Prolyl hydroxylation is necessary for proper localization of cell wall proteins and pollen germination in Arabidopsis thaliana

To produce fertilization, pollen tubes have to travel along the pistil and then deliver sperm cells upon reaching the ovules. To sustain the polarized growth of pollen tubes, the role of the cell wall, which is constantly being remodeled in the apical region, is crucial. Different polysaccharides such as callose, pectin and cellulose together with structural proteins that belong to the family of hydroxyprolyl-rich glycoproteins (HRGP) are involved in cell wall organization. Members of HRGPs family are the Leucine-rich repeat extensins (LRXs), hybrids proteins that contain an N-terminal domain involved in protein-ligand interactions and a C-terminal extensin-like 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 cell wall integrity since polarized growth of pollen tubes in loss of function lrx9-2 lrx10-1 lrx11-1 triple mutant is altered both in vitro and in vivo. The lack of LRXs caused severe abnormalities in pollen tube morphology, a decrease in pollen germination rate and a skewed pollen segregation ratio. Moreover, microscopy analysis showed an altered deposition of polysaccharides, such as callose and pectin, in the cell wall of triple mutant pollen tubes. To determine whether post-translational modifications are required for the functionality of LRXs, we aim to study the importance of proline hydroxylation, catalyzed by prolyl-4-hydroxylases (P4H), necessary to define future O-glycosylation sites. We hypothesize that pollen-specific P4H4 and P4H6 catalyze the hydroxylation of prolines at the extensin domain of LRXs. Simple loss of function p4h4 and p4h6 mutants and p4h4p4h6 double mutant showed a reduction in pollen germination rates; similar results were obtained by applying specific P4Hs inhibitors to the pollen germination medium. Transgenic plants expressing the construction pP4H4::P4H4-YFP showed that P4H4 is localized in the Golgi apparatus and/or endoplasmic reticulum. In addition, pollen tubes from transgenic plants expressing pLRX11::LRX11-GFP in the p4h4p4h6 background showed a re-localization of LRX11-GFP from the tip to the cytoplasm. Together these results suggest that LRXs are putative targets of the P4H4 and P4H6 enzymes since the lack of hydroxylation and subsequent glycosylation in the p4h4ph6 double mutant, prevents LRX11 from proper cross-linking at the pollen tube cell wall. Fil: Sede, Ana Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Wengier, Diego Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina