Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

Rebuilt 3D structure of the chloroplast f1 ATPase-tentoxin complex

The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.     

Viruslike particles in tentoxin-producing strains of Alternaria alternata.

Double-stranded (ds) RNAs associated with viruslike particles have been found in six isolates of Alternaria alternata which produce tentoxin. Isolates had from one to three dsRNAs ranging in size from 1.0 to 5.1 kilobase pairs. In two isolates the dsRNAs were associated with 30-nm particles. No dsRNA was detected in any of six other tentoxin-producing isolates or nine isolates which did not produce tentoxin.     

Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana Sylvestris by fusion with X-irradiated N. tabacum protoplasts

Summary Restoration of male fertility was achieved by fusing protoplasts from male sterile (CMS) Nicotiana sylvestris plants with X-irradiated protoplasts derived from fertile N. tabacum plants. The CMS N. sylvestris plants were derived from a previous somatic hybridization experiment and contained alien (Line 92) cytoplasm. About one quarter of the regenerated plants were found to be cybrids. i.e. they consisted of N. sylvestris nuclei combined with all or some components of N. tabacum cytoplasm. In one half of these cybrids male fertility was restored to different levels. The chloroplasts of the two parental donors differ in respect to tentoxin sensitivity: chloroplasts of CMS N. sylvestris are sensitive while those of N. tabacum are insensitive. It could therefore be demonstrated that there was an independent segregation of chloroplast type and male fertility/sterility: several somatic cybrids were male fertile but tentoxin sensitive and others were tentoxin insensitive yet they were male sterile. Only in about one half of the somatic cybrids was male fertility restored together with restoration to tentoxin insensitivity.     

Tentoxin effects on variable fluorescence and P515 electrochromic absorbance changes in tentoxin-sensitive and -resistant plant species

The effects of the cyclic tetrapeptide phytotoxin, tentoxin, on variable chlorophyll fluorescence quenching and the P515 electrochromic absorbance change were examined in tentoxin-sensitive and -resistant species and an interspecies hybrid. Immediately after infiltration of leaf discs, up to 100 μM tentoxin had no effect on the O, I, D, or P portions of the fluoresence transients of either tentoxin-sensitive or -resistant plants. However, the quenching of fluorescence following maximal fluorescence was reduced by approximately fourfold in tentoxin-treated tissues of tentoxin-sensitive plants, but was unaffected in resistant plants. Tentoxin significantly increased the magnitude of the P515 electrochromic absorbance change in all tentoxin sensitive plants by an average of approximately twofold. However, it was unaffected by tentoxin in resistant species. These data suggest that there is a close correlation between interaction of tentoxin with CF₁ ATPase in vivo and the ability to cause chlorosis in developing chloroplasts.     

An insight into the mechanism of inhibition and reactivation of the F(1)-ATPases by tentoxin

The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungal cyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kinetic analysis using synthetic analogues of tentoxin. The alpha-subunit of chloroplast F(1)-ATPase (CF(1)) was specifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxin binding to the alpha-subunit, and 3D homology modeling was used to locate tentoxin in its putative binding site at the alpha/beta interface. The non-photosynthetic F(1)-ATPase from thermophilic bacterium (TF(1)) proved to be also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to its inhibitory site, contrary to what was previously observed with epsilon-depleted CF(1) [Santolini, J., Haraux, F., Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxin preferentially binds to an ADP-loaded alpha beta pair, and mechanically blocks the catalytic cycle, perhaps by the impossibility of converting this alpha beta pair into an ATP-loaded alpha beta pair. Using (14)C-tentoxin and selected synthetic analogues, we found that toxin binding to the tight inhibitory site of CF(1) exerts some cooperative effect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-binding sites are filled in reactivated F(1)-ATPase, they do not exchange their role during catalytic turnover, indicating an impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysis provides a mechanical interpretation of the inhibition of F(1)-ATPase by tentoxin and a clue for understanding the reactivation process.     

Comparative chemical screening and genetic analysis reveal tentoxin as a new virulence factor in Cochliobolus miyabeanus,the causal agent of brown spot disease on rice

Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non‐ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra‐high‐performance liquid chromatography (UHPLC) coupled to high‐resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem‐quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild‐type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin‐insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.     

