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

Abstract

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-richglycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-richdomain and a C-terminal domain with repetitive Ser–Pro3–5 motifs that are potentially to be Oglycosylated.It has been demonstrated that pollen-specific LRX8–LRX11 from Arabidopsis thalianaare necessary to maintain the integrity of the pollen tube cell wall during polarized growth. InHRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are convertedto hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In thiscontext, we aimed to determine whether hydroxylation and subsequent O-glycosylation ofArabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollentubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of theproline units present in Ser–Pro3–5 motifs of LRX8–LRX11. Here, we show that the p4h4-1 p4h6-1double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tubelength. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation isrequired for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1background show partial re-localization of LRX11–green fluorescent protein (GFP) from the pollentube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem MSmass spectrometryanalysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11–GFP in the p4h4-1p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together,these results suggest that P4H4 and P4H6 are required for pollen germination and for properhydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.