The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

Tentoxin effects on infrastructure and greening of ivyleaf morningglory (Ipomoea hederacea var. hederacea) cotyledons

The effects of 20 μM tentoxin on mesophyll chloroplast ultra-structural development, chlorophyll organization and accumulation, and pigment transformations in cotyledons of dark-grown, 4-day-old ivyleaf morningglory [Ipomoea hederacea (L.) Jacq. var. hederacea]were monitored. After 6 h of white light (200 μEm^?2T.s^?1), many plastids of tentoxin-treated tissues contained prolamellar bodies or inconsistent internal membrane orientation in contrast to the uniform internal membrane orientation and absence of prolamellar bodies in controls. Grana stacking did not progress beyond three to four disc loculi in tentoxin-treatments, and fret membranes were usually discontinuous and reduced. Cylindrical or cupped grana appeared in many chloroplasts after 3 days of light, while other chloroplasts in which disruption was more pronounced had few grana except for remnants, but usually did possess vesicles or structures resembling prolamellar bodies. Tentoxin had no apparent effect on stroma density or plastoglobuli size and number. No starch grains appeared in any of the tentoxin treatments, whereas they appeared after 24 h in controls. Initial protochlorophyllide content and its photoconversion to chlorophyllide and subsequent Shibata shift were not affected by tentoxin. Chlorophyll accumulation rates in tentoxin-treated cotyledons were about 10% of control rates during the first 24 h of greening and about 20% of controls from 48 to 72 h of greening. Chlorophyll alb ratio and PSU size (total Chl/P700) were not significantly affected by tentoxin.     

Predicting the conformational states of cyclic tetrapeptides

Biologically active cyclic tetrapeptides, usually found among fungi metabolites, exhibit phytotoxic or cytostatic activities that are likely to be governed by specific conformations adopted in solution. For conformational studies and drug design, there is a strong interest in using fast and reliable methods to determine correctly the conformational population of cyclotetrapeptides. We show here that standard molecular mechanics computational approach gives satisfactory results. The method was validated step by step by experimental data either obtained after synthesis and NMR analysis, or found in the literature. The cyclo(Gly)(4), cyclo(Ala)(4), cyclo(Sar)(4), and cyclo(SarGly)(2) peptides were used to evaluate the prediction of the peptide backbone conformation, and the detailed conformational analysis of tentoxin, a natural phytotoxic cyclotetrapeptide in which N-alkylated peptide bonds alternate with regular secondary ones, was used to validate the computation of conformers proportions. From the knowledge of an initial cyclic primary structure and of the D or L configuration of the amino acids, we show that it is possible to determine the exact orientation of carbonyl groups and to predict the nature of conformers present in solution. The proportion of each conformer can be inferred from a statistical thermodynamics approach by using the potential energy values of each conformer, computed by molecular mechanics methods with the TRIPOS force field, which allowed us to account for the solvent. The solvent contribution was processed by two different methods according to the nature of the interactions: whether through the dielectric constant introduced in the electrostatic potential, when interaction with solute molecules are weak or negligible, or through the computation of free energy of solvation using the algorithm SILVERWARE for solvents explicitly interacting with the solute. When applied to tentoxin, this conformational analysis yielded results in very good agreement with the experimental data reported by Pinet et al. (Biopolymers, 1995, Vol. 36, pp. 135-152), on both the nature of existing conformers and their relative proportions, whatever the nature of the considered solvent.     

High yield synthesis of tentoxin, a cyclic tetrapeptide.

