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Steroidal glycoalkaloids (SGAs) are specialized metabolites in Solanaceae that serve as defensive compounds and undergo significant compositional changes during fruit ripening. This study explored the roles of transcription factors SlNOR and SlNOR-like1 in SGAs biosynthesis during tomato fruit development. UPLC-MS/MS revealed dynamic changes in four major SGAs: tomatidine, β-tomatine, α-tomatine, and Esculeoside A. Transgenic studies with knockout and overexpression lines demonstrated that both SlNOR and SlNOR-like1 positively regulated SGAs accumulation. RT-qPCR analysis showed that these transcription factors modulated multiple GAME genes in the SGAs biosynthetic pathway. Through EMSA and DLR assays, we established that SlNOR and SlNOR-like1 directly bound to and activated GAME25 and GAME40 promoters, two key genes involved in tomatidine synthesis and α-tomatine conversion, respectively. These findings reveal a previously unknown regulatory mechanism of SGAs metabolism and suggest potential strategies for optimizing tomato fruit quality through molecular breeding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

A well-known defense-associated steroidal glycoalkaloid (SGA) metabolic shift eliminates the bitterness and toxicity of ripe tomato fruits. This study was conducted to clarify the effects of MADS-RIN (RIN) and its cofactors on SGA metabolism in tomato fruits. Using a CRISPR/Cas9-based gene-editing system, we mutated RIN and two cofactor genes (FUL1 and FUL2). The observed changes to fruit color and size in the mutants reflected the overlapping and distinct effects of RIN, FUL1, and FUL2 on fruit ripening. According to a UPLC-MS/MS analysis, the RIN and cofactor mutants had decreased levels of the relatively non-toxic metabolite esculeoside A, but they accumulated toxic SGA pathway intermediates, suggesting RIN and its cofactors are directly involved in esculeoside A biosynthesis. Transcriptome and qPCR analyses detected the downregulated expression of GAME5, which encodes a key enzyme mediating esculeoside A biosynthesis. ChIP-seq and ChIP-qPCR analyses confirmed GAME5 is targeted by RIN. RIN was observed to activate GAME5 transcription by binding to two non-canonical CArG-boxes in the GAME5 promoter. Additionally, RIN promotes SGA metabolism independently of ethylene. Collectively, these findings enhance our understanding of the molecular mechanism governing tomato fruit ripening and SGA biosynthesis. Furthermore, they may be useful for improving tomato fruit quality and safety., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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, β-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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 Sl3βHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3βHSD1 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 Sl3βHSD1 protein revealed that Sl3βHSD1 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 Sl3βHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3βHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3βHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3βHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 3β-hydroxysteroid dehydrogenase/Δ 5,4 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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2016 CSIRO. New Phytologist © 2016 New Phytologist Trust.)

α-Tomatine and chlorophyll (a and b) decreased, and β-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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Variation in resource inputs to plants may trigger bottom-up effects on herbivorous insects. We examined the effects of water input: optimal water vs. limited water; water salinity: with vs. without addition of 100 mM NaCl; and their interactions on tomato plants (Solanum lycopersicum), and consequently, the bottom-up effects on the tomato leaf miner, Tuta absoluta (Meytick) (Lepidoptera: Gelechiidae). Plant growth was significantly impeded by limited water input and NaCl addition. In terms of leaf chemical defense, the production of tomatidine significantly increased with limited water and NaCl addition, and a similar but non-significant trend was observed for the other glycoalkaloids. Tuta absoluta survival did not vary with the water and salinity treatments, but the treatment "optimal water-high salinity" increased the development rate without lowering pupal mass. Our results suggest that caution should be used in the IPM program against T. absoluta when irrigating tomato crops with saline water.

The Colorado potato beetle Leptinotarsa decemlineata (Say) (CPB) is a coleopteran herbivore that feeds on the foliage on Solanum species, in particular, potato. Six resistant wild Solanum species were identified, and two of these species had low levels of glycoalkaloids. Comparative analysis of the untargeted metabolite profiles of the foliage using UPLC-qTOF-MS was done to find metabolites shared between the wild species but not with Solanum tuberosum (L.) to identify resistance-related metabolites. It was found that only S. tuberosum produced the triose glycoalkaloids solanine and chaconine. Instead, the six wild species produced glycoalkaloids that shared in common tetrose sugar side chains. Additionally, there were non-glycoalkaloid metabolites associated with resistance including hydroxycoumarin and a phenylpropanoid, which were produced in all wild species but not in S. tuberosum.

