| Pectin is a major component of the primary cell wall in plants.It is also an important component of pollen wall and pollen tube wall.Pectin contributes significantly to cellular structural integrity,cell adhesion,and the mediation of defense responses.The synthesis and degradation of pectin is performed by a wide range of hydrolytic enzymes.Pectin methylesterases(PMEs)are the first kind of enzymes that catalyze pectin,whose activity is controlled by pectin methylesterase inhibitors(PMEIs).The PMEI family has many members.At present,PMEI gene has been identified in multiple species and has been proved to be involved in many processes of plant growth and development,such as hypocotyl elongation,pollen development and pollen tube growth,seed germination,fruit ripening,and various stress reactions.In evolution,many duplicated PMEI genes are produced due to the occurrence of whole genome duplication and tandem duplication events.In addition,whole genome duplication event is also accompanied by diploidization which could cause considerable gene loss,leading to the fate divergence of duplicated PMEI genes.The evolution mechanism and expression profile of PMEI family members of some species have been analyzed in previous studies.However,the expansion pattern of PMEI family members in the evolutionary process,the expression characteristics of’repeated PMEI genes,the retention and loss mechanism of PMEI family members,and the comparison and functional identification of orthologous PMEI genes are still unclear.In this study,combined with the genome-wide sequencing data published previously,we conducted an in-depth analysis on the evolution and expression patterns of PMEI gene family in B.camnpestris and B.oleracea using Brassica campestris L.ssp.chinensis Makino(syn.B.rapa ssp.chine,nsis)and B.oleracea as experimental materials,.In addition,we compared the expansion patterns,retention rates,expression differences of homologous genes,and the distribution of collinear PMEI genes between two species in three subgenomes.To further investigate the potential functions of the PMEI genes in pollen development,we identified and analyzed two pollen-specific genes in B.campestris,namely BcPMEI1(gene ID:Bra013821)and BcPMEI85(gene ID:Bra017515),and a pollen-specific expression gene in B.oleracea,namely BoPMEI1(Gene ID:Bol039450).The main results are summarized as following:(1)A total of 100 PMEI genes were identified in the whole genome of B.campestris.Chromosomal localization analysis showed that 96 members were unevenly distributed on 10 B.campestris chromosomes,and the remaining 4 were located on scaffold000164,scaffold000215,scaffold000257,and scaffold000305,respectively.A total of 9 tandem repeat clusters were detected,containing 20 B.ca,npestris PMEI genes.The synteny analysis of PMEI genes between B.campestris and Arabidopsis thaliana detected 80 pairs of syntenic PMEI orthologs.These results indicated that the events of whole genome triplication and tandem duplication were the main driving forces accounting for the expansion of the B.campestris-PMEI gene family.During evolution,the retention rate of B.campestris PMEI genes was up to 52%,in line with the"gene balance hypothesis".The retention rate in LF subgenome(60%)was significantly higher than that in MF1 and MF2 subgenomes(50%and 42%,respectively).This phenomenon is consistent with the gene retention level of B.campestris on the whole genome,which can be explained by the "two-step theory".The nonsynonymous substitution rate,synonymous substitution rate,and the ratio between them of homogenous PMEI gene pairs were computed,suggesting that the B.campestris PMEI gene family was mainly undergone purifying selection in evolution.By the analyses of qRT-PCR and transcriptomic data,we found that 10 B.campestris PMEI genes were expressed highly and specifically in mature pollen grains.A number of cis-acting elements related to hormone regulation and stress responses were found through the analysis of the cis-elements in promoter region of each PMEI gene.