ER-/Golgi-localized P4Hs that hydroxylate EXTs are crucial for cell elongation in Arabidopsis root hairs

SILVIA MELINA VELÁSQUEZ, MARTINIANO M. RICARDI, JAVIER GLOAZZO DOROSZ, NORBERTO D. IUSEM, JOSE M. ESTEVEZ.

La Plata, Argentina

Congreso; XXVIII Reunion Argentina de Fisiologia Vegetal; 2010

Asociacion Argentina de Fisiologia Vegetal

Precise biological roles for posttranslational modifications on plant O-glycoproteins have been elusive. Here we demonstrate in Arabidopsis thaliana that proline hydroxylation on root hair extensins (rhEXTs) by prolyl 4-hydroxylases (AtP4Hs) is crucial for root hair growth. P4H2, P4H5 and P4H13 are highly root- expressed prolyl 4-hydroxylases (rhP4Hs). It has already been shown by this group that T-DNA mutants from these P4Hs have drastic reducing effects on root hair growth and also that these rhP4Hs hydroxylate root hair EXTs. When analyzing P4Hs sub-cellular location, rhP4H5 is highly co-localized with ER-marker and with medial-Golgi marker. In contrast, rhP4H2 and rhP4H13 co-localized almost exclusively with ER. All 3 rhP4Hs are similar in terms of protein sequence, except that rhP4H5 and rhP4H 13 have a [RK]X[RK] motif (ERG, linked to ER-Golgi transport) on the N-terminus, and rhP4H2 does not posses that domain but has a C-terminus rich-cystein domain (CD). The removal of the CD domain from rhP4H2 showed no effect (it fully complemented the mutant phenotype) when it was expressed under its respective promoter in p4h2. When P4H2+CD+ERG and P4H2–CD+ERG constructs were expressed in p4h2 mutant background, no changes in the root hair growth were observed. However, when the ERG domain was deleted from rhP4H5, no complementation of p4h5 mutant was observed. Also, there was no co-localization of P4H5-GFP with Cherry tagged Golgi markers. When P4H5–ERG+CD was expressed in p4h5 no complementation was observed either. This suggests that Golgi localization is necessary for the function of rhP4H5. Other proteins of the O-glycosylation network were analyzed in search for a similar reduced root hair growth, in order to determine possible interactions with rhP4Hs. Mutant analysis of four recently discovered glycosyltransferases that can arabinosylate EXTs (rra1-3 and xeg113-2) phenocopy rhp4hs thus supporting the hypothesis of a crucial role of O-glycans on rhEXTs as key regulators of cell elongation in root hairs. By differential microarrays analysis of several root hair mutants and also by co-expression analysis, several two type-II rhEXTs were identified with obvious root hair phenotypes suggesting low level of genetic redundancy in modulate root hair elongation. All of the previously described data help further characterize rhP4Hs, their function and their subsequent implication on the network of O-glycosylation. The control of growth by O-glycosylated EXTs may represent a more general mechanism for regulating cell expansion in different plant cell types.