Grass-Specific EPAD1 Is Essential For Pollen Exine Patterning In Rice

The male gametophyte of flowering plants,pollen,is enclosed within a multi-layered cell wall whose structure directs successful pollination and provides physical and chemical resistance against environmental stresses.Pollen wall surface patterning exhibits vast morphological diversity among species and constitutes an important feature of plant taxonomic classification.However,its structure and development share common features across plant taxa.The pollen surface patterns are formed by sporopollenin accumulation.The template for sporopollenin deposition and polymerization is the primexine,a thin microfibrillar matrix that appears on the tetrad surface.Primexine composition is similar to the primary cell wall,containing cellulose,pectin and xylan,as well as arabinogalactan-proteins and lipoproteins.But the detailed components and the mechanism(s)by which primexine guides exine patterning remain elusive.Comparative phylogenomics and biochemical experiments have revealed that the genes that regulate sporopollenin biosynthesis and primexine formation are highly conserved among land plants.Although many molecular factors required for primexine formation are identified,the biological functions of most of them remain unknown.Several Arabidopsis(Arabidopsis thaliana)mutants exhibit abnormal,delayed or absent primexine formation,for example mutations affecting: calcium-binding protein DEX1;plastidic integral membrane protein NEF1;sugar transporter RPG1/SWEET8 and its paralog RPG2;plasma membrane localized protein NPU;and de novo DNA methyltransferase EFD.Polysaccharide biosynthetic components,such as ?-1,3-galactosyltransferase UPEX1/KNS4 and putative xylosyl transferases SPG2/IRX9 L and its paralog IRX14 L,are also required for primexine formation.In rice,mutation of Os DEX1 impairs callose degradation and primexine formation,while DPW3 is a membrane alpha integrin-like protein whose mutation affects callose deposition and primexine formation,resulting in pollen abortion.These genes are conserved in flowering plants,while the factors that regulate species-specific wall patterning remain unknown.In addition,most of these genes are exclusively or predominantly expressed in the tapetum,indicating that the tapetum plays an essential role in the exine patterning.Pollen mother cells(PMCs)have also been implicated in exine patterning,but there is little evidence for their direct involvement in regulating exine patterning.Here,we report that Poaceae-specific EXINE PATTERN DESIGNER 1(EPAD1),which encodes a non-specific lipid transfer protein,is required for primexine integrity and pollen exine patterning in rice.EPAD1 and its orthologs appear to have evolved together with the emergence of Poaceae,and they may play conserved roles in grass pollen development,evidenced by similarities in the protein sequences,such as the conserved eight-cysteine LTP domain,preferential expression in young spikelets,and similar pollen wall phenotypes in the Ta MS1 and epad1 mutants.Detailed phenotypic analysis suggested that disruption of EPAD1 leads to abnormal exine pattern and complete male sterility,although sporopollenin biosynthesis is unaffected.By contrast to the mutant phenotypes of sporopollenin related genes,the cuticle and Ubisch body development were normal in epad1 anthers.These observations suggest that EPAD1 is not required for the sporopollenin biosynthesis and translocation.In addition,the callose wall development and degradation were normal in the epad1 PMCs,which differs from most primexinedefective mutants.These results suggest that EPAD1 is specifically involved in controlling primexine deposition,which subsequently influence the location of assembly units and final exine pattern.EPAD1 is specifically expressed in male meiocytes,indicating that reproductive cells exert genetic control over exine patterning.EPAD1 is re-localized to the plasma membrane and function in pollen exine patterning at tetrad to early microspore stage.These results are consistent with exine pattern determinants being present in PMCs in prophase I.Our results thus indicate that the deposition and assembly of sporopollenin is regulated,not only by tapetal cells,but also by the microspore.EPAD1 possesses an N-terminal signal peptide and three redundant glycosylphosphatidylinositol(GPI)-anchor sites at its C-terminus,segments required for its function and localization to the microspore plasma membrane.In vitro assays indicate that EPAD1 can bind phospholipids,that are found in microspores.Therefore,it is likely that EPAD1 may bind to specific PI-rich plasma membrane domains that can facilitate recruitment of proteins,such as SAPs,involved in primexine development.Alternatively,EPAD1 may provide docking sites for PI molecules in the plasma membrane,which go on to recruit primexine-regulatory proteins.A third possibility is that the lipid molecules carried by EPAD1 are required to maintain the homogeneity of primexine components that drive correct exine deposition.Our results demonstrate that EPAD1 is a meiocyte-derived determinant that controls primexine patterning in rice,and its orthologs may play a conserved role in the formation of grass-specific exine pattern elements.