| ABSTRACT The pollen wall of higher plants,which has rich chemical compositions and complex structures,plays important roles in the pollination process.The outermost layer of maize pollen wall is pollen coat.Its materials participate in pollen adhesion,hydration,germination and growth in the stigma by interacting with stigma components.It contains lipids,proteins and pigments.It is known that pollen coats are mainly involved in pollen hydration processes.The function of pollen coat proteins is not yet fully understood.Therefore,the mechanism of how the pollen coat protein participated in the pollen-stem interaction has become an urgent problem to be studied in plant reproductive biology.Maize pollen coat proteins contain xylanase,polygalacturonase,glucanase,cysteine protease,etc.The structural characteristics and evolution of maize pollen cysteine proteases and their genes,as well as possible interactions between enzymes and stigma proteins were analyzed in this study through bioinformatics methods.The localization of cysteine proteases in maize mature pollen walls and the expression levels of enzymes at different stages of pollen development have been studied.The purpose of this study was to analyze the evolutionary process and location of cysteine proteases in maize pollen coat,and to clarify its function in the interaction between the pollen and the stigma.The results are as follows:1.Expression characteristics of maize pollen coat cysteine protease(Zm PCP)homologues and structural characteristics of their encoded proteinsA total of 32 Zm PCP homologues were retrieved from the maize genome.The encoded proteins belong to the protease C1 family.Their active site follows the structure of GlnX5/6CysX133-139HisX20/21Asn.They have two conserved domains,namely protease inhibitor 129 and protease C1.The sequences and structures of both domains in the 32 Zm PCP genes are highly conserved.Their encoded cysteine proteases,including SAG39,RD19,Mirl,XCP,and Vignain,are involved in different biological processes.RNA-seq data in database were used to analyze the expression patterns of 19 maize cysteine protease genes in different tissues.Eight genes showed relatively high expression levels in at least one tissue.Aging-related SAG39 expression was found to be the lowest.In addition,Zm PCP was not detected with high expression level in seedlings nor the roots using RT-PCR or immunoblotting,which was inconsistent with the results of gene assay.The matching rate between cloned maize leave-PCP and the reported Zm PCP was high.However,a 84 bp insertion was found at the site of 478 bp of the reported Zm PCP.The results indicated the tissue specificity of Zm PCP expression.2.The evolution analysis of maize pollen coat cysteine protease in plantsThe evolution of Zm PCP is conserved in plants.Zm PCPs of most plant are highly homologous,except for Selaginella.In evolutionarily conserved plants,cysteine proteases do not show significant species specificity.Cotton SAG39 has a more homologous relationship with Zm PCP.3.The probale function of maize pollen coat cysteine proteaseZm PCP was found to be similar to ragweed cysteine protease in 3D structure and be most similar to macrodontain-1 protein of maize in sequence.Both cysteine proteases of are associated with allergic reactions.It is suggested that Zm PCP of maize pollen may also participate in allergic reaction.Maize pollen coat PCP and cotton SAG39 were found to be close homologous,which indicated the potentially similar aging-specific cell death related function of Zm PCP.The Arabidopsis thaliana SAG 12 gene was found to be orthologous to Zm PCP.The homology of cotton SAG39 with Arabidopsis SAG12 was 60%.SAG12 is mainly involved in organ aging and external stimuli resistance.Therefore,Zm PCP expressed in early stage of anther development is very likely to participate in the process of tapetum cell death.SAG12 and ubiquitin-like activating enzyme E1 APG7 may be co-expressed and interacted.The ubiquitin-proteasome system is specifically or predominantly expressed in the mature stigma of maize and may play a role in corn pollen-stigma interactions.The ubiquitin-like activating enzyme E1 may interact with Zm PCP,which may lead to ubiquitinating of Zm PCP.The ubiquintous Zm PCP might participate in pollen tube identification on the stigma.4.Localization of maize pollen cysteine proteaseZm PCP of mature pollen was observed in the pollen coat,especially in the inner layer of pollen outer wall.In addition,a few were found in the pollen cytoplasm.Zm PCP was expressed in the early stage of pollen development before tetrasporidium microsporulation.The expression levels increased along with the formation of microspores.However,gene expression was not detected in pollen and embryo.During the development of the tapetum,the pollen mother cell meiosis and the tapetum cells begin to transform into secretory cells.The tapetum cells are disintegrated when the microspores form the outer wall.Therefore,Zm PCP may have been synthesized in the early stage of pollen development and secreted from the tapetum during microsporogenesis.It accumulates in the inner layer of pollen outer wall,also accumulates in the pollen coat with the tapetum cells completely disintegrated.The way Zm PCP entering the intracellular pollen remains to be studied.Maize pollen cysteine protease is one of the few proteolytic enzymes present on the surface of pollen.It is detected in pollen coat,inner wall of cell wall,as well as cytoplasm.ZmPCP was found to be evolutionarily conserved in plants and orthologous to Arabidopsis thaliana SAG12.It might be involved in the process of senescence of tapetum cells.Moreover,The enzyme might interact with the ubiquitin-activating enzyme on the stigma surface to participate in the interaction of pollen and stigma.This study systematically analyzed the function of maize cysteine proteases through bioinformatics methods,which would provide experimental evidence for elucidating the mechanism of maize pollen-stem interaction. |
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