Isolation And Functional Characterization Of Target Genes Of Rice Stress-Related Transcription Factor SNAC1

Abiotic stresses such as drought, high salinity and heat cause extensive losses to agricultural production worldwide. Enhanced drought and salt resistance by overexpression of some stress-inducible genes have been shown in many reports. But very few studies have shown enhanced stress resistance and increased crop yield under drought stress in the field conditions. Previous study identified a NAC transcription factor SNAC1that had significantly effect on improving drought resistance in rice. Overexpression of SNAC1significantly enhanced drought resistance in field under severe drought stress condition at the reproductive stage while showing no phenotypic change or yield penalty, indicating great promise for genetic improvement of stress tolerance in rice. However, the SNAC1-mediated stress responsive pathway and stomatal closure mechanism remain unclear, and the molecular and physiological basis of the enhanced drought resistance by SNAC1need further study.OsSRO1c and OsPP18, two downstream target genes of SNAC1, were identified in this study by analysis of the chip data of SNAC1-overexpressing plant and yeast one-hybrid pairwise test. Based on the functional characterization of these two genes, the mechanism of SNAC1-mediated stomatal closure was preliminarily clarified. We found that reduced water loss and ROS accumulation under drought stress are the important physiological basis of the enhanced drought resistance in SNAC1-overexpressing plants. The main results are as follows:1. OsSRO1c is a member of plant-specific SRO (similar to RCD one) protein family. SNACl could bind to the promoter of OsSRO1c and activate the expression of OsSRO1c. OsSRO1c was induced by multiple stress and phytohormone treatment. Under drought stress, OsSRO1c was predominantly induced in guard cells. The ossrolc mutant and OsSRO1c amiRNA plant was also sensitive to drought and osmosis stress at seedling stage. OsSRO1c-overexpressing plants had more stomata closed than WT, and overexpression of OsSRO1c decreased transpirational water loss and reduced stomatal conductance. Higher level of H2O2accumulation was detected in the OsSRO1c-overexpressing plants. In addition, more H2O2accumulation was detected in the guard cells of the CsSRO1c-overexpressing plants. Expressions of DST, a reported zinc finger gene negatively regulating H2O2-induced stomatal closure, and its downstream gene were significantly suppressed in the OsSRO1c-oversxpressing plants. Overexpression of OsSRO1c enhanced the sensitivity to oxidative stress, and the expression of ROS-scavenging enzyme genes and activity of ROS-scavenging enzymes were reduced in OsSRO1c-overexpressing plants. Six OsSROlc-interacting proteins (SRIPs) were isolated by yeast two-hybrid screening, and some of the OsSRO1c-interacting proteins are predicted to be involved in the control of stomatal aperture and oxidative stress tolerance.2. The ossro1c mutant and OsSRO1c amiRNA plant showed enhanced drought resistant at reproductive stage. The ossro1c mutant and OsSRO1c amiRNA plants have less chlorophyll degradation and higher seed setting compared to the control under drought stress. OsSRO1c-overexpressing plant was more sensitive to drought stress at reproductive stage. The expression of OsSRO1c increased along with the development and senescence of leaves. Expression of many genes related to stress responses, senescence, phytohormone and secondary metabolism were altered in the ossro1c mutant. Many genes related to ROS-scavenging were up-regulated in the ossro1c mutant. The ossrolc mutant and OsSRO1c amiRNA plant showed enhanced resistance to oxidative stress. The activities of ROS-scavenging enzymes were increased in the ossro1c mutant and H2O2content of ossro1c mutant was lower than WT. Leaves of ossro1c mutant showed delay of senescence under dark and oxidative stress. Expression of OsDOS, a reported zinc finger gene negatively regulating JA-induced leaf senescence, was up-regulated in ossro1c mutant. Leaves of ossro1c mutant also showed delay of senescence under JA treatment.3. OsPP18encodes an enzymatically active type2C serine/threonine protein phosphatase (PP2C). SNAC1could bind to the promoter of OsPP18and activate the expression of OsPP18. OsPP18was induced by drought, salt, cold and heat stress, but not induced by ABA treatment. ospp18mutant was sensitive to drought stress at both seedling and reproductive stage. OsPP18amiRNA plant was sensitive to drought and osmosis stress. Expression of many genes related to stress responses and secondary metabolism were down-regulated in the ospp18mutant, and the same for some ROS-scavenging genes. osppl8mutant was sensitive to oxidative stress, and the activity of ROS-scavenging enzymes were reduced in ospp18mutant. Overexpression of OsPP18improved the tolerance to osmosis and oxidation stress. No interaction between OsPP18and SAPK2was detected, and the ABA sensitivity of osppl8mutant and OsPP18-overexpressing plants was not altered, indicating that OsPP18medicated drought stress response through ABA-independent pathway.