Biosynthesis and degradation of α-tomatine in developing tomato fruits

Fruits of tomato incorporated [2-¹⁴C]mevalonic acid lactone into the steroidal glycoalkaloid α-tomatine. Young fruits showed the greatest alkaloid-synthesizing ability but this decreased as the fruits developed. Analysis of sap exuded from fruit stalks and also application of[4-¹⁴C]cholesterol to leaves confirmed that tomatine is not transported into fruits from vegetative organs. Accumulation of this alkaloid in fruits thus appears entirely due to synthesis. Excised fruits of all developmental stages degraded injected [¹⁴C]tomatine and rates were directly related to fruit age. The pattern of accumulation/decline in fruit tomatine may be explicable on the basis of changing capacity for synthesis/degradation during development. Label from injected [¹⁴C]tomatine was present mainly in chlorophylls and carotenoids where it increased with time as that in tomatine decreased. The significance of the relationship between tomatine disappearance and carotenoid development is briefly discussed. The aglycone tomatidine was not detected in green fruits but a Δ¹⁶-5α-pregnenolone-like compound was.     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

Biosynthesis of steroidal alkaloids in Solanaceae plants: Incorporation of 3β-hydroxycholest-5-en-26-al into tomatine with tomato seedlings

The C-26 amino group of tomatine, a representative Solanaceae steroidal alkaloid, is introduced in an early step of its biosynthesis from cholesterol. We recently proposed a transamination mechanism for the C-26 amination as opposed to the previously proposed mechanism involving a nitrogen nucleophilic displacement. In the present study, a deuterium labeled C-26 aldehyde, (24,24,27,27,27-²H₅)-3β-hydroxycholest-5-en-26-al, was synthesized and fed to a tomato (Solanum lycopersicum) seedling. LC–MS analysis of the biosynthesized tomatine indicated that the labeled aldehyde was incorporated into tomatine. The finding strongly supports the intermediacy of the aldehyde and the transamination mechanism during C-26 amination.     

番茄碱对人红细胞膜Na+-K+-ATPase活性影响的研究

以低渗法从新鲜健康人红细胞中制得膜Na^ -K^ -ATPase,研究了番茄碱(tomatine)X~人红细胞膜Na^ -K^ -ATPase活性的影响。实验结果表明,反应体系中的tomatine浓度低于1mmol／L时,对不依赖钙调蛋白(CAM)激活的Na^ -K^ -ATPase(称之为基本酶活)影响不大,浓度达1mmol／L时,该酶活性仍保持在95％左右;而在此浓度范围内,tomatine对依赖CaM的Na^ -K^ -ATPase有明显的抑制作用,其IC50值为0．16mmol／L．说明tomatine对膜酶Na^ -K^ -ATPase活性的影响可能是通过阻断CaM激活的途径而起作用,从而为进一步研究番茄碱的作用机制奠定了基础。     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

番茄碱对棉铃虫的毒性作用机理初探

采取荧光光谱法分析了番茄碱及虫体内钙调蛋白的图谱,从钙调蛋白的角度探讨了番茄碱的作用机理.结果显示:(1)番茄碱对棉铃虫具有毒性,随浓度的增加及饲喂时间的延长,对棉铃虫的杀伤力增强;(2)番茄碱对虫体内钙调蛋白的影响较大,随番茄碱浓度的增加,钙调蛋白的含量逐渐降低;(3)钙离子的加入增强了钙调蛋白的刚性,荧光图谱出现了红移(由350 nm移至416.58 nm);(4)番茄碱的加入破坏了钙调蛋白-钙离子复合物的刚性,荧光图谱出现了蓝移(416.58 nm移至377.65 nm).以上可以说明,番茄碱可能作为钙调蛋白的拮抗剂,拮抗钙调蛋白被钙离子激活的位点,影响其与靶酶的结合而发挥作用.此项的研究为探讨番茄碱的杀虫机理提供了科学依据.     

