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.

Protein-binding partners of the tobacco syntaxin NtSyr1.

Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.

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.

Systemic acquired resistance.

This paper examines induced resistance (SAR) in plants against various insect and pathogenic invaders. SAR confers quantitative protection against a broad spectrum of microorganisms in a manner comparable to immunization in mammals, although the underlying mechanisms differ. Discussed here are the molecular events underlying SAR: the mechanisms involved in SAR, including lignification and other structural barriers, pathogenesis-related proteins and their expression, and the signals for SAR including salicylic acid. Recent findings on the biological role of systemin, ethylene, jasmonates, and electrical signals are reviewed. Chemical activators of SAR comprise inorganic compounds, natural compounds, and synthetic compounds. Plants known to exhibit SAR and induced systemic resistance are listed.

Systemic Responses in Arabidopsis thaliana Infected and Challenged with Pseudomonas syringae pv syringae.

Attack of plants by necrotizing pathogens leads to acquired resistance to the same or other pathogens in tissues adjacent to or remotely located from the site of initial attack. We have used Arabidopsis thaliana inoculated with the incompatible pathogen Pseudomonas syringae pv syringae on the lower leaves to test the induction of systemic reactions. When plants were challenged with Pseudomonas syringae pv syringae in the upper leaves, bacterial titers remained stable in those preinfected on the lower leaves. However, there was a distinct decrease in symptoms that correlated with a local and systemic increase in salicylic acid (SA) and in chitinase activity. Peroxidase activity only increased at the site of infection. No changes in catalase activity were observed, either at the local or at the systemic level. No inhibition of catalase could be detected in tissue in which the endogenous levels of SA were elevated either naturally (after infection) or artificially (after feeding SA to the roots). The activity of catalase in homogenates of A. thaliana leaves could not be inhibited in vitro by SA. SA accumulation was induced by H2O2 in leaves, suggesting a link between H2O2 from the oxidative burst commonly observed during the hypersensitive reaction and the induction of a putative signaling molecule leading to system acquired resistance.

Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

Vacuolar chitinases of tobacco: a new class of hydroxyproline-containing proteins.

The fungicidal type I chitinases contribute to the defense response of plants against pathogens. Two tobacco chitinases represent a different class of hydroxyproline-containing proteins. Hydroxyproline-rich proteins are predominantly extracellular, structural glycoproteins proteins that lack enzymatic activity and contain many hydroxyproline residues. In contrast, type I chitinases are vacuolar enzymes. They are not glycosylated and contain a small number of hydroxyproline residues restricted to a single, short peptide sequence.

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins.

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.

In vitro processing of tomato proteinase inhibitor I by barley microsomal membranes: a system for analysis of cotranslational processing of plant endomembrane proteins.

A plant-derived in vitro system for the study of cotranslational processing of plant endomembrane proteins has been developed and used to investigate cotranslational proteolytic processing of tomato proteinase inhibitor I. Translation of the inhibitor I precursor in wheat germ lysate supplemented with barley aleurone microsomal membranes resulted in cotranslational import of the protein into microsomal vesicles and cleavage of the signal sequence. NH(2)-terminal sequence analysis of the translocated inhibitor I processing intermediate showed that the signal sequence was cleaved between Ala(23) and Arg(24) of the precursor protein. Parallel experiments using dog pancreas microsomal membranes indicated an identical site of cleavage, suggesting that the substrate determinants for signal sequence processing are conserved across kingdoms. The plant-derived processing system used for this study may be valuable for analysis of cotranslational processing of other plant preproteins and for characterizing the components of the cotranslational import machinery in plants.

alpha-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer.

The subcellular site of the posttranslational modification of alpha-amylase was investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of alpha-amylase isoforms, referred to as low-isoelectric point (low-pl) or HAMY1 and high-pl or HAMY2, when incubated in gibberellic acid and CaCl(2). Whereas homogenates of aleurone layers contain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incubation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of HAMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endoplasmic reticulum and Golgi apparatus (GApp) marker enzyme activities and a peak of light membranes with characteristics of the GApp. The heavy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isoforms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that membranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibits the secretion of alpha-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membranes isolated from monensin-treated aleurone layers indicating that the GApp is involved in the conversion of the precursor to the secreted forms of the enzyme.

Intracellular transport and processing of a tobacco vacuolar ß-1,3-glucanase.

