| Maize is one of the most important cereal crops,used for both grain and feed,and as an industrial raw material.In recent year,the consumption of maize in China has continued to increase,and there is an urgent need to increase maize production,which depends on the research of its growth and development process.Maize aerial tissues are all developed from the shoot apical meristem(SAM),and the steady state of SAM stem cells needs to be maintained in many ways,the most important of which is the CLV-WUS pathway.The CLV-WUS feedback signaling pathway relies on communication between a series of receptors,peptide ligands,and transcription factors expressed in different regions.FEA3(FASCIATED EAR3)is a new leucine-rich repeat receptor-like protein(LRR-RLP).FEA3 mediates signaling from leaf primordium to inhibit ZmWUS1 gene expression from the bottom of the meristem,which is in parallel to the CLV-WUS pathway.Significantly,the weak allele of fea3 increased the KRN and yield per ear.Therefore,this new feedback regulatory pathway can be used to discover new genetic loci for yield genetic improvement.However,the downstream components of FEA3 receptor and how to inhibit the expression of ZmWUS remains unclear.In addition,the genetic function of ZmWUS gene in maize is not characterized.Therefore,by searching for FEA3-interacting proteins,we can clarify the functions of the interacting proteins and the genetic function of ZmWUS,we hope to increase the understanding of the downstream pathways of FEA3.This study was based on the previous immunoprecipitation and mass spectrometry analysis of the inflorescence meristem of RFP-FEA3 transgenic plants.The protein with a relatively large number of specific peptides specifically detected in RFP-FEA3 samples were selected.BIP1(Binding Protein 1),BIP2(Binding Protein2),CDC48D(Cell Division Cycle 48 homolog D)and GRF1(General Regulator Factorl)were selected as candidate interacting proteins.Firstly,the expression profiles of BIP1,BIP2,CDC48D and GRF1 in meristem were clarified by analyzing the published transcriptome data.Similar to the expression pattern of FEA3 in meristem,the identified candidate genes are also expressed in different meristems.Subcellular localization of these proteins in tobacco leaves was detected to determine whether the candidate protein has the similar subcellular localization with FEA3.BIP1、BIP2 and CDC48D are localized on cell membrane and endoplasmic reticulum,these candidate proteins colocalize locally with FEA3.GRF1 is localized on the cell membrane,which coincided with the location of FEA3.The similarity of localization indicates the possibility of interaction between candidate protein and FEA3.Secondly,the interaction between the candidate protein and FEA3 was further verified by Co-IP.Finally,mutants of the above genes were constructed by CRISPR-Cas9 technique or obtained from maize public EMS mutant library,and were crossed with fea3 mutant,meanwhile double mutant and high order mutant were constructed,in order to further investigate their relationship between FEA3 and candidate proteins and whether the candidate proteins were involved in the regulation of meristem development.Comparing the wild-type and mutant phenotypes in the double mutant segregated population,bip1 and bip2 single mutant have no obvious phenotype in plant morphology and ear,bip1 bip2 homozygous double mutant is lethal,and the lethal phenotype of the double mutant needs to be further explored.The fea3 bip1,fea3 bip2 double mutants and fea3 bip1 bip2 triple mutant obtained by crossing still need to be further characterized in terms of SAM,IM size and number of ear rows;cdc48d and grfl single mutant have no obvious plant and ear development phenotype in the phenotyping.fea3 cdc48d and fea3 grfl double mutants need to be further identified and phenotypic statistics.In addition,mutants of ZmWUSl and its homologous genes identified from maize public EMS mutant library.The obtained homozygous single mutants of Zmwus1,Zmwus2,Zmwus3,Zmwox3a and Zmwox3b had no obvious phenotype.Considering the potential functional redundancy,it is necessary to construct high-order mutants for further functional verification,and construct double mutants with fea3 and high-order mutants for genetic verification with FEA3.Our study provides a basis for the study on FEA3 regulation and its downstream signaling mechanism pathway. |
