Expression, Subcellular Localization And Functional Study Of Arabidopsis RabD2b GTPase

Rab proteins constitute a largest family of small GTPases (Ras-related) in plants, mammals and microorganisms. They regulate different steps during vesicle trafficking in cells. Rab1-related GTPases regulate the vesicle trafficking from endoplasmic reticulum (ER) to Golgi in mammals and yeasts. Plant Rab1-related GTPases are ascribed to the RabD subfamily. In Arabidopsis, the RabD subfamily could be further divided into two functionally distinct groups, AtRabD1(including AtRabD1) and AtRabD2(including AtRabD2a, AtRabD2b and AtRabD2c) subclasses. The in vivo roles of AtRabD1and AtRabD2a in vesicle trafficking have been investigated in tobacco leaf epidermal cells using their dominant-negative N121I mutants. They regulate the similar ER-to-Golgi vesicle traffic step. The cellular and physiological functions of AtRabD2b have only recently begun to be unraveled. To gain further insight into the role of AtRabD in planta, we investigated the expression pattern and subcellular localization of AtRabD2b. We also generated transgenic plants that constitutively expressed YFP-AtRabD2b [Q67L], YFP gene fused with a constitutively active form of AtRabD2b, and examined their morphological and developmental phenotypes. The followings are the main results:1. Rab1-related GTPases from different specials share high amino acid homology with each other and they all contain several conserved functional domains. Yeast complementary assay revealed that AtRabD2b could rescue the yeast yptl mutant and substitute Yptl as a functional ortholog.2. AtRabD2b is localized on Golgi stacks and a second subpopulation of punctate structures in tobacco abaxial epidermis cells. The mutated protein, AtRabD2b [Q67L], which contains the mutation from glutamine (Q) to leucine (L) at the67th amino acid, was defective in the ability to hydrolyze GTP and thus kept in a constitutively active GTP-bound state. AtRabD2b [Q67L] did not markedly alter the membrane distribution of AtRabD2b. AtRabD2b [N121I], containing the mutation of asparagine (N) at position121to isoleucine (I) in the G4motif, is predicted to reduce the affinity of Rab proteins for both GDP and GTP. AtRabD2b [N121I] is localized in the cytosol. These results indicate that the nucleotide state alters the membrane localization of AtRabD2b.3. RT-PCR results showed that mRNA transcripts of AtRabD were detected in all examined tissues including the roots, stems, rosette leaves, cauline leaves, buds, flowers and siliques, indicating AtRabD genes are ubiquitously expressed in various plant tissues. The detail expression pattern of AtRabD2b was examined by histochemical staining of GUS in AtRabD2bpro::GUS transgenic plants. It was showed that GUS activity was detected in both seedlings and mature plants and dominantly expressed in active growth and differentiated tissues.4. Loss-of-function mutants of AtRabD2b and/or AtRabD2c exhibit no obvious phenotypes.Transgenic plants constitutively expressing YFP(yellow fluorescent protein)-AtRabD2b [Q67L] fusion gene exhibited altered morphology of rosette leaves and reduced silique angles in the shoots. Down-regulation of AtRabDl, AtRabD2b and AtRabD2c, due to AtRabD2b gene co-suppression in transgenic plants, resulted in stunted bushy growth phenotype, very low fertility and the necrosis at the apical region of the stem, suggesting functional redundancy within AtRabD2subclass and between AtRabD1and AtRabD2subclasses.5. Microscopic observations revealed that.necrotic cells firstly appeared in the periphery epidermal cell layer and spread from cell to cell throughout the whole section. Transmission electron microscopic observation showed that chloroplasts in these cells exhibited developmental defects such as fewer internal thylakoid membranes and large starch grains. Moreover, in most of the dying cells, cellular components or organelles were found to be invaginated inside the vacuoles by autophagy. These data suggested that AtRabD genes are essential for plant cell viability in certain tissues.6. The two conserved cysteins at C terminus of AtRabD2b protein is important for the prenylation modification of AtRabD2b. Two mutated genes, AtRabD2c199S and AtRabD2bΔCC, were created by PCR amplification. It was showed that the two mutant proteins are localized in the cytoplasm. Yeast complementary assay showed that the two mutant genes can not complement the yeast temperature-sensitive mutant strain ASY01. Transgenic plants overexpressing AtRabD2bC199s or AtRabD2bΔCC produced different phenotypes with transgenic plants overexpressing AtRabD2b. These results revealed that the mutations of the conserved cysteins at C terminus of AtRabD2b protein lead to the alteration of the subcellular localization and function of AtRabD2b.