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Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

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 anti-nutritional 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/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.

α-tomatineis 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-tomatineinto 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-tomatineinto 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, Δcftom1 mutants of C. fulvum are more sensitive to α-tomatineand are less virulent than the wild-type fungus on tomato. Although α-tomatineis 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.

Nuclear factor-kappa B (NF-κB) plays a role in prostate cancer and agents that suppress its activation may inhibit development or progression of this malignancy. Alpha (α)-tomatine is the major saponin present in tomato (Lycopersicon esculentum) and we have previously reported that it suppresses tumor necrosis factor-alpha (TNF-α)-induced nuclear translocation of nuclear factor-kappa B (NF-κB) in androgen-independent prostate cancer PC-3 cells and also potently induces apoptosis of these cells. However, the precise mechanism by which α-tomatine suppresses NF-κB nuclear translocation is yet to be elucidated and the anti-tumor activity of this agent in vivo has not been examined. In the present study we show that suppression of NF-κB activation by α-tomatine occurs through inhibition of I kappa B alpha (IκBα) kinase activity, leading to sequential suppression of IκBα phosphorylation, IκBα degradation, NF-κB/p65 phosphorylation, and NF-κB p50/p65 nuclear translocation. Consistent with its ability to induce apoptosis, α-tomatine reduced TNF-α induced activation of the pro-survival mediator Akt and its inhibition of NF-κB activation was accompanied by significant reduction in the expression of NF-κB-dependent anti-apoptotic (c-IAP1, c-IAP2, Bcl-2, Bcl-xL, XIAP and survivin) proteins. We also evaluated the antitumor activity of α-tomatine against PC-3 cell tumors grown subcutaneously and orthotopically in mice. Our data indicate that intraperitoneal administration of α-tomatine significantly attenuates the growth of PC-3 cell tumors grown at both sites. Analysis of tumor material indicates that the tumor suppressing effects of α-tomatine were accompanied by increased apoptosis and lower proliferation of tumor cells as well as reduced nuclear translocation of the p50 and p65 components of NF-κB. Our study provides first evidence for in vivo antitumor efficacy of α-tomatine against the human androgen-independent prostate cancer. The potential usefulness of α-tomatine in prostate cancer prevention and therapy requires further investigation.

Unlike most plants, members of the genus Solanum produce cholesterol and use this as a precursor for steroidal glycoalkaloids. The production of the compounds begins as a branch from brassinosteroid biosynthesis, which produces cholesterol that is further modified to produce steroidal glycoalkaloids. During the cholesterol biosynthesis pathway, genetic engineering could alter the formation of cholesterol from provitamin D3 (7-dehydrocholesterol) and produce vitamin D3. Cholesterol is a precursor for many steroidal glycoalkaloids, including α-tomatine and esculeoside A. Alpha-tomatine is consumed by mammals and it can reduce cholesterol content and improve LDL:HDL ratio. When there is a high α-tomatine content, the fruit will have a bitter flavor, which together with other steroidal glycoalkaloids serving as protective and defensive compounds for tomato against insect, fungal, and bacterial pests. These compounds also affect the rhizosphere bacteria by recruiting beneficial bacteria. One of the steroidal glycoalkaloids, esculeoside A increases while fruit ripening. This review focuses on recent studies that uncovered key reactions of the production of cholesterol and steroidal glycoalkaloids in tomato connecting to human health, fruit flavor, and plant defense and the potential application for tomato crop improvement.

