Sucrose And ABA Regulate Starch Biosynthesis In Maize Endosperm Through Transcription Factors,ZmEREB156 And ZmEREB17

Maize(Zea mays L.),one of the main crops in the world,due to its kernel storage of a large number of starch,is widely used in food,feed and other industries.Therefore,starch biosynthesis and accumulation is an important process that not only determines grain yield but also influences grain quality.Starch biosynthesis in the cereal endosperm requires the coordinated activities of several major enzymes,including adenosine 5’ diphosphate-glucose(ADP-Glc)pyrophosphorylase(AGPase),starch synthase(SS),starch branching enzyme(SBE),and starch debranching enzyme(DBE).Starch biosynthesis is a complex process whose regulation mechanism is unclear yet.The transcription factors play a wide variety of roles in the regulation of plant secondary metabolism,responsing to hormone and environmental factors,and plant growth and development.Some reports suggest that the transcriptional regulation of starch synthetic genes is important for the pattern of starch synthesis and accumulation.In recent years,studies have also shown that sucrose and ABA act as signaling molecules involved in the regulation of gene expression related to starch synthesis,but the molecular mechanism is not fully understood.In the present study,endosperms 10 DAP(days after pollination)of maize inbred line B73 were treated with sucrose,ABA,or both,and then analysed by RNA sequencing(RNA-seq).Analysis of these gene sets identified different treatments of gene expression,including hundreds of transcription factor genes.We found that some transcription factor genes were affected by Sucrose or/and ABA,and then select and analyse some of them involved in maize endosperm starch synthesis.The aim of this paper is to ananlyze the roles of transcription factors in maize starch biosynthesis pathway,and elucidate their molecular mechanisms,through identification and functional analysis of starch synthesis-related transcription factors.The results will provide theoretical,technical,and material basis for further comprehensive analysis of the regulatory network of starch biosynthesis,artificialcontrol starch synthesis,and cultivating new varieties of high yield and quality of corn.The main results as follows:1.Maize endosperms were collected ten DAP and treated with sucrose(Suc),abscisic acid(ABA),or both(Suc+ABA).Mannitol was added to the samples without sugars as an osmotic control.Four cDNA libraries were constructed from total RNAs extracted and analysed sequences by RNA-seq on the Illumina HiSeqTM 2000.After quality control,approximately roughly 19.3 Gb of nucleotides and 214.54 million valid reads were obtained,and about 4.8 Gb of nucleotides and 53.6 million valid reads per sample library were obtained.At least 75%of the reads from each sample perfectly matched the maize reference genome.The sequencing data analysis is available after evaluating the distribution and coverage of statistical reads on the reference sequence.2.There were 2838,9396 and 4537 significantly differentially expressed respectively in the Suc,ABA and Suc+ABA treatment by comparing the Suc,ABA,and Suc+ABA libraries with the control.In the Suc-treated endosperm,1201 genes were up-regulated,1637 genes were down-regulated and 805 genes were specifical-expressed.In the ABA-treated endosperm,3181 genes were up-regulated,6215 genes were down-regulated and 6275 genes were specifical-expressed.In the Suc+ABA-treated endosperm,1661 genes were up-regulated,2876 genes were down-regulated and 1494 genes were specifical-expressed.There were 897 significantly differential genes that were commonly shared in Suc,ABA and Suc+ABA groups.The hierarchical heatmap display revealed that maize endosperm treated with a combination of exogenous Suc and ABA had a different transcript pattern from that treated with Suc or ABA alone and the synergistic and antagonistic effect between sucrose and ABA signalling involving in regulating gene expression.GO and KEGG pathway enrichment were analysed from differentially expressed genes,there were mainly biosynthesis of secondary metabolites,plant hormone signal transduction,starch and sucrose metabolism,flavonoid biosynthesis,fatty acid metabolism,and so on.This suggested that Sucrose and ABA signaling involved in multiple metabolic pathways in the developing maize endosperm.3.In our RNA-seq analysis,741 DEGs were annotated as transcription factor genes and classified into 50 TF families according to the criteria of PlantTFDB 3.0.47 candidate transcription factors were selected based upon their relatively high expression levels in the libraries.Twenty-one transcription factors and seven genes related to starch synthesis were confirmed by qPCR,the results consistent with the RNA-seq data.In our research,whether and how sucrose/ABA regulate starch biosynthesis in maize through the two transcription factors ZmEREB156 and ZmEREB17.4.The fusion construct p35S:eGFP-ZmEREB156 and p35S:eGFP-ZmEREB17 were introduced into onion epidermal cells via particle bombardment.GFP fluorescence was observed under a confocal microscope and was localized solely in the nuclei of the onion epidermal cells.These results indicated that ZmEREB156 and ZmEREBl 7 localized to the nucleus in vivo.We fused the encoding region of ZmEREB156 and ZmEREBl 7 in frame to the GAL4 DNA binding domain in the pGBKT7 vector,and transformed the resulting construct into yeast strain AH 109.The results showed that cells transformed with pGBKT7-ZmEREB156 and ZmEREB17 grew well on synthetic SD-Trp-His-Ura selection media and turned blue in X-a-gal indicator,demonstrating that ZmEREB156 and ZmEREB17 was a transcriptional activator in yeast.5.To analyse whether overexpression of ZmEREBl56/l7 enhances the promoter activity of starch genes in maize endosperms,the effector constructs(pUbi-ZmEREB 156 and pUbi-ZmEREB 17)were co-bombarded into maize endosperm with the respective reporter constructs(pPromotor-Luc)and internal construct(pUbi-Gus)and LUC/GUS relative activity was measured.Results showed that the luciferase(LUC)activity driven by the pSh2:Luc and pSSIII3a:Luc construct in the ZmEREB 156-overexpressing endosperms was about 7.6 times and 2.4 times higher than that of endosperms not expressing ZmEREB156 and LUC activity also increased about 1.8 times in the pSSI:Luc construct and-bombarded endosperms with pUbi-ZmEREB 17.These findings indicate that the expression of ZmSh2 and ZmSSlIIa were regulated by ZmEREB156 and the expression of ZmSSI was regulated by ZmEREBl 7.6.To confirm whether the ZmEREBl56/17 protein could bind to the promoter region of the three starch genes,a yeast one-hybrid assay was performed.The pGADT7-Rec2-ZmEREB156/17 plasmid(containing the putative DNA-binding domain of the two transcription factors fused to the GAL4 activation domain)and the construct pHIS-cis(promoters of the three putative target genes)were co-transformed into yeast strain Y187.As indicated by the activation of the reporter genes,ZmEREB156 can bind to the promoters of ZmSSIIIa gene and ZmEREB17 can bind to the promoters of ZmSSl gene.These results implied that ZmEREB156 and ZmEREB17 have DNA binding activity and may directly regulate the expressions of the target gene ZmSSIIIa and ZmSSI respectively;however,ZmEREB156 may indirectly regulate the expressions of the target gene ZmSh2.