Title:Functional Identification Of Two Salt Stress Response Genes TaHB30and TaCHP From Wheat Introgression Line Shanrong3

Wheat has the most extensive distribution, the largest amount of acreage and the most abundance of processed products among major crops. Its yield and quality were adversely affected by the scarcity of water resources, land desertification and increasingly serious of abiotic stresses such as drought and salt, which promotes us to dissect its rules of stress response with the aim to stress-tolerance cultivar breeding. However, the response upon abiotic stress is governed by multi-genes, and so many physiological evens are involved, including cellular defense, ion transport, secondary metabolism, energy flow and so on. On account of the different chromosome type to Arabidopsis and rice (allohexaploid to diploid), the salt tolerance mechanism of wheat would be more complicated, such research progress in wheat is also much slower than in rice, corn and other crops.Wheat is a glycophyte with limited salt tolerance, but its wild relatives have been demonstrated to be the pivotal gene pool for salt tolerance improvement breeding via genetic and cell engineering technologies. In our previous work, a new somatic hybrid introgression line Shanrong No.3(SR3) had been generated from hybrids of common wheat Jinan177(Triticum aestivum2n=42, JN177) with Thinopyrum ponticum (2n=70), a salt and drought tolerant grass. The salinity tolerance indices as well as physiological, biochemical and genetic parameters of SR3were significantly better than JN177, suggested that major salt tolerance genes and some minor genes controlled the salt tolerance of SR3.Based on the transcriptomic and proteomic data of SR3, we found two genes showing obvious stress-responsive patterns, and isolated their full-length sequences via PCR from the SR3salt-stressed cDNA library and RACE cloning, respectively. Then, their functions in stress tolerance were performed. The main research contents and results were summarized as follows: 1. Cloning and preliminary functional analysis of TaHB30gene involved in salt stressBased on the screening of SR3salt-stressed cDNA library, we found a gene that was up-regulated in response to salinity treatment, which was named TaHB30according to the library number. Its deduced protein sequence possesses a typical active site of Cu/Zn superoxide dismutase, including four Cu2+and four Zn2+binding sites. Therefore, TaHB30was classified into the Cu/Zn superoxide dismutase gene family.The transcription of TaHB30was gradually induced both in SR3and JN177when treated with200mM NaCl. After exposure to10mM H2O2, the expression of TaHB30was down-regulated at the early phase and then gradually resumed in roots of two cultivars; in leaves, the gene appeared the comparable expression profile in SR3, while it was down-regulated during the whole treatment course. Moreover, SR3accumulated more TaHB30transcripts in SR3than in JN177under the two treatments.Cu/Zn superoxide dismutase is found to be ubiquitously distributed in plant cells. To determine the action site of TaHB30, a subcellular localization assay was conducted by transiently expressing TaHB30-GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts. The GFP signals traced that TaHB30located in both cytoplasm and nucleus.TaHB30was transformed into Arabidopsis and wheat for determining its role in abiotic stress tolerance, and transgenic Arabidopis overexpression (OE) lines were used to further analyze. Under the control conditions, OE lines and vector control (VC) line had no developmental and reproductive difference during the whole life cycle. Under more than50mM NaCl treatment, OE lines showed more vigorous growth ability than VC line, and they had longer main roots and more amounts of lateral roots. Moreover, OE lines had higher tolerance capacity to H2O2. By contrast, OE and VC lines had unconspicuous phenotype in medium plate containing different concentrations of mannitol. Besides, TaHB30overexpression did not change the sensitivity to phytohormone such as IAA, JA, ABA, and GA3. These results speculates that TaHB30enhance the plant salt tolerance through the ROS pathway, which seems to have no interplay with the hormone pathways. TaHB30was further transformed into Arabidopsis defective in the homologue of TaHB30. In comparison with VC line, the mutants overexpressing TaHB30had shorter main roots under the control conditions, but similar ones under NaCl or