Abstract
Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.
Highlights
Rab proteins are key components of the vesicular traffic machinery found in all eukaryotes
Female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule
Signal was not obvious in the developing functional megaspore (FM) or female gametophyte (FG) (Fig. 9E–G), but was detected in most sporophytic ovule tissues including the funiculus (Fig. 9E–G).As the ovule approached maturity, signal was maintained in the funiculus and was abundant in the egg apparatus (Fig. 9H).These results indicate that RGTB1 mRNA partially overlaps with the sites of PIN accumulation, but is not abundant in the megaspore mother cell (MMC) or nucellus from meiosis until gametophyte cellularization
Summary
Rab proteins are key components of the vesicular traffic machinery found in all eukaryotes. They reside on the cytosolfacing leaflet of lipid bilayers of organellar membranes. Rabs undergo post-translational modification with two geranylgeranyl moieties on cysteine residues close to the protein C-terminus. This modification enables stable anchoring of Rabs to the membranes, which results in a 10 times higher affinity for the membranes (Silvius and l’Heureux, 1994; Shahinian and Silvius, 1995).The enzyme catalyzing the prenylation of Rab proteins is rab geranylgeranyl transferase (RGT), a complex of catalytic RGTA and lipid substratebinding RGTB subunits and the accesory Rab escort protein (REP) (Seabra et al, 1992; Thoma et al, 2001). Single geranylgeranylated or unmodified Rab proteins are mistargeted and non-functional (Gomes et al, 2003)
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