Functional Analysis Of Abiotic Stress Responsive Genes TaCHP And TaDSU

The response to abiotic stress such as salt and drought is governed by a complicated network, which is made up of phospolipids and other signaling pathways. Phospholipids are a class of signaling molecules, and play crucial roles in the response to abiotic stress in plants. Diacylglycerol (DAG) is an important signaling molecule in mammals; however its target signaling proteins have not been identified, so its regulatory mechanism is still unclear. The response to abiotic stress is characterized by a drastic alteration in diverse modifications of vast proteins, of which sumoylation functions in protein stabilization. Protein sumoylaion is dynamic and reversible, but the role of protein desumoylation in the response to abiotic stress has not been well addressed.Wheat is an important crop, but exhibits moderate resistance to abiotic stress such as salt and drought. The increasing serious situation of soil salinization and drought pushes us to identify salt and drought resistant genes, with aim to breed salt and drought resistant cultivars. Our lab bred a wheat introgression cultivar SR3with high salt and drought resistance via the asymmetric somatic hybridization method, and it is a near isogenic line of common wheat cultivar JN177, so SR3is an excellent cultivar for mining salt and drought resistant genes. Our group previously identified a salt resistant gene TaCHP from SR3, and this gene is suggested to participate in phospholipids signaling pathway. In this thesis, we conducted the following two studies:1. The association between TaCHP and phospholipids signaling pathwayTaCHP possesses three C1domains that specially bind DAG, have transcriptional activation activity, and localizes in cytoplasmic membrance, cytoplasm, and nucleus. These match the characteristics of protein kinase D, the important DAG binding protein in mammals, suggesting that TaCHP is a putative DAG bindin protein, and enhances salt resistance via modulating phospholipids signaling pathway. In comparison with Col-0, TaCHP overexpression lines exhibited differential transcription patterns of some genes encoding PLCs, NPCs and DGKs under the control or after exposure to NaCl, H2O2and ABA. Transient expression assay in onion epidermis showed that the distribution of TaCHP in cytoplasm was elevated when the synthesis of DAG was partially inhibited by adding PLC inhibitor, implying that TaCHP is recruited onto cytoplasmic membrane by binding with DAG However, fat western blot assay showed that TaCHP did not bind in vivo with16C-DAG, speculating that TaCHP may bind with some other DAGs rather than16C-DAG.TaCHP did not influence the effect of either16C-DAG or16C-PA on plant development. The inhibition of DAG synthesis by adding PLC inhibitor blocked plant growth, and the inhibition of DAG’s conversion to PA by adding DGK inhibitor blocked the formation of lateral roots; TaCHP overexpression attenuated these blocking effects. However, TaCHP overexpression lines have similar phenotype to Col-0in the presence of PLD inhibitor. These results indicate that TaCHP specially participate in the effect on plant growth by blocking both DAG synthesis and metabolism.In the presence of16C-DAG or16C-PA, TaCHP overexpression enhanced the salt resistance. However, in the presence of PLC inhibitor or PLC inhibitor/16C-DAG, TaCHP overexpression lines had similar salt resistance ability to Col-0. Similar result was also found in the presence of DGK inhibitor or DGK inhibitor/16C-DAG. These findings demonstrate that the role of DAG in enhancing TaCHP’s contribution to salt resistance by converting to PA.2. Functional analysis of wheat SUMO protease gene TaDSUWe screened the cDNA microarray, and found an abiotic stress responsive probe with annotation as ULP. Based on this probe, we isolated a novel wheat SUMO protease gene TaDUS. The transcription of TaDSU was responsive to NaCl, mannitol or ABA, and the responsive patterns were differential between SR3and JN177. TaDSU shared the highest identity to the orthologous gene of Triticum urartu, the ancestor of wheat A genome, showing that TaDSU originated from T. urartu. TaDSU possibly localized in chromosome5AL, which embeds a salt resistance QTL of SR3.To confirm the function of TaDSU in abiotic stress response, we constructed TaDSU overexpression Arabidopsis lines. TaDSU overexpression did not influence the development of Arabidopsis. TaDSU overexpression lines have pronounced resistance to salt, osmotic stress and drought, but lower sensitivity to ABA. TaDSU overexpression lowered the expression level of some abiotic stress responsive genes, elevated K+content and K+/Na+ratio. In comparison with Col-0, TaDSU overexpression lines have lower ROS level under the control, but higher ROS level under the drought. These results primarily indicate that TaDSU enhance abiotic stress resistance through modulating ionic and redox homeostasis.