The class I ß-1,3-glucanases are basic, vacuolar enzymes implicated in the defense of plants against pathogen infection. The tobacco (Nicotiana tabacum L.) enzyme is synthesized as a preproprotein with an N-terminal signal peptide for targeting to the lumen of the endoplasmic reticulum and an N-glycosylated C-terminal extension which is lost during protein maturation. The transport and processing of ß-1,3-glucanase in cellsuspension cultures of the tobacco cultivar Havana 425 was investigated by pulse-chase labelling and cell fractionation. We verified that mature ß-1,3-glucanase is localized in the vacuole of the suspension-cultured cells. Comparison of the time course of processing in homogenates, the soluble fraction, and membrane fractions indicates that proglucanase is transported from the endoplasmic reticulum via the Golgi compartment to the vacuole. Processing to the mature form occurs in the vacuole. Treatment of cells with tunicamycin, which inhibits N-glycosylation, and digestion of the (35)S-labelled processing intermediates with endoglycosidase H indicate that ß-1,3-glucanase has a single N-glycan attached to the C-terminal extension. Glycosylation is not required for proteolytic processing or correct targeting to the vacuole.

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro.

Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.

A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes.

The effects of heat shock on the synthesis of alpha-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25 degrees C to 40 degrees C for 3 hours, inhibits the accumulation of alpha-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca(2+). When ER is isolated from heat-shocked aleurone layers, less newly synthesized alpha-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca(2+) transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

The expression of barley α-amylase genes in Xenopus laevis oocytes.

The synthesis and secretion of α-amylase (EC 3.2.1.1) from Xenopus laevis oocytes injected with plasmids containing barley α-amylase complementary DNA (cDNA), genomic DNA, or synthetic α-amylase mRNA were studied. α-Amylase accumulated within the oocytes beginning 12 h after injection of DNA and in the medium 12 h later as a result of secretion. S1 mapping showed that the transcription of genomic DNA was initiated at the same site in oocytes as in barley aleurone, but that the transcription of cDNAs was less precise than that of genomic DNA. The α-amylase secreted by oocytes injected with either RNA or DNA had a molecular mass (Mr) of 44000 daltons (Da) and was indistinguishable from native barley α-amylase in size, isoelectric point, antigenicity and enzymatic activity. Isoelectric focussing showed that two enzymatically active isoforms of α-amylase were synthesized and secreted from oocytes injected with synthetic RNA or DNA. The results permit us to assign specific electrophoretic bands to specific cDNA clones. We conclude that the Xenopus oocyte is a promising surrogate system for the study of transcription and translation of plant genes.

The calcium requirement for stability and enzymatic activity of two isoforms of barley aleurone alpha-amylase.

alpha-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca2+-containing metalloenzymes. We studied the effect of Ca2+ on the activity and structure of the two major groups of aleurone alpha-amylase by incubating affinity purified enzyme in solutions containing Ca2+ from pCa 4 to 7. Both groups of isoforms required one atom of Ca2+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca2+. Both groups of alpha-amylase were irreversibly inactivated by incubation in low Ca2+ (pCa 7). This inactivation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone alpha-amylase indicates that the Ca2+-binding region of barley amylases is structurally similar to that of mammalian alpha-amylases. Our data indicate that micromolar levels of Ca2+ are required to stabilize the structure of barley alpha-amylases in the endoplasmic reticulum of the aleurone layer where these enzymes are synthesized.

Evidence for precursor forms of the low isoelectric point alpha-amylase isozymes secreted by barley aleurone cells.

Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two alpha-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430-438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of alpha-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse-chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra- to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70 degrees C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation.

Calcium requirement for the secretion of peroxidases by plant cell suspensions.

Spinach (Spinacia oleracea, L.) cells in liquid culture release peroxidases. This release is reduced by EGTA and promoted by calcium ions. In a medium deprived of calcium the rate of peroxidase release is low, but immediately after addition of I mM calcium there is a sudden increase of the extracellular peroxidase activity. Extracellular calcium apparently penetrates into the cultured cells rather freely and, as a consequence, the rate of peroxidase secretion by these cells is directly correlated with the concentration of calcium in the medium. Magnesium, at twice the concentration used for calcium, has no effect on the release of peroxidases. Cells treated with Na azide, Na hydrogenarsenate of fluphenazine secrete less peroxidase upon addition of calcium.