(2)The PMEI gene is a large gene family in B.oleracea,similar to B.campestris,with a total of 95 B.oleracea PMEI genes identified.The analysis of chromosomal localization showed that 79 B.oleracea PMEI genes were distributed on 9 chromosomes unevenly,and the remaining 16 members located on scaffolds.A total of 10 tandem repeat clusters were detected,containing 21 B.oleracea PMEI genes.The synteny analysis of PMEI genes between B.oleracea and Arabidopsis was performed,and a total of 77 syntenic orthologous gene pairs were found.These results implied that the main driving forces for the expansion of B.oleracea PMEI gene family were whole genome triplication and tandem duplication events,in according with B.campestris.During evolution,the retention rate of PMEI genes in B.oleracea was slightly lower than that in B.campestris,but it was still as high as 50%.The retention rate of B.oleracea PMEI genes in LF subgenome(60%)was significantly higher than those in MF subgenomes.But.the retention rate in MF1 subgenome(42%)was lower than that in MF2 subgenome(47%).The analysis of nonsynonymous substitution rate and synonymous substitution rate illustrated that,similar to the B.campestris PMEI genes,the B.oleracea PMEI genes was mainly experienced purifying selection during evolution,and the divergence time was consistent with the time of whole genome triplication event.The qRT-PCR analysis showed that B.oleracea PMEI genes were closely related to the growth and development of B.oleracea,and five members were expressed highly and specifically in mature stamens.In addition,a number of cis-acting elements related to hormone regulation and stress responses were identified irn the promoter regions of B.oleracea PMEI genes.(3)Based on the genomic information from the BRAD,the DNA sequence and CDS of BcPMEI1 and BoPMEIl were cloned from B.campestris and B.oleracea,respectively.Both BcPMEI1 and BoPMEI1 contain two exons and one intron,and the amino acid sequences encoded by them are very similar.Signal peptide prediction and subcellular localization experiment indicated that BcPMEI1 and BoPMEI1 were secretory proteins located in the plasma membrane and cell wall.Through qRT-PCR analysis,we found that both BcPMEI1 and BcPMEI1 had high expression levels in mature stamens,but their expression patterns were different.(4)Through the over-expression analysis of BcPMEI1,we found that approximately 50%of pollen grains in transgenic plants were inactive,collapsed and wrinkled,and the volume was reduced.The transgenic Arabidopsis had the shorter mature siliques and less seeds compared with the wild-type plants.Transmission electron microscopy observation of semi-thin sections and ultra-thin sections showed that the collapse of aberrant microspore started at the uninucleate microspore stage and was accompanied with no pollen intine formation;at later stages of development,the collapse of microspore was even more remarkable due to the degeneration of all the contents including the nuclei.These results imply that BcPMEI1 may affect pollen development by regulating pectin metabolism during pollen intine formation.The similarity of promoter sequences between BcPMEI1 and BoPMEI1 was 84.35%.Both promoter sequences of BcPMEI1 and BoPMEI1 contain some pollen-specific motifs and classic cis-regulating elements.The promoter-GUS analysis showed that the GUS signals driven by both BcPMEI1 and BoPMEI1 were mainly present in mature anthers and pollen grains.(5)Based on the genomic information from the BRAD,the DNA sequence and CDS of BcPMEI85 were cloned.This gene contains one exon.Subcellular localization experiments and signal peptide and transmembrane domain predictions indicated that BcPMEI85 was a secreted protein localized to plasma membrane and cell wall.The promoter sequence of BcPMEI85 contained some pollen-specific motifs and multiple cis-acting elements involved in regulation of hormones and responses to various kinds of stresses.The promoter-GUS analysis showed that the GUS signals were mainly expressed in mature anthers and pollen grains.The qRT-PCR analysis showed that BcPMEI85 had high and specific expression levels in stamens.At5G46940 is a homologous gene of BcPMEI85.The microarray data of A thaliana indicated that At5G46940 was mainly expressed in mature stamens and pollen grains.Mentioned above indicated that BcPMEI85 belonged to the class of "late" pollen genes.The functional interruption of BcPMEI85 by antisense RNA technology had no effect on the floral organ morphology,pollen activity,and pollen germination,indicating that there might be functional redundancy from other PMEI genes. |
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