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)-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., (Copyright © 2014. Published by Elsevier Ltd.)

· 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 β-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., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

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., (© 2012 Society of Chemical Industry.)

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.

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.

The composition of glycoalkaloids in tomato fruit changes with ripening. However, it has not been clarified whether the accumulation of glycoalkaloids is controlled by the ripening-inducing phytohormone, ethylene. Here, we report the effect of ethylene on the accumulation of tomato fruit glycoalkaloids. We investigated the effect of exogenously applied ethylene. In response to ethylene treatment, the content of alpha-tomatine decreased, whereas the content of esculeoside A increased. Next, we analyzed the fruits of ripening mutants, rin, nor, and Nr. In fruits of these mutant lines, the level of accumulation of esculeoside A decreased, whereas alpha-tomatine accumulated to higher levels than in wild-type fruit. These results demonstrated that the esculeoside A accumulation was associated with production and perception of ethylene. Additionally, the accumulation profiles of the intermediate metabolites of esculeoside A biosynthesis in ripening mutant fruits suggest that a glycosylation step in the putative pathway from alpha-tomatine to esculeoside A depends on ethylene.

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.

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.

Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid alpha-tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves alpha-tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of alpha-tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension-cultured tomato cells. A tomatinase-negative F. oxysporum strain inherently non-pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of alpha-tomatine but also for suppression of induced defense responses of host.

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from alpha-tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.

Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.

Tomatoes, a major food source for humans, accumulate a variety of secondary metabolites including phenolic compounds, phytoalexins, protease inhibitors, and glycoalkaloids. These metabolites protect against adverse effects of hosts of predators including fungi, bacteria, viruses, and insects. Because glycoalkaloids are reported to be involved in host-plant resistance, on the one hand, and to have a variety of pharmacological and nutritional properties in animals and humans, on the other, a need exists to develop a better understanding of the role of these compounds both in the plant and in the diet. To contribute to this effort, this integrated review presents data on the history, composition, and nutrition of tomatoes, with special focus on the assessment of the chemistry, analysis, composition, nutrition, microbiology, and pharmacology of the tomato glycoalkaloids comprising alpha-tomatine and dehydrotomatine; their content in different parts of the tomato plant, in processed tomato products, and in wild and transgenic tomatoes; their biosynthesis, inheritance, metabolism, and catabolism; plant-microbe relationships with fungi, bacteria, viruses, insects, and worms; interactions with ergosterol and cholesterol; disruption of cell membranes; tomatine-induced tomatinases, pantothenate synthetase, steroid hydroxylases, and cytokines; and inhibition of acetylcholinesterase. Also covered are tomato-human pathogen relationships and tomatine-induced lowering of plasma cholesterol and triglycerides and enhancement of the immune system. Further research needs in each of these areas are suggested. The overlapping aspects are discussed in terms of general concepts for a better understanding of the impact of tomato glycoalkaloids in the plant in general and in food in particular. Such an understanding can lead to the creation of improved tomatoes and to improved practices on the farm and in the consumption of tomatoes.

Plant disease resistance can be conferred by constitutive features such as structural barriers or preformed antimicrobial secondary metabolites. Additional defence mechanisms are activated in response to pathogen attack and include localized cell death (the hypersensitive response). Pathogens use different strategies to counter constitutive and induced plant defences, including degradation of preformed antimicrobial compounds and the production of molecules that suppress induced plant defences. Here we present evidence for a two-component process in which a fungal pathogen subverts the preformed antimicrobial compounds of its host and uses them to interfere with induced defence responses. Antimicrobial saponins are first hydrolysed by a fungal saponin-detoxifying enzyme. The degradation product of this hydrolysis then suppresses induced defence responses by interfering with fundamental signal transduction processes leading to disease resistance.

The degradation fo steroid glycoalkaloids (SGAs) has been studied in model solutions. The number of colony forming units (CFU) was determined using a nondirect (cultivation) method during all stages of fermentation. The changes in SGAs content were observed by HPLC on the Supelcosil LC-NH2 column. The changes in alpha-tomatine concentration added to fermented Lactobacillus MRS broth have been studied. A mathematical model of steroid glycoalkaloids degradation during lactic fermentation was proposed. The mathematical model was based on the experimental data of SGA and glucose concentration and should be used for study and prediction of SGA concentration changes of fermented samples. The ratio of SGA degradation rate by fermentation and by lactic acid hydrolysis was calculated. The experimental data evaluated by proposed mathematical model for the selected strain of Lactobacillus plantarum 976H show real feasibility for SGA degradation by lactic acid fermentation.

