The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three – GDP, IDP and UDP – were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca²⁺, Mg²⁺ and Mn²⁺ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent K_m values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.     

Disruption of apyrases inhibits pollen germination in Arabidopsis

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive beta-glucuronidase staining in the pollen of apyrase promoter:beta-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

Effects of chemical inhibitors and apyrase enzyme further document a role for apyrases and extracellular ATP in the opening and closing of stomates in Arabidopsis

In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results further confirm the role of extracellular ATP in regulating stomatal apertures.     

A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.     

Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB,Rab 3, trans-golgi network 46, and mannosidase II

Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.     

Dynamic response of prevacuolar compartments to brefeldin a in plant cells

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.     

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.     

Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles. Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence. Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures. By contrast, wortmannin caused YFP-labeled PVCs but not YFP-labeled Golgi stacks to vacuolate. VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from high-pressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs. MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining. Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells. Thus, we have unequivocally identified MVBs as PVCs in N. tabacum BY-2 cells. Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells.     

OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.     

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.     

Rab-A2 and Rab-A3 GTPases define a trans-golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate

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-A2a 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-A2a 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.     

On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 approximately 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 approximately 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 approximately 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 approximately 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

Small GTP-binding proteins in Plasmodium falciparum

Summary— During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-relate protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.     

Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-rich mucilage

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

Apyrases (nucleoside triphosphate-diphosphohydrolases) play a key role in growth control in Arabidopsis

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.     

Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells

The role of endocytosis in nutrient uptake by Toxoplasma gondii is unknown. To explore this issue, we characterized an endosomal compartment by identifying a T. gondii Rab5 homologue, a molecular marker for early endosomes in eukaryotic cells. The deduced amino acid sequence of the T. gondii Rab5 gene encodes a protein of 240 amino acids, which we termed TgRab51. TgRab51 was epitope-tagged at the N-terminus, expressed in the parasite, and localized by immunofluorescence and immunoelectron microscopy to tubulovesicular structures anterior to the parasite nucleus and adjacent to, but distinct from the Golgi. By immunofluorescence analysis, TgRab51wt-HA staining partially overlapped with Golgi/TGN markers, but not with the T. gondii secretory organelles. A dominant positive mutant, TgRab51Q103L-HA, enhanced uptake of exogenous cholesterol analogues in intracellular parasites, augmented formation of lipid droplets and accelerated parasite growth. Brefeldin A disrupted the TgRab51 compartment, and altered the distribution of fluorescent exogenous cholesterol in cells expressing TgRab51Q103L-HA. These results suggest that TgRab51 facilitates sterol uptake, possibly through a Golgi-dependent pathway.     

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.