Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids

The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2- 1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues

Tomato Rab11a Characterization Evidenced a Difference Between SYP121-Dependent and SYP122-Dependent exocytosis

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat β-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory event

Intracellular vesicle transport in "Arabidopsis thaliana": functional characterization of the t-SNARE homologue AtSNAP33

Alle eukaryontischen Zellen benötigen für die Sekretion ein funktionierendes endomembranes System. Proteine, die sekretiert werden besitzen ein Signalpeptid und werden am Endoplasmatischen Retikulum (ER) synthetisiert. Das Signalpeptid initiiert den Transport in den Lumen des ER und wird danach enzymatisch vom Protein abgetrennt. Korrekt gefaltete Proteine werden in Vesikel gepackt, die sich vom ER abschnüren und mit der cis Seite des Golgi Apparats fusionieren. Die Proteine durchlaufen den Golgi Apparat und an der trans Seite des Golgis werden die Proteine, die für die Prevakuole, lytische Vakuole oder Proteinvakuole bestimmt sind getrennt von den Proteinen, die ihre Funktion im interzelluläre Raum haben. Der Transport zwischen den einzelnen Organellen, die in der Sekretion involviert sind, findet via Vesikel statt, die sich von einer Geber-Membran abschnüren und mit der entsprechenden Ziel-Membrane fusionieren. Die korrekte Fusion der einzelnen Vesikel mit der entsprechenden Organelle ist von höchster Wichtigkeit, ansonsten verliert die Zelle ihre Kompartimentierung und kollabiert. Verschiedene SNARE-Proteine (Soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors) und die entsprechenden Faktoren sind verantwortlich für die einzelnen Fusionen von Vesikeln mit den entsprechenden Membranen. Das allgemein akzeptierte Modell postuliert, dass ein Membran assoziertes Protein auf dem Vesikel (v-SNARE) mit einem Membran assozierten Protein auf der entsprechenden Organelle (t-SNARE) wie Schlüssel und Schloss interagieren. SNARE Proteine sind detailliert beschrieben in Nervenzellen. Die Neurotransmitter werden via Vesikelfusion mit der Plasmamembran, beim Eintreffen eines Aktionspotential ausgeschüttet. In diesem Fall interagiert das Synaptobrevin, ein v- SNARE welches auf dem Vesikel lokalisiert ist, mit zwei t-SNAREs SNAP-25 und Syntaxin 1A, beide mit der Plasmamembran verbunden, zu einem ungewöhnlich stabilen SNARE-Komplex. NSF und α-SNAP, zwei SNARE bindende Faktoren sind in der Lange unter Hydrolyse von ATP den Komplex aufzulösen. In dieser Arbeit wurde ein Arabidopsis Homolog von dem t-SNARE SNAP-25, AtSNAP33 isoliert. AtSNAP33 interagiert mit KNOLLE, ein Zytokinese spezifisches Syntaxin, welches an der Plasmamembran lokalisiert ist und essentiell für die korrekte Bildung der neuen Zellmembran während der Zytokinese ist. AtSNAP33 ist ein allgegenwärtig exprimiertes Membranprotein. Pflanzen reagieren nach Befall von Mikroorganismen wie Bakterien, Pilze oder Viren mit der Produktion von Abwehrproteinen (Pathogenesis related) PR Proteinen. Einige von den PRs werden am ER synthetisiert und in den interzellularen Raum sekretiert. Infizierte, wie auch mechanisch beanspruchte Blätter von Arabidopsis haben eine erhöhte Menge von AtSNAP33 im Vergleich zu nicht behandelten Blätter. Eine T-DNA Insertion im AtSNAP33 Gen, welches den Verlust von AtSNAP33 bedeutet, resultiert in einem letalen Zwerg-Phänotyp der Pflanze. Sieben Tage nach der Keimung kann die Bildung von ROI (reactive oxygen intermediates) und Nekrosenbildung auf den Kotyledonen beobachtet werden. Die Nekrosen breiten sich auf die ganze Pflanze aus und nach 3 bis 4 Wochen stirbt die atsnap33 Mutante. Das Wurzelwachstum der Mutante ist reduziert verglichen mit dem Wildtyp, welche unter den gleichen Bedingungen gewachsen sind. In sieben Tag alten atsnap33 Mutanten sind die Abwehrgene PR-1, PR-2 und PDF1.2 exprimiert im Gegensatz zu den beiden Homologen von AtSNAP33, AtSNAP29 und AtSNAP30, welche nicht exprimiert sind. Atsnap33 Mutanten wurden mit einer Gerste α- Amylase, welche konstitutiv sekretiert wird, transformiert. In den ersten sieben Tagen war kein Unterschied zwischen der Mutante und Columbia die je mit der gleichen α-Amylase transformiert wurde, festzustellen. Aber in den folgenden Tagen wurde die α-Amylase Sekretion in der atsnap33 Mutante inhibiert. Diese Inhibition korreliert mit der Bildung der Nekrosen. Die Reduktion des Wurzelwachstums und der Inhibierung der α-Amylase Sekretion in der atsnap33 Mutante zeigt, dass AtSNAP33 für eine funktionelle Sekretion nötig ist. Mit Hilfe eines 'Yeast two hybrid Screen’ mit AtSNAP33 als Köder, führte zur Isolation von zwei noch nicht beschriebenen Syntaxinen (t-SNARE) AtSYP122 und AtSYP43. AtSYP122 ist konstitutiv in den Wurzeln exprimiert, nicht aber in Stengeln, Blätter, Blüten oder Schoten. Die Transkription von AtSYP122 wurde durch Inokulation von Krankheitserregern induziert. AtSYP43 ist weder in Wurzeln, Blätter, Stengeln, Blüten noch in den Schoten exprimiert. Auch das Inokulation von Krankheitserregern in die Blätter stimuliert nicht die Transkription von AtSYP43. Interessanterweise sind AtSYP122 und AtSYP43 in der atsnap33 Mutante exprimiert, welche keine Expression von AtSNAP33 zeigt. Die biologische Funktion und Lokalisation von AtSYP122 und AtSYP43 sind nicht bekannt.The secretory pathway is a complex endomembrane system essential for all eukaryotic cells. It transports proteins to the extracellular space or to the vacuole. All proteins which are secreted are synthesized at the endoplasmic reticulum (ER) and have a signal peptide in common. The signal peptide is necessary for the transport into the lumen of the ER where the correct folding of the protein takes place. Then proteins are packaged into vesicles which bud of from the ER and transported to the cis-Golgi and fuse with it. The proteins go through the Golgi apparatus and at the trans Golgi network (TGN) they are sorted into vesicles destined for several organelles including the prevacuolar compartment (PVC), lytic vacuole, protein storage vacuole, (PSV) or for secretion to the plasma membrane (PM). Transport between the organelles of the secretory pathway occurs by budding of vesicles from a donor membrane and fusion with an acceptor membrane. The fusion of the appropriate vesicles with the correct target membrane is of outmost importance, otherwise the cell will lose its compartmentalisation and collapse. Soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors (SNAREs) and their associated factors were described to be responsible for these fusions steps. The general hypothesis postulates that a membrane associated protein on the vesicle, the v-SNARE, interacts with another membrane associated protein on the target membrane, the t-SNARE. SNAREs are well studied in neuronal cells where vesicles fuse with the plasma membrane to release neurotransmitters after an action potential. In this case the v-SNARE synaptobrevin which is located on the vesicle interacts with the two t-SNAREs SNAP-25 and syntaxin1A at the plasma membrane and form a SNARE core complex which is unusual stable. NSF and α-SNAP two SNARE binding proteins are able to disassociate the complex by hydrolysis of ATP. In this work an Arabidopsis homologue of the t-SNARE SNAP-25, AtSNAP33 was identified as an interactor of KNOLLE. KNOLLE is a cytokinesis-specific syntaxin which is localized at the phragmoplast and is needed for proper cell plate formation during cytokinesis. AtSNAP33 is an ubiquitously expressed membrane-associated protein. Plants respond to bacterial, fungal and viral pathogen attack by the synthesis of several pathogenesis related (PR) proteins. Some of these PR’s are synthesized at the ER and transported to the extracellular space by the secretory pathway. AtSNAP33 is induced in infected leaves as well after mechanical stimulation such as touch and wind and wounding. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. Seven days after germination reactive oxygen intermediates (ROI) and lesion formation can be observed on the cotyledons. The lesions spreads out over the whole plant expect the roots and after 3 - 4 weeks the upper green part of the plant is dead. The root growth rate in the mutants was lower compared to the wild type which were grown in the same conditions. In 7 day old atsnap33 mutants the defence genes PR-1, PR-2, and PDF1.2 were upregulated whereas the two homologues of AtSNAP33, AtSNAP30 and AtSNAP29 were not expressed. Atsnap33 mutants and Columbia plants as a control were transformed with a barley α-amylase which is constitutively expressed and secreted. In the first 7 days there was no difference in secretion of α-amylase and the mutant expressing α-amylase. But after 7 days the secretion of α-amylase was inhibited in the atsnap33 mutant. This correlates exactly with the development of the lesion mimic phenotype. The reduction of the root growth together with inhibited secretion of α- amylase indicate that AtSNAP33 is necessary for successful secretion of proteins. A yeast two hybrid screen with AtSNAP33 as a bait, led to the isolation of two not yet described syntaxins (t-SNARE) AtSYP122 and AtSYP43. AtSYP122 is constitutively expressed in roots but not in inflorescence stems, leaves, flowers and siliques. However AtSyp122 transcripts were induced in leaves after inoculation with pathogens. AtSyp43 transcripts were not expressed in roots, inflorescence stems, leaves, flowers and siliques as analysed by RNA blot hybridisation. Neither pathogen inoculation nor mechanical stimulation led to the induction of the expression of AtSyp43. Interestingly, the AtSyp122 and AtSyp43 transcripts were highly expressed in atsnap33 plants which show no expression of AtSNAP33. The biological function and localization of AtSYP122 and AtSYP43 are still unknown

