Mechanisms Of OsNSL2 And OsCPP1 Regulating Rice Leaf Development

Rice(Oryza sativa L.)is one of the most important cereal crops worldwide and a major source of food for humans.According to the statistical data of the Food and Agriculture Organization of the United Nations,over 3 billion people worldwide rely on rice as their primary source of food.Especially in the Asian region,rice is an indispensable food on people’s tables and play an important source in income for farmers.However,due to the impact of climate change,population growth,and other factors,global food security issues are becoming increasingly severe.Therefore,it has become an important goal for global research to improve rice yield and quality.Moreover,as a model plant,rice has made significant contributions to the development of plant science in genetics and molecular biology research.Therefore,in-depth study of the biological characteristics,growth and development rules,yield and quality regulation mechanisms of rice is not only helpful to improve rice yield and quality,ensure global food security,but also to promote the development of plant science,providing reference and inspiration for the research of other economic crops such as rapeseed and maize.The two mutants used in this study were independently induced in the laboratory and were treated with ethyl methane sulfonate(EMS).One of the mutants,osnsl2(narrowstripe leaf2),was obtained by screening the indica restorer line Jinhui 10(J10)after treatment with EMS,and the other mutant oscppl(carbohydrate partitioning protein 1)was screened by EMS treatment of a maintainer line rice Xinong 1B.On the other hand,we investigated the cause of narrowed leaves in osnsl2 through phenotype analysis,cytological observation,map-based cloning,subcellular localization,dNTP content measurement,flow cytometry analysis,and qRT-PCR analysis.has been genetically identified,map-based cloning,and complementation verification in our laboratory.It was found that oscppl is a mutant with a dominant carbohydrate allocation defect,and OsCPP1 has previously controlled the allocation of carbohydrates.Functional analysis was carried out.This paper will conduct related research on how OsCPP1 regulates the leaf angle.Through phenotypic analysis and cytological observation,it was found that the number and number of sclerenchyma cells in the occipital part of the oscppl leaf were reduced.At the same time,qRT-PCR analysis it was found that oscppl was highly expressed in the leaf occipital,and it was verified by yeast two-hybrid and bimolecular fluorescence complementation experiments that there was an interaction between OsCPPl and the cellulose synthase family protein CSLA6.The main research results are as follows:1.The narrow leaf mutant gene OsNSL2.(1)The narrow leaf stripe mutant osnsl2 was successfully isolated from the mutant population of rice J10 induced by EMS in the early stage of the laboratory.Through statistical observation,it was found that compared with WT,the leaf width of flag leaf,inverted second leaf and inverted third leaf of osnsl2 was significantly reduced,while the leaf length had no significant difference.In order to study the reason for the decrease of leaf width,we observed the morphology and histology of the middle part of the leaf at the booting stage,and found that the average number of large and small veins of osnsl2 was significantly reduced by paraffin section and scanning electron microscope.By light microscopy,it was found that the number of cells along the leaf width was significantly reduced in osnsl2,while the cell width remained unchanged.These observations suggest that the reduced leaf width may be due to a reduced number of cells along the width of the leaf.(2)The OsNSL2 gene was previously mapped to the short arm region of chromosome 6 in our laboratory.In this study,we used 1500 recessive individuals from the F2 generation to perform fine mapping.The OsNSL2 gene was previously mapped to the short arm of chromosome 6 in our laboratory.In this study,we performed fine mapping using 1500 recessive individuals from an F2 population.Eventually,OsNSL2 was mapped to a 51 kb interval between Indel6-1 and Indel6-2 on the short arm of chromosome 6.DNA sequencing was performed for all 10 annotated genes within this region.We found a single nucleotide substitution from G to A in the LOC_Os06g14620 gene encoding a ribonucleotide subunit,resulting in a change from glycine(Gly)to serine(Ser)in the encoded amino acid sequence,which is responsible for the narrow leaf phenotype observed in osnsl2.Further investigation by transforming a segment of LOC_Os06g14620 from WT(including the upstream 2000 bp sequence,1020 bp coding sequence,and downstream 899 bp sequence)into osnsl2 callus tissue led to complementation of the osnsl2 phenotype in positive transgenic plants,indicating that LOC_Os06g14620 is the OsNSL2 gene.(3)Based on map-based cloning and genetic complementation experiments,OsNSL2 encodes a small subunit of ribonucleotide reductases(RNRs).By constructing a phylogenetic tree,the ribonucleotides encoded by OsNSL2 were found in monocotyledonous and dicotyledonous plants The small subunit of reductase has a relatively conservative biological function,and OsNSL2 has a close homologous relationship with LOC_Os06g03720,another small subunit of ribonucleotide reductase,in rice.(4)Using qRT-PCR to analyze the expression pattern of OsNSL2,it was found that OsNSL2 was expressed in roots,stems,leaves,sheaths and panicles,but the expression level was the highest in leaves;at the same time,a p35S::OsNSL2-GFP fusion expression protein was constructed and passed through rice protoplasts Transient expression in vivo found that green fluorescent signals could be detected in the nucleus and cytoplasm,indicating that OsNSL2 was localized in the nucleus and cytoplasm.(5)By measuring the dNTPs content in WT and osnsl2,it was found that the dNTPs content of osnsl2 decreased.Further analysis by flow cytometry and expression analysis of cell cycle-related genes showed that the cell cycle of osnsl2 decreased.The results showed that dNTPs content was reduced,leading to a blockage of DNA synthesis,which in turn affected the cell cycle,eventually resulting in a decrease in cell number and narrowing of leaves.2.OsCPP1 regulates leaf angle in rice.(1)Based on the observation and statistical analysis of leaf angle in WT and oscppl at seedling,tillering,and maturity stages,we found that oscppl had a significantly increased leaf angle compared to WT.Specifically,at the seedling stage,the leaf angle of oscppl was 44.5°,which was 161%higher than that of WT(27.6°);at the maturity stage,the average leaf angle of oscppl was 53.1°,which was 283%higher than that of WT which was 18.7°(n=20).The difference in leaf angles between oscppl and WT was extremely significant.Through observation with paraffin sections and transmission electron microscopy,it was found that the number of thick-walled cells,cell layers,and the distance between near and far axis ends in the leaf sheath of oscppl were significantly smaller than those in WT,indicating that the increase in leaf angle of oscppl was due to a decrease in the number of cells,cell layers,and distance between near and far axis ends at the leaf cushion.(2)A yeast screening library was performed by constructing the pGBKT7-CPP1 bait vector.And by comparing the cloned sequences,the cellulose synthase family protein CSLA6 interacting with OsCPPl was finally screened.Therefore,OsCPP1 synergistically regulates cellulose synthesis by interacting with CSLA6,and then participates in the regulation of leaf angle formation.(3)Through yeast two-hybrid assays,we identified that OsCPP1 interacts with three cellulose synthesis-related domains of CSLA6.Furthermore,BiFC assays showed that OsCPPl interacts with CSLA6,indicating that OsCPP1 affects cellulose synthesis and ultimately leads to an increase in leaf angle.(4)The analysis of the cellulose content in the occipital of oscppl showed that the cellulose content in the occipital of oscppl was only 70.7%of that of WT,which was significantly lower than that of WT;at the same time,the cellulose synthesis-related genes CESA1,CESA4,CESA7,CESA8,and CESA9 were found in oscppl leaves.The expression levels in the occipital region all decreased significantly.