Tentoxin is a naturally occurring phytotoxic cyclic tetrapeptide excreted by fungi of the Alternaria alternata family. The four total syntheses of tentoxin published to date give poor total yields, mainly owing to two difficulties, the introduction of the dehydro amino acid and more especially the cyclization step. Here we describe a method that stereospecifically introduces Z-dehydrophenylalanine (deltaZPhe) by a modified Erlenmeyer aldolization reaction. The linear tetrapeptide, Boc-R1Ala-Leu-R2deltaZPhe-G1y-OMe (R1, R2: CH3, 14CH3), the precursor of tentoxin, was obtained in a 72% yield from Boc-Leu-Gly-OH. This linear tetrapeptide, labelled with carbon-14, was used for a comparative study of four cyclization reagents DPPA, DCC-PfpOH, HBTU and HATU. This last was the most effective and gave tentoxin in a 81% cyclization yield. The activated ester formed with this reagent displayed an enhanced capacity for cyclization, permitting cyclization in concentrated medium (10 mM). This new synthetic route gave tentoxin in a 60% yield from Boc-Leu-Gly-OH and offers a means of achieving the synthesis of hitherto elusive analogues.     

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.     

链格孢Alternaria alternata侵染导致瓜类幼苗白化的机制

用分离自无症状瓜类植物的27株链格孢属Alternaria真菌接种黄瓜,有14株引起了黄瓜幼苗的白化,这些菌株均属于链格孢A. alternata。为揭示A. alternata引起黄瓜幼苗白化的机制,对代表菌株5F29经固体发酵后提取其次级代谢产物,将粗提物按一定量混入基质进行盆栽实验。结果表明,黄瓜、丝瓜和甜瓜对该粗提物敏感,出现白化苗;小白菜和辣椒对粗提物不敏感,生长正常。植物幼苗用粗提物处理的结果与用产生菌接种的结果相一致。粗提物经硅胶柱层析、葡聚糖凝胶柱层析及高效液相色谱（HPLC）等方法分离纯化得到一种活性化合物,经测定该化合物引起黄瓜幼苗白化的最低浓度为12.5μmol/L。该化合物在此浓度下可引起黄瓜幼苗生长点白化,在15μmol/L的浓度下可引起黄瓜幼苗大部分组织白化,致使幼苗很快死亡。应用核磁共振和质谱技术,确定了该化合物的结构,鉴定为一种植物毒素——腾毒素（tentoxin）。研究结果揭示了A. alternata引起瓜类幼苗白化的机制以及不同植物对于腾毒素的不同敏感性。     

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.     

Effect of tentoxin on K+transport in winter wheat seedlings of different K+-status

Klotz, M. G. and Erdei, L. 1988. Effect of tentoxin on K⁺ transport in winter wheat seedlings of different K⁺-status. The influence of the phytoeffective mycotoxin, tentoxin, [cyclo-(L-leucyl-N-methyltrans-dehydronhenyl-alanyl-glycyl-N-methyl-L-alanyl)] (in K⁺ uptake and on translocation of K⁺ from roots to shoot was studied in 14-day-old winter wheat plants (Triticum aestivum L. cv. Martonvásári-8) grown at different levels of K⁺ supply. For comparison, the effects of 2,4-dinilrophcnol and valinomycin were also investigated. In I-h experiments I pM tentoxin reduced K⁺ influx in the routs over the external K⁺ concentration range 0.1 to 1 m^M (low-K⁺ plants), whereas stimulation was observed al lower and higher K⁺ concentrations. On the other hand, in plants grown at 0.3 m^M K⁺, tentoxin stimulated the translocation of K⁺ from roots to shoots in 5-h experiments. Valinomycin affected K⁺ transport only al high K⁺-status (slight stimulation). In low-K⁺ plants 2,4-dinitrophenol (DNP) caused drastic inhibition of K⁺ uptake, but in high-K⁺ plants uptake was only slightly inhibited and translocation slightly stimulated, It is concluded that the opposite effects of tentoxin on K⁺ uptake and translocation agree1 with the directions of the H⁺-ATPases pumping H⁺ towards the apoplast and located at the cortex plasmalemma and the xylem parenchyma plasma-membrane, respectively. These effects should probably be attributed to the interaction between tentoxin and the K⁺-carrier protein rather than to a direct influence of tentoxin on H⁺-ATPase.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.