Intrinsic or acquired resistance of tumor cells to multiple cytotoxic drugs (multidrug resistance MDR) is a major cause of failure of cancer chemotherapy. MDR is often caused by elevated expression of drug transporters such as P-glycoprotein (P-gp) or multidrug resistance protein (MRP). A number of compounds, termed chemosensitizers, have little or no cytotoxic action of their own, but inhibit (P-gp) or MRP-mediated drug export and are capable of sensitizing MDR cells to the cytotoxic effects of chemotherapeutic drugs. Here we examined the ability of steroidal alkaloids of plant origin, namely the Veratrum sp. alkaloid cyclopamine and the Lycopersicon sp. alkaloid tomatidine, to act as potent and effective chemosensitizers in multidrug resistant tumor cells. Drug uptake was determined by measuring accumulation of tetramethylrosamine in multidrug resistant NCI AdrR human adenocarcinoma cells. Resistance to adriamycin and vinblastine was determined by utilizing the MTT cell survival assay. Cyclopamine and tomatidine elevate tetramethylrosamine uptake by NCI AdrR cells and sensitize the cells to the cytotoxic action of adriamycin and vinblastine. In both cases these agents are comparable in patency and efficacy to verapamil, a reversal agent commonly used in MDR research. It is concluded that steroidal alkaloids of plant origin act as inhibitors of P-gp-mediated drug transport and multidrug resistance and therefore may serve as chemosensitizers in combination chemotherapy with conventional cytotoxic drugs for treating multidrug resistant cancer.

Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms.

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.

The anti-fungal, steroidal, glycoalkaloid saponin, alpha-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to alpha-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify alpha-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to alpha-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, alpha-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.

Lycopersicon species often contain the toxic glycoalkaloid alpha-tomatine, which is proposed to protect these plants from general microbial infection. however, fungal pathogens of tomato often are tolerant to alpha-tomatine and detoxification of alpha-tomatine may be how these pathogens avoid this potential barrier. As an initial step to evaluate this possibility, we have purfied to homogeneity a beta-1,2-D glucosidase from the tomato pathogen Septoria lycopersici that hydrolyzes the beta-1,2-D glucosyl bond on the tetrasaccharide moiety of alpha-tomatine to produce beta2-tomatine. The enzyme is a 110-kDa protein with a pI of 4.5 and a Km for alpha-tomatine of 62 microM. Little or no activity was detected on a variety of other glycosides. The gene encoding this protein was isolated and contains an open reading frame of 803 amino acids that shares sequence homology with several other beta-D-glucosidases. When S. lycopersici was incubated with alpha-tomatine, beta2-tomatinase mRNA accumulated, suggesting that the enzyme is substrate inducible. Aspergillus nidulans expressed ¿beta2-tomatinase¿ activity when transformed with this gene but transformants were still sensitive to alpha-tomatine.

Pretreatment of tomato seeds with pendimethalin or metribuzin and inoculation of seedlings with the antagonistic Streptomyces corchorusii or/and Streptomyces mutabilis were tested for the formation of tomatine in roots and stems of tomato, infested with Pseudomonas solancearum or/and Fusarium oxysporum f. sp. lycopersici. All treatments induced the formation of variable quantities of tomatine, compared with untreated control. The variation was proportional to: the pathogen, Fusarium was more stimulating than Pseudomonas; the antagonistic organism, S. corchorusii being more eliciting than S. mutabilis; the herbicide and its concentration, pendimethalin at 2 x 10(-3) M being the most eliciting of tomatine; and according to the soil, plants grown in non-sterilized soil accumulated more tomatine than did these grown in sterilized soil. In all treatments, stems had more tomatine than roots and non-sterilized soil was better than sterilized soil. The antagonistic streptomycetes induced accumulation of tomatine more than did the herbicides. The highest amounts of tomatine were detected in plants pretreated with pendimethalin at 2 x 10(-3) M, grown in non-sterilized soil, infested with F. oxysporum, and inoculated with S. corchorusii and S. mutabilis. The effect of the extracted tomatine on the growth of Fusarium and Pseudomonas was examined in vitro. The crude extract of tomatine from all treatments reduced growth and sporulation of F. oxysporum and growth of P. solanacearum in defined media. The reduction varied according to the treatment and was proportional to the quantities of extracted tomatine, the highest amounts being the most effective. The mechanism of phytoalexins in controlling tomato wilt pathogens was also discussed.

