Study On Octadecanoid Pathway Genes Of Triticum Aestivum Responding To Abiotic Stress

In recent years, the agricultural ecological environment is deterisrating and the high salinity stress becomes more and more serious, which adversely affect the crop production. Therefore, to cultivate salt tolerant crops are an important and urgent task of modern agriculture and have becoming one of the world's research focuses. Wheat is one of the most important crops worldwide, but its growth and productivity are obviously inhibited by abiotic stresses such as drought and salt. In our previous work, a new wheat introgression line Shanrong No.3(SR3) was generated using common wheat Jinan177(JN177) and Thinopyum ponticum, a salt and drought tolerant grass, via asymmetric somatic hybridization. Former results showed that SR3's genome were integrated with some chromatin fragments of T. ponticum, and took place a high frequency of allelic variation, indicating that SR3is an excellent material for investigating genetic variation, isolating stress tolerance associated genes, and dissecting the mechanisms underlying stress response. Transcriptomic and proteomic analysis showed that the SR3's resistance is closely related to its strong antioxidant capacity.Salt stress could induce cells to produce large amount of reactive oxygen species (ROS), which perform as intracellular secondary oxidative stress. Plants have evolved a complex and effective ROS scavenging system to cope with such stress, including enzymatic and non-enzymatic antioxidants as well as complicated signaling molecules and transcription factors to regulate their expression. ROS-induced lipid oxidation (lipid peroxidation) form a variety of oxidation products, some of which contain unsaturated bonds and cause membrane oxidative damage and other toxic injuries, so the oxidized fatty acids (also known as oxylipids) is the indicator of membrane oxidative damage degree, and serve as signals for cells to perceive oxidative stress. There have many types of oxylipids, of which the most characterized are the12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid, two intermediates of octadecanoid pathway, suggesting the possible role of this pathway in SR3's high salt tolerance. OPDA reductases (OPRs) catalyze the reduction of OPRA, the first step of its conversion to JA, and they are classified into two subgroups, OPRI and OPRII; OPRII participates in JA synthesis, but OPRI's function is still not known. Here, we performed a comprehensive comparison on the transcriptional patterns of genes involved in octadecanoid synthesis pathway, from which we cloned three octadecanoid pathway associated genes TaOPR1, TaOPR2, and TaAOCl from SR3, ascertained their roles in development and tolerance to abitoic stresses through overexpression in Arabidopsis and wheat, and uncovered the mechanisms of these genes in governing development and abiotic stress response in plants. The main research contents and results achieved in this work were summarized as follows.1. Screening expression patterns of octadecanoid pathway genesIn order to further know the relationship of oxylipids synthesized by octadecanoid pathway in the plant response to salt stress, we performed a screening of the expression patterns of genes involved in the pathway during abiotic stress. Firstly, we analyzed the expression profiles of genes encoding key enzymes in the pathway such as AOS, AOC, and OPR, according to the data from cDNA microarray of SR3and JN177. Bioinformatic analysis showed that AOS, AOC and OPR encoding genes have multiple copies in wheat genome, and the probes referring to these genes in cDNA microarray appeared differential expression patterns. We then divided these genes into several classes based on their stress responsive modes, and found there has one class of OPR genes that were dramatically induced by more than20or even100fold under salt stress, and the induction strength were much stronger in SR3than in JN177. This implies that these OPR genes may play important roles in salt stress response in wheat.In addition, we analyzed the expression patterns of Arabidopsis AOS, AOC and OPR genes under NaCl and PEG stresses with the online transcriptomic data at TAIR website. The results show that all of three OPR copies were significantly induced, with a more induction extent in OPRI subfamily AtOPR1and AtOPR1than in OPRII subfamily gene AtOPR3. Besides, all of four AOC copies were stress-responsive with different patternsonly, but one copy of AOS genes was slightly up-regulated.These results suggest that octadecanoid synthesis pathway associated genes of wheat and other plants certainly participate in plant salt stress response, of which the linkage between OPR genes, especially OPRI genes, and the response may be closer.2. Cloning and function analysis of TaOPRl and TaOPR1in abiotic stress toleranceComprehensive