Oxidative Signal-inducible 1 Research Articles

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

OXIDATIVE SIGNAL-INDUCIBLE1 induces immunity by coordinating N-hydroxypipecolic acid, salicylic acid, and camalexin synthesis.

Expression of OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1) is induced by a number of stress conditions and regulates the interaction of plants with pathogenic and beneficial microbes. In this work, we generated Arabidopsis OXI1 knockout and genomic OXI1 overexpression lines and show by transcriptome, proteome, and metabolome analysis that OXI1 triggers ALD1, SARD4, and FMO1 expressions to promote the biosynthesis of pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP). OXI1 contributes to enhanced immunity by induced SA biosynthesis via CBP60g-induced expression of SID2 and camalexin accumulation via WRKY33-targeted transcription of PAD3. OXI1 regulates genes involved in reactive oxygen species (ROS) generation such as RbohD and RbohF. OXI1 knock out plants show enhanced expression of nuclear and chloroplast genes of photosynthesis and enhanced growth under ambient conditions, while OXI1 overexpressing plants accumulate NHP, SA, camalexin, and ROS and show a gain-of-function (GOF) cell death phenotype and enhanced pathogen resistance. The OXI1 GOF phenotypes arecompletely suppressed when compromising N-hydroxypipecolic acid (NHP) synthesis in the fmo1 or ald1 background, showing that OXI1 regulation of immunity is mediated via the NHP pathway. Overall, these results show that OXI1 plays a key role in basal and effector-triggered plant immunity by regulating defense and programmed cell death via biosynthesis of salicylic acid, N-hydroxypipecolic acid, and camalexin.

Dimethylthiourea Alleviates Drought Stress by Suppressing Hydrogen Peroxide-Dependent Abscisic Acid-Mediated Oxidative Responses in an Antagonistic Interaction with Salicylic Acid in Brassica napus Leaves.

In plants, prolonged drought induces oxidative stress, leading to a loss of reducing potential in redox components. Abscisic acid (ABA) is a representative hormonal signal regulating stress responses. This study aimed to investigate the physiological significance of dimethylthiourea (DMTU, an H2O2 scavenger) in the hormonal regulation of the antioxidant system and redox control in rapeseed (Brassica napus L.) leaves under drought stress. Drought treatment for 10 days provoked oxidative stress, as evidenced by the increase in O2•- and H2O2 concentrations, and lipid peroxidation levels, and a decrease in leaf water potential. Drought-induced oxidative responses were significantly alleviated by DMTU treatment. The accumulation of O2•- and H2O2 in drought-treated plants coincided with the enhanced expression of the NADPH oxidase and Cu/Zn-SOD genes, leading to an up-regulation in oxidative signal-inducible 1 (OXI1) and mitogen-activated protein kinase 6 (MAPK6), with a concomitant increase in ABA levels and the up-regulation of ABA-related genes. DMTU treatment under drought largely suppressed the drought-responsive up-regulation of these genes by depressing ABA responses through an antagonistic interaction with salicylic acid (SA). DMTU treatment also alleviated the drought-induced loss of reducing potential in GSH- and NADPH-based redox by the enhanced expression of glutathione reductase 1 (GR1) and up-regulation of oxidoreductase genes (TRXh5 and GRXC9). These results indicate that DMTU effectively alleviates drought-induced oxidative responses by suppressing ABA-mediated oxidative burst signaling in an antagonistic regulation of SA.

The apple MdPTI1L kinase is phosphorylated by MdOXI1 during S-RNase-induced reactive oxygen species signaling in pollen tubes

Apple (Malus domestica) exhibits classic S-RNase-mediated gametophytic self-incompatibility. Previous studies have shown that the S-RNase secreted from style cells could trigger signal transduction and defense responses mediated by Ca2+ and reactive oxygen species (ROS) after entering into the pollen tube. In this study, we investigated the downstream genes activated by ROS during S-RNase-mediated gametophytic self-incompatibility in pollen tubes. A substantial increase in ROS, as well as up-regulated expression of a serine-threonine protein kinase gene, OXIDATIVE SIGNAL-INDUCIBLE1 (MdOXI1), was detected in apple pollen tubes treated with self-S-RNase. A kinase assay-linked phosphoproteomics (KALIP) analysis suggested that MdOXI1 could bind and phosphorylate the downstream protein kinase Pto-interacting protein 1-like (MdPTI1L). The phosphorylation level of MdPTI1L was significantly reduced after silencing MdOXI1 with antisense oligonucleotides in the pollen tube. Silencing of either MdOXI1 or MdPTI1L alleviated the inhibitory effect of self-S-RNase on pollen tube growth. Our results thus indicate that MdPTI1L is phosphorylated by MdOXI1 in the pollen tube and participates in the ROS signaling pathway triggered by S-RNase.

