Prolyl hydroxylation is necessary for pollen germination and proper cell wall assembly during pollen tube growth in Arabidopsis thaliana

SEDE A. R.; ESTEVEZ J. M.; WENGIER, DIEGO L.; MUSCHIETTI J. P.; SOMOZA, SOFÍA C.

Congreso; Reunion Conjunta SAIB/SAMIGE; 2020

Major constituents of the plant cell walls are structural proteins that belong to the Hydroxyprolyl-rich glycoprotein (HRGP) family. Members of HRGPs are divided according to their glycosylation pattern in: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Leucine-rich repeat extensins (LRXs) are hybrid EXTs that contain a C-terminal domain with Ser-Pro(3-5) repetitions plausible to be glycosylated. We have previously demonstrated that Arabidopsis pollen-specific LRXs (LRX8-11) are necessary to maintain the integrity of the pollen tube cell wall during polarized growth 1; loss of function lrx9-2 lrx10-1 lrx11-1 triple mutant pollen displayed severe abnormalities both in vitro and in vivo. The lack of LRXs caused a decreased in pollen germination rate, the early bursting of pollen tubes and impaired fertilization. Moreover, an altered deposition of callose and pectins in pollen tube cell walls of lrx9-2 lrx10-1 lrx11-1 triple mutants was found preventing of normal tip elongation. It is well known that in classical EXTs, proline residues are converted to hydroxyproline by Prolyl-4-hydroxylases (P4Hs) and this post-translational modification (PTM) is necessary to define novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation, and subsequent glycosylation, of Arabidopsis pollen LRXs are required for their function and proper localization at the cell wall of pollen tubes. We hypothesize that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of proline units present in Ser-Pro(3-5) motifs of LRX8-11 extensin domain. The analysis of loss of function p4h4 and p4h6 mutants and p4h4p4h6 double mutant showed a drastically reduction in pollen germination rates and a slightly reduction in the pollen tube length. Pollen germination was also inhibited by applying specific P4Hs inhibitors to the pollen germination medium, suggesting that prolyl hydroxylation is mainly necessary at early stages of pollen tube development. Transgenic pollen tubes expressing pP4H4::P4H4-YFP construct revealed that P4H4 localized in dots-like structures that resemble to Golgi apparatus. Interestingly, transgenic plants expressing pLRX11::LRX11-GFP construct in p4h4p4h6 background showed a re-localization of LRX11-GFP from the tip to the cytoplasm of pollen tubes. All together, these results suggest that P4H4 and P4H6 are involved in pollen hydroxylation and this PTM is necessary for pollen germination and proper localization and crosslink of LRX11 (and might also LRX8-10) at the cell wall of growing pollen tubes.