Map-based Cloning And Functional Analysis Of Leaf Shape Gene CFL2 And Plant Height Gene SD38 In Rice

As one of the most important food crops in the world,rice yield is related to global food security and social stability.Rice is the first cereal crop to be sequenced,as well as a relatively simple genome,it has been used as a model plant for monocotyledons,as important as Arabidopsis thaliana for dicotyledons.The emergence of a large number of dwarf and semi-dwarf cultivars led to the first"green revolution"in rice production,which greatly increased crop yields in the 1960s.With the development of society,the deterioration of environmental and the reduction of available cultivation area resulte in the increasingly serious contradiction between food production and the continuous growth of population.Leaf morphology and plant height are the important components of rice ideal plant phenotype,which are related to the improvement of rice yield.The identification of related trait mutants and clone of regulatory genes provides breeding materials and theoretical references for the construction of ideal plant phenotype and the breeding of high-yield rice.The curled flag leaf mutant cfl2(curled flag leaf 2)and semi-dwarf mutant sd38(semi dwarf 38)were isolated from EMS(Ethyl methane sulfonate)mutagenesis library of indica rice restorer line,,Jinhui 10".We performed phenotypic and cytological analysis of cfl2 and sd38 mutant,and completed the mapping cloning of two candidate genes,and also analyzed and verified their functions through a series of biological experiments.The main research results are as follows:1.Curled flag leaf gene CFL2(1)The leaf morphology of the cfl2 mutant was identical to that of the WT during the vegetative growth period.At the booting stage,the flag leaf base of the cfl2 mutant was abnormal,in that the basal half of the leaf was curled inward,whereas the middle and distal portions were normally developed.In addition,the blade of other leaves of the cfl2 mutant,including the second and third upper leaves,were normally unfolded as in the WT.The main agronomic traits of the cfl2 mutant,including plant height,effective panicle,and grain were normal compared with those of WT.The observation of paraffin sections and scanning electron microscopy showed that the abnormal epidermal structure and the enlarged bulliform cells may be the reasons for the curled flag leaf of cfl2 mutant.(2)Genetic analysis showed that the curled flag leaf trait of the cfl2 mutant was controlled by a single recessive nuclear gene.A total of 837 F₂individuals consistent with the mutant phenotype were used for mapping analysis,and CFL2 was fine mapped to the long arm of the chromosome 3 between In Del marker Ind03-11 and Ind03-6 with a physical interval of 78 kb,which containing 15 annotated genes.DNA sequencing revealed a single-nucleotide transition(C to T,casing a substitution of Ala to Val)within LOC?Os03g04680(encodes a cytochrome P450 protein)in the cfl2 mutant.To verify that LOC?Os03g04680 is equivalent to the CFL2 gene,the WT LOC?Os03g04680genomic fragment,consisting of 2877 bp upstream of the start codon,1617 bp open reading frame,and 899 bp downstream of the stop codon,was introduced into the cfl2mutant,and the flag leaf base of the complemented transgene lines were recovered.These results confirm that LOC?Os03g04680 is the CFL2 gene.(3)qRT-PCR analysis and promoter+GUS test showed that CFL2 was expressed in diverse tissues,and the high level of expression was observed in the leaf blade and sheath,especially in flag leaf sheath.In situ RNA hybridization revealed that the CFL2m RNA signal was detected in the outmost cells of shoot apical meristem and leaf,which showed that CFL2 is an outermost cell layer-specific gene.Subcellular localization analysis indicated that CFL2 is located on the endoplasmic reticulum membrane.(4)qRT-PCR analysis found that CFL2 was extremely significant down-regulated in the clf1 mutant(a T-DNA insertion mutant of the HD-ZIP IV transcription factor Roc5).Yeast one-hybrid and dual-luciferase assays confirm that Roc5 could directly bind to the cis-element L1 box in the promoter of CFL2 before activating CFL2expression.