The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome-derived signaling in plant development.

Hydrogen peroxide (H2 O2 ) is generated in many metabolic processes. As a signaling molecule, H2 O2 plays important roles in plant growth and development, as well as environmental stress response. In Arabidopsis, there are three catalase genes, CAT1, CAT2, and CAT3. The encoded catalases are predominately peroxisomal proteins and are critical for scavenging H2 O2 . Since CAT1 and CAT3 are linked on chromosome 1, it has been almost impossible to generate cat1/3 and cat1/2/3 mutants by traditional genetic tools. In this study, we constructed cat1/3 double mutants and cat1/2/3 triple mutants by CRISPR/Cas9 to investigate the role of catalases. The cat1/2/3 triple mutants displayed severe redox disturbance and growth defects under physiological conditions compared with wild-type and the cat2/3 double mutants. Transcriptome analysis showed a more profound transcriptional response in the cat1/2/3 triple mutants compared to the cat2/3 mutants. These differentially expressed genes are involved in plant growth regulation as well as abiotic and biotic stress responses. In addition, expression of OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1) and several MAPK cascade genes were changed dramatically in the catalase triple mutant, suggesting that H2 O2 produced in peroxisomes could serve as a peroxisomal retrograde signal.

Melatonin is required for H2 O2 - and NO-mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana.

Melatonin influences plant innate immunity through the mitogen-activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)-knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal-inducible1) kinases are responsible for triggering melatonin-induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H2 O2 and NO individually, but dependent on the combination of H2 O2 and NO. Moreover, melatonin-mediated induction of the expression of defense-related genes, such as PR1 and ICS1, was not altered in the H2 O2 -deficient rbohD/F-knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin-mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.

MARIS plays important roles in Arabidopsis pollen tube and root hair growth.

In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes. Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood. In this study, we characterized the receptor-like cytoplasmic protein kinase (RLCK) gene, MARIS (MRI) that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tube-specific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXI1 in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXI1.

Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase.

Studies of the singlet oxygen ((1)O2)-overproducing flu and chlorina1 (ch1) mutants of Arabidopsis (Arabidopsis thaliana) have shown that (1)O2-induced changes in gene expression can lead to either programmed cell death (PCD) or acclimation. A transcriptomic analysis of the ch1 mutant has allowed the identification of genes whose expression is specifically affected by each phenomenon. One such gene is OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) encoding an AGC kinase that was noticeably induced by excess light energy and (1)O2 stress conditions leading to cell death. Photo-induced oxidative damage and cell death were drastically reduced in the OXI1 null mutant (oxi1) and in the double mutant ch1*oxi1 compared with the wild type and the ch1 single mutant, respectively. This occurred without any changes in the production rate of (1)O2 but was cancelled by exogenous applications of the phytohormone jasmonate. OXI1-mediated (1)O2 signaling appeared to operate through a different pathway from the previously characterized OXI1-dependent response to pathogens and H2O2 and was found to be independent of the EXECUTER proteins. In high-light-stressed plants, the oxi1 mutation was associated with reduced jasmonate levels and with the up-regulation of genes encoding negative regulators of jasmonate signaling and PCD. Our results show that OXI1 is a new regulator of (1)O2-induced PCD, likely acting upstream of jasmonate.

The OXI1 Kinase Pathway Mediates Piriformospora indica-Induced Growth Promotion in Arabidopsis

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

The Arabidopsis protein kinase Pto‐interacting 1‐4 is a common target of the oxidative signal‐inducible 1 and mitogen‐activated protein kinases

In Arabidopsis thaliana, the serine/threonine protein kinase oxidative signal-inducible 1 (OXI1), mediates oxidative stress signalling. Its activity is required for full activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, in response to oxidative stress. In addition, the serine/threonine protein kinase Pto-interacting 1-2 (PTI1-2) has been positioned downstream from OXI1, but whether PTI1-2 signals through MAPK cascades is unclear. Using a yeast two-hybrid screen we show that OXI1 also interacts with PTI1-4. OXI1 and PTI1-4 are stress-responsive genes and are expressed in the same tissues. Therefore, studies were undertaken to determine whether PTI1-4 is positioned in the OXI1/MAPK signalling pathway. The interaction between OXI1 and PTI1-4 was confirmed by using in vivo co-immunoprecipitation experiments. OXI1 and PTI1-4 were substrates of MPK3 and MPK6 in vitro. Although no direct interaction was detected between OXI1 and MPK3 or MPK6, in vitro binding studies showed interactions between MPK3 or MPK6 with PTI1-4. In addition, PTI1-4 and MPK6 were found in vivo in the same protein complex. These results demonstrate that PTI1-4 signals via OXI1 and MPK6 signalling cascades.

