The Ypt3/Rab11/Rab25 subfamily of Rab GTPases has expanded greatly in Arabidopsis thaliana, comprising 26 members in six provisional subclasses, Rab-A1 to Rab-A6. We show that the Rab-A2 and Rab-A3 subclasses define a novel post-Golgi membrane domain in Arabidopsis root tips. The Rab-A2/A3 compartment was distinct from but often close to Golgi stacks and prevacuolar compartments and partly overlapped the VHA-a1 trans-Golgi compartment. It was also sensitive to brefeldin A and accumulated FM4-64 before prevacuolar compartments did. Mutations in RAB-A2 a that were predicted to stabilize the GDP-or GTP-bound state shifted the location of the protein to the Golgi or plasma membrane, respectively. In mitosis, KNOLLE accumulated principally in the Rab-A2/A3 compartment. During cytokinesis, Rab-A2 and Rab-A3 proteins localized precisely to the growing margins of the cell plate, but VHA-a1, GNOM, and prevacuolar markers were excluded. Inducible expression of dominant-inhibitory mutants of RAB-A2 a resulted in enlarged, polynucleate, meristematic cells with cell wall stubs. The Rab-A2/A3 compartment, therefore, is a trans-Golgi compartment that communicates with the plasma membrane and early endosomal system and contributes substantially to the cell plate. Despite the unique features of plant cytokinesis, membrane traffic to the division plane exhibits surprising molecular similarity across eukaryotic kingdoms in its reliance on Ypt3/Rab11/Rab-A GTPases.

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We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

SummaryWe have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jelly®sh green uorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin ®laments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) signi®cantly slowed down the recovery of Golgi¯uorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.

We describe a green fluorescent protein (GFP)-based assay for investigating membrane traffic on the secretory pathway in plants. Expression of At Rab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase At Rab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type At Rab1b but not At Rab8c. When a Golgi-targeted and N -glycosylated variant of GFP was coexpressed with At Rab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H-sensitive population appeared. Unexpectedly, expression of At Rab1b(N121I), but not of the wild-type At Rab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that At Rab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement. INTRODUCTIONThe structure and biosynthetic activities of the endoplasmic reticulum (ER) and Golgi apparatus in higher plants are well described, but the mechanisms that promote and regulate membrane traffic between these compartments are not well understood (reviewed in Robinson and Kristen, 1982;Staehelin and Moore, 1995;Dupree and Sherrier, 1998;Vitale and Denecke, 1999). The Golgi apparatus in higher plants exists as a large number of independent Golgi stacks distributed throughout the cytoplasm. Each Golgi stack typically comprises five to eight flattened cisternae, whose protein and polysaccharide composition may vary in a cis to trans direction across the stack (Zhang and Staehelin, 1992; Wee et al., 1998;Nebenführ et al., 1999). Analysis of tobacco cells in which the Golgi stacks were labeled with green fluorescent protein (GFP) revealed that each stack can travel as much as 2 to 4 m sec Ϫ 1 on an actin network that is coextensive with the dynamic ER network (Boevink et al., 1998;Nebenführ et al., 1999). Consequently, despite the dynamic organization of both the ER and Golgi, both organelles remain in close association. The movement of individual Golgi stacks appears to alternate between two modes: a directed vectorial mode, probably dependent on myosin motors, and a nonvectorial mode involving apparently random oscillations reminiscent of Brownian motion at particular points in the cytoplasm (Nebenführ et al., 1999). Video microscopy of GFP-labeled Golgi stacks in cultured tobacco cells suggests that the control of Golgi movement is devolved to the individual stacks because adjacent stacks can enter independently into phases of vectorial movement, and a stack undergoing vectorial movement can travel past another that is exhibiting the nondirected oscillatory type of motion (Nebenführ et al., 1999).In contrast, the mammalian Golgi apparatus in general forms a single interconnected cluster of membranes at the microtubule organizing center. ER-derived COP...

These authors contributed equally to this work. SummaryTo facilitate glucocorticoid-inducible transgene expression from the pOp promoter in Arabidopsis the ligandbinding domain of a rat glucocorticoid receptor (GR LBD) was fused to the amino terminus of the synthetic transcription factor LhG4 to generate LhGR-N. Fusions bearing the GR LBD at other positions in LhG4 exhibited incomplete repression or inefficient induction. LhGR-N was stringently repressed in the absence of exogenous glucocorticoid but was fully activated by addition of 2 lM dexamethasone which resulted in 1000-fold increase in GUS reporter activity. Half maximal induction was achieved with 0.2 lM dexamethasone. Reporter transcripts were detectable within 2 h of dexamethasone application and peaked 4-10 h later. Neither LhGR-N nor dexamethasone affected seedling development although ethanol retarded development when used as a solvent for dexamethasone. The efficiency of the pOp target promoter was improved 10-to 20-fold by incorporating six copies of the ideal lac operator with sufficient inter-operator spacing to allow simultaneous occupancy. Introduction of the TMV X sequence into the 5¢UTR resulted in a further 10-fold increase in dexamethasone-inducible reporter activity and an increase in the induction factor to 10 4 . Although promoters containing the TMV X sequence exhibited slightly increased basal expression levels in the absence of dexamethasone, stringent regulation of the cytokinin biosynthetic gene ipt was achieved with all promoters. Despite the severity of the induced ipt phenotypes, transcripts for the KNOX homoeodomain transcription factors BREVIPEDICELLUS and SHOOTMERISTEMLESS were not significantly increased within 48 h of dexamethasone application to seedlings.

Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom, but documented examples are rare. ARF-GEFs (guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases) are essential for vesicular trafficking in all eukaryotic kingdoms, but of the eight ARF-GEF families, only the ancestral BIG and GBF types are found in plants. Whereas fungal and animal GBF proteins perform conserved functions at the Golgi, the Arabidopsis thaliana GBF protein GNOM is thought to act in only the process of recycling from endosomes. We now show that the related Arabidopsis GBF protein GNOM-LIKE1 (GNL1) has an ancestral function at the Golgi but is also required for selective internalization from the plasma membrane in the presence of brefeldin A (BFA). We identified gnl1 mutants that accumulated biosynthetic and recycling endoplasmic reticulum markers in enlarged internal compartments. Notably, in the absence of functional GNL1, Golgi stacks were rendered sensitive to the selective ARF-GEF inhibitor BFA, which caused them to fuse with the endoplasmic reticulum. Furthermore, in BFA-treated gnl1 roots, the internalization of a polar plasma-membrane marker, the auxin efflux carrier PIN2, was selectively inhibited. Thus, GNL1 is a BFA-resistant GBF protein that functions with a BFA-sensitive ARF-GEF both at the Golgi and in selective endocytosis, but not in recycling from endosomes. We propose that the evolution of endocytic trafficking in plants was accompanied by neofunctionalization within the GBF family, whereas in other kingdoms it occurred independently by elaboration of additional ARF-GEF families.

A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines. The promoter, pOp, consists of lac operators cloned upstream of a minimal promoter. No expression was detected from this promoter when placed upstream of a ␤-glucuronidase (GUS) reporter gene in transgenic plants. Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4. This factor comprised transcription-activation domain-II from Gal4 of Saccharomyces cerevisiae fused to a mutant lac-repressor that binds its operator with increased affinity. When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter. The LhG4͞pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed. The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention. It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.