The escalating demand for sustainable, nutrient-dense feeds underscores the need to valorize the agro-industrial byproducts utilizing innovative bioconversion strategies. In this context, we have studied the feasibility of incorporating tomato (Solanum lycopersicum) cultivation residues into Black Soldier Fly (BSF) larvae diets to produce high-protein insect meals. These residues are known to contain the naturally occurring toxic steroidal alkaloids tomatidine and α-tomatine, prohibiting their incorporation into human and animal diets. Herein, the tomato cultivation biomass was dried and mill-ground, and its varying volumes were incorporated into standard poultry feed (seven diet levels with 0–100% biomass inclusion) and tested in BSF-larvae-rearing trials to produce insect meals. The optimal results with respect to larvae growth, protein accumulation (highest value = 30.61%), lipid–fiber content, and antioxidant capacity were determined for insect meals obtained from BSF larvae reared with a ration composed of 40% tomato plant biomass. In addition, the toxicity of this insect meal was substantially low, as a consequence of the observed groundbreaking reduction in the contained toxic steroidal alkaloids α-tomatine and its aglycone tomatidine. The results herein reveal the efficacy of the BSF-larvae-rearing process in acting as a biological filter for the bioconversion of the toxic tomato cultivation waste into a functional, safe, and protein-rich livestock feed, supporting the principles of a circular economy.

Steroidal glycoalkaloids and saponins are plant cholesterol‐based steroid metabolites with antimicrobial activities and potential pharmacological value. The saponin uttroside B from black nightshade (Solanum nigrum) plays an important role in defence against herbivorous insects and exhibits anti‐hepatocellular carcinoma activity. The tomato (Solanum lycopersicum) glycoalkaloid α‐tomatine has been studied because of its antinutritional effects; however, its role in protecting plants from fungal pathogens remains understudied. The biosynthetic pathway of α‐tomatine involves multiple clustered genes designated as glycoalkaloid metabolism (GAME) genes. In this study, we generated single knockout mutants of SlGAME4 and SlGAME2 by CRISPR/Cas9‐based genome editing. The SlGAME4 mutants did not accumulate glycoalkaloids but instead redirected resources towards steroidal saponin (uttroside B) synthesis. SlGAME2 mutants contained unaltered α‐tomatine contents, indicating that the SlGAME2 gene, previously reported to catalyse the transfer of xylose to β1‐tomatine, is not involved in α‐tomatine biosynthesis. Infection assays with four fungal tomato pathogens demonstrated that the SlGAME4 mutant plants were slightly more susceptible to Botrytis cinerea, but equally susceptible to the other three fungi. Up‐regulation of α‐tomatine‐responsive genes in B. cinerea was observed during infection on SlGAME4 mutant tomato, as well as on S. nigrum, suggesting that uttroside B induces a fungal transcriptional response similar to α‐tomatine. Furthermore, we observed that tolerance mechanisms to plant saponins mediated by glycosyl hydrolases and a glycosyltransferase contribute to the virulence of B. cinerea on SlGAME4 mutant plants and S. nigrum. This indicates that uttroside B also contributes to defence against fungal pathogens and can be detoxified by B. cinerea. We generated knockout mutants of two tomato genes proposed to be involved in α‐tomatine biosynthesis by genome editing. SlGAME2 mutants contained unaltered α‐tomatine contents; this gene is not involved in its synthesis. SlGAME4 mutants did not accumulate any glycoalkaloids but instead redirected resources towards steroidal saponin synthesis.

