Summary HYDROXYPROLINE O ‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana , HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft‐like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed‐set phenotype, we identified a missense mutation in exo70a2 , a predicted member of the vesicle‐tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a , indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst‐mediated secretion. To monitor the trafficking of Hyp‐Ara modified proteins, we generated an HPAT‐targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.
SUMMARY 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.
In plant cells, linkage between the cytoskeleton, plasma membrane and cell wall is crucial to maintain 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 Pro-rich extracellular domain (ECD) and are candidate coordination factors. Here, 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 ECD of both is necessary for their function and identify distinct O-glycans attached to these sequences, AtFH5 being Hyp-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of Hyp-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 where AtFH5 and AtFH3, restricted to specific plasma membrane domains by their ECDs and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.
Hydroxyproline O-arabinosylation is a highly-conserved and plant-specific post-translational modification found on extensins and other structural proteins in the cell wall, and is catalyzed by Hydroxyproline O-arabinosyltransferases (HPATs). In Arabidopsis, loss of HPAT1 and HPAT3 (hpat1/3) causes reorganization of components in the pollen tube (PT) cell wall, which compromises cell wall structural integrity and decreases PT growth and fertility. We have previously shown that reduced secretion (caused by loss-of-function mutations in secretory genes EXO70A2, SEC15A, and SEC1A) suppressed cell wall defects and strongly rescued poor growth and fertility in hpat1/3 PTs. Here, we show that a missense mutation in PHOSPHOLIPASE C6 (PLC6) also rescues hpat1/3 PT growth and fertility. Transgenic insertion mutations that disrupt PLC6 expression did not improve hpat1/3 pollen fertility, and did not affect PT growth or fertility in the wild type background. This data suggests that our missense allele (plc6-4) does not function like a true loss-of-function allele, and that PLC6 is not required for wild type PT growth. However, in the absence of hpat1/3, plc6-4 PTs have defects in transmission and polarized growth, as indicated by meandering growth paths and a resulting crooked appearance. plc6-4 PT elongation and straightness are more sensitive to elevated levels of calcium than wild type. This may be due the nature of the plc6-4 mutation, which causes an E569K amino acid substitution in the lipid-binding C2 domain. The 569 position is located among conserved residues that bind calcium. The resulting charge inversion caused by the E569K substitution may disrupt the lipid binding and phospholipase activities of PLC6. Here, we show that PLC6 influences polarized PT growth and HPAT-mediated PT growth and fertility, and future studies are necessary to better understand the relationship between calcium and PLC6 in PT growth.
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