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Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3.

ABSTRACT: Pollen in angiosperms plays a critical role in double fertilization by germinating and elongating pollen tubes rapidly in one direction to deliver sperm. In this process, the secretory vesicles deliver cell wall and plasma membrane materials, and excessive materials are sequestered via endocytosis. However, endocytosis in plants is poorly understood. AP180 N-terminal homology (ANTH) domain-containing proteins function as adaptive regulators for clathrin-mediated endocytosis in eukaryotic systems. Here, we identified 17 ANTH domain-containing proteins from rice based on a genome-wide investigation. Motif and phylogenomic analyses revealed seven asparagine-proline-phenylalanine (NPF)-rich and 10 NPF-less subgroups of these proteins, as well as various clathrin-mediated endocytosis-related motifs in their C-terminals. To investigate their roles in pollen germination, we performed meta-expression analysis of all genes encoding ANTH domain-containing proteins in Oryza sativa (OsANTH genes) in anatomical samples, including pollen, and identified five mature pollen-preferred OsANTH genes. The subcellular localization of four OsANTH proteins that were preferentially expressed in mature pollen can be consistent with their role in endocytosis in the plasma membrane. Of them, OsANTH3 represented the highest expression in mature pollen. Functional characterization of OsANTH3 using T-DNA insertional knockout and gene-edited mutants revealed that a mutation in OsANTH3 decreased seed fertility by reducing the pollen germination percentage in rice. Thus, our study suggests OsANTH3-mediated endocytosis is important for rice pollen germination.

SUBMITTER: Lee SK

PROVIDER: S-EPMC8076639 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Understanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development. RESULTS:We performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer. CONCLUSION:Through the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis and storage reserves of pollen. In addition, these candidates might be useful targets for future examinations of late pollen development, and will be a valuable resource for accelerating the understanding of molecular mechanisms for pollen maturation and germination processes in rice.

Project description:Background and aimsA pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.MethodsThe esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.Key resultsOlive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.ConclusionsIn this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

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Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3.

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