(5)The transcriptome sequencing of the flag leaf base from the cfl2 mutant and WT were performed.A total of 1033 differentially expressed genes between the cfl2 mutant and WT were identified,of which 789 genes were up-regulated and 244 genes were down-regulated.The results of GO enrichment analysis showed that genes were significantly over-represented in carbohydrate metabolic process,polysaccharide metabolic process,lipid transport,cellulose biosynthetic and cell-wall-related processes.The expression level of several cell wall-related genes was verified by qRT-PCR,which showed a similar expression pattern with the transcriptome data.The main cell wall component,lignin and cellulose were increased significantly,and the monosaccharide,such as mannose,glucose and xylose were increased at an extremely significant level in the cfl2 mutant.These results indicate that CFL2 may be involved in cell wall development.2.Semi-dwarfism gene SD38(1)The sd38 mutant displayed a semi-dwarf phenotype during all stages of development from seedling to maturity.the panicle length and all internodes of the sd38mutant were significantly shorter than those of the WT.Except for the 1000-seed weight and seed setting rate were significantly reduced in the sd38 mutant,no significant change in other agronomic traits were observed between the WT and sd38 mutant.Microscopic observations found that the lengths of the parenchyma cells in the leaf sheath epidermis of the sd38 mutant were 20%shorter than those of the WT.The length of the parenchyma cells in the middle portion of the second internode were significantly reduced in the sd38 mutant.The area of the first and second internodes below the SAM was also significantly reduced in the sd38 mutant.These results suggest that reduced cell length was the underlying cause of the semi-dwarf phenotype of the sd38 mutant.(2)The genetic analysis of the progeny population from hybrid combination of,,Xinong 1A"and sd38 mutant showed that the semi-dwarfism traits of the sd38 mutant was controlled by a single recessive nuclear gene.A total of 886 semi-dwarf individuals in the F₂ progeny were sampled and used for mapping analysis,and the SD38 gene was localized on the long arm of chromosome 10 between the SSR markers RM25546 and RM25553 with a physical interval of 99 kb,which containing 11 annotated genes.A single-nucleotide transition from G to A,casing a substitution of Ser to Asn,was identified in the sd38 mutant at the locus LOC?Os10g33370 by DNA sequencing.The phenotypes of complementary transgenic and CRISPR/Cas9 knockout transgenic plants confirmed that LOC?Os10g33370 was the SD38 gene.(3)Expression pattern analysis showed that SD38 was not expressed in the root,but was expressed in the abovementioned part,and the expression level was higher in the caulicle,tiller bud and leaf sheath.Subcellular localization showed that SD38 was located on the membrane of endoplasmic reticulum.SD38 encodes a fatty acid elongase,?-ketoacyl-Co A synthase(KCS),which is involved in the synthesis of very-long-chain fatty acids(VLCFAs).Phylogenetic analysis showed that SD38 protein had the highest identity with two unknown Arabidopsis KCS proteins,At KCS3 and At KCS12,and had lower homology with four KCS proteins reported in rice.(4)A functional complementarity test in Saccharomyces cerevisiae indicated that SD38 was capable of complementing the deficiency of Elo3p activity in BY4741-elo3knockout yeast cells by controlling the synthesis of C24:0 VLCFA.The content of C24:0 VLCFA in the sd38 mutant was also significantly lower than that of WT.Lipid omics analysis revealed that no significant change in total fatty acid content in the sd38mutant,but the amounts of the major components of the cellular membranes,lyso PE and lyso PC wre significant decrease in the sd38 mutant.These results indicated that SD38 was involved in the extension of C24:0 VLCFA and affects the composition of fatty acids in the rice.(5)Exogenous supplied C24:0 VLCFA and cafenstrole(CS),an inhibitor of VLCFAs biosynthesis,can promote or inhibite plant height of WT and sd38 mutant seedlings to different degrees,respectively.These results indicate that the defective C24:0 VLCFA was responsible for the semi-dwarf phenotype of the sd38 mutant.q PCR analysis found that the expression of genes associated with to ethylene synthesis were down-regulated,especially Os ACS2,Os ACS3,Os ACS4,and Os ACO7,which attained significant differences,leading to a significant decrease ACC(ethylene synthesis precursor)in the sd38 mutant seedlings when compared to WT.Exogenous application of ACC and ethephon partially recovered the semi-dwarf phenotype of sd38 mutant seedlings.The transcript level of Os ACS3 was induced by the application of C24:0.In contrast,the CS suppressed expression of Os ACS3.These results indicated that SD38was involved in the synthesis of C24:0 VLCFA and influenced rice plant height by regulating ethylene content VLCFAs may influence ethylene biosynthesis by regulating the transcription of Os ACS3.