The phosphoproteome of Arabidopsis plants lacking the oxidative signal‐inducible1 (OXI1) protein kinase

The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.

ABA overly-sensitive 5 (ABO5), encoding a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis

To study the molecular mechanism of abscisic acid (ABA) regulation of root development, we screened the root growth of Arabidopsis mutants for sensitivity to ABA. ABA overly-sensitive 5 (ABO5/At1g51965) was identified, and was determined to encode a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3 (nad2 is one subunit in complex I). Under constant light conditions (24-h light/0-h dark photoperiod), abo5 mutants exhibited various phenotypes and expressed lower transcripts of stress-inducible genes, such as RD29A, COR47 and ABF2, and photosynthesis-related genes proton gradient regulation 5 (PGR5) and PGR5-likephotosynthetic phenotype (PGRL1), but higher levels of nuclear-encoded genes alternative oxidase 1a (AOX1a) and oxidative signal-inducible 1 (OXI1). Prolonged ABA treatment increased the expression of the cox2 gene in complex IV and nad genes in complex I to a higher level than no ABA treatment in the wild type, but only to a moderate level in abo5, probably because abo5 already expressed high levels of mitochondrial-encoded cox2 and nad genes under no ABA treatment. More H(2) O(2) accumulated in the root tips of abo5 than in the wild type, and H(2) O(2) accumulation was further enhanced by ABA treatment. However, these growth phenotypes and gene-expression defects were attenuated by growing abo5 plants under short-day conditions (12-h light/12-h dark photoperiod). Our results indicate that ABO5 is important in the plant response to ABA.

OXI1 protein kinase is required for plant immunity against Pseudomonas syringae in Arabidopsis

Expression of the Arabidopsis Oxidative Signal-Inducible1 (OXI1) serine/threonine protein kinase gene (At3g25250) is induced by oxidative stress. The kinase is required for root hair development and basal defence against the oomycete pathogen Hyaloperonospora parasitica, two separate H2O2-mediated processes. In this study, the role of OXI1 during pathogenesis was characterized further. Null oxi1 mutants are more susceptible to both virulent and avirulent strains of the biotrophic bacterial pathogen Pseudomonas syringae compared with the wild type, indicating that OXI1 positively regulates both basal resistance triggered by the recognition of pathogen-associated molecular patterns, as well as effector-triggered immunity. The level of OXI1 expression appears to be critical in mounting an appropriate defence response since OXI1 overexpressor lines also display increased susceptibility to biotrophic pathogens. The induction of OXI1 after P. syringae infection spatially and temporally correlates with the oxidative burst. Furthermore, induction is reduced in atrbohD mutants and after application of DPI (an inhibitor of NADPH oxidases) suggesting that reactive oxygen species produced through NADPH oxidases drives OXI1 expression during this plant–pathogen interaction.

Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis

Hydrogen peroxide (H 2O 2) is now recognised as a key signalling molecule in eukaryotes. In plants, H 2O 2 is involved in regulating stomatal closure, gravitropic responses, gene expression and programmed cell death. Although several kinases, such as oxidative signal-inducible 1 (OXI1) kinase and mitogen-activated protein kinases are known to be activated by exogenous H 2O 2, little is known about the proteins that directly react with H 2O 2. Here, we utilised a proteomic approach, using iodoacetamide-based fluorescence tagging of proteins in conjunction with mass spectrometric analysis, to identify several proteins that might be potential targets of H 2O 2 in the cytosolic fraction of Arabidopsis thaliana, the most prominent of which was cytosolic glyceraldehyde 3-phosphate dehydrogenase (cGAPDH; EC 1.2.1.12). cGAPDH from Arabidopsis is inactivated by H 2O 2 in vitro, and this inhibition is reversible by the subsequent addition of reductants such as reduced glutathione (GSH). It has been suggested recently that Arabidopsis GAPDH has roles outside of its catalysis as part of glycolysis, while in other systems this includes that of mediating reactive oxygen species (ROS) signalling. Here, we suggest that cGAPDH in Arabidopsis might also have such a role in mediating ROS signalling in plants.