Tentoxin sensitivity of citrus: cotyledon-dependency of growth inhibition and reversibility of chlorosis

Tentoxin is a cyclic tetrapeptide, produced by the fungus Alternariaalternata, that induces chiorosis in germinating seedlings ofsome angiosperms. Since the most pronounced chiorotic effectof tentoxin is at the initial stages of germination most studieshave evaluated the effects of tentoxin on cotyledons. In thispreliminary work a unique biological system was establishedfor the study of the mechanism of tentoxin induced chiorosisin developing citrus seedlings. This system was used to comparethe effects of tentoxin on the in vitro germination of intactversus decotyle donized embryos. It is demonstrated here thatthe chlorotic effect of tentoxin is reversible and that ten-toxin blocks the ability of decotyledonized embryos to utilizenutrients from the growth medium and, there fore, to compensatefor the lack of cotyledons. The citrus system offers a uniqueway to study the relation between the effect of tentoxin onthe activity of choloplast ATPase and the induction of chlorosis. Key words: Tentoxin, citrus, chiorosis     

Hybrid Rhodospirillum rubrum F(0)F(1) ATP synthases containing spinach chloroplast F(1) beta or alpha and beta subunits reveal the essential role of the alpha subunit in ATP synthesis and tentoxin sensitivity

Trace amounts ( approximately 5%) of the chloroplast alpha subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores of Rhodospirillum rubrum with the chloroplast beta subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the alpha subunit in the rebinding of beta, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast alpha and beta subunits. The treated chromatophores were found to have lost, in addition to most of their beta subunits, approximately a third of the alpha subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum alpha and chloroplast beta subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast alpha and beta subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum alpha subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast alpha subunit is required together with the chloroplast beta subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the beta subunit on sensitivity to tentoxin.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Growth and internal ion concentrations in seedlings of winter wheat are affected by 1 pM tentoxin

The long-term effect of tentoxin on K^+;, Ca²⁺ and total phosphorus (P) concentrations in the roots and shoots of 7- and 14-day-old seedlings of winter wheat ( Triticum aestivum L. cv. Martonvásári-8) was studied. Growth (dry weight) and shoot to root ratios (dry weight and mineral concentrations) were also estimated. One p M tentoxin increased the shoot to root ratio for dry weight after a 14-day period of application. The concentration of Ca²⁺ slightly increased in the shoot. In roots, tentoxin caused a 30% higher accumulation of Ca²⁺ after 7 days, which did not change with treatment during the following 7 days. The accumulation of Ca²⁺ was enhanced by increasing concentrations of tentoxin. K⁺ and total P levels increased in roots but decreased in shoots after 7 days. However, they were redistributed between root and shoot during days 8–14 of tentoxin treatment. The effect of tentoxin is explained as a stimulation of ion transport mainly into the vacuoles of the immature metaxylem elements. It is suggested that tentoxin and other microbial products effective at very low concentrations may have a general significance in promoting plant infection or symbiosis via the modification of physiological or biochemical processes.     

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.     

Evidence for a catalytic function of the coupling factor 1 protein reconstituted with chloroplast thylakoid membranes.

The effects of tentoxin on the ATPase activities of coupling factor 1 proteins (CF1) and photophosphorylation with isolated chloroplasts and chloroplasts reconstituted with coupling factor proteins have been examined. 1. The calcium-dependent ATPase activities of coupling factors isolated from spinach, lettuce and Nicotiana otophora are completely inhibited by tentoxin. The ATPase activities of coupling factors isolated from Nicotiana tabacum and Nicotiana knightiana are not affected by tentoxin. 2. Phenazine methosulfate-catalyzed cyclic photophosphorylation with chloroplasts isolated from spinach, lettuce and N. otophora is completely inhibited by tentoxin, whereas chloroplasts isolated from N. knightiana and N. tabacum are relatively insensitive to tentoxin. 3. Spinach chloroplasts, partially depleted in CF1, can be reconstituted with coupling factors isolated from a wide variety of plants including lettuce, radish, N. tabacum, N. knightiana and N. otophora. 4. Spinach chloroplasts reconstituted with spinach, lettuce and N. otophora CF1 retain their sensitivity to tentoxin; however, when reconstituted with N. knightiana and N. tabacum coupling factor proteins, a significant fraction of the reconstituted rate remains tentoxin insensitive. These data are interpreted as evidence that coupling factors that reconstitute with spinach thylakoid membranes have both a catalytic and structural function.     

Potassium transport through lipid bilayer membranes facilitated by tentoxin dimers: A new mechanism of ion carrier transport?

The cyclic tetrapeptide tentoxin at concentrations greater than 5 X 10(-7) M selectively increases the ion conductivity for potassium of lipid bilayer membranes, while the naturally occurring derivative dihydrotentoxin has no influence on this property. Current-voltage curves, zero-current potential and charge-pulse measurements were used to characterize the action of tentoxin. The results suggest that a new mechanism of facilitated ion transport operates. The model of tentoxin dimerization and tentoxin-K+ association developed is in contradiction to the model of tentoxin pore formation described recently by Heitz et al. (Biophys. Chem. 23 (1986) 245).     

Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin.

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1). An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F(1) [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding. A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179-2186]. In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.     

