Biological Function And Molecular Mechanism Of ZmEXPB15 Regulating Maize Kernel Size

The maize kernel consists of three parts:embryo,endosperm and maternal tissue.However,the key genes and underlying mechanisms that control early endosperm development and the maternal nucellus elimination are largely unknown.It is known that kernel size regulation genes are mostly isolated from defective mutants and have limited application potential.It is therefore particularly important to isolate elite alleles that control maize kernel size for genetic improvement of kernel size related traits.We previously identified a major QTL HKW9 for maize hundred-kernel weight on chromosome 9.In this study,we carried out cloning and functional analysis of candidate genes,elite alleles mining and evaluation of breeding effects on qHKW9,the main results are as follows:ZmEXPB15 is a key candidate gene of qHKW9.The ZmEXPB15 encoding EXPANSIN B15 protein was identified by combining the initial localization interval of HKW9 and transcriptomic data of near-isogenic line（NILs）.There were important variation sites associated with hundred-kernel weight in the ZmEXPB15 promoter region,and the expression level of ZmEXPB15 was significantly positively correlated with hundred-kernel weight.The function of ZmEXPB15 in positive regulation of kernel size and weight was verified by transgenic technology.ZmEXPB15 mainly affects the early endosperm development by regulating the elimination process of the nucellar tissue,thereby determining the kernel size and weight.Both ZmEXPB15 gene expression and its protein localization were restricted to maize nucellus tissue,with subcellular localization showing expression in the cell wall,cytoplasm and also nucleus.The observation of early developmental kernel sections showed that the programmed cell death（PCD）process of nucellus in the small-kernel NIL line and the zmexpb15 mutant was significantly slower than that in the control,and the expression of PCD-related genes was also significantly down-regulated,and also in the overexpression（OE1）line which showed the opposite.IP-MS analysis found that ZmEXPB15 interacts with multiple proteases.ZmNAC11 and ZmNAC29 are upstream regulators of ZmEXPB15.The association site of ZmEXPB15 gene promoter region contains the difference of CACG motif of NAC transcription factor binding site.In this study,two NAC transcription factors,ZmNAC11and ZmNAC29,are highly expressed in the nucellus.It was verified that ZmNAC11 and ZmNAC29 can bind to the CACG motif in the promoter region of ZmEXPB15 and activate its expression.Both the single and double mutants of ZmNAC11 and ZmNAC29caused a decrease in hundred kernel weight,kernel length and kernel width.The zmnac11;zmnac29 double mutants had a delay in nucellus cell elimination,impaired endosperm development and severely down-regulated expression of PCD-related genes,indicating that ZmNAC11 and ZmNAC29 positively regulate kernel size and weight by promoting nucellus elimination,similar to ZmEXPB15.Genetic analysis of 3 genes and 8genotypes showed that ZmEXPB15 may act,at least in part,in a common genetic pathway with ZmNAC11 and ZmNAC29 to control kernel weight.ZmEXPB15 has the breeding potential to optimize maize kernel size.The overexpression lines of ZmEXPB15 and the corresponding wild-type lines were crossed with 4 different backbone inbred lines,respectively,and eight hybrid combinations were constructed.All four F₁ hybrids harboring transgenic positive plants had significantly higher hundred kernel weight,kernel length and kernel width compared to control non-transgenic hybrids.In summary,this study elucidated the biological functions of the expansin ZmEXPB15and two NAC transcription factors,ZmNAC11 and ZmNAC29,in controlling nucellus elimination and early kernel development,and revealed that the NAC-EXPANSIN module is an unknown molecular pathway regulating early endosperm and kernel development,while confirming the breeding value of ZmEXPB15 and providing an important genetic resource for genetic improvement of maize kernel traits.