Transcriptome Analysis Of Flower Buds And Characterization Of Two MYB Transcription Factors Involved In Pollen Development In Brassica Campestris

Pollen plays a key role in plant reproduction by producing male gametes and delivering them to the embryo sac for double fertilization.The processes of pollen development are quite complex,involving the expression and regulation of numerous genes and proteins.However,the existing research results on pollen development are not enough to fully understand the molecular mechanisms regulating these processes.Comparative transcriptome analysis of mutant defective in pollen development and its wild-type is a powerful approach to detect genes involved in pollen development and to investigate their dynamic expression characteristics during this biological process,and ultimately to help to understand the characteristic of pollen development and the corresponding molecular mechanism,thus providing an important theoretical basis for the control of plant fertility in agricultural production.‘Aijiaohuang’ genic male sterility AB line(ajhGMS ‘Bcajh97-01A/B’)in Brassica campestris L.subsp.chinensis Makino,syn.B.rapa subsp.chinensis were used in the present study.Using the sterile and fertile floral buds of ‘Bcajh97-01A/B’ as materials,a detailed gene expression profile at five typical developmental stages(Ⅰ ~ Ⅴ),namely,pollen mother cells,tetrad,uninucleate pollen,binucleate pollen,and mature pollen stage,were examined by RNA sequencing(RNA-seq).The analysis of stage-specific differentially expressed genes combined with detailed phenotypic observation of ‘Bcajh97-01A/B’ digged out a number of functional genes and transcription factors involved in pollen development and pollen wall formation in Brassica campestris.Subsequently,the two MYB genes,BcMF28 and BcMYB1R203,were selected for further research.Their sequence structures,protein properties and spatiotemporal expression pattern were analyzed.Furthermore,artificial miRNA technology or RNA interference technology and heterologous expression experiment were adopted to verify the biological functions of the genes.The morphological,molecular biological and cytological aspects of the corresponding transgenic plants were characterized to understand the functional mechanisms of the genes.The main results are summarized as following:(1)Detailed phenotypic observation of ‘Bcajh97-01A/B’ revealed that the karyokinesis during meiosis was demonstrated to be normal,whereas callose cound not deposite normally in the cell plate of tetrads in ‘Bcajh97-01A’,leading to failed formation of tetrads.And the premature tapetal programmed cell death in ‘Bcajh97-01A’ caused defects in development of tapetosome and elaioplasts,leading to abnormal pollen exine development.(2)Using the sterile and fertile floral buds of ‘Bcajh97-01A/B’ as materials,a detailed gene expression profile at five typical developmental stages(Ⅰ ~ Ⅴ),namely,pollen mother cells,tetrad,uninucleate pollen,binucleate pollen,and mature pollen stage,were examined by RNA sequencing(RNA-seq).In total,8,288 genes(11.48% of Unigenes)were differentially expressed in at least one stage of the sterile floral buds compared with the fertile ones,and these genes were designated herein as DEGs.Functional analysis of these DEGs identified stage-specific genes associated with the main events associated with pollen wall development,including callose metabolism,pollen exine formation and cell wall modification.Twenty-five genes encoding different kinds of proteins were screened as candidate regulators of tapetum development or function.There were 493 transcription factor-encoding genes differentially expressed in sterile flower buds and fertile flower buds.Most of these genes were expressed in both of the sterile and fertile lines,but 70 genes were expressed specifically in the fertile floral buds,whereas 18 genes were specific to the sterile floral buds.(3)By sequence comparison and expression analysis,it was found that BcMF28 is an anther-specific R2R3-MYB gene.Sequence analysis indicated that BcMF28 encodes a R2R3-MYB protein,which shares highly similar amino acid sequences with Arabidopsis R2R3-MYB 18 subgroup members.Subcellular localization analysis and transactivational activity assay showed that BcMF28 was localized in the nucleus and cytoplasm,and had transactivational activity.The spatiotemporal expression analysis showed that BcMF28 was predominantly expressed in inflorescence with the highest expression level in fertile flower buds at the binucleate pollen stage.And promoter activity analysis revealed that the expression of BcMF28 was specifically detected in tapetum,developing microspores,anther endothecium,and filaments during late stamen development.