An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains β-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response.     

A Point Mutation in the Ethylene-Inducing Xylanase Elicitor Inhibits the β-1-4-Endoxylanase Activity But Not the Elicitation Activity

Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the β-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.     

Isolation of plasma membrane and binding of the Ca2+ antagonist nimodipine in Chlamydomonas reinhardtii

Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg²⁺-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg²⁺-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [³H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K_D= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (K_D= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B_max= 440 and 470 fmol (mg protein)^-1, respectively. [³H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [³H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl₂.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in theplasma membrane from rice leaf and root cells

Presence of a high-affinity binding protein for N-acetylchitooligosaccharide (fragments of chitin) elicitor in the plasma membrane from rice leaf and root cells was shown by affinity labeling experiments with an ¹²⁵I-labeled N-acetylchitooligosaccharide derivative. Binding studies also showed that binding site in the leaf cells has a high affinity to highly elicitor-active, larger chitin fragments but much lower or no affinity to less elicitor-active or elicitor-inactive oligosaccharides. The amount of the binding protein in the leaf cells was slightly smaller than that in the suspension-cultured cells but much larger compared to that in the root cells. These results indicate the possible- involvement of the elicitor binding protein in the perception of the elicitor signal in intact rice plant.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.     

Alterations in Nicotiana tabacum L. cv Xanthi Cell Membrane Function following Treatment with an Ethylene Biosynthesis-Inducing Endoxylanase

An ethylene biosynthesis-inducing xylanase (EIX) produced by the fungus Trichoderma viride elicited enhanced ethylene biosynthesis and leakage of potassium and other cellular components when applied to leaf disks of tobacco (Nicotiana tabacum L. cv Xanthi). Suspension-cultured cells of Xanthi tobacco responded to EIX by rapid efflux of potassium, uptake of calcium, alkalization of the medium, inhibition of ethylene biosynthesis, and increased leakage of cellular components. EIX-treated cell suspensions released 1-aminocyclopropane-1-carboxylate (ACC) into the surrounding medium, resulting in a reduction of cellular pools of ACC. The responses of both cell suspensions and leaf disks were inhibited (50-80%) by the preincubation of the tissues with the calcium channel blocker La³⁺. High concentrations of EGTA inhibited the alkalization of the medium by cell suspensions responding to EIX, but EGTA alone caused extensive loss of K⁺ and ACC and inhibited ethylene biosynthesis by tobacco cells. Alterations in membrane function appear to be important in the mode of action of EIX in Xanthi cells.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Reevaluation of the role of auxin binding site II

Binding of 1-naphthylacetic acid (1-NAA) was assayed in microsomal membranes from Zea mays coleoptiles and from hypocotyls of Cucurbita pepo. Auxin binding site II was differentiated from site I binding by using phenylacetic acid (PAA) to saturate site I binding capacity. The amount of type-II binding sites, per gram original fresh weight, was 34 pmol with Zea and 6.4 pmol with Cucurbita. When maize membranes were separated by dextran gradient centrifugation, auxin binding site II migrated coincident with tonoplast marker enzymes. The physiologically active auxin 4-chloroindoleacetic acid (4-Cl-IAA) competed very poorly with 1-NAA binding to both site I and site II. This result suggests that sites I and II are not involved in the regulation of growth. When comparing isolated outer epidermis with intact coleoptile of Zea, similar amounts and ratios of site I and site II binding activities were observed.     

<Emphasis Type="Italic">Trichoderma viride</Emphasis> cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

Background  

Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Characteristics of ethylene biosynthesis-inducing xylanase movement in tobacco leaves

¹²⁵I-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active ¹²⁵I-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. ¹²⁵I-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.     

Heterogeneity of the S-100 Protein Specific Binding Sites in Synaptosomal Particulate Fractions and Subfractions

The specific interaction of S-100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under various conditions. The results suggest that S-100 binds to two populations of membrane sites: high -affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low-affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S-100 binding to SYN does not result from heterogeneity of the S-100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high-affinity binding.     

Specific binding of a hypersensitive lignification elicitor from Puccinia graminis f. sp. tritici to the plasma membrane from wheat (Triticum aestivum L.)

We have recently reported the isolation and characterization of a glycoprotein (M_r 67 000) from germ-tube walls of Puccinia graminis f. sp. tritici which elicits the cellular hypersensitive lignification response in wheat (G. Kogel et al., 1988, Physiol. Mol. Plant Pathol. 33, 173–185). The present study uses this glycoprotein, referred to as Pgt elicitor, to identify putative elicitor targets in wheat cell membranes. In enzyme-linked immunosorbent assays using anti-Pgt elicitor antibodies, specific binding sites for Pgt elicitor were detected in highly purified plasma-membrane vesicles of wheat (Triticum aestivum L.) primary leaf cells. Binding proved to be independent of the presence or absence in wheat of the Sr5 gene for rust resistance, and also occurred on barley (Hordeum vulgare L.) plasma membrane. The binding sites have an Mr of 30 000 and 33 000, respectively, and binding activity was not lost in the presence of sodium dodecyl sulfate. [¹⁴C]imido-Pgt elicitor was used to determine the apparent K _d value for specific binding, found to be 2.0 M, and the maximum content of binding sites, found to be 250 pmol per mg of plasma-membrane protein. The relevance of the elicitor binding for the outcome of the interaction of P. graminis and wheat is discussed.Abbreviations BSA bovine serum albumin - ELISA enzyme linked immunosorbent assay - IDPase inosine 5-diphosphatase - MPLC medium-pressure liquid chromatography - MF microsomal fraction - Pgt elicitor elicitor of Puccinia graminis f. sp. tritici - SDS sodium dodecyl sulfate - Pre U₃, Pre U₁ pure plasma membrane from wheat cultivar Prelude and plasma membrane contaminated by intracellular membrane, respectively This work was supported by the Deutsche Forschungsgemeinschaft. We wish to thank C. Larsson, Lund, Sweden for his kind support in the preparation of plasma membrane.     

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase‐activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock‐out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root‐tip‐specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone‐directed adaptation of root architecture in response to salinity.     

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.