OsEMF2b Participates In The Molecular Mechanism Of Controlling Anther Development In Rice

Rice is an important food crop in China.Its flower organs are both breeding organs and the basis of rice grain formation.The development of flower organs is the key to affect rice yield and quality.The study found that PcG(polycomb group)gene EMBRYONIC FLOWER 2b(OsEMF2b)is very important for the development of flower organs in rice,and its mechanism for flowering time,flower organ morphology,and the regulation of seed development have been studied in depth,but In some of the related mutants,flower organ development is seriously deficient,resulting in the lack of anthers.Therefore,the study of OsEMF2b involvement in male reproductive organ anthers and pollen development control mechanisms is still relatively small.In this study,we identified a weak mutant of osmf2b-4,the T-DNA insertion of the OsEMF2b gene,which is characterized by severe floral organ development defects.Using semi-thin sections,in situ hybridization,subcellular localization,and RT-q PCR methods,the biological function of OsEMF2b in the development of rice anthers was studied,revealing that OsEMF2b plays an important role in the development of anthers and pollen after meiosis.The main results are as follows:(1)The obtained OsEMF2b gene T-DNA insertion mutant osemf2b-4 was detected and co-segregated.The results showed that there was a 200 bp insertion in the promoter region of OsEMF2b gene,and the mutant phenotype was co-segregated from the T-DNA insertion site,a binary expression vector fused with the rice ACTIN promoter(strong promoter)and the OsEMF2b coding sequence was transformed into the mutant osemf2b-4,for complementation experiments.The results showed that the positive plants with successful recovery of OsEMF2b expression showed normal anther development and pollen,indicating that the phenotype of the mutant osemf2b-4 was indeed caused by the weakened function of the OsEMF2b gene.(2)The homozygous plants were slightly shorter than the wild-type plants.The flower organs showed different degrees of developmental defects,and the pollen development decreased to varying degrees.Among them,minor defects were divided into Small Husk,Beak-Like,Open-Husk,and long sterile lemma,accounting for 34.5%,34.5%,13.8%,and 6.9%;severe defect types included double caries 3.4%,glomerulus curl 3.4%,absence of internal malleolus or lateral malleolus 2%,and leafy spikelets 1.4%.Real-time quantitative PCR(RT-q PCR)analysis revealed that the OsEMF2b gene was significantly reduced in the mutant osemf2b-4 in spikelets and leaves;By slicing the anthers of mutant osemf2b-4 at different pollen development stages,it was found that the mutants showed various types of developmental defects,such as tapetum swelling or delayed degradation,lack of middle layer,etc.,which ultimately cause varying degrees of fertility defects.(3)The results of in situ hybridization showed that the OsEMF2b gene was highly expressed in tapetum and microspores during meiosis and in tapetum during microsporation;subcellular localization in rice protoplasts showed that OsEMF2b was localized in the nucleus.(4)A total of 15 nuclear localization genes involved in anther development or pollen development were detected in the mutant osemf2b-4 by RT-q PCR.The results showed that 14 genes including GAMYB were up-regulated in mutants.Only PTC1 expression was detected in the mutants.This indicates that OsEMF2b may participate in the development of rice anthers and pollen by regulating the expression of these genes.