Study Of Transcription Factor MYB30 In Regulating Stress Responses Through Cytosolic Calcium Signaling In Arabidopsis

The calcium ion(Ca2+)is an important intracellular secondary messenger in plant,it is critical for regulating downstream responses during plant encountering unfavorable environmental conditions.Various abiotic and biotic stimuli,including oxidative stress,heat,osmotic stress and pathogen attack,trigger elevation in the cytosolic free Ca2+([Ca2+]cyt).Such elevated[Ca2+]cyt varies in duration,magnitude,frequency and spatial distribution depending on the stimuli,which is referred to as "calcium signature".Calcium signature is decoded by intracellular Ca2+ sensors,and leads to the downstream phosphorylation and transcriptional events.Current studies of calcium signal focus on the calcium channels,calcium transporter proteins and the downstream calcium signal cascades,but the generation of specific calcium signal and its role in stress responses remain elusive.To search for key factors in calcium signal generation,this study undertook a forward-genetic screening of mutants defective in stress-induced[Ca2+]cyt elevation,the Arabidopsis with stable expression of AEQUORIN was used as wild type.Through the screening,the mutant of transcription factor MYB30 was found to exhibit greater H2O2-induced[Ca2+]cyt elevation.Arabidopsis MYB30 is an important R2R3-MYB transcription factor.The complementation lines rescued the H2O2-induced[Ca2+]cyt elevation in myb30 to the wild-type level,and the over-expression of MYB30 repressed the H2O2-induced[Ca2+]cyt elevation to a very low level.These results suggest that MYB30 is a negative regulator of calcium signaling under H2O2 treatment.Real-time RT-PCR results indicated that the expression of a number of ANNEXINs(ANNs),encoding calcium-dependent and membrane-binding proteins and regulating cytosolic calcium signature,were up-regulated in myb30 mutants.Luciferase assay,ChIP(Chromaitin Immunoprecipitation),EMSA(Electrophoretic Mobility Shift Assay)and yeast one-hybrid assay showed that MYB30 bound to the promoters of ANN1 and ANN4 and repressed the expression both in vitro and in vivo.Phenotype tests showed that the myb30 mutants were sensitive to methyl viologen(MV)and heat stresses.While the H2O2-and heat-induced abnormal[Ca2+]cyt elevation in myb30 was suppressed by the defective of ANNs,the MV and the heat sensitivity of myb30 was suppressed by the deletion of ANNs or the LaCl3,a calcium channel blocker.These results indicate that MYB30 regulates oxidative and heat stress responses through calcium signaling at least partially mediated by ANN1 and ANN4.Further analysis showed that abnormal calcium signal in myb30 could be induced by various stresses,but some of them were independent of ANNs.Some stress sensitivity of myb30,such as ABA sensitivity,could not be suppressed by the deletion of ANNs or the LaCl3.These results indicate that the calcium signal is specific to each stress,and leads to specific stress response.In conclusion,this study finds that transcription factor MYB30 regulates oxidative and heat stress responses through ANNs-mediated specific cytosolic calcium signaling in Arabidopsis.This study contributes to the knowledge towards the generation and important role of specific stress-induced cytosolic calcium signaling in stress response in plant.