Response of Tissues2

Efflux of K⁺ from tomato fruit pericarp disks was not affectedby 100 µM tomatine, and only slightly by 1 mM. No differentialresponses to the alkaloid were observed in disks obtained fromfruits at different stages of development, and there was noevidence of any correlation between the level of endogenoustomatine and the effect of exogenous tomatine. Growth of tomatoseedlings in liquid nutrient medium was unaffected by 1 mM tomatinewhereas the growth oflettuce seedlings was stunted in the presenceof 100 µM alkaloid. The polyene antibiotic nystatin gavesimilar resultsto tomatine, having little effect on tomato tissuesbut causing marked inhibition of lettuce seedling development.     

Isolation of tomatine from cultured excised roots and callus tissues of tomato

Tomatine was present in cultured excised tomato roots but in lower concentrations than in seedling radicles of the same age. The alkaloid was not detected in 'spent' root medium. Newly-initiated callus cultures of hypocotyl, radicle and cotyledon origin produced roots, and tomatine was isolated from both roots and callus. Roots contained more tomatine than callus, but neither contained as much as the organ explants from which the cultures were initiated. The number of roots produced decreased with time, as did also the tomatine content of the callus tissues. After 447 days, when no organized structures were produced by callus cultures, tomatine was not detected. An established hypocotyl callus contained small amounts of tomatine when grown on certain nutrient media, but a chlorophyllous sub-isolate of this callus did not produce detectable quantities of the alkaloid. Tomatine was not detected in an established root callus isolate or in suspension cultures initiated from established, tomatine-containing hypocotyl callus.     

Effect of {alpha}-Tomatine on the Response of Wheat Coleoptile Segments to Exogenous Indole-3-acetic Acid

A concentration of 10^–5 M tomatine had no effect on leakagefrom, or elongation of, wheat coleoptile segments, but consistentlyreduced IAA-enhanced extension growth by c. 50 per cent. Therewas no evidence of chemical interaction between the alkaloidand the auxin in solution, and IAA action was not affected bypre-treatment for up to 3 h with 10^–5 M tomatine. Studieswith [2-¹⁴C]IAA revealed that 10^–5 M tomatine did notinhibit uptake of auxin into segments. The effect of pre-treatingsegments for up to 3 h with IAA could be virtually nullifiedby 10^–5 M tomatine, as could also IAA-induced changesin properties of coleoptile cell walls. Results are discussedin relation to the ability of tomatine to disrupt membrane functionand to current hypotheses implicating membranes in the primaryaction of auxin.     

Tomato roots secrete tomatine to modulate the bacterial assemblage of the rhizosphere

Saponins are the group of plant specialized metabolites which are widely distributed in angiosperm plants and have various biological activities. The present study focused on α-tomatine, a major saponin present in tissues of tomato (Solanum lycopersicum) plants. α-Tomatine is responsible for defense against plant pathogens and herbivores, but its biological function in the rhizosphere remains unknown. Secretion of tomatine was higher at the early growth than the green-fruit stage in hydroponically grown plants, and the concentration of tomatine in the rhizosphere of field-grown plants was higher than that of the bulk soil at all growth stages. The effects of tomatine and its aglycone tomatidine on the bacterial communities in the soil were evaluated in vitro, revealing that both compounds influenced the microbiome in a concentration-dependent manner. Numerous bacterial families were influenced in tomatine/tomatidine-treated soil as well as in the tomato rhizosphere. Sphingomonadaceae species, which are commonly observed and enriched in tomato rhizospheres in the fields, were also enriched in tomatine- and tomatidine-treated soils. Moreover, a jasmonate-responsive ETHYLENE RESPONSE FACTOR 4 mutant associated with low tomatine production caused the root-associated bacterial communities to change with a reduced abundance of Sphingomonadaceae. Taken together, our results highlight the role of tomatine in shaping the bacterial communities of the rhizosphere and suggest additional functions of tomatine in belowground biological communication.

