Pectin Methylesterase Gene PME21 Modulates Pollen Development And Pollen Tube Growth In Arabidopsis

During double fertilization,two sperm cells are transferred into the embryo sac in angiosperms through the growth of the pollen tube,where they are fused with the egg and central cells to form the fertilized egg and endosperm,respectively.Exocytosis and endocytosis play an important role in maintaining pollen tube tip growth.Pectin methylesterase?PME?demethylyzes the high-methylated pectin.And then the low-methylated pectin formed by cross-linking with Ca²⁺to maintain the hardness of cell wall,which plays an important role in pollen development and pollen tube growth.PME functions by reaching the cell wall at the top of the pollen tube via exocytosis,but it is unclear whether it will participate in the endocytosis process.Arabidopsis TML gene encodes a subunit of endocytosis complex and plays role by binding the cargo proteins in clathrin-mediated endocytosis.In our early study,we found that Arabidopsis PME21 interacted with TML through yeast double hybrid screening.PME21 was highly expressed in pollen from the 12th stage and in growing pollen tubes,which was consistent with the expression patterns of TML in pollen and pollen tube.In pollen grains and pollen tubes,PME21 was localized near the germ pore of germinating pollen grain and the tip of the growing pollen tube.In pme21-1/+,a T-DNA insertion mutant of PME21,about half of pollen morphology was abnormal.The pollen grains of this mutant showed abnormal viability since the 12th stage judged by Alexandria staining,and the accumulation of callose and pectin in the pollen grains was also abnormal,which were like those of TML mutant.We obtained another mutant of PME21,pme21-2,by CRISPR/Cas9 technology.In this mutant,a thymine T was inserted350 bp downstream of the initiation of ATG,which resulted in translation termination at 120th amino acid.As the pme21-1,pme21-2 mutation also resulted in abnormal male and female gametophytes.To study the role of PME21 in the growth of pollen tubes,an estrogen-induced construct pER8::amiRNA-PME21 was constructed and transferred into Arabidopsis.We found that the down-regulation of PME21 expression by estrogen treatment caused the burst of the growing pollen tubes in vitro.In contrast,the transgenic pollen tubes grew normally on the wild-type stigmas treated by estrogen,while some of the pollen tubes could not pass through the stigmas into the transmitting tract.We found that,under the condition of limited pollination,the abnormal pollen tube in the pistil treated with estrogen was about 10%more than that in the untreated pistil.These results indicated that the down-regulation of PME21 expression would inhibit the growth of pollen tube in pistil to some extent.PME21 belongs to Type2 PME,containing a PRO region?including a signal peptide and a PMEI domain?at its N-terminal,and a PME domain at its C-terminal.It is found that the Type2 PME was cleaved into two truncated proteins between the PMEI domain and the PME domain.To investigate the localization of these two truncated proteins,we made the pPME21::mCherry-PME21-GFP construct to transform Arabidopsis and observed the distribution of red fluorescence and green fluorescence in the pollen tube of transgenic lines.We found that mCherry-PME21_N-ter-ter was located in the plasma membrane and cell wall of the pollen tube,while PME21_C-ter-GFP was located in the apical plasma membrane and cell wall.At the same time,the dynamic process of PME21-mcherry and mCherry-PME21-GFP localization in the pollen tube under the growth state was analyzed.Our previous studies indicated a direct interaction between TML and PME21,suggesting that PME21 may be a cargo protein of TML in endocytosis.Thus,we observed the co-localization of PME21-mCherry and TML-YFP,and found that TML and PME21 had co-localization on the side of the top of the pollen tube.In addition,the localization of PME21at the top of the pollen tube disappeared or extended backward after the down-regulation of TML expression,indicating that the localization of PME21 at the top of the pollen tube may be regulated by the TML-involved endocytosis.Subsequently,we treated the transgenic pollen tubes with TyrA23,an endocytosis inhibitor,and found that the localization of PME-domain of PME21 was disturbed,which further confirmed that endocytosis was involved in the location of PME21.Thus,TML plays roles in the regulation of pollen development and pollen tube growth via the endocytosis of mature PME.