Arabidopsis pollen prolyl-hydroxylases P4H4/6 are relevant for correct hydroxylation and secretion of LRX11 in pollen tubes.

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRXs) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro(3-5) motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGP including classical extensins (EXTs) and likely in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4Hs inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-GFP from the pollen tube tip apoplast to the cytoplasm. Finally, IP-MS-MS analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared to lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization at the cell wall of pollen tubes.