The Rtos1Protein And Transcription Factor Rap2.6L Regulate Plant Stress Responses And Accelerate Flowering In Arabidopsis

RTOS1(response to oxidative stress1; At5g27830) encodes an unknown function protein, which has been predicted to contain a folate-receptor domain. Folate, as a necessary substance in maintaining the normal function of immune system in Eukaryotes, affects plant growth and development process through biosynthesis, metabolism and especially folate-mediated redox reactions. RAP2.6L (At5g13330) belongs to the AP2/ERF family transcription factor which plays a vital role in plant growth and development process. This study reveals that RTOSI and RAP2.6L regulate plant disease resistance, abiotic stress tolerance and accelerate flowering in Arabidopsis.1. RTOSI overexpression enhances resistance to Pst DC3000in ArabidopsisFolate, as a necessary substance in maintaining the normal function of immune system in Eukaryotes, affects plant growth and development process through biosynthesis, metabolism and especially folate-mediated redox reactions. RTOSI encodes a protein of unknown function, which has been predicted to contain a folate-receptor domain, and the function of RTOSI in plant defense, growth and development has not been studied. In this study, Paraquat and Pst DC3000upregulated the expression of RTOS1, suggesting that RTOSI may involve in oxidative stress and plant defense processes. T-DNA insertion mutants enhanced the growth of Pst DC3000, displayed increased disease symptom severity, and decreased expression of PR-1and increased expression of growth-related AtEXP-1. However, the overexpression plants displayed decreased disease symptom, increased expression of PR-1and decreased expression of AtEXP-1after three days post Pst DC3000inoculation. We also measured pathogen resistance and defensive responses through H2O2accumulation, cell death, lipid peroxidation and defense gene expression after Pst DC3000infection. Our studies indicate that RTOSI mediates generation of H2O2and expression of PR-1in plant defense.2. RTOSI overexpression enhances tolerance to osmotic and oxidative stresses in ArabidopsisAbiotic stresses affect normal plant growth and development. In response to abiotic stresses, plants produce and accumulate ROS that may cause oxidative stress. RTOS1encodes a protein of unknown function, which has been predicted to contain a folate-receptor domain, and folate affects plant growth and development process through folate-mediated redox reactions, but little attention has been devoted to understanding RTOS1function in plant responses to abiotic stresses. In this study, ABA, osmotic and oxidative stresses upregulated the expression of RTOS1. RTOS1localized exclusively in the nucleus and extracellularly in the onion epidermal cells. RTOS1overexpression enhanced tolerance to osmotic and oxidative stresses via a reduction in hydrogen peroxide concentration and increases in the accumulation of ABA. Furthermore, up-regulation of antioxidant enzyme activities and transcripts of ROS-scavenging genes were observed. RTOS1overexpression caused significant increases in the expression of stress-responsive genes. In overexpression plants, pretreatment with a ROS-scavenging inhibitor or an ABA biosynthesis inhibitor arrested the decrease in ROS levels and led to an enhancement of oxidative damage. Thus, these results suggest that RTOS1enhances tolerance to osmotic and oxidative stresses by decreasing hydrogen peroxide levels and this gene possibly functions in an ABA-dependent pathway.3. RTOS1overexpression accelerates flowering in ArabidopsisFlowering is an important event from vegetative phase to reproductive stage in flowering plants. Up to2000gene expression levels have changed in Arabidopsis growth and flowering processes, there are more than80genes involved in flowering directly. Folate-mediated redox reactions affect plant growth and development process. RTOS1overexpression increases folate content in overexpression plants and RTOS1also has a function in plant defense.The overexpression plants flowered earlier than wild-type and mutants under long or short days. RTOS1overexpression changed the expression of genes involved in flowering time. The expression levels of CO, FT, SOC1and CCA1gene in overexpression plants were increased and the expression of LFY was decreased. Further bimolecular fluorescence complementation, yeast two-hybrid and screening in flowering library of Arabidopsis assays suggest that RTOS1interacts with FT. Genetic hybridization results showed that mutation of FT gene delays RTOS1overexpression accelerated early flowering. Based on the above evidence on molecular biology and genetic hybridization, we suggest that RTOS1accelerates flowering by interacting with FT.4. Mutation of RAP2.6L enhances plant defense against Pst DC3000in ArabidopsisERF transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. We analyzed the role of the RAP2.6L in plant defense against the bacterial pathogen Pseudomonas syringae. RAP2.6L localized in the nucleus. Analysis of Pst DC3000-induced RAP2.6L in the defense signaling mutants abil-1, jar1-1, ein2-1and npr1-1further indicated that this gene is positively regulated by the SA and JA signaling pathway and negatively regulated by ET signaling pathway. T-DNA insertion mutants reduced growth of Pst DC3000and displayed reduced disease symptom severity as compared to wild-type plants. Mutants also displayed increased expression of the PR-1and PR-2genes after the pathogen infection. Based on analysis of the mutants, stress-induced RAP2.6L functions as a negative regulator of SA-mediated defense responses to Pst DC3000. RAP2.6L was a transcriptional activator and was able to activate the expression of genes involved in plant defense.5. RAP2.6L overexpression retards waterlogging-induced premature senescenceWaterlogging usually results from overuse or poor management of irrigation water and is a serious constraint in damaging effects. RAP2.6L overexpression enhances plant resistance to drought and salt stresses in Arabidopsis. However, it is yet unknown whether RAP2.6L overexpression in vivo improves plant tolerance to waterlogging stress. The expression of RAP2.6L was induced by waterlogging or ABA treatment and reduced by pretreated with ABA biosynthesis inhibitor tungate (TUN). RAP2.6L overexpression inhibited water loss and membrane leakage under waterlogging. Time-course analyses of ABA content, production of H2O2, and activities of antioxidant enzymes showed that increased ABA precedes the increase of H2O2, and follows by a marked increase in the activities of antioxidant enzymes. Increased ABA promoted stomatal closure and made the leaves exhibited a delayed waterlogging-induced premature senescence. Transcripts of ROS-scavenging enzymes genes APX1(ascorbate peroxidase1) and FSD1(Fe-superoxide dismutase1), ABA biosynthesis gene ABA1(ABA deficient1) and signaling ABH1(ABA-hypersensitive1) and waterlogging stress-responsive gene ADH1(alcohol dehydrogenase1) were increased in overexpression plants, but the transcript of ABI1(ABA insensitive1) was reduced. Mutation of ABI1reduced the hypersensitive phenotype of RAP2.6L overexpression plants to ABA (germination and growth response). These suggest that RAP26L retards waterlogging-induced premature through an ABA-dependent pathway.