O-glycosylation of the extracellular domain of pollen class I formins modulates their plasma membrane mobility.

In plant cells, linkage between the cytoskeleton, plasma membrane, and cell wall is crucial for maintaining cell shape. In highly polarized pollen tubes, this coordination is especially important to allow rapid tip growth and successful fertilization. Class I formins contain cytoplasmic actin-nucleating formin homology domains as well as a proline-rich extracellular domain and are candidate coordination factors. Here, using Arabidopsis, we investigated the functional significance of the extracellular domain of two pollen-expressed class I formins: AtFH3, which does not have a polar localization, and AtFH5, which is limited to the growing tip region. We show that the extracellular domain of both is necessary for their function, and identify distinct O-glycans attached to these sequences, AtFH5 being hydroxyproline-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of hydroxyproline arabinosylation altered the plasma membrane localization of AtFH5 and disrupted actin cytoskeleton organization. Moreover, we show that O-glycans differentially affect lateral mobility in the plasma membrane. Together, our results support a model of protein sub-functionalization in which AtFH5 and AtFH3, restricted to specific plasma membrane domains by their extracellular domains and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

SEC1A is a major Arabidopsis Sec1/Munc18 gene in vesicle trafficking during pollen tube tip growth.

Pollen tubes (PTs) grow by the targeted secretion of new cell wall material to their expanding tip region. Sec1/Munc18 (SM) proteins promote membrane fusion through regulation of the SNARE complex. We have previously shown that disruption of protein glycosylation in the Arabidopsis thaliana hpat1 hpat3 double mutant leads to PT growth defects that can be suppressed by reducing secretion. Here, we identified five point mutant alleles of the SM protein SEC1A as hpat1/3 suppressors. The suppressors increased seed set, reduced PT growth defects and reduced the rate of glycoprotein secretion. In the absence of the hpat mutations, sec1a reduced pollen germination and PT elongation producing shorter and wider PTs. Consistent with a defect in membrane fusion, sec1a PTs accumulated secretory vesicles. Though sec1a had significantly reduced male transmission, homozygous sec1a plants maintained full seed set, demonstrating that SEC1A was ultimately dispensable for pollen fertility. However, when combined with a mutation in another SEC1-like SM gene, keule, pollen fertility was totally abolished. Mutation in sec1b, the final member of the Arabidopsis SEC1 clade, did not enhance the sec1a phenotype. Thus, SEC1A is the major SM protein promoting pollen germination and tube elongation, but in its absence KEULE can partially supply this activity. When we examined the expression of the SM protein family in other species for which pollen expression data were available, we found that at least one Sec1-like protein was highly expressed in pollen samples, suggesting a conserved role in pollen fertility in other species.