ELONGATED HYPOCOTYL 5 regulates steroidal glycoalkaloid biosynthesis and fungal tolerance in tomato.

Tomato (Solanum lycopersicum L.) is rich in nutrients and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defense but are toxic to humans and considered antinutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomatoes. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of structural and regulatory genes. SlHY5 complemented Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) hy5 mutants at molecular, morphological, and biochemical levels. CRISPR/Cas9-based knockout tomato plants, SlHY5CR, showed downregulation of SGA and phenylpropanoid pathway genes, leading to a significant reduction in SGA (α-tomatine and dehydrotomatine) and flavonol contents, whereas plants overexpressing SlHY5 (SlHY5OX) showed the opposite effect. Enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR lines were susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes.

α-Tomatine degradation to tomatidine by food-related Aspergillus species belonging to the section Nigri.

α-Tomatine is a steroidal glycoalkaloid in tomato plants and degrades with ripening. The aglycone form, tomatidine, is reported to have beneficial effects. In this study, the ability of food-related microorganisms to produce tomatidine from α-tomatine was evaluated. A total of 11 strains of Aspergillus species belonging to the section Nigri exhibited tomatinase activity, and Aspergillus luchuensis JCM 22302 was selected for optimization due to its high activity in its mycelia, conidia, and non-mycotoxin-producing property. Next, using A. luchuensis JCM22302 conidia, the highest yield was obtained in a 24-h reaction with 50 m m of acetic acid-sodium acetate buffer (pH 5.5) at 37 °C. Similar to the tomato pathogen Fusarium oxysporum f. lyceopersici, the time course analysis suggested that A. luchuensis JCM 22302 removed the entire sugar moiety in a single step. Future research will focus on utilizing conidia for large-scale tomatidine production because of their high tolerance and manageability.

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.

Tomato E8 Encodes a C-27 Hydroxylase in Metabolic Detoxification of α-Tomatine during Fruit Ripening.

Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3ß-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

Identification of α-Tomatine 23-Hydroxylase Involved in the Detoxification of a Bitter Glycoalkaloid.

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Identification of a 3ß-Hydroxysteroid Dehydrogenase/ 3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato.

α-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the Δ5,6 double bond; the dehydro-type). The aglycone of α-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3ßHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3ßHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3ßHSD1 protein revealed that Sl3ßHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3ßHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3ßHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3ßHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3ßHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.

A tomatinase-like enzyme acts as a virulence factor in the wheat pathogen Fusarium graminearum.

During their interactions with plants, fungal pathogens employ large numbers of pathogenesis-associated molecules including secreted effectors and enzymes that can degrade various defence compounds. However, in many cases, in planta targets of pathogen-produced enzymes remain unknown. We identified a gene in the wheat pathogen Fusarium graminearum, encoding a putative enzyme that shows 84% sequence identity to FoTom1, a tomatinase produced by the tomato pathogen Fusarium oxysporum f. sp. lycopersici. In F. oxysporum f. sp. lycopersici, FoTom1 is a virulence factor involved in the degradation of tomato defence compound tomatine, a saponin compound. Given that wheat is unknown to produce tomatine, we tested the ability of F. graminearum to degrade tomatine and found that F. graminearum was unable to degrade tomatine in culture. However, FgTom1 degraded tomatine in vitro when heterologously expressed. To determine the possible function of FgTom1 in pathogen virulence, we generated FgTom1 knockout mutants (ΔTom1). ΔTom1 mutants were not different from wild type when grown in culture but showed significant reduction in pathogen virulence in root rot and head blight assays. In an attempt to identify possible in planta targets of FgTom1, the metabolomes of wheat heads infected with wildtype pathogen and ΔTom1 were compared and several peaks differentially abundant between treatments identified. Although the exact identity of these peaks is currently unknown, this result suggested that FgTom1 may have in planta targets in wheat, possibly tomatine-like saponin compounds. Overall, our results presented here show that FgTom1 is a new virulence factor in F. graminearum.

Enzyme-driven metabolomic screening: a proof-of-principle method for discovery of plant defence compounds targeted by pathogens.

Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes. Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed. Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted. The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies.

