Functional Characterization Of Arabidopsis Annexin5and ADF5in Pollen Development And Growth

The regulation of pollen development and pollen tube growth is complicated and crucial for sexual reproduction in flowering plants. In this study, we are interested in the effect of vesicles, the actin cytoskeleton and Ca2+on a pollen cell. The annexin family consists of a class of proteins that can bind to the membrane, phospholipids and actin in a Ca2+-dependent manner. Members share an evolutionarily conserved structure that exists in a wide variety of eukaryotic cells. Therefore, Annexins may function as a "linker" between the actin cytoskeleton, membrane and Ca2+. The actin cytoskeleton system is highly dynamic and must be precisely regulated during pollen germination and pollen tube growth processes because it maintains specific structures and performs distinct functions at different times and locations. Furthermore, it is generally accepted that actin turnover is the major driving force for pollen germination and pollen tube growth. ACTIN-DEPOLYMERIZING FACTORS (ADFs) play critical roles in enhancing the turnover of actin filaments. The pollen-specific AtADF7has been well characterized to promote the turnover of the longitudinal actin cables through severing actin filaments in pollen tubes. To date, the precise roles of other At ADFs in pollen germination and pollen tube growth are less studied. Here, we functionally characterized Arabidopsis Annexin5(Ann5) and ADF5in pollen cells using biochemical and genetic evidence.1. Ann5was strongly expressed in open flowers and cotyledons, while barely detected in other tissues, using tissue RT-PCR and GUS staining. In addition, the expression of Ann5initiated at the bicellular stage followed by a considerable increase at the tricellular stage and in mature pollen. Ann5-GFP was evenly distributed in the cytosol of pollen grains and tubes.2. The downregulation of Ann5by RNAi interference caused severely sterile pollen grains beginning from the bicellular stage. Furthermore, this phenotype could be rescued by exogenetic Ann5. The cytoplasm of pollen developed from the bicellular stage in the Ann5-RNAi line became grainy and gradually detached, and the plasma membrane lost integrity and broke on the membrane surface. After a gradual course of autolysis, the pollen exhibited a large-scale loss of cytoplasm and disintegration of the basic organelle structure. Only debris from degraded pollen was found, and no recognizable membrane structures were detected. These observations suggested a functional role of Ann5in pollen development.3. Purified recombinant Ann5could bind to phospholipid membranes in a Ca2+-dependent or-independent manner in vitro. In vivo, pollen overexpressing Ann5increased the Ca2+-dependent resistance to Brefeldin A (BFA) treatment during pollen germination and pollen tube growth and promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in response to BFA. Recombinant Ann5bound to actin filaments in vitro. However, unlike BFA treatment, both pollen germination and pollen tube growth in the Ann5-overexpressing lines were the same as in the control lines when treated with LatB. Ann5has been postulated to be involved in intracellular trafficking events, and actin filaments serve as molecular tracks in pollen grains and tubes. The defects observed in pollen grains of the Ann5UTR-RNAi lines could be partially recovered by Ann5mutants at Ca+-binding sites, in comparison to the complete rescue by wild-type Ann5.4. As an atypical actin depolymerization factor, ADF5could bind, bundle and stabilize actin filaments in a pH-sensitive manner in vitro biochemical analyses. The actin binding, bundling and stabilizing activities of ADF5were more efficient under acidic conditions. ADF5co-localized with actin filaments in vivo, which suggests that ADF5might serve as an actin-binding protein during pollen germination and pollen tube growth.5. ADF5was abundantly expressed in mature pollen and pollen tubes. The loss of ADF5resulted in delayed pollen germination and pollen tube growth defects due to altered dynamic organization of the actin array. The actin filaments of pollen grains and pollen tube shanks in the adf5mutants were less bundled and more sensitive to LatB treatment, which was consistent with the biochemical activities of ADF5.6. There were two actin-binding domains (ABDs) located within ADF5. The N-terminal domain of ADF5was found to interact directly with actin filaments and to be crucial for its actin-bundling activity. The opposing biochemical characteristics of ADF1and ADF5might be due to the differences in their N-terminal domains. As a marked proton gradient exists in pollen and regulates the activity of ADFs, we demonstrated that ADF5acted synergistically or antagonistically with other ADFs to regulate the organization and dynamics of actin in pollen grains and pollen tubes, e.g., ADF5competed with ADF1for actin binding.These results demonstrated that Ann5participates in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking in a calcium-dependent manner. Our results provided preliminary and reliable molecular mechanisms that underlie the function of Ann5in pollen. Furthermore, ADF5is an actin-bundling protein that bundles actin filaments and plays a critical role in the formation, organization and maintenance of actin bundles during pollen germination and pollen tube growth processes. These findings provide important insights into the functions of ADF5.