Identification Of Regulatory Factors In Maize Shoot Apical And Inflorescence Meristem

Maize is one of the three major crops in China and plays an essential role both in daily life as well as in industrial production.The study of the regulatory mechanisms of maize growth and development is an important guideline for the improvement of yield traits in maize.The aboveground part of maize is derived from shoot apical meristem(SAM),which can directly and indirectly form maize inflorescence meristem(IM).And the female inflorescence meristem of maize develops to form the female ears of maize.Previous studies have shown that the size of IM in maize is positively correlated with the kernel row numbers,which is an important factor for maize yield.So,the development of IM directly affects the yield of maize.Therefore,the study of SAM and IM regulatory mechanism could help us to provide new resources for the maize breeding to improve maize yield to meet the urgent demand for maize in China.Previous studies have shown that maize meristem development is maintained by the classical CLAVATA-WUSCHEL(CLV-WUS)negative feedback pathway.The CLV receptors TD1 and FEA2,as well as the ligand peptide CLE7,play important roles in maize SAM and IM development.FEA3 is a recently identified new receptor protein in maize.FEA3 perceives FCP1 signaling from the primordia and inhibit WUS from downward gene expression.Studies on FEA3 reveals a novel pathway in which signals from differentiating cells can provide feedback to the stem cell niche,which is in parallel to the classical CLV-WUS feedback pathway that occurs at the center of the meristematic tissue,by repressing WUS gene expression from the bottom of the meristematic tissue.Thus,FEA3 could maintain the balance between cell division and differentiation in meristem.We took the receptor protein FEA3 as a starting point to explore the possible regulatory factors involved in the SAM and IM regulatory pathways.We found the interactions between calnexin homolog(CLX)and FEA3 receptor protein in maize and speculated that CLX might be involved in FEA3 mediated pathway.Therefore,we used CRISPR to create CLX mutant and obtained clx1,clx2,and clx1;clx2 double mutants by crossing.Based on the plant and ear phenotypic analysis,no significant differences were found between clx1.clx2 single mutants and the wild type.But,the size of ear was slightly smaller in clx;clx2 double mutants than that in wild type.Once we have enough seeds for the mutants,we will carry out detailed phenotypic analysis by measuring the SAM and IM size and statistically analyzing kernel row number to further clarify whether the clx mutation effects meristem development.FEA3 is a newly identified receptor protein in maize.To explore whether there are FEA3 homologs with compensatory effects and redundancy in their functions,we identified four homologs of FEA3 in maize by phylogenetic analysis,which are F3H1,F3H2,F3H3,F3H4,with different levels of expression in meristem tissue.We generated loss-of function mutants of these four genes using CRISPR knockout technology and obtained f3h1;f3h2,f3h3;f3h4 quadruple mutants by crossing and self-crossing.Preliminary morphological observation of the ears showed that no significant differences were found between the quadruple mutants and the wild type.We also crossed the fea3 mutants and constructed the fea3;f3h1;f3h2;f3h3;f3h4 quintuple mutants.Mutations in f3h1;f3h2;f3h3;f3h4 did not significantly enhance the fasciated phenotype of fea3.Next,we will expand the sample size and perform SAM,IM size and ear row number data statistics as well as analysis to further investigate the functions of these homologous genes and their genetic relationships with FEA3.Finally,we also investigated whether the transcription factor ZmHAIRY MERISTEM(ZmHAM)family proteins are involved in the regulation of maize meristem development.Studies in Arabidopsis have shown that HAM is another transcription factor other than WUS involved in the regulation of meristem development.HAM interacts with WUS and participates in the regulation of meristem by inhibiting activation of CLV3 by WUS.Whether it is involved in the signaling regulation pathway mediated by FEA3 remains to be clarified.Therefore,for three ZmHAM genes with high expression in the meristem,we identified their single mutants from the EMS mutant library.We found that mutation in any single Zmham gene did not affect normal plant growth and development,suggesting redundancy among these three genes in maize.We started to construct multiple mutants of these genes,and further investigate the function of these ZmHAM genes in the regulatory pathway of meristem.