The surface distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells was investigated by freeze-fracture. Replicas of fractured smooth muscle cell plasma membrane showed caveola-free regions with few intramembrane particles, interspersed with caveola-populated areas with a higher intramembrane particle density. Electron microscopy of thin sections of quail gizzard smooth muscle revealed the regions free of caveolae to be occupied by membrane-associated dense plaques; anchoring sites for the contractile filaments of the cell. Demarcation between the caveola-populated and caveola-free regions on the relicated intramembrane surface was not clear and thus provided little information concerning the distribution of dense plaque sites. However, treatment of the smooth muscle tissue with the cholesterol-binding agents filipin or tomatin prior to freeze-fracture allowed the dense plaque sites to be easily observed as the sites remained free of the membrane deformations characteristic of these agents. The dense plaque sites consist of caveola-free oval areas juxtaposed in regular bands that traverse the long axis of the cell. The dense plaque sites on the freeze-fracture replica were confirmed by electron microscopy of thin sections of filipin-treated quail gizzard smooth muscle cells, which showed the plasma membrane associated with the dense plaques to be unaffected by the actions of filipin, whereas that of the caveola-populated region was severely deformed. The observations presented in this study provide evidence for a highly ordered distribution of dense plaques at the cell surface of intact quail gizzard smooth muscle cells and thus corroborate existing evidence for an organized substructure of smooth muscle cells.

The microbial dehydrogenation of steroid alkaloids follows the dehydrogenation pattern of steroids until the 3-keto-1,4-diene stage. No side-chain cleavage or degradation of the steroid nucleus is observed. Side-chain cleavage of tomatidine is achieved only by previous induction of side-chain splitting enzymes.

The bioconversion of steroid alkaloid tomatine by Nocardia restricta yields the conjugate with lactic acid. We studied the bioconversion of some steroid glycosides without a nitrogen atom in the molecule to determine the effect of the nitrogen atom. The glycosides were of three different types: sterol glycosides, bufadienolide rhamnoside and steroid saponine. The results of bioconversions showed that Nocardia restricta converts steroid glycosides differently according to the sugar bound to the steroid aglycone. It can be concluded that in the absence of a nitrogen atom in the steroid molecule no conjugation with lactic acid by Nocardia restricta occurs.

In the degradation pathway of the steroid alkaloid tomatidine by Gymnoascus reesii the A-ring of tomatidine is opened with the formation of the 4-hydroxy-3,4-secotomatidine-3-oic acid, which was identified in the form of N-acetyl-3,4-tomatidine-carbolactone by mass, IR and 1H NMR spectra. Cleavage of the A-ring in the starting reaction indicates that an alternative pathway must be operating, instead of the general oxidative one.

(22S,25S)-5alpha-tomatanin-3beta-ol, N-acetyl-(22S,25S)-5alpha-tomatanin-3beta-ol, (22R,25R)-5alpha-tomatanin-3beta-ol and (22R, 25S)-22, 26-epimino-5alpha-cholestane-3beta, 16beta, 16beta-diol are transformed by Nocardia restrictus into corresponding 3alpha-ol compounds with yields from 7o to 5%.

The planar distribution of 3-beta-hydroxysterols in the plasmalemma of the green flagellate Chlamydomonas reinhardii has been studied with the freeze-fracture technique using the polyene antibiotic filipin and the saponin tomatin as cytochemical markers. Filipin-sterol complexes were predominantly observed as 25 to 30 nm protuberances on the E-face of the plasmalemma with corresponding invaginations on the P-face. Generally filipin-sterol and tomatin-sterol complexes were randomly distributed, but were virtually absent from sites of intramembrane particle arrays (IMP-arrays, i.e. the flagellar necklace, the flagellar bracelet including strut arrays, the eyespot membrane). The results suggest that IMP-arrays of Chlamydomonas are deficient in 3-beta-hydroxysterols and may therefore be regarded as special lipid arrays. IMP-arrays might require an altered lipid environment for proper function. Some functional aspects of IMP-arrays in Chlamydomonas are discussed.