The ABC Family Member OsABCG3 Is Involved In Pollen Wall Formation In Rice

Utilization of hybrid rice heterosis is an effective way to increase rice yield,and the male sterility is the basis of the extension and application of hybrid rice.Therefore,it is very important to study rice male sterility.Studies in recent years have shown that pollen fertility is closely related to the development of pollen walls.Normally developed pollen walls can protect male gametophytes effectively and contribute to pollen germination.However,researches on the development mechanism of rice pollen wall is still lagging behind that of Arabidopsis.To further understand the development mechanism of rice pollen wall,the EMS-induced mutant library of an indica cultivar 9311 were screened and a new male sterile mutant was identified in this study.The gene that control male fertility was successfully cloned and its function was verified.The main research results are as follows:1.Phenotypic analysis of mutants.A new male sterile mutant was obtained by screening the EMS-induced mutant library of an indica cultivar 9311.The mutant showed normal vegetative growth and floral organ morphology,while the pollen grains were significantly reduced and shrunken,and had not been stained by I₂/KI.Therefore,we named this mutant lsp1?Less and shrunken pollen 1?.Anther semi-thin section results showed abnormally swollen of the tapetum in lsp1 at the stage 11 of the anther development,and hollow and empty pollen was formed at the stage 12.Electron microscopy observation showed an abnormal development of the bacula and the absent of intine formation.2.Gene mapping and cloning.Using the BSA re-sequencing and MutMap method,a specific SNP linkage site?G>A?located on the exon of LOC_Os01g61940 gene was identified.This mutation resulted in an amino acid change of the encoded protein(Ser⁵⁸⁷>Asn⁵⁸⁷).Based on the co-segregation analysis of F₂ generation of lsp1,we preliminarily determined the candidate gene as LOC_Os01g61940.3.Validation of the candidate gene.We then used CRISPR/Cas9 genome editing technology to disrupt LOC_Os01g61940,and obtained five knockout?KO?plants with homozygous mutation?in the background of japonica cultivar Nipponbare?.The phenotype of these KO plants were basically consistent with which of lsp1.Based on the co-segregation analysis of F₂ population of KO plants,we confirmed that LOC_Os01g61940 was LSP1controlling the male sterility.4.Protein structure analysis.LOC_Os01g61940 encodes OsABCG3,a member of ABCG transporter family,which is highly conserved between monocots and dicots.This finding suggests that the biological function of OsABCG3 may be similar to its homologous genes in Arabidopsis?AtABCG1 and AtABCG16?.Protein 3D modeling analysis showed that,in lsp1,the point mutation in LSP1/OsABCG3 caused the change of a?-sheet and two?-helices in its protein structure.5.Expression pattern analysis.qPCR and promoter-GUS analysis showed that LSP1/OsABCG3 was highly expressed in the somatic cell layer and microspores of anther at stage 9,followed by a specific expression in microspores at stage 11.These results indicate that LSP1/OsABCG3 is involved in anther and pollen development.6.Subcellular localization.Transient expression of LSP1/OsABCG3-GFP construct in rice protoplast revealed that this protein was located on the cytoplasmic membrane,indicating that LSP1/OsABCG3 may function on the cytoplasmic membrane.In summary,we cloned a new male sterility controlling gene LSP1,which encodes a transport protein of the ABCG family,OsABCG3,and plays an important role in the development of rice pollen wall.