H2O2treatments, which is possibly attributed to the modulating effect of TaHB30in ROS signaling pathway.Semi-quantitative RT-PCR and real-time quantitative PCR indicated that OE lines had higher abundance of endogenous SOD (superoxide dismutase) transcripts than VC line under the control conditions. The genes encoding ROS scavenging enzymes catalyzing in downstream steps of SOD were also up-regulated, of which the transcription of GPX (glutathione peroxidase) was notably elevated, and APX (ascorbate peroxidase) and CAT (catalase) also had a higher expression levels. It was noteworthy that the expression of AtRBOH-D and AtRBOH-F, encoding two subunits of NADPH oxidase and functioning in H2O2generation, was significantly raised, what's more, OE lines did not show more vigorous growth ability than VC line to H2O2and inhibitor of NADPH oxidase; The expression of AtRBOH-D did not change and GPX had a higher expression levels in the mutants overexpressing TaHB30than VC line under the control conditions. The results above showde that Wheat Cu/Zn superoxide dismutase gene TaHB30played a pivotal role in salt tolerance through regulating NADPH oxidase of ROS signaling pathways.Following the transcriptional profiles, the activities of these ROS generating and scavenging enzymes were also improved in TaHB30overexpressors. Of them, the activities of total SOD and Cu/Zn SOD were both remarkably increased in OE lines, the CAT and GPX activities of transgenic plants were higher approximately1.5-2and1.3times respectively than those of VC line, and the activity of nonspecific NADPH oxidase (NOX) was also increased by1.6fold. DAB staining showed OE lines had less H2O2content than VC line, while NBT staining exhibited that they had higher amount of superoxide anion.These results confirmed that wheat Cu/Zn superoxide dismutase enhanced salt tolerance through ROS signaling pathways.2. Functional analysis of TaCHP gene involved in salt stress In our previous study, we identified a salt stress response gene TaCHP, which encoded a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene. TaCHP possesses three divergent C1domains; C1domain was found to specifically bind to phospholipid signaling molecule diacylglycerol (DAG) in animals, and most of proteins with this domain have kinase activity (localize at cytoplasm) and/or act as transcriptional factors (localize in nucleus). Here, TaCHP was found to localize at cytoplasm and in nucleus, suggesting that TaCHP might serve as both a transcription factor and a putative DAG binding protein to confer salt tolerance. The in vitro PKC kinase activity result showed that TaCHP had no kinase activity. Transactivation assay showed that the full-length TaCHP and C-terminal two C1domains had transcriptional activation activity, which indicated that TaCHP might function as a transcription factor.TaCHP was majorly transcribed in roots of seedlings at the three-leaf stage with a more remarkable level in SR3than in JN177. in situ hybridization further showed that the transcript localized in the cells of the root tip cortex and meristem. Its transcript abundance was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA, but SR3overall expression maintained at a significantly higher level than JN177.When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performances in the face of salinity, drought and the indexes of physiological and biochemical indicators all improved, proving the abiotic stress tolerance of TaCHP. To study the mechanism of its action, TaCHP overexpression Arabidopsis lines(BS, A5) were constructed. In addition to the salt tolerance, the tolerance about H2O2and ABA increased in overexpression Arabidopsis lines. The expression levels of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABIl) were raised in the transgenic lines in the presence of salinity stress, while ABA synthetic genes AtAAO3, AtABA2and AtMYB15reduced. Real-time PCR displayed that the average increase expression of AtRBOH-D, which encoded a component of NADPH oxidase required for ROS production, was about7times in the overexpression lines, the ROS scavenging enzymes CAT and APX encoding genes AtCAT2and AtAPX2were induced, combining with the improved enzymatic activity results of SOD and CAT, indicating that ROS production and scavenging capacity were increased in BS and A5lines. These data indicated that TaCHP participates in abiotic stress response through the ABA-dependent, ABA-independent and ROS signaling pathways.