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.

Molecular Characterization, Evolutionary Analysis, and Expression Profiling of BOR Genes in Important Cereals

“…ZmBOR6, however, showed a distinct pattern, as it had an additional ANTH domain. This protein might be involved in pollen germination processes, as the ANTH domain is associated with pollen germination [ 32 ]. Motif analysis was performed to study the functional similarity pattern of BOR genes.…”

Membrane trafficking functions of the ANTH/ENTH/VHS domain‐containing proteins in plants

“…2). For Arabidopsis and rice ANTH proteins, we followed the nomenclatures proposed by Muro et al [39] and Lee et al [40], respectively, and the other published names for each gene and protein are also included in Fig. 2.…”

“…[39] and Lee et al. [40], respectively, and the other published names for each gene and protein are also included in Fig. 2.…”

“…ANTH proteins from Homo sapiens and Saccharomyces cerevisiae were included as outgroups [76]. The ANTH proteins of A. thaliana and O. sativa analysed in previous studies [3,36,40] were also included in this analysis. For the ANTH proteins in other plant species, we performed a BLASTP search using the amino acid sequence of the ANTH domain of Arabidopsis PICALM1a (a.a. 1–314) as a query in the following databases: MarpolBase ( https://marchantia.info/) for O. lucimarinus, O. tauri, C. reinhardtii, K. nitens, C. braunii, S. muscicola, M. polymorpha, A. agrestis and P. abies , and Phytozome ( https://phytozome‐next.jgi.doe.gov/) for V. carteri, P. patens, S. moellendorffii and A. trichopoda .…”

“…Seventeen ANTH proteins have been found in the genome of rice, among which OsANTH3 , OsANTH11 , OsANTH7 , OsANTH2 , and OsANTH14 are preferentially expressed in the pollen. Among these OsANTH members, the effect of mutations in Os ANTH3 has been investigated, revealing that fertility is substantially decreased in the osanth3 mutant, which could be due to a reduced pollen germination rate caused by compromised clathrin‐mediated endocytosis [40]. GORI , a rice REN4 homolog, has also been identified as a gene required for the germination and elongation of pollen tubes [65].…”