α-Tomatine is the major toxic saponin secreted from tomato roots at high levels during early growth stages and plays an important role in the formation of bacterial communities in the rhizosphere.     

Biosynthesis and distribution of tomatine in cultured excised Lycopersicon esculentum roots

Growth of cultured excised tomato roots in the presence of ¹⁴C-mevalonic acid lactone results in labelling of tomatine. In the main axis of the root, incorporation was greatest in the apical meristematic region. Tomatine was present in equal concentrations in all parts of the cultured root system.     

Effect of dietary tomatine on cholesterol metabolism in the rat

Tomatine is a virtually nonabsorbable saponin which has been used as an antifungal agent and analytically as a cholesterol precipitant. It was used in this study to determine whether or not it can form a complex with cholesterol in vivo in the rat intestine and what effects such complex formation would have on cholesterol metabolism. Rats that were fed tomatine as 1% of the diet had a decreased uptake of dietary cholesterol by the liver, an increased rate of hepatic and intestinal cholesterol synthesis as well as a partial offsetting of the dietary cholesterol-induced decrease in hepatic cholesterogenesis, and an apparent increase in sterol excretion without an effect on bile acid excretion. In vitro, tomatine did not sequester cholic acid as did cholestyramine. The results show that tomatine has an effect on cholesterol absorption and on other aspects of lipid metabolism in the rat similar to that of cholestyramine, with the notable exception that tomatine increased sterol excretion while cholestyramine increased bile acid excretion. It was suggested that tomatine forms a nonabsorbable complex with cholesterol in the rat intestine.     

Changes in the Alkaloid Content of Developing Fruits of Tomato (Lycopersicon esculentum Mill.): II. EFFECTS OF ARTIFICIAL ACCELERATION AND RETARDATION OF RIPENING

Fruit ripening in normal red-, orange- and yellow-fruited cultivarsof tomato was accelerated by treatment with Ethrel and suchfruits had lower tomatine levels than untreated controls. Fruitsin which ripening was retarded by incubating under reduced pressurehad higher alkaloid levels than controls. In each case, fruitripeness (as measured by pigmentation) showed a strong negativecorrelation with fruit tomatine. Ethrel treatment of fruitsof the non-ripening mutants nor and rin caused only a smalldevelopment of carotenoid pigment but markedly enhanced tomatinedisappearance. Pigmentation and tomatine were again negativelycorrelated although the quantitative relationship differed.Under normal circumstances, tomatine disappearance from tomatofruits-is apparently governed by the physiological (cf. chronological)age of the fruit rather than by its growth or ripening characteristicsalone. Key words: Tomato, Fruit ripening, Acceleration, Retardation, Tomatine     

The influence of secondary plant substances on the growth and development of Myzus persicae of Beijing

Green peach aphids Myzus persicae from the Beijing region, maintained on greenhouse tobacco plants, were reared on modified artificial diet; sinigrin, tomatine, nicotine, gossypol, phlorizin, inulin and malic acid were added separately in concentrations of 0.1% or 0.2%. In preference tests none of the diets enriched by secondary substances was more acceptable than the basal diet. Diet uptake during larval development was determined quantitatively by using C¹⁴ glycine in the diets. For the basal diet the total uptake during larval development was about 1.16 l/aphid. The addition of secondary plant substances to the diet caused reductions of uptake to different degrees in the order: nicotine>phlorizin>gossypol>tomatine>malic acid>inulin>sinigrin. The larvae feeding on diets containing sinigrin or tomatine from the second day after birth seemed to have a much higher tolerance for sinigrin than tomatine, with a mortality of 9.6% within 7 days of rearing for the former and 89.4% for the latter. Hence this aphid differs from that occurring in the Netherlands in that the latter has a higher tolerance towards tomatine. The addition of inulin, malic acid or sinigrin did not cause a significant difference in the rate of larval development or in adult longevity. Diets containing these substances could support the aphid's development and reproduction for at least three generations.

