Mechanism Investigation Of Wheat Membrane-Bound Transcription Factor TaNTLB3 In Plant Drought Stress Response

As the second most important food in China,wheat is distributed all over the country.In recent years,with the global warming,drought has become one of the most important factors limiting agricultural,which seriously affects the growth and development of plants and the yield of crops.Stomata are the gateway for gas exchange and water loss between plants and the outside environment.Under drought stress,plants can reduce water loss by regulating the closure of stomata,so as to improve the drought-resistant ability of plants.Therefore,through the study of the stomatal movement regulation mechanism and related signal transduction,we can explore the drought-resistant functional genes and cultivate new drought-resistant varieties,which provides an important theoretical basis for the development of modern agriculture.As an important stress hormone,ABA induces the expression of stress responsive genes.Under drought stress,the content of endogenous ABA in cells increases,which changes the turgor of cells and makes guard cells to shrink,thereby inducing the stomatal closure.In addition,ROS is an important second messenger molecule that participates in ABA-mediated stomatal closure.Studies have shown that NADPH oxidases AtRbohD and AtRbohF in plants are major sources of ROS production in guard cells,which are necessary for ABA-induced ROS increase and stomatal closure.NAC transcription factors,as plant specific transcription regulators,can be involved in a variety of biological and abiotic stress responses.As a special class of NAC membrane binding transcription factors,NTLs have been reported to play an important role in various abiotic stress responses in Arabidopsis thaliana,maize and rice.However,there are few reports on related NTLs genes in wheat,and the mechanism related to drought stress has not been reported yet.In this study,we used wheat cultivar Jinan 177(JN177)and Arabidopsis thaliana ecotype Col-0 as experimental materials to analyze the mechanism of wheat NAC membrane-binding transcription factor TaNTLB3 in drought stress response.We transformed the full length(FL)and the constitutive active form(ΔC)of TaNTLB3 into Arabidopsis wild-type Col-0 plants,and explored their development phenotypes under normal growth conditions.As a result,no significant developmental difference was found between TaNTLB3FL-OE and Col-0,whereas TaNTLB3ΔC-OE plants showed an obvious dwarf and late flowering phenotype,so TaNTLB3AC-OE lines was used for further study thereafter.To explore the role of TaNTLB3 in drought stress,the tissue and inductive expression pattern of TaNTLB3 were analyzed.The results showed that TaNTLB3 highly expressed in root,stem and leaf tissues in wheat,and its expression was induced greatly in leaves by both 50 μM ABA and exposure to drought stress.The TaNTLB3ΔC-OE plants showed reduced sensitivity to ABA in cotyledon greening,root length and stomatal movement.Further study showed that the TaNTLB3ΔC-OE plants lost water faster and had much lower survival rate in response to drought stress than Col-0.These findings suggest that TaNTLB3 may mediate the drought stress response through negatively regulating ABA-mediated stomatal movements.To further explore the molecular mechanism of altered ABA responses observed in TaNTLB3AC-OE plants,we analyzed the H2O2 content by DAB staining,and fluorescence intensity statistics.It showed that the content of ROS in the leaves of transgenic lines was higher than that of Col-0 under normal conditions.In contrast,the accumulation of ROS in the leaves of TaNTLB3AC-OE plants significantly decreased in the presence of ABA.qRT-PCR analysis of ROS related genes indicated that TaNTLB3AC could significantly down-regulate ROS synthesis related genes AtRbohD,AtPRX33,AtPRX34 and AtRbohF.Further luciferase report gene assay showed that TaNTLB3 could dierectly bind to the promoter of AtRbohD to regulate the expression of AtRbohD.In summary,our current findings showed that wheat NAC membrane-bound transcription factor TaNTLB3 negatively regulates the plant’s tolerance to drought stress and ABA-mediated stomatal movements by regulating the transcription of ROS related genes and thus ROS accumulation.