Biosynthesis of steroidal alkaloids in Solanaceae plants: incorporation of 3ß-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-(2)H5)-3ß-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.

Detoxification of α-tomatine by Cladosporium fulvum is required for full virulence on tomato.

· α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds ß-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine. · Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. · We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. · Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.

A long-term comparison of the influence of organic and conventional crop management practices on the content of the glycoalkaloid α-tomatine in tomatoes.

BACKGROUND: α-Tomatine, synthesized by Lycopersicon and some Solanum species, is a steroidal glycoalkaloid which functions to protect against pathogens and insects. Although glycoalkaloids are generally considered toxic, α-tomatine appears to be well tolerated in humans. α-Tomatine has numerous potential health benefits including the ability to inhibit cancer cell growth in in vitro studies. α-Tomatine is influenced by numerous agronomic factors including fertilization and nitrogen availability. Herein, the levels of α-tomatine were compared in dried tomato samples (Lycopersicon esculentum L. cv. Halley 3155) produced in organic and conventional cropping systems that had been archived over the period from 1994 to 2004 from the Long Term Research on Agricultural Systems project (LTRAS) at UC Davis. RESULTS: The α-tomatine levels of tomatoes in both cropping systems ranged from 4.29 to 111.85 µg g(-1) dry weight. Mean levels of α-tomatine were significantly higher in the organically grown tomatoes than conventional ones (P < 0.001). In the organic management system, α-tomatine content was also significantly (P < 0.001) different between cropping years, suggesting that other influencing factors such as environmental conditions also affect α-tomatine content in tomato. CONCLUSIONS: The organically produced tomatoes had higher average α-tomatine content than their conventional counterpart over the 10-year study. Significant annual variability in the α-tomatine content in tomatoes was also observed and suggests that environmental factors, external to nitrogen fertilization, influence α-tomatine content in tomatoes.

Alpha-tomatine and the two sides of the same coin: An anti-nutritional glycoalkaloid with potential in human health.

Discovery of new selective anticancer, anti-inflammatory, and anti-microbial agents is a crucial and necessary step to ensure a pipeline for innovative products to improve disease management. Several new bioactive agents derived from plants have been investigated and an example is the steroidal glycoalkaloid (SGA) class of natural products found in plants, investigated for their health-beneficial biological activities. Among them, α-tomatine is a SGA derived from the plant parts of unripe green tomatoes. In this review we aimed at searching for two different perspectives to study α-tomatine from green tomatoes, namely from its dual action point of view: as an anti-nutrient and as a health promoter. The aspects associated to its synthesis and degradation were considered. Finally, the current strategies for its extraction from natural sources and the methodologies commonly used for its identification and quantification were discussed.

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

Applications of femtochemistry to proteomic and metabolomic analysis.

Femtosecond laser pulses have been widely used as a tool to study molecular ionization and fragmentation. This article bridges the application of femtosecond laser technology in early research focused on small isolated molecules with that in modern biological mass spectrometry for proteomics and metabolomic analysis on large (140+ atoms) biomolecules. The single-shot interaction of a femtosecond laser with neutral para-nitrotoluene (pNT) is investigated with time-of-flight mass spectrometry and compared with the ultrafast photodissociation of protonated pNT in an ion trap mass spectrometer accumulated over ∼1000 pulses. The ion trap experiment is then extended to longer biomolecules. As demonstrated in the examples of vasopressin and tomatine, this novel ion activation method provides greater sequence coverage and nonstatistical fragmentation, leading to valuable information complementary to conventional methods for structural analysis.

SlMYC2 are required for methyl jasmonate-induced tomato fruit resistance to Botrytis cinerea.

