Studies On Mechanism Of The Rapid Response To Sugar Signal In Arabidopsis Thaliana

Sugars are not only the primary energy resource in most life activities,and also are the substrate for a variety of anabolic metabolism.Recently,sugars are recognized as important molecular signal to regulate growth and development.In plants,the mechanism of sugar signaling transduction is serious complicated.While the dynamic change of sugar concentration caused by several factors,like circadian rhythm,growth and development status,biotic and abiotic stress.Sugar signal plays importance in maintaining normal life process in growth,mature and senescence.Increased sugar signal in cells especially the glucose signal will induce genes expression in several developmental programs,including glycolysis,starch biosynthesis,and anthocyanin accumulation,but depress gene expression about photosynthesis.While the molecular basis of regulation in sugar rapid response is still unknown in plants.In this study,we deeply investigate the regulation pattern of sugar induced gene GPT2(Glucose-6-phosphate /phosphatetranslocator2)to illuminate the sugar signaling transduction mechanism in pattern plant Arabidopsis thaliana,and discovering the crucial cis-element as W-Box and histone acetylation regions in GPT2 promoter.GPT2 encodes a translocator which localized on the inner envelope membrane of chloroplast.GPT2 is considered to participate in cell division and differentiation,seeds mature and germination,and acclimation of metabolism to daily environmental changes through transporting of G6 P from cytosol to plastid.By using several strategies of molecular biology,biochemistry and genetic manners,we explain the function of sugar signal in regulating sugar-induced gene expression.At the same time,we find WRKY18,WRKY53 and HAC1 proteins act as novel positive regulators in rapid sugar response,and straighten out the molecular mechanism in sugar signal transduction.The main results are as follows:(1)According to RNA-Seq and RT-qPCR assays,we confirmed GPT2 as sugar rapid response marker gene because of its expression changed most obvious to exogenous glucose treatment in short time.Additionally,to generate the sugar response reporter system,we ligase the GPT2 promoter to GUS or LUC.Based on Cis-elements analysis,mutation analysis and kinetic luciferase reporter system,we recognize the W-Box cis-element involved in the promoter region of GPT2 is the crucial element for sugar rapid response;(2)We construct GPT2pro-P3::LacZ as a bait,and demonstrate that several WRKY transcription factors could bind to the promoter of GPT2 in vitro through yeast one hybrid.In addition,according to RT-qPCR,transcriptional activation assays,bioluminescence assays and genetic approach,we demonstrate the positive of WRKY18 and WRKY53 in rapid sugar response.(3)After treated with high concentration of glucose to Arabidopsis seedling,mutant phenotype observation showed repressed anthocyanin accumulation in wrky18,wrky53 and wrky18 wrky53 after high glucose treatment for a long time.These results indicate WRKY18 and WRKY53 play critical role in both short-term and long-term sugar response.In addition,ChIP assays are demonstrated that WRKY18 and WRKY53 could directly bind to the promoter of sugar induced genes,including GPT2,CHS and DFR,and activate their expression thus rapidly response to evaluated sugar contents in the cytosol.(4)We first take advantage of yeast two hybrid assays to demonstrate that HAC1 interact with WRKY18,and WRKY53 in vitro,and then using BiFC,LCI and Co-IP assays to confirm the direct interaction occuring in vivo.(5)According to phenotype observation and RT-qPCR analysis,the depletion of hac1-4 mutant shows the glucose induced sugar rapid response genes and anthocyanin accumulation significantly repressed,compared to Col-0.(6)To clarify the character of WRKY18,WRKY53 and HAC1 in sugar signaling transduction,we performed comparative transcriptome analysis in the w18 w53,hac1-4 mutant seedlings with low level glucose treatment,and identify several genes expression are activated by WRKY18-WRKY53-HAC1.The co-activated genes are involved in numerous pathways including starch synthesis and anthocyanin accumulation,which is associated with phenotype observation results.(7)Because of the positive regulation of HAC1 in sugar response,Western Blot and ChIP-qPCR assays are first demonstrate that the activity of glucose induces genes expression is dependent on histone acetylation,especially the elevated level of H3K27 ac.Additionally,ChIP-qPCR assays revealed that glucose induced H3K27 ac enrichment associated with the special promoters is depressed in hac1-4 mutant.Transcriptional activation assays show that HAC1 activate downstream genes expression required the function of WRKY18 and WRKY53,indicating HAC1 coordinate with WRKY18 and WRKY53 to rapidly regulate sugar induced gene expression.Therefore,our finding establishs a molecular link from the sugar signal,WRKY transcription factors and HAC1 to the downstream sugar response,and revealed the novel function of histone acetylation modification and WRKY transcription factors in sugar signaling,metabolism,and plant growth.This work provide new theoretical support to further understand the sugar storage and utilization in plant and improve the quality of crops,vegetables and fruits..