Molecular Mechanism Of Sugar Signal Regulating Zein Genes Expression In Maize Kernel

Maize is an important food,economic and industrial crop.Zein is the most abundant storage protein in maize kernel.Sugar signaling plays an essential role in plant growth and development,but it is unclear how sugar signaling regulates zein gene expression.It will be helpful to the genetic improvement of maize kernel quality to study the mechanism of zein accumulation regulated by sugar signaling.In this study,a small kernel mutant,designed as small kernel 3(smk3)was used as a research materials.smk3 mutant kernels display the smaller,thinner,and the tip shrunk phenotype.Kernel weight of mutant is lighter 77% than wild type(WT).Although mutant plants are shorter than WT during seedling stage,the height of mature plant is similar to wild-type control.Genetic analysis showed that smk3 mutant was controlled by a single recessive locus.Further phenotypic analysis revealed that embryo and endosperm in mutant have retarded development,smaller starch granules,and increased protein body content phenotypes compared to wild type kernels.The content of 19Kd-? zein,22Kd-? zein and 50Kd-? zein were increased in the endosperm of smk3 mutant.The soluble sugar content in kernel was also determinated.The results showed that the sucrose content of the mutant smk3 was higher than that of WT.The result of map-based cloning showed that the guanine at position 1447 in the second exon of the candidate gene SMK3 was changed to adenine(G?A),causing the conversion from aspartate(Asp)to asparagine(Asn).Gene expression analysis showed that SMK3 gene was dominantly expressed in the kernel and highly in the kernel at 5 day after pollination(DAP).Amino acid sequence analysis showed that the SMK3 gene encodes a cell wall invertase containing three conserved domains,the cysteine catalytic site MWECPD,the glycosylation motif NAT,and the glycosylation motif NES.The mutation site of SMK3 gene is located on the conserved domain of the cysteine catalytic site.At the same time,we also obtained an allelic mutant smk3-1 with Huang Zao 4 background.In smk3-1 mutant,a 37-bp nucleotide from position 923 to position 959 in SMK3 gene was deleted,which resulted in early termination of protein translation.Analysis of smk3-1 mutant revealed that the phenotype of smk3-1 mutant was identical to that of smk3 mutant.Allelic analysis of target gene indicated that SMK3 gene is Mn1 gene.Differential gene expression profiling revealed that four genes encoding 19Kd-? zein and four genes encoding 22Kd-? zein were up-regulated in the mutant smk3,whereas sugar metabolism related genes were down-regulated.Promoter analysis revealed that all three zein(19Kd-?,22Kd-?,and 50Kd-?)gene promoters contain cis-acting elements that respond to sugar signals.To further determine the regulation of zein gene by sugar signal,wild type 16 DAP fresh endosperms were harvested and cultured in MS medium containing mannitol,sucrose,glucose and fructose,respectively.And then,the expression levels of 19Kd-?,22Kd-? and 50Kd-? zein genes were detected.Three genes expression were significantly up-regulated in the endosperm treated with sucrose.These results were further confirmed in the maize endosperm transient expression experiment by particle bombardment.Expression profile data showed that a WRKY transcription factor SRF1(SUGAR RESPONSE FACTOR1)gene was up-regulated in mutants.Moreover,this gene promoter region(2Kb)contains 4 sugar signal response elements G-box.Dual-luciferase assays demonstrated that SRF1 can activate the expression of ?-zein genes in leaf cell of tobacco.More interestingly,both sucrose levels and SRF1 gene expression were decreased,and zein levels were significantly reduced in the kernels overexpressing Mn1 gene.Taken together,the relationship between sugar signaling and zein content could be outlined based on our results in this study.Mutation of Mn1 gene leads to elevated sucrose levels,which in turn induced expression of SRF1 gene.And then,transcriptional factor SRF1 might directly bind the promoter of zein genes to promote zein gene expression,which eventually results in increament of zein content in mutant kernel.