Action Mechanisms And Regulation Of Reactive Oxygen Species,Phytohormone Interactions, Transcription Factors And Autophagy In Tomato And Arabidopsis Stress Response

China has a vast territory, vegetable production are often affected by low temperature, heat stress, salt stress, drought and a variety of diseases all over the country. Especially in recent years, extreme weather events occur more frequently under the common action of greenhouse effect and human activities. Chilling injury of winter and spring season, hot climate of summer and fall season and drought in north and southwest of China are also on the increase. These questions greatly restrict the production and supply of vegetables in our country. Therefore, analyzing the mechanism of plant in response to stress, and using molecular genetics, physiology and biochemistry, and environmental regulation to improve resistance to adverse environmental conditions in vegetable crops have very significant sense in both science and realism to improve the vegetable production, quality and economic benefit, and to establish sustainable agriculture. This paper uses tomato as material to study the involvement of hydrogen peroxide (H2O2) in the cold acclimation-induced chilling tolerance. Then, using virus induced gene silencing technology (VIGS), we further investigate the critical role of reactive oxygen species (ROS) in induction of cross-acclimation to abiotic stresses. Through use of genetics, physiology, molecular biology, we research dynamic interplays between brassinosteroids (BRs)-and abscisic acid (ABA)-induced H2O2in tomato stress tolerance. And then, we use Arabidopsis as material to study NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. Through genetics, we analyze the function of CHIP E3ubiquitin ligase in plants stress response. Finally, by comparing the evolution and function of WRKY33homologous genes in Arabidopsis and tomato, we discuss regulation mechanism of WRKY33transcription factors in plant stress responses. The main results are as follows:1. We have studied the the involvement of H2O2in the cold acclimation-induced chilling tolerance of tomato plants. Cold acclimation increases plant tolerance to a more-severe chilling and in this process an accumulation of H2O2in plants is often observed. To examine the role of H2O2in cold acclimation in plants, the accumulation of H2O2, antioxidant metabolism, the glutathione redox state, gas exchange and chlorophyll fluorescence were analyzed after cold acclimation at12/10℃and during the subsequent chilling at7/4℃in tomato(Solanum lycopersicum) plants. Cold acclimation modestly elevated the levels of H2O2, the gene expression of respiratory burst oxidase homolog1(RBOH1) and NADPH oxidase activity, leading to the up-regulation of the expression and activity of antioxidant enzymes. In non-acclimated plants chilling caused a continuous rise in the H2O2content, an increase in the malondialdehyde (MDA) content and in the oxidized redox state of glutathione, followed by reductions in the CO2assimilation rate and the maximum quantum yield of photosystem Ⅱ (Fv/Fm). However, in cold-acclimated plants chillinginduced photoinhibition, membrane peroxidation and reductions in the CO2assimilation rate were significantly alleviated. Furthermore, a treatment with an NADPH oxidase inhibitor or H2O2scavenger before the plants subjected to the cold acclimation abolished the cold acclimation-induced beneficial effects on photosynthesis and antioxidant metabolism, leading to a loss of the cold acclimation-induced tolerance against chilling. These results strongly suggest that the H2O2generated by NADPH oxidase in the apoplast of plant cells plays a crucial role in cold acclimation-induced chilling tolerance.2. We have studied RBOH1-dependent H2O2production and subsequent activation of MPK1/2play an important role in acclimation-induced cross tolerance in tomato. H2O2and MAPKs cascades play important functions in stress response in plants, but their roles in acclimation response remains unclear. Here, we examined the roles of H2O2and MPK1/2in acclimation-induced cross tolerance in tomato plants. Mild cold, paraquat or drought for acclimation all enhanced the tolerance to more severe subsequent chilling, photooxidative and drought stress. The acclimation-induced cross tolerance was associated with increased transcripts of RBOH1and stress-and defense-related genes, elevated apoplastic H2O2accumulation, increased activity of NADPH oxidase and antioxidant enzymes, reduced glutathione redox state and activation of MPK1/2in the plants. Virus-induced gene silencing of RBOH1, MPK1, and MPK2or MPK1/2all compromised acclimations-induced cross tolerance and associated stress response. Taken together, these results strongly suggest that acclimation-induced cross tolerance is largely dependent on RBOH1-dependent H2O2production at the apoplast, which may subsequently activate MPK1/2to induce stress