A novel cucumber gene associated with systemic acquired resistance

Several genes were isolated by differential display of mRNAs from cucumber leaves inoculated with the bacterium, Pseudomonas syringae pv. lachrymans. A full-length cDNA encoding a novel pathogen-induced gene, Cupi4, was cloned and characterized in detail. While Cupi4 did not share evident homology with known sequences in the database at the nucleotide level, the predicted amino acid sequence of Cupi4 shared homology with the pathogen-inducible proteins, pMB57-10G 5′ of Brassica napus (21%) and CXc750/ESC1 of Arabidopsis thaliana (16%). Cupi4 transcripts accumulated after 12 h in leaves inoculated with P. s. lachrymans and after 48 h in the systemic upper leaves of the inoculated plants. Treatment with the chemical inducers of systemic acquired resistance (SAR), salicylic acid, 2,6-dichloroisonicotinic acid and benzothiadiazole as well as inoculation with different pathogens, P. s. syringae, Colletotrichum lagenarium and tobacco necrosis virus also led to the accumulation of Cupi4 transcripts. The increase of Cupi4 transcripts in both the inoculated first leaf and in systemic upper leaves suggested that the Cupi4 gene product is associated with systemic acquired resistance in cucumber. Induced expression of CUPI4 in different host strains of a bacterium, Escherichia coli, led to death of bacterial host cells, suggesting that CUPI4 might have antibacterial properties