinity stress, which induces both ionic and osmotic damage, impairs plant growth and causes severe reductions in crop yield. Plants are equipped with defense responses against salinity stress such as regulation of ion transport including Na(+) and K(+), accumulation of compatible solutes and stress-related gene expression. The initial Ca(2+) influx mediated by plasma membrane ion channels has been suggested to be crucial for the adaptive signaling. NADPH oxidase (Nox)-mediated production of reactive oxygen species (ROS) has also been suggested to play crucial roles in regulating adaptation to salinity stress in several plant species including halophytes. Respiratory burst oxidase homolog (Rboh) proteins show the ROS-producing Nox activity, which are synergistically activated by the binding of Ca(2+) to EF-hand motifs as well as Ca(2+)-dependent phosphorylation. We herein review molecular identity, structural features and roles of the Ca(2+)-permeable channels involved in early salinity and osmotic signaling, and comparatively discuss the interrelationships among spatiotemporal dynamic changes in cytosolic concentrations of free Ca(2+), Rboh-mediated ROS production, and downstream signaling events during salinity adaptation in planta.

Comprehensive analysis of transcriptome response to salinity stress in the halophytic turf grass Sporobolus virginicus.

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H(+)-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes.

Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice.

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15)N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

Roles of autophagy in male reproductive development in plants.

Autophagy, a major catabolic pathway in eukaryotic cells, is essential in development, maintenance of cellular homeostasis, immunity and programmed cell death (PCD) in multicellular organisms. In plant cells, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes such as abiotic and biotic stress responses. However, its roles during reproductive development had remained poorly understood. Quantitative live cell imaging techniques for the autophagic flux and genetic studies in several plant species have recently revealed significant roles of autophagy in developmental processes, regulation of PCD and lipid metabolism. We here review the novel roles of autophagic fluxes in plant cells, and discuss their possible significance in PCD and metabolic regulation, with particular focus on male reproductive development during the pollen maturation.

Growth and physiological adaptation of whole plants and cultured cells from a halophyte turf grass under salt stress.

Understanding the mechanisms used by halophytic members of the Poaceae to cope with salt stress will contribute to the knowledge necessary to genetically engineer salt-tolerant crops. In this study, we identified a genotype of Sporobolus virginicus, a halophytic turf grass collected in Japan, and investigated its growth rate, ion concentration and secretion, and proline concentration in comparison with the reported properties of genotypes collected from the USA, South Africa and Egypt. Surprisingly, the Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a 3-fold higher concentration than seawater salinity. Shoot growth was stimulated by 100 mM NaCl and root growth was stimulated at salinities of up to 1 M NaCl. Accumulation of Na(+) and CI(-) in shoots and roots was rapidly elevated by salinity stress but did not exceed the levels required for osmotic adjustment, due in part to ion secretion by salt glands, which are present in genotypes of S. virginicus. However, the Japanese genotypes accumulated K(+) to a higher level than other genotypes, resulting in a relatively high K(+)/Na(+) ratio even under salinity stress. An increase in proline concentration was observed that was proportional to the NaCl concentration in the culture solution and might partially account for osmotic adjustment in the shoots. We also generated and characterized cultured cells of S. virginicus. In 500 mM NaCl, the cultured cells showed an enhanced growth compared with cultured cells of rice. The concentration of Na(+) and CI(-) in the cultured cells in 300-500 mM NaCl was lower than in 100 mM NaCl. Cultured cells of S. virginicus accumulated proline to higher levels than rice cells cultured under salinity stress. The active regulation of Na(+), Cl(-) and K(+) influx/efflux and proline accumulation might be involved in salt tolerance mechanisms at the cellular level as well as in planta.

Involvement of elevated proline accumulation in enhanced osmotic stress tolerance in Arabidopsis conferred by chimeric repressor gene silencing technology.

Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PHD) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PHD gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development.

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

An S-type anion channel SLAC1 is involved in cryptogein-induced ion fluxes and modulates hypersensitive responses in tobacco BY-2 cells.

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl(-) and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl(-) efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl(-) efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.