Tentoxin. An uncompetitive inhibitor of lettuce chloroplast coupling factor 1.

The interaction of tentoxin [cyclo-(-L-leucyl-N-methyl-(Z)-dehydrophenylalanyl-glycyl-N-methyl-L-alanyl-)] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 . 10(8) M-1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.     

Inhibition of light-induced stomatal opening and of guard-cell-protoplast swelling in Vicia faba L. by tentoxin,an inhibitor of photophosphorylation

The fungal phytotoxin tentoxin and its natural derivative dihydrotentoxin impair light-induced stomatal opening in epidermal strips of broad bean (Vicia faba L.) incubated in a potassium-rich medium. Swelling of guard-cell protoplasts (GCPs) of the same species is inhibited in the presence of both substances. Swollen GCPs shrink after tentoxin or dihydrotentoxin treatment and these effects cannot be fully compensated by the phytoeffector fusicoccin. A comparison with the potassium carrier valinomycin shows that tentoxin acts in a different manner, because it is effective in the light only, whereas valinomycin causes shrinkage of GCPs also in the dark. Determination of adenine nucleotides in GCPs indicates a reduced ATP content and an enhanced ADP level after addition of tentoxin. At the same time, tentoxintreated GCPs contain more NADPH and less NAD⁺ than the control (NADP⁺ and NADH content does not differ). The results presented are consistent with the hypothesis that tentoxin closes stomata as a consequence of its inhibitory action on photophosphorylation.Abbreviations FC fusicoccin - GCP guard-cell protoplast - KIDA potassium iminodiacetate     

Tentoxin-induced binding of adenine nucleotides to soluble spinach chloroplast coupling factor 1.

The effect of tentoxin on the binding of adenine nucleotides to soluble chloroplast coupling factor (CF1) has been studied and the following results have been obtained: 1. Tentoxin (400 micron) increases the maximum attainable tight binding of ADP to CF1. In the absence of tentoxin, the maximal binding observed by the method employed is about 0.3 nmol ADP/mg protein, whereas in the presence of tentoxin this ranges from 1.5 to 2.0 nmol ADP/mg protein. 2. Tentoxin-induced binding of ADP to CF1 is severely inhibited by divalent cations (50% inhibition at about 2 mM) but only weakly inhibited by monovalent cations (less than 50% inhibition at 100 mM). 3. The binding of ADP to CF1 induced by tentoxin is inhibited by ATP and adenylyl imidodiphosphate but is not inhibited by other nucleotides including AMP, GDP, CDP, IDP, or beta, gamma-methylene ATP. 4. The ADP-CF1 complex induced by tentoxin is quite stable. 75% remains bound to CF1 even after passage of the complex through a gel filtration column. An additional 25% can be removed by incubation in the presence of ADP, and all of the bound ADP can be removed only after incubation in the presence of both tentoxin and ADP. The latter result is interpreted as a tentoxin-induced exchange of bound ADP for medium ADP.     

Ferredoxin-NADP+ reductase,a nuclearly-coded enzyme unaffected by tentoxin treatment

Previous studies in our laboratory have shown that tentoxin prevents the incorporation of polyphenol oxidase (PPO), a nuclearly-coded protein, into the chloroplasts of sensitive species. In this study, we show, by comparison of electrophoretically separated isozymes, that ferredoxin-NADP⁺ reductase (FNR) is nuclearly coded in Nicotiana. Electrophoresis of FNR isozymes from tentoxin treated seedlings of a sensitive and a resistant species demonstrated that, unlike PPO, ferredoxin-NADP⁺ reductase was unaffected by tentoxin treatment. These data indicate that tentoxin selectively inhibits transport of cytoplasmically synthesized proteins into the chloroplast, and does not produce a generalized disruption of cellular integration.This research was supported, in part, by funding under cooperative agreement number 58-7B30-3-548, and is published with the approval of the Director of Arkansas Agr. Exp. Stn. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the US Dep. Agric. or cooperating agencies and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Effects of tentoxin on enzymic activities in cucumber and cabbage cotyledons

Tentoxin, produced by Alternaria alternata (Fr.) Keissl. causes severe variegated chlorosis in germinating seedlings of certain dicotyledonous species. However, it does not impair radicle and hypocotyl elongation or cotyledon expansion. Effects of the toxin on the activity of selected enzymes from both chloroplasts and cytoplasm were determined. Cucumber (Cucumis sativus L.), which is highly sensitive to tentoxin and cabbage (Brassica oleracea L.), which is resistant, were used as test plants. The activities of chloroplastic, but not cytoplasmic, enzymes were decreased by treatment of cucumber cotyledons with tentoxin. Neither group of enzymes was affected by the toxin in cabbage cotyledons. The decreased enzymic activities are probably related to reported inhibition of photophosphorylation by tentoxin.