| Rice(Oryza sativa L.)is a staple food for over half the global population and has a pivotal position in ensuring food security.Rice plant architecture is the foundation of yield and represents a collection of genetically controlled agronomic traits.Plant height and panicle morphology are two major agronomic traits in rice plant architecture,reasonable rice plant height and panicle traits such as the number of panicles per unit area,the number of grains per panicle,and the thousand-grain weight are important factors in determining the growth and yield potential of rice.Currently cloned rice plant height-related genes mostly introduce hormonal pathways to elucidate gene functions.However,studies about how the intracellular vesicle transport pathway regulates rice plant architecture at the cell level are still scarce,relevant genes are still less identified and the molecular mechanism remains unclear.In addition,the degeneration of spikelets in panicles often occurs during the natural planting or breeding of rice,which seriously affects the final yield of rice.Spikelet degeneration is susceptible to environmental factors and unstable in phenotype,which makes it difficult to clone genes that responsible for this trait.Nowadays,the molecular mechanism or genetic mechanism revealing the spikelets degeneration remains little understanding.In this study,we identified a plant architecture mutant dph1-1(decreased plant height 1-1)derived from the progeny of tissue culture,and its defect phenotype can stabilize inheritance.We analyzed cytological detail of the dwarf and spikelets degeneration phenotype in the dph1-1 mutant during rice development.By map-based cloning,we isolated the target gene DPH1 and investigated the molecular mechanism of rice dwarf and spikelets degeneration controlled by DPH1 in further experiments.The main results of our research are listed as follows:1.The growth and development of the dph1-1 is arrested from the seedling stage to the mature stage compared with the wild-type,which is manifested by the shortening of root length,decreased plant height,shorter panicle length,smaller grain,leaf color fades,and delayed heading date.The spikelets in the panicles are seriously degraded phenotype,the number of primary and secondary branches decreased,the number of grains per panicle decreased significantly,and the yield per plant decreased in the dph1-1.2.Observation of young panicles at rice different developmental stages showed that the spikelet degeneration of the dph1-1 occurred in the early stage of young panicle development.The reactive oxygen species in dph1-1 degenerated spikelet cells were unbalanced and the cells produced programmed cell death(PCD).The results of flow cytometry and microscopic observation showed that compared with the wild-type,the cell number of the dph1-1 remained unchanged,and there was no obvious abnormality in cytokinesis.Cytological observation and statistics found that the cell area reduced and cell expansion was affected in the dph1-1.3.Using the map-based cloning method,the mutant gene was finely mapped to a range of about 73 kb region at the long arm of rice chromosome 1.Genomic sequencing revealed that the donor splicing site of the sixth intron of ORF7(LOC＿Os01g70320)was found to produce a single-base mutation with G to T change,resulting in intron cleavage errors and subsequently led to abnormal transcripts and premature terminations proteins.Transgene complementation,RNA interference(RNAi)and gene knockout experiments verified that ORF7 is the target gene to control the phenotype of the dph1-1 mutant.DPH1 is plant-specific and encodes a protein containing two coiled-coil domains and an unknown functional domain.Amino acid sequence alignment found that DPH1 is a homologous gene of Arabidopsis SCD2(Stomatal Cytokinesis Defective 2),thus we also designate DPH1 as OsSCD2.4.Consistent with the pleiotropic phenotype of the dph1 mutants,quantitative RT-PCR analysis revealed that OsSCD2 was widely expressed in all tissues examined including roots,leaves,leaf sheaths,stems,and young and mature panicles,with relative higher expression in leaves and mature panicles.OsSCD2 is a peripheral membrane protein localized to the plasma membrane and cytoplasm of the cell.The immunogold labeling experiment and the co-localization experiment of organelles showed that OsSCD2 is mainly localized to clathrin-coated vesicles(CCVs)5.The uptake and intracellular accumulation result of the endocytosis marker FM4-64 indicated that the endocytosis in the dph1-1 was affected.After the treatment of the vesicle trafficking inhibitor inhibitor BFA with the auxin export carrier OsPIN2 as the post-Golgi trafficking protein,it was found that the exocytosis and endosomal recycling of PIN2 was altered in the dph1-1.Protein mass spectrometry assays results and protein interaction verification experiments showed that OsSCD2 physically interacts with OsSCD1.The phenotype of the dph1-1 Osscd1 double mutant indicates that OsSCD1 and OsSCD2 function genetically in a common pathway.6.Transmission electron microscopy observation revealed that the sclerenchyma and parenchymal cell walls of dph1-1 mutant internodes was significantly thinner than those of the wild-type.Co-localization experiments show that OsSCD2 and cellulose synthase co-localize in the plasma membrane and cytoplasm.A subcellular fractionation assay to separate the plasma membrane and endomembrane fractions detected a higher proportion of OsCESA4 distributed to the endomembrane fraction in the dph1-1 mutant.Cell wall component determination showed that cellulose content was decreased in dph1-1 and several primary monosaccharide components of cell wall were significantly altered in the dph1-1 mutant compared with the wild-type.The dph1-1 mutant was also more sensitive to cellulose synthesis inhibitor isoxaben treatment.These results collectively suggest that the OsSCD2 mutation impaired cellulose synthesis,most likely by disruption of CESAs transport in the dph1-1. |
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