(4)Through gene knockdown and heterologous expression exprements,BcMF28 was found to be involved in the regulation of pollen wall formation and pollen development.When the expression of BcMF28 was knockdown by artificial miRNA technology,the pollen viability of the transgenic plants was not affected,but the pollen exine structure was disordered,and the deposition of tryphine was abnormal.Detailed observation showed that the tapetal cells in bcmf28 distributed abnormal lipid droplets at the binucleate pollen stage,and the bacula of pollen exine became densely packed with an increase in length.The heterologous expression of BcMF28 under the control of its own promoter in Arabidopis caused shorter filaments,non-dehiscent anthers and fewer viable pollen grains,leding to complete male sterility.In addition,both the pollen grains in bcmf28 and transgenic Arabidopsis plants became larger.Combined with the phenotype of bcmf28 and transgenic Arabidopsis plants,BcMF28 was speculated to participate in pollen development by regulating the construction of pollen wall,and also involve the regulation of pollen maturation,filament elongation and anther dehiscence.(5)Bioinformatics analysis indicated that the expression of BcMF28 was regulated by Bra-miR319,which was further confirmed by 5’ RACE assay and transient tobacco co-expression system.Through the online software psRNATarget,BcMF28 was predicted to be the only target regulated only by Bra-miR319 among all the nine homologous genes in B.campestris corresponding to Arabidopsis R2R3-MYB subgroup 18 genes.5’ RACE experiment demonstrated that the transcript of BcMF28 was cleaved by Bra-miR319.Transient tobacco co-expression experiments further demonstrated that Bra-miR319,instead of Bra-miR159,regulated the expression of BcMF28.And different copies of Bra-miR319 were found to play differernt roles in regulation of BcMF28 expression.(6)A total of 190 members of 203 1R-MYB genes in B.campestris were expressed in roots,leaves,inflorescences or siliques.Among them,nearly 40% of the genes(77/190)exhibited high expression levels in the inflorescences.Transcriptome analysis of the sterile and fertile floral buds of ‘Bcajh97-01A/B’ at five typical developmental stages during pollen development indicated that 61 1R-MYB genes were expressed in flower buds at different stages.Twenty genes were differentially expressed in at least one stage of the sterile floral buds compared with the fertile ones.A total of 61 members of 68 1R-MYB genes in A.thaliana were expressed in roots,leaves,inflorescences or siliques.Among them,nearly 40% of the genes(24/61)exhibited inflorescence-preferential expression.Transcriptome analysis of pollen development and tapetum development in Arabidopsis indicated that 51 1R-MYB genes were expressed in pollen or tapetum at different stages.Throuth the comprehensive comparison of gene expression data in B.campestris and A.thaliana,eight 1R-MYB genes that might be involved in pollen development were screened.(7)An anther-specific 1R-MYB gene,BcMYB1R203,was involved in the regulation of pollen development.Sequence analysis showed that BcMYB1R203 shares highly similar nucleic acid sequences with its Arabidopsis homologous gene AtMYBR57,both of which encode the CPC-like 1R-MYB protein.Subcellular localization analysis and transactivational activity assay showed that both BcMYB1R203 and AtMYBR57 were localized in the nucleus and cytoplasm,and both had transactivational activity.The spatiotemporal expression analysis showed that BcMYB1R203 was specifically expressed in the flower buds at stage Ⅱ of ajhGMS ‘Bcajh97-01B’.And BcMYB1R203 promoter-driven GUS expression was detected in meiotic sporocyte,tetrads and tapetum.AtMYBR57 exhibited a similar spatiotemporal expression pattern with BcMYB1R203.The heterologous expression of BcMYB1R203 under the control of CaMV 35 S promoter in Arabidopis resulted in partial male sterility.Nearly 50% pollen grains in transgenic plants were aborted due to the degradation of cytoplasm and defects in pollen intine formation.