Genetic Regulatory Network Of MAPK-MYB86-CAD2 Controls The Leaf Morphogenesis In Rice

Crop plant type is one of the main factors determining yield.Moderate curling of leaf is conducive to shaping an ideal plant type,improving light utilization efficiency,and improving the light conditions at the base of the population in the middle and late stage of rice growth.So far,many genes related to curl leaf have been reported and preliminary studies on their molecular regulation have been carried out.Still,most of these works study on a single gene for regulating leaf morphological development,so little is known about the coordination relationship of these regulation genes.The analysis of their cellular basis,molecular regulation mechanism,and interaction regulation network is insufficient.It hinders the exploration and utilization of high light efficiency rice which can improve leaf morphology and shape ideal plant type through molecular regulation.This study screened a mutation with ideal plant type characteristics from the mutant rice library,named rlm1-D.The mutant had moderately curly leaves,upright leaves,and a compact plant type.An R2R3-type MYB transcription factor was cloned by a map-based cloning strategy.A T-DNA insertion at the rlm1 promoter region induced the ectopic overexpression of rlm1,which improved lignin synthesis in rice,increased the thickness of the secondary cell wall and affected water transport from root to leaf,and finally leaded to the curly leaf.Transcriptional activation analysis of rlm1 in yeast showed that rlm1 was an MYB with transcriptional activation activity.It was further found that a peptide with 18 amino acid sequences was closely related to its transcriptional activation activity,and the acidic amino acids in the peptide were closely related to its transcriptional activation activity.Transcriptome analysis showed that the genes with up-regulated expression in rlm1 mutant were mainly enriched in the phenylpropanoid metabolic pathway,an important lignin synthesis pathway.Through the TF-centered Y1 H assays multiple motifs were found that could be bound with RLM1.Combining with transcriptome results,it was found that there was an MYB1 AT motif in the promoter of OsCAD2.OsCAD2 is a key enzyme of the lignin synthesis pathway specifically expressed in stem nodes.RLM1 can activate OsCAD2 expression and increase lignin content.The RLM1 mutation was generated by CRISPR-Cas9,named rlm1.The secondary cell wall of the stem node of rlm1 was significantly thinner and the pore diameter increased,but the lodging resistance of rlm1 was not affected.The plant height of rlm1 was like that of the wild type,the number of grains per panicle per plant increased significantly,and although there was no significant change in 1000-grain weight,it also showed a trend of yield increase,but its molecular mechanism still needs to be further studied.OsMAPK10,a protein interacting with RLM1,was obtained by Yeast TwoHybrid screening.The interaction between OsMAPK10 and RLM1 was proved by DNA-binding assay and EMSA experiments.Further research found that OsMAPK10 could phosphorylate RLM1,but the phosphorylation site was not been obtained.The phenotype of OsMAPK10 overexpressed was like that of rlm1,in which the lignin content of the stem increased,and the secondary cell wall thickened.The transcriptional activation experiment in rice protoplast also proved that phosphorylated RLM1 could enhance the activation ability of downstream genes.Therefore,a new OsMAPK10-RLM1-OsCAD2 gene regulatory network was preliminarily constructed to shape the ideal plant type of rice.The finding that the secondary cell wall thickness of stem nodes is increased by increasing the lignin content to affect the leaf morphology provide a new idea for the study of regulating leaf morphology.The cloning of RLM1 also provides a new gene for shaping the ideal plant type of rice by modifying the leaf morphology.The materials of Rlm1 expression and knockout also provide new genetic material for molecular breeding.