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Résumé Des Myzus persicae de la région de Beijing, conservés sur tabac en serre, ont été élevés sur un régime artificiel voisin de celui de Dadd & Mittler (1966); sinigrine, tomatine, nicotine, gossypol, phlorizine, inuline et acide malique ont été ajoutés séparément aux concentrations de 0,1 et 0,2%. En comparant les préférences, aucun des régimes enrichis en substances secondaires n'était supérieur au régime de base. La prise alimentaire pendant le développement larvaire a été quantifiée en utilisant de la glycine C¹⁴ dans le régime. Les èlevages étaient conduits à 25±1 °C et H. R. 85% et une photopériode J/N: 16/8. Le prélèvement total de régime de base pendant le développement larvaire était environ 1,16 l/puceron. L'addition de substances secondaires végétales au régime induit de façon inégale le prélèvement, dans l'ordre suivant: nicotine>phlorizine>gossypol>tomatine>acide malique>inuline>sinigrine. Les larves consommant sur des régimes contenant de la sinigrine ou de la tomatine à partir du second jour après la naissance semblent prèsenter une plus grande tolèrance pour la sinigrine que la tomatine, avec une mortalité de 9,6% en 7 jours d'élevage pour la première et 89,4% pour la seconde. Ainsi ces pucerons diffèrent de ceux de Hollande, puisque ces derniers tolèrent plus la tomatine (Schoonhoven & Derksen-Koppers, 1976). L'addition d'inuline, d'acide malique ou de sinigrine ne provoque pas de différence significative dans le taux de développement larvaire ou la longévité des adultes. Des régimes contenant ces substances pourraient permettre le développement et la reproduction de pucerons pendant au moins 3 générations.

     

The effect of selected allelochemicals on germination of conidia and blastospores and mycelial growth of the entomopathogenic fungus,Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Selected allelochemicals that protect plants from invasion by plant pathogenic fungi were investigated for their activity against the entomopathogenic fungus, Paecilomyces fumosoroseus. The alkaloids tomatine, solanine, and camptothecin; the furanocoumarin, xanthotoxin; and the phenolic, tannic acid were tested for their effects on germination of conidia and blastospores and growth of mycelia. The LC50 values (corresponding to 50% inhibition of germination) for tomatine, solanine, camptothecin, xanthotoxin and tannic acid were 51.6, 95.9, 55.9, 83.0 and 72.8 mg/l respectively. When blastospores were placed on media containing a concentration of the individual allelochemicals that inhibit germination in approximately 50% of conidia, all but blastospores on tomatine had significantly less germination than did aerial conidia. Growth rates of mycelia were slowest in the camptothecin medium, followed by those of tomatine and xanthotoxin and were not significantly different from controls in the media containing solanine and tannic acid. A multitude of biotic and abiotic factors are responsible for specificity and degree of pathogenicity of entomopathogens. The effect of crop plant chemistry on the efficacy of entomopathogens should be quantified further in order to maximize their potential when used concomitantly with resistant plant varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of chlorogenic acid- and tomatine-fed caterpillars on performance of an insect predator

Two diet experiments addressed the effects of allelochemical-fed prey (Manduca sexta caterpillars), temperature, and gender on performance of the insect predator, Podisus maculiventris. Two of the major allelochemicals in tomato were used: chlorogenic acid and tomatine. Predator performance was negatively affected by both chlorogenic acid-fed and tomatine fed-prey, and there were allelochemical by thermal regime interactions for both. Relative consumption rate and growth rate decreased at the higher levels of tomatine at the warmer thermal regime (summer conditions) but were unaffected at the cooler thermal regime (spring conditions). At the cooler thermal regime, stadium duration was prolonged when the predators were given chlorogenic acid-fed prey, but at the warmer thermal regime there was no such effect. There were several effects of gender: biomass gained, food consumed, relative growth rate and efficiency of conversion of ingested food to biomass were higher for females than males. Furthermore, the effects of thermal regime and tomatine on food consumption and biomass gained differed for females and males. In general, the hypothesis that generalist insect predators may be a selective pressure shaping host plant range of insect herbivores was supported by these results. But the occurrence of allelochemical by thermal regime interactions means that it will be difficult to determine the relative importance of plant chemistry versus predators on patterns of feeding specialization by herbivores without taking into account a third factor, temperature. Received: 20 March 1995 /Accepted: 2 August 1996     

