Cloning And Functional Characterization Of Abiotic Stress-responsive Genes From Tomato

Tomato(Solanum lycopersicuum) is generally considered as a nutritive vegetable worldwide and plays very important role in the vegetable industry of China. It is also a robust model plant for research. Using tomato to reveal the mechanism of abiotic-resistance and produce germplasm of abiotic tolerance is important in plant research.Previously, we isolated two drought-tolerant introgression line (IL). By microarray transcriptional analysis under drought stress, we selected13candidate genes in response to drought for functional analysis using transgenic approach.4genes were characterized related to abiotic stress in tomato, including HyPRP (hybrid proline-rich protein) and DBB(Double B-box zinc finger protein) gene, two novel gene which have not been reported in abiotic stress as far as we known, and other two genes SlDREB and SljmjC, which can cause tomato GA-sensitive dwarf and GA-insensitive dwarf respectively. The dwarf phenotype would benefit plants through avoidance of high energy cost by producing stress-tolerance proteins when plants encounter adverse ambiance. This research is performed by analyzing transgenic plants, deployed on phenotype, cytology, physiological and biochemical and molecular level. We try to functional analyzing the candidate genes, focused on abiotic resistance related metabolic pathways and tomato signal transduction factors on transcription and protein level, and trying to explain the role of genes under abiotic stress in systems biology. The main results are as follows:1. Isolation and functional characterization of HyPRP gene in tomato.A HyPRP encodes a protein containing proline-rich domain and eight-cysteine motif (8CM). Its expression was suppressed under various abiotic stresses, such as drought, high-salinity, cold, heat and oxidation, and treatment of phytohormone abscisic acid in tomato. Functional analysis of HyPRP was performed through over-expression and RNA interference (RNAi) approaches in tomato. Transgenic plants had enhanced tolerance to various abiotic stresses (e.g., oxidative stress, dehydration, and salt). Interestingly, HyPRP-interacting proteins can detoxify sulfur dioxide (SO2) revealed in Yeast Two-Hybrid screening. These proteins include sulfite oxidase (SO), ferredoxins (Fds), and methionine sulfoxide reductase (Msr). During biosynthesis, SO and Fds catalyze the transformation of sulfites (main component of acid rain converted by SO2) to non-toxic sulfates or hydrogen sulfides, and Msr reduces the production of ROS (reactive oxygen species). When SO2toxicity occurs, HyPRP knockdown lines can accumulate more sulfates and transcripts of Msr and Fds than wild-type plants. Our findings show that the tomato HyPRP is a negative regulator of abiotic-stress, which may modulate Msr, SO, and Fds to enhance tolerance to SO2and oxidative toxicity.2. Isolation and functional characterization of DREB gene in tomato.SlDREB, a tomato DREB gene, is a transcription factor cloned from cultivated tomato M82. It plays a negative role in tomato plant architecture and enhances drought tolerance. Tissue expression profiles indicate that S1DREB is expressed mainly in stem and leaf. It can be induced by abscisic acid (ABA), but suppressed by GA and ethylene. SlDREB can alter plant morphology by restricting leaf expansion and internode elongation when over-expressed, and the resulting dwarfism of tomato plants can recover by the application of exogenous GA3. Transcriptional analysis of transgenic plants reveals that over-expression of SlDREB caused dwarfing phenotype by down-regulating key genes involved in GA biosynthesis such as ent-copalyl diphosphate synthase (SICPS) and GA20-oxidases (SlGA20oxl,2, and4), thereby decreasing endogenous GA levels in transgenic plants. A yeast activity assay demonstrates that SlDREB specifically binds to dehydration-responsive element/C-repeat (DRE/CRT) elements of the SICPS promoter region. Taken together, these data demonstrates that SlDREB can down-regulate the expression of key genes required for GA biosynthesis and acts as a positive regulator in drought stress responses by restricting leaf expansion and internode elongation.3. Isolation and functional characterization of DBB gene in tomato.A DBB (Double B-Box) gene is a novel type of transcription factor, and its function is rarely reported. The DBB protein contains two B-Box zinc finger domains, and its protein sequence shares high homology with other species. Expression analysis demonstrates that SpDBB is up-regulated rapidly by drought, salt, wounding, MV, cold and ABA, tissue specially expressed in stem and leaf. We find that SIDBB-KNAi tomatoes can significantly improve the tolerance to drought stress, and its over-expressed lines behave sensitive under drought condition comparing to wild types. As far as we know, this is the first report on the RNAi-knockout of SIDBB, which shows tolerance to drought stress in tomato. Moreover, RNA-seq transcnptional analysis reveals that genes encoding proteinase inhibitor, UDP-glucosyltransferase and oxidative stress-related proteins were found up-regulated in SIDBB-RNAi lines and down-regulated in SpDBB over-expression lines under drought condition. 4. Isolation and functional characterization of jmjC gene in tomato.The SljmjC shares high homology with other species, and exists in almost all species. Expression analysis shows that SljmjC is negatively regulated by GA, and decrease rapidly after GA treatment. RNAi-knockdown of SljmjC causes severe dwarf phenotype of tomato, smaller shrink leaves and shorter internode, particularly in reproductive growth stage of dwarf phenotype is more severe, and this dwarf phenotype can not be rescued by exogenously spraying GA3. RNA-seq transcriptome analysis on the SljmjC-RNAi dwarf plants and wild-type plants, we find that GA biosynthesis genes including GA20oxs and GA3oxs are significantly unregulated in SljmjC-RNAi transgenic plants, and the content of the various types of plant endogenous GAs such as:GA53, GA20and GA4are higher in transgenic plants. This is another evidence of SlimjC-RN Ai knockdown plant is not a GA-deficient dwarf. In order to further reveal the mechanism of SljmjC-RNAi knockdown causing dwarfism, we analyzed GA response genes using RNA-seq transcriptome data and find that two putative DELL A proteins370and380which missed DELLA domain are higher expression in transgenic plants. We cloned370and380and over-expressed them in tomato by transgenic approach. The result shows that the over-expression of370also leads to dwarf tomato, while the over-expression of380has not led to obvious dwarf phenotype. We further performed DNA methylation analysis by DNA bisulfite sequencing PCR approach to detect the DNA CpG methylation in transgenic plant and wild-types, and the result reveals that the degree of CpG methylation of370in SljmjC-RN Ai knockdown lines is lower than wild-type. Taken together, SljmjC can upregulate the expression of the370gene by reducing its degree of coding region CpG methylation, so that result in the GA-insensitive dwarf.5. Identification and expression pattern of a ZPR1gene in wild tomato (Solatium pennellii)We isolated a SpZPR1from the wild tomato species S. pennellii LA0716and found that its protein sequence shares homology with ZPR1genes from other organisms. SpZPR1mRNA was found to accumulate in tomato apical meristem in response to ABA (abscisic acid) treatment and heat stress. Transactivation activity assay in yeast revealed that SpZPRl functions as a transcriptional activator while the results of our gel electrophoresis mobility shift assay showed that SpZPR1was able to bind ABRE (ABA-responsive element) sequence in vitro. Our study is the first to provide evidence that SpZPRl is involved in the ABA signaling network and plays a potential role in plant cell development and abiotic stress response.