The mechanism of SlMYC2, involved in methyl jasmonate (MJ)-induced tomato fruit resistance to pathogens, was investigated. The data indicated that MJ treatment enhanced the accumulation of total phenolics and flavonoids, as well as individual phenolic acids and flavonoids, which might be caused by the increased phenylalanine ammonia-lyase and polyphenol oxidase activities, induced pathogenesis-related gene (PR) expression, ß-1,3-glucanase and chitinase activities, as well as α-tomatine, by inducing GLYCOALKALOID METABOLISM gene expression. These effects, induced by MJ, partly contributed to tomato fruit resistance to Botrytis cinerea. Nevertheless, the induction effects of MJ were almost counteracted by silence of SlMYC2, and the disease incidence and lesion diameter in MJ + SlMYC2-silenced fruit were higher than those in MJ-treated fruit. These observations are the first evidence that SlMYC2 plays vital roles in MJ-induced fruit resistance to Botrytis cinerea, possibly by regulating defence enzyme activities, SlPRs expression, α-tomatine, special phenolic acids and flavonoid compounds.

Evidence for allosteric interactions of antagonist binding to the smoothened receptor.

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Streptomyces scabies 87-22 possesses a functional tomatinase.

The actinomycete Streptomyces scabies 87-22 is the causal agent of common scab, an economically important disease of potato and taproot crops. Sequencing of the S. scabies 87-22 genome revealed the presence of a gene with high homology to the gene encoding the alpha-tomatine-detoxifying enzyme tomatinase found in fungal tomato pathogens. The tomA gene from S. scabies 87-22 was cotranscribed with a putative family 1 glycosyl hydrolase gene, and purified TomA protein was active only on alpha-tomatine and not potato glycoalkaloids or xylans. Tomatinase-null mutants were more sensitive to alpha-tomatine than the wild-type strain in a disk diffusion assay. Interestingly, tomatine affected only aerial mycelium and not vegetative mycelium, suggesting that the target(s) of alpha-tomatine is not present during vegetative growth. Severities of disease for tomato seedlings affected by S. scabies 87-22 wild-type and DeltatomA1 strains were indistinguishable, suggesting that tomatinase is not important in pathogenicity on tomato plants. However, conservation of tomA on a pathogenicity island in S. acidiscabies and S. turgidiscabies suggests a role in plant-microbe interaction.

Tomatinase from Fusarium oxysporum f. sp. lycopersici is required for full virulence on tomato plants.

Saponin detoxification enzymes from pathogenic fungi are involved in the infection process of their host plants. Fusarium oxysporum f. sp lycopersici, a tomato pathogen, produces the tomatinase enzyme Tom1, which degrades alpha-tomatine to less toxic derivates. To study the role of the tom1 gene in the virulence of F. oxysporum, we performed targeted disruption and overexpression of the gene. The infection process of tomato plants inoculated with transformants constitutively producing Tom1 resulted in an increase of symptom development. By contrast, tomato plants infected with the knockout mutants showed a delay in the disease process, indicating that Tom1, although not essential for pathogenicity, is required for the full virulence of F. oxysporum. Total tomatinase activity in the disrupted strains was reduced only 25%, leading to beta(2)-tomatine as the main hydrolysis product of the saponin in vitro. In silico analysis of the F. oxysporum genome revealed the existence of four additional putative tomatinase genes with identities to tomatinases from family 3 of glycosyl hydrolases. These might be responsible for the remaining tomatinase activity in the Deltatom1 mutants. Our results indicate that detoxification of alpha-tomatine in F. oxysporum is carried out by several tomatinase activities, suggesting the importance of these enzymes during the infection process.

Detoxification and function of immature tomato.

α-Tomatine and chlorophyll (a and b) decreased, and ß-carotene and lycopene increased with ripening of tomatoes. α-Tomatine was localised in peel of immature green tomatoes. The dose-response curve of α-tomatine determined by WST-1 (water soluble tetrazolium) assay was the same as that by LDH (lactate dehydrogenase) assay, suggesting that the cytotoxicity of α-tomatine depends on the destruction of plasma membrane. Immature green tomatoes had little cytotoxic effect after one month-incubation with 25% ethanol or 4.5% acetate at 7°C, and α-tomatine was decomposed by crude enzymes extracted from immature green tomatoes. Immature green tomatoes incubated with 4.5% acetic acid inhibited the accumulation of lipid in adipocytes. From the above facts the detoxification and the anti-obesity effect of immature green tomatoes are expected to be controlled by the removal of peel, the enzymatic decomposition or the incubation with 4.5% acetate or 25% ethanol.