| Rice is not only one of the most important food crops in the world,but also a model plant to study flower development of grasses.The spikelet is the unique characteristic unit of inflorescence architecture in grasses and contains a fertile floret and two pairs of glume-like structures(the sterile lemmas and rudimentary glumes)in rice.Rice spikelets are the main source of human and livestock rations,and their formation and development are also important factors in determining yield and quality.Therefore,it is of great significance to study molecular mechanisms of spikelet development in-depth.Recent studies have identified several key genes regulating rice spikelet development,but molecular mechanisms still remain largely unknown.Therefore,in order to further comprehensively explain the molecular regulation mechanism of rice spikelet development,we need to identify new genes involved in spikelet development.In this study,we systematacially characterized a deformed floral organ recessive mutant dfo2 in the background of indica rice and analyzed the intrinsic molecular mechanism for defects of floral organs.The main results are as follows:(1)A mutant of deformed spikelets was isolated from a 60Co-irradiated mutant pool of the indica rice cultivar 9311.The mutant displayed various defects in spikelets development and we named the mutant as deformed floral organ2(dfo2)accorrding to the phenotypic.Genetic analysis showed that the deformed spikelet trait is controlled by a single recessive muclear gene.Sequence analysis revealed that the dfo2 mutant contains a 15-nucleotide deletion of Os08g0500100,which encodes OsPEX5,an ortholog of Arabidopsis PEX5.OsPEX5 had two transcripts(OsPEX5L and OsPEX5S),and functional complementation confirmed that OsPEX5 is the target gene we are looking for to control spikelet development.(2)OsPEX5 was a ubiquitously expressed gene and strikingly accumulated in the spikelet primordium.Subcellular localization results indicated that OsPEX5 is localized to the cytoplasm and peroxisome.Yeast two-hybrid assays showed that OsPEX5 protein interacted with peroxisome membrane protein OsPEX14,and this result was supported by BiFC and CoIP experiments.These results suggest that OsPEX5 indeed likely acts as bona fide peroxisome receptor protein responsible for import of peroxisomal proteins.(3)Yeast two-hybrid assay showed that OsPEX5 can interact with the 12-oxo-phytodienoic acid reductase OsOPR7 which involved in JA synthesis and localized to peroxisome.Subcellular localization revealed that OsOPR7 could not be positively located to peroxisome in the mutant,and the C-terminus of OsOPR7 protein contained the recognition site of OsPEX5,indicating that OsOPR7 is the cargo protein of OsPEX5.application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7.Knockout of OsOPR7 showed a phenotype of spikelet development defects,while the jasmonic acid content in the mutants dfo2 and Osopr7 was decreased,and application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7.These data suggested that OsPEX5 affects the spikelet development through regulating the transport of OsOPR7 to control the biosynthesis of JA.(4)OsMYC2,as a positive regulator of JA in rice,displayed a severe phenotype of spikelet development when knocked out,and OsMYC2 interacted with OsJAZ1,OsJAZ3 and OsJAZ6 in the OsJAZ family.Yeast one-hybrid,LUC,EMSA and ChIP assays indicated that OsMYC2 directly bind to the promoters of OsMADS1,OsMADS7 and OsMADS14 to activate their expression and that the activity of OsMYC2 is antagonized by OsJAZ proteins in regulating spikelet development.Our results suggest that OsPEX5 plays a critical role in regulating spikelet development through transporting OsOPR7 into peroxisomal,thus providing new insights into regulation of JA biosynthesis in plants and expanding our understanding of of the role and molecular mechanism of plant reproductive development. |
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