transcription comparison revealed the close relation between OPRI genes and salt stress response. Therefore, we cloned an OPRI subfamily gene TaOPR1and TaOPR2from SR3based on its most obvious induction by salt stress in cDNA microarray.RT-PCR and Real-time PCR showed that TaOPR1was induced by under NaCl, PEG6000, ABA and H2O2stresses in both SR3and JN177, with a more significant degreee in SR3. The induction of TaOPR1by NaCl can be inhibited by ABA biosynthesis inhibitor norflurazon, indicating the NaCl-induced TaOPR1expression is ABA-dependent.To know its role in salt stolerance, TaOPR1was transformed into wheat and Arabidopsis. TaOPR1overexpression did not affect the growth and reproduction ability of wheat and Arabidopsis. Under NaCl stress, wheat overexpression lines (CE) had a superior growth ability than the wildtype, and Arabidopsis overexpression lines (OE) also grew more vigorously than the empty vector control (VC) lines, indicating the positive contribution of TaOPR1in salt tolerance.In TaOPR1overexpressors, the activities of ROS scavengers superoxide dismutase (SOD) and catalase (CAT) were significantly increased, but ROS and malondialdehyde (MDA) contents were decreased evidently. Consistently, under H2O2stress, Arabidopsis OE seedlings were stronger than the VC line, and their seeds also had higher germination rate. This indicates that the contribution of TaOPR1in salt tolerance is partially achieved through its role in enhancement of ROS removal.Arabidopsis OE lines were hypersensitive to exogenous ABA, and their seedling root length and seed germination rate were both restricted more obviously than the VC line. Real-time PCR analysis showed that in ABA biosynthesis pathway associated genes AtABA1, AtABA2, AtNCED3and AtAAO3and ABA-dependent stress response pathway genes AtRD22, AtMYB2and AtMYC2all had higher transcript levels in OE lines. These results provide convinced evidence that besides through helping in ROS removal, TaOPR1enhanced stress tolerance by promoting the ability of ABA synthesis and ABA-dependent stress response pathway.In addition, Arabidopsis OE lines and the VC line showed similar response to exogenous JA. RT-PCR analysis demonstrated that JA biosynthesis and signal transduction pathway associated genes except for AtAOCl had no differential transcription patterns between the OE and VC lines. This indicates that TaOPR1has no crosstalk with JA pathway, which is in agreement with the knowledge that the OPRI subfamily genes are not involved in JA biosynthesis.Real-time PCR analysis showed that TaOPR2can be activated by NaCl, PEG, H2O2, and ABA in SR3, indicating that TaOPR2is also involved in abiotic stress responses. In comparison with TaOPR1, the response of TaOPR2to abiotic stresses was more distinguishable; TaOPR2accumulated more transcripts under PEG than NaCl stresses.Alike TaOPR1, TaOPR2overexpression also enhanced tolerance to salt, PEG and H2O2stresses, and sensitivity to exogenous ABA. But unlike TaOPR1, TaOPR2overexpressors not only had longer roots, but also larger leaves, indicating its excellent potential for crop breeding.Physiological and molecular analysis indicated that TaOPR2also promote the activities of ROS scavengers and ABA-dependent stress response pathway. But unlike TaOPRl, TaOPR1overexpression inhibited the expression of AtABI1, whose encoding protein is an inhibition factor for ABA signaling pathway, indicating that the acceleration of ABA-dependent stress response pathway by TaOPR2is accomplished through releasing its repression. Besides, TaOPR2but not TaOPR1induced the expression of DREBs, key components of ABA-independent stress response pathway. The similarities of TaOPRl and TaOPR2as well as their specificities in role of abiotic stress response indicating their functional conservation and differentiation during evolution 3. Cloning and function analysis of TaAOCl in abiotic stress toleranceAOC catalyzes the last step of OPDA biosynthesis, during which AOC can convert cis-OPDA to trans-OPDA, the in vitro substrate of OPRIs, suggesting its putative coordinated role in TaOPR1/TaOPR2-induced salt tolerance. Based on this speculation, we first cloned a wheat AOC gene TaAOC1from SR3, which shares the highest identity to AtAOCl, the AOC copy induced in Arabidopsis TaOPR1OE lines, among available wheat ESTs annotated as AOC. The gene was induced at early stage of salt and PEG600treatments. TaAOC1overexpression Arabidopsis (OE) lines showed a growth-inhibited and lateral root-increased phenotype, which is similar to the wild-type seedlings exposed to exogenous JA. TaAOC1OE lines had superior salt tolerance to salt stress and the lateral root-increased phenotype was controled. These results provide a primary glimpse to the role of AOC and octadecanoid pathway metabolites in plant tolerance to abiotic stresses, but how they perform this role needs to be further studed.