Effect of {alpha}-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Respiration in isolated mung bean shoot mitochondria was notaffected by either of the tomatine treatments (10^–3 M,pH 5, and 10^–5 M, pH 8) but was reduced in the excisedshoots by both treatments, although only at the higher of thetwo temperatures (5 °C and 25 °C). Inhibition was gradualand took at least 2 h. Tomatine treatment of excised shootsalso resulted in an increased leakage of K+. PS II activityin isolated spinach leaf chloroplasts was reduced only by thehigh pH tomatine treatment at 25 °C. Again, about 2 h treatmentwas required before significant effects were observed but thealkaloid did not cause disintegration of the chloroplast asmeasured by pigment release. Disruption of lysosomes isolatedfrom cauliflower inflorescence tissue and release of acid phosphatasewas enhanced by tomatine. Initially only 10^–5 M tomatineat pH 8 was effective but, later, effects could only be obtainedwith 10^–3 M alkaloid at pH 5. The differential susceptibilityof these organelles to tomatine is discussed in relation tomembrane structure and to the mode of toxicity of the alkaloid.     

Dietary mixing in grasshoppers: Changes in acceptability of different plant secondary compounds associated with low levels of dietary protein (Orthoptera: Acrididae)

Schistocerca americanasixth-instar nymphs were examined for a change in diet acceptance, in which insects experiencing an unfavorable diet subsequently become predisposed to eat relatively less of that diet and more of diets with a novel flavor than they would had they previously fed on a more adequate diet. Insects were pretreated for 4 h on either low-protein (2 % wet wt) or higherprotein (4%) artificial diets flavored with a plant secondary compound (tomatine or rutin). They were then offered, in choice or no-choice tests, the lowprotein diet with the familiar or a novel (tomatine, rutin, or NHT) flavor. When tomatine was the familiar and rutin the novel flavor in a no-choice test, the insects previously fed low-protein diets took relatively long meals on the novel and relatively short meals on the familiar diets compared with the insects that had previously eaten higher-protein diets. A similar, but in this case considerably less pronounced and statistically nonsignificant, pattern existed in the reciprocal design experiment in which rutin was the familiar and tomatine the novel flavor. Similarly, insects fed low-protein diets flavored with rutin subsequently showed an increased relative preference for the novel flavor (NHT) in a choice test, compared with the high protein-pretreated insects. It is concluded that insects fed protein-deficient diets may subsequently show a preference for novel foods through different mechanisms, the importance of which may differ in different circumstances.     

Destabilization of liposome membranes by the steroidal glycoalkaloid α-tomatine

The effect of the steroidal glycoalkaloid α-tomatine on the leakage of peroxidase from liposomes was studied. At pH 7.2, the optimum pH for disruption, tomatine had little effect at concentrations less than 10μM but was increasingly disruptive at concentrations of 10–100μM. Liposome destabilization was pH-dependent declining with decreasing pH until at pH 5 lysis was achieved only at tomatine concentrations of 500–1000μM. At all pH values tested (pH 5–8), tomatine caused significant disruption only if the membrane contained sterol. The extent of membrane damage was correlated with the concentration of sterol in the liposomes but not with the nature of the sterol or of the phospholipid. These findings are inconsistent with claims that surface glycosidases, which convert the glycoside to the aglycone, are prerequisites for tomatine action and that the aglycone is the active moiety.     

Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.