Liver fibrosis is a progressive pathological process triggered by chronic liver disorders, which may progress to hepatocellular carcinoma (HCC) if left untreated. Currently, there are no specific therapeutic agents for liver fibrosis, highlighting an urgent need for novel pharmacological strategies. Tomatidine (TD), a major steroidal glycoal-kaloid abundant in immature tomato fruits, leaves, and stems, exhibits diverse biological activities including anti-inflammation, anti-tumor effects, and autophagy regulation. However, its role in liver fibrosis and the underlying molecular mechanisms remain incompletely understood. In this study, we combined network pharmacology, molecular docking, and experimental validation to investigate the potential effects of TD against liver fibrosis and its associated mechanism of action. In vitro experiments using the human hepatic stellate cell (HSC) line LX-2 demonstrated that TD inhibited HSC proliferation in a dose- and time-dependent manner, and downregulated the expression of fibrosis-related markers α-smooth muscle actin (α-SMA) and collagen type I α1 chain (COL1A1) at the gene, protein, and cellular levels. Network pharmacology analysis identified 18 common targets between TD and liver fibrosis, with core targets including MAPK3, RELA, and MAPK1 involved in intracellular signal transduction and stress-activated MAPK cascade. Although no autophagy-related targets were identified in the current database among these common targets, pharmacological evidence and experimental validation confirmed that TD promoted autophagy in LX-2 cells, as indicated by reduced P62 expression, increased LC3-II/LC3-I ratio and Beclin-1 levels, and enhanced autophagic flux. Further mechanism exploration revealed that TD exerted its autophagy-promoting effect by regulating the ERK/MAPK-mTOR-ULK1 signaling pathway: TD suppressed the phosphorylation of ERK and mTOR, while activating ULK1 phosphorylation. Molecular docking verified stable binding affinity between TD and key proteins in this pathway (ERK, MAPK, mTOR, ULK1) as well as autophagy-related proteins (P62, Beclin-1, LC3) and fibrosis-related protein COL1A1, with specific amino acid residues mediating hydrogen bond formation. Collectively, our findings demonstrate that TD modulates fibrosis-related markers in hepatic stellate cells by promoting autophagy in HSCs via the ERK/MAPK-mTOR-ULK1 pathway. This study enriches the biological function research of TD and provides a novel potential candidate and theoretical basis for the development of anti-liver fibrosis therapeutics.

High-performance liquid chromatography (HPLC) analysis of three commercial tomatine samples and another isolated from green tomatoes revealed the presence of two small peaks in addition to those associated with the glycoalkaloids dehydrotomatine and α-tomatine. The present study investigated the possible structures of the compounds associated with the two small peaks using HPLC–mass spectrophotometric (MS) methods. Although the two peaks elute much earlier on chromatographic columns than the elution times of the known tomato glycoalkaloids dehydrotomatine and α-tomatine, isolation of the two compounds by preparative chromatography and subsequent analysis by MS shows the two compounds have identical molecular weights, tetrasaccharide side chains, and MS and MS/MS fragmentation patterns to dehydrotomatine and α-tomatine. We suggest the two isolated compounds are isomeric forms of dehydrotomatine and α-tomatine. The analytical data indicate that widely used commercial tomatine preparations and those extracted from green tomatoes and tomato leaves consist of a mixture of α-tomatine, dehydrotomatine, an α-tomatine isomer, and a dehydrotomatine isomer in an approximate ratio of 81:15:4:1, respectively. The significance of the reported health benefits of tomatine and tomatidine is mentioned.

Head and neck squamous cell carcinoma (HNSCC) is frequently associated with cisplatin resistance, which limits the therapeutic efficacy of conventional chemotherapy. In this study, we investigated whether α-tomatine could enhance cisplatin sensitivity and augment its antitumor efficacy in HNSCC cells. Treatment with 20 μM cisplatin alone induced relatively low cytotoxicity in FaDu and YD38 cells (18.45 ± 2.59% and 9.40 ± 2.33%, respectively). In contrast, co-treatment of FaDu and YD38 cells with cisplatin (20 μM) and a non-cytotoxic concentration of α-tomatine (2 μM) significantly increased cell death to 52.98 ± 7.84% and 40.40 ± 3.06%, respectively, compared with cisplatin monotherapy. The combination treatment markedly suppressed colony formation, indicating reduced clonogenic survival, and significantly enhanced apoptosis through the simultaneous activation of intrinsic and extrinsic apoptotic pathways. The enhanced apoptosis was driven by the activation of the mitogen-activated protein kinase (MAPK) signaling cascade. Furthermore, the enhanced antitumor effect of α-tomatine and cisplatin was confirmed in a xenograft tumor model. These findings demonstrate that α-tomatine enhances cisplatin-induced apoptosis via MAPK-mediated signaling, supporting its role as a chemosensitizing agent for HNSCC.