| Polygalacturonase (PG) is a kind of cell wall hydrolases. It plays an important role in cell separation events during the plant development process by degrading pectin. The structure and expression among PG members have numerous variations and differences. Moreover, some PG genes may exhibit functional redundancy. Whole genome duplication (WGD) event occurs when accompanied with fractionation and rearrangement of genome fragments, resulting in the gain and loss of PG genes. The WGD and tandem duplication events also generate many duplicated PG genes. The evolution and classification of PG members in some species have been discussed in previous studies. However, the analysis of the patterns and extent of expansion of the PG gene family, the mechanisms that contribute to the expansion of the family, and the degree of both spatial and temporal expression divergence between duplicated PG genes are still inadequate. Furthermore, the possible mechanisms of duplicate retention and loss and the function characterized of collinear PG gene between species are still insufficient. Pollen plays an important role in the sexual reproduction process of plants. Insufficient pollen directly affects pollination, fertilization, and offspring multiplication. The major component of pollen intine and pollen tube wall is pectin. PG may be closely related to the development of pollen and the formation of pollen tubes. A possible function of a single PG gene in pollen development and pollen tube formation has been characterized in various species. However, research for the expression and functional divergence of duplicated PG genes is still few.Using Brassica campestris (syn. B. rapa ssp. chinensis) and B. oleracea as experimental materials, combined with their whole-genome sequencing data and the expression data of the B. campestris PG genes obtained in our previous study, we conducted an in-depth analysis on the expression and evolution of PG genes in B. oleracea. We also compared and discussed the patterns and extent of the expansion, possible mechanisms of retention, and loss of PG genes; the degree of both spatial and temporal expression divergence between duplicated PG genes; and the distribution of collinear PG genes between two species in three sub-genomes.In addition, for problems with collinear PG gene becoming lost between two species and the duplicated PG genes having diverged expression, we analyzed the expression of B. oleracea PG gene BoMF25 (gene ID:Bo1018697). Its collinearity PG gene in B. campestris was found to be missing. We also characterized the expression and functional divergence of duplicated PG genes BcMF26a (gene ID: Bra011440) and BcMF26b (gene ID:Bra037005) in B. campestris genome. The main results are summarized in the following:(1) A total of 102 full-length genes encoded putative PG proteins were identified in B. oleracea. The phylogenetic tree showed that all the PG genes formed eight distinct clades, Clade A to Clade H. The analysis of copy number changes of B. oleracea and Arabidopsis PG gene in different clades showed that the PG gene in Clades A to F have a high proportion of gain and loss in genes. The analysis of expression characteristic suggested that members in Clade A and Clade E were expressed in almost all the detected organs. PG members in Clade B could be expressed in all detected organs, except in leaves. PG members in Clades C, D, and F were mainly expressed in inflorescences and siliques. The statistics for the number of most recent common ancestor (MRCA) indicated that at least 82 MRCAs exist between B. oleracea and A. thaliana PG gene family, and at least 76 MRCAs between B. campestris and A. thaliana PG gene family.This finding also suggested that the PG gene family in B. campestris genome expanded more rapidly than B. oleracea. In addition, the analysis for the percentage of gene retention after whole-genome triplication revealed that the percentage of B. campestris PG gene family is higher than B. oleracea. The comparison of the expression characteristic between duplicated PG genes showed the characteristics exhibited in four modes:the duplicated genes had similar expression patterns or were not expressed in all the detected organs; the expression partially overlapped, except for distinct expression profiles; one gene was not expressed in any tissue examined, while the other was selectively expressed in some organs; and duplicated genes completely expressed in different organs. The statistics for gene numbers in the three sub-genomes also suggested that the PG members of B. campestris and B. oleracea had similar distributions; however, some of collinear PG was missing in one of the species.(2) Phylogenetic analysis showed that Brassica oleracea Male Fertility 25 (BoMF25) was the orthologous gene of At4g35670. BoMF25 was localized in chromosome 6 in the B. oleracea genome. It could be aligned to the position where At4g35670 was localized in the A. thaliana genome. Collinear analysis between At4g35670, BoMF25, and B. campestris genome showed that the collinear gene of BoMF25 in B. campestris genome was lost by genome deletion and reshuffling related to fractionation during the genome duplication processes. qRT-PCR analysis suggested that BoMF25 was mainly expressed in anther at the late stage of pollen development. The putative promoter sequence of BoMF25, containing classical cis-acting elements and pollen-specific motifs, could drive GUS expression in the anther at the late stage of pollen development. In situ hybridization analysis indicated that the strong and specific expression signal of BoMF25 existed in pollen grains at the mature pollen stage. Moreover, subcellular localization showed that the fluorescence signal was observed in the cell wall of onion epidermal cells, which suggested that BoMF25 may be a secreted protein localized in the pollen wall.(3) Phylogenetic analysis showed that Brassica campestris Male Fertility 26a(BcMF26a) and BcMF26b were both orthologous genes of At4g33440. Collinear analysis indicated that they originated from a segmental chromosomal duplication. qRT-PCR analysis showed that the expression level of BcMF26b was higher than that of BcMF26a in almost all the tested organs and tissues of B. campestris. Promoter expression analysis showed that at the reproductive development stages, BcMF26b could drive the expression of GUS gene in tapetum, pollen, and pistils, whereas BcMF26a was expressed only in pistils. Therefore, BcMF26a and BcMF26b may be the result of neofunctionalization or subfunctionalization during the evolution process. Sequence analysis indicated that the intron and promoter sequences had lower similarity; their expression differences might have been caused by the differences in their transcriptional regulatory sequences and introns. Moreover, the analysis of knockout mutants showed that the absence of At4g33440 delayed the growth of pollen tubes. When amiRNA technology was used to co-inhibit the expression of the two genes, the pollen intine of the transgenic plants formed abnormally and the pollen tubes were unable to grow or stretch. In subcellular localization experiment, BcMF26a and BcMF26b proteins can be localized to the cell wall. Combined with the results that the expression level of BcMF26b was much higher than that of BcMF26a, which could drive the expression of GUS gene in tapetum and pollen, we conclude that BcMF26b may be the major actor for pollen development in transgenic plants. |
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