response.3. We have studied Dynamic interplays between brassinosteroids (BR)-and abscisic acid (ABA)-induced H2O2in tomato stress tolerance. The production of H2O2is critical for BR-and ABA-induced stress tolerance in plants. In this study, the relationship between BR and ABA in the induction of H2O2production and their role in stress tolerance were studied in tomato. BR induced only a transient increase in RBOH1gene expression, NADPH oxidase activity, apoplastic H2O2accumulation, and stress tolerance in the ABA biosynthetic mutant notabilis(not), whereas ABA induced a strong and prolonged increase in these responses in the BR biosynthetic mutant d∧tm compared with wild-type plants. ABA levels were reduced in the BR biosynthetic mutant but could be elevated by exogenous BR. Silencing of RBOH1compromised BR-induced apoplastic H2O2production, ABA accumulation, and stress responses; however, ABA-induced stress responses were largely unchanged in the RBOH1-silenced plants. BR induces stress tolerance involves a positive feedback mechanism in which BR-induced rapid and transient H2O2production by NADPH oxidase. The process in turn triggers increased ABA biosynthesis, leading to further increases in H2O2production and, prolonged stress tolerance. ABA induces H2O2production in both the apoplastic and chloroplastic compartments.4. We have studied NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62and NBR1. We isolated two nbr1knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5and atg7mutants. Like ATG5and ATG7, NBR1is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5and ATG7, however, NBR1is dispensable in age-and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and deliver}’systems. The compromised heat tolerance of atg5, atg7, and nbr1mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.5. We have studied E3ubiquitin ligase CHIP and NBR1-mediated selective autophagy protect additively against proteotoxicity in plant stress responses. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbrl mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble protein aggregates under heat stress. To determine functional interactions between CHIP and NBR1, we also generated chip nbrl double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating that the roles of CHIP and NBR1were additive. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Based on these results, we propose CHIP and NBR1mediate two distinct but complementary anti-proteotoxic pathways in which CHIP ubiquitinates misfolded proteins and targets their degradation by26proteasomes, while a different unknown E3ubiquitin ligase ubiquitinates stress-induced protein aggregates and targets their degradation by NBR1-mediated selective autophagy. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbrl single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbrl mutant while a number light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbrl mutants and, to a greater extent, in the chip nbrl double mutant. Based on these results, we propose that CHIP and NBR1mediate two distinct but complementary anti-proteotoxic pathways and protein’s proneness to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.6. We have studied evolutionary and functional analysis of WRKY33homologs in plant stress responses. In the present study, we analyze the structural and evolutionary basis for the uniquely important roles of the WRKY transcription factor in plant stress responses. When compared to its close homologs such as AtWRKY25, AtWRKY33has an extended C-terminal domain (CTD). Both AtWRKY25and AtWRKY33ACTD (CTD deletion mutant) genes driven by the strong CaMV35S promoter restored both the resistance to necrotrophic fungal pathogen Botrytis cinerea and heat tolerance to the atwrky33mutant plants. By contrast, AtWRKY25and AtWRKY33ACTD driven by the AtWRKY33gene promoter or AtWRKY33driven by the AtWRKY25gene promoter failed to restore resistance to B. cinerea and could only partially restore heat tolerance of atwrky33mutant plants. Thus, both the protein structure, as exemplified by its unique CTD, and expression pattern conferred by the gene promoter are critical for the important biological roles of AtWRKY33in plant stress responses. Phylogenetic analysis identified close homologs of AtWRKY33in both dicot and monocot plants including two in tomato (SlWRKY33A and S1WRKY33B). As with AtWRKY33, expression of both SIWRKY33A and SlWRKY33B was highly induced by Botrytis infection and heat stress. Virus-induced silencing of SIWRKY33A and SIWRKY33B compromised both the resistance to B. cinerea and heat tolerance in tomato. Both S1WRKY33A and SIWRKY33B driven by the AtWRKY33promoter fully complemented Arabidopsis atwrky33mutant plants for Botrytis resistance and heat tolerance. These results indicate that WRKY33proteins are evolutionarily conserved transcription factors with a critical role in plant responses to broad biotic and abiotic stresses.