Map-based Cloning And Functional Analysis Of An Narrow And Rolled Leaf Gene NRL1 In Rice

Leaf is the most important organ for photosynthesis in rice plant,which directly affects the grain yield and the accumulation of dry matter.Moreover,the molecular regulation of leaf shape and development is also very important for basic research of plant sciences.Therefore,leaf shpe mutant is important for both basic research and rice breeding.In the present study,we identified a narrow and rolled leaf mutant,nrl1.we analyzed the NRL1 gene function based on phenotypic and microscopic analyses,genetic mapping,positional cloning,gene expression and RNA-seq,etc.The main results are as follows:1.The nrl1 mutant showed several mutative phenotype,such as dwarfism,narrow and rolled leaf,shorter root system,increased tiller number and elongated grain length,etc.Microscopic analysis showed that the nrl1 mutant has reduced size of leaf bulliform cells,decreased number of leaf veins and reduced area and length of cells in stem.2.Genetic analysis revealed that the nrl1 is controlled by a single recessive nuclear gene.Using molecular markers,the NRL1 gene was mapped to 90-kb physical region on chromosome 12.Map-based cloning revealed that the NRL1 gene encodes a member of cellulose synthesis protein D4,Os CSLD4 protein.In the nrl1 mutant,a point mutation(C to T)in the second exon region leads to the loss-of-function of NRL1 gene.The NRL1 gene function was further confirmed by complementation experiment.3.Gene expression analysis revealed that the NRL1 gene was higher expression in roots of the seedling stage and tillering stage.Moreover,it was revealed that the expression of NRL1 could be induced by treatment with GA,Me JA,ABA or drought stress.Sub-cellular location analysis showed that the NRL1 protein located in entire cell.4.In the nrl1 mutant,it was showed that the expression levels of RL14,Os MYB103 L,Roc5,Os LAC17,SND1,Os CESA1,2,3,5,6 and 8 were down-regulated,further indicating the NRL1 function in regulating cellulose synthesis in rice.5.The NRL1 protein has a conserved cellulose-synt domain structure and belongs to a branch with At Csl D5 and Bd Csl D4 in evolutionary relationship.The evolutionary relationship suggesting that they may have similar functions.6.Transcriptome sequencing results showed that a total of 848 genes were down-regulation and 1531 genes were up-regulation in the nrl1 mutant.Further analysis revealed that the molecular functions of the differentially expressed genes(DEGs)were mainly concentrated in catalytic activity,molecular binding and transport activity.In terms of cellular components,the DEGs are mainly involved in the formation of membrane components,organelle components,and macromolecular complexes.In terms of gene pathway annotation,the DEGs are involved in metabolic processes,cellular processes,single-tissue processes and biological processes.The DEGs were further enriched in multiple KEGG pathways,such as carbon metabolism,amino acid synthesis,and cysteine and methionine metabolism.Taken together,our results suggest that NRL1/Os CSLD4 gene play an important role in regulating leaf shape and development.The present study also enriched the germplasm resource related to leaf shape.