The Regulation Mechanism Of MSCA1 And Paralogous Genes In Maize Ear Development

Ear type directly affects maize yield,so it is of great significance to study the molecular regulation mechanism of ear morphogenesis in maize.The redox state also makes a major contribution to the development of meristems.Glutaredoxins（GRXs）are small oxidoreductases which can modify the redox（reduction/oxidation）state of the target protein activities as the electron donor.However,the mechanism by which GRXs modulate the activity of target proteins in vivo is missing.In this study,we show that three maize CC-type glutaredoxin（GRX）genes,including MALE STERILE CONVERTED ANTHER1（MSCA1）and its two paralogs Zm GRX2 and Zm GRX5,and found they played important role in meristem development and ear morphogenesis through modifying the redox state and activity of the putative target,a TGA transcription factor FASCIATED EAR 4（FEA4）that acts as a negative regulator of inflorescence meristem development.The main results of this study are as follows:Using CRISPR/Cas9 to knocked out Zm GRX2 and Zm GRX5 which are two paralogous genes of MSCA1,and loss-of-function mutants zmgrx2 and zmgrx5 were obtained,which were crossed with msca1-ref mutant to obtain multiple mutants,the phenotypic investigation was performed.We find the development of the shoot Apical Meristem（SAM）and Inflorescence Meristem（IM）of the msca1;zmgrx5 double mutants were significantly suppressive,resulting in a significantly shorter plant height and decreased in the tassel branches number and suppressed ear development.In the msca1;zmgrx2;zmgrx5 triple mutants,these phenotypes are more prominent,indicating that the GRX plays a key role in the development of maize meristems.Spatiotemporal expression and in situ analysis found that the gene expression levels and expression patterns of MSCA1,Zm GRX2,and Zm GRX5 were similar.They are specifically expressed in Spikelet-Pair Meristem（SPM）and vascular tissues in maize ears.In addition,MSCA1 and Zm GRX5 are also expressed in the inflorescence meristem（IM）and have complementary positions,while Zm GRX2 is not expressed in IM,implying that MSCA1 and Zm GRX5 play a leading role in IM development in addition to the functional redundancy of the three genes.Through Yeast Two Hybrid（Y2H）and Luciferase Complementation Image（LCI）experiments,the interaction between FEA4 protein and MSCA1 subfamily protein was verified,and the key amino acid position which impact on interaction was found through point mutations.Crossing the grx-triple mutant（msca1;zmgrx2;zmgrx5）with the loss-of-function mutant fea4 of the TGA family transcription factor FASCIATED EAR4（FEA4）to obtain four mutants:fea4;grx quadruple mutant,and its ears also appear the abnormal increase in IM leads to the fascinated phenotype,indicating that FEA4 is downstream of these three GRXs.This genetic interaction relationship validates the proteins interaction results.Through Biotin labeling assay and Biotin switch assay,we found that FEA4 protein can be oxidized by H₂O₂,showing sensitivity to changes in redox state,and glutaredoxin MSCA1 can act as an electron donor to inhibit Oxidation of FEA4.FEA4 exists as monomer and dimer in vivo.It was found that the ratio of FEA4 monomer/dimer was sensitive to the redox state by application of redox reagent in vitro and in vivo.The YFP-FEA4 transgenic plant and the grx-triple mutant were hybridized to obtain the material of YFP-FEA4 in the grx-triple mutant background,and compared with the YFP-FEA4 in the wild-type background,we found that the proportion of the dimer form increased significantly in vivo,indicating that the GRX could reduce the FEA4 dimer to maintain its balance between monomer and dimer,then regulate its activity.The change of redox state can influence the ratio of FEA4 dimer/monomer.Chromatin immunoprecipitation sequencing（Ch IP-seq）by young ears of YFP-FEA4in the wild-type and grx-triple mutant background,we found that FEA4 has stronger DNA binding ability in the grx-triple mutant background,meanwhile,through contrast the RNA-seq analysis between lack of FEA4 in wild-type and grx-triple mutant background showed that the transcriptional activity of FEA4 in grx-triple mutant was also significantly enhanced.The further Ch IP-q PCR and RT-q PCR experiments verified more dimeric form（in the grx-triple mutant background）of FEA4 protein can enhance its ability to regulate downstream gene transcription.Using the young ears of the wild-type,fea4 mutant and the grx-triple mutant for RNA-seq analysis.We found large common downstream response genes between fea4 mutant and the grx-triple mutant,and a part of these common different expression genes which related the development were opposite regulated,suggesting FEA4 could be the main downstream regulatory protein of the three GRX proteins.Combining the opposite phenotypes of the fea4 and the grx-triple mutant in ears,We speculate that the MSCA1 and its paralogs mainly regulate the redox state of FEA4 to affect its activity in the inflorescence meristem to regulate the development of ear.Our findings reveal the important role of glutaredoxin in maize meristem development and ear morphogenesis,also provide direct evidence for glutaredoxin mediated redox modification of target proteins in plants and the related regulatory mechanism,which has guiding significance for maize plant architecture and yield improvement.