Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a 'high-cholesterol' model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication.Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying transfer of wild species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci significantly linked with levels of hundreds of transcripts and metabolites. These associations occurred in functionally coherent hotspots that represent coordinated perturbation of metabolic pathways and ripening-related processes. We demonstrated the efficacy of our approach by discovering yet undisclosed components of the renowned Solanum alkaloids pathway, identifying new genes and metabolites involved in pathogen defence and linking fungal resistance with changes in the fruit ripening regulatory network. Together, our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.

The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.

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Plants have evolved to produce a blend of specialized metabolites that serve functional roles in plant adaptation. Among them, triterpenoids are one of the largest subclasses of such specialized metabolites, with more than 14,000 known structures. They play a role in plant defense and development and have potential applications within food and pharma. Triterpenoids are cyclized from oxidized squalene precursors by oxidosqualene cyclases, creating more than 100 different cyclical triterpene scaffolds. This limited number of scaffolds is the first step towards creating the vast structural diversity of triterpenoids followed by extensive diversification, in particular, by oxygenation and glycosylation. Gene duplication, divergence, and selection are major forces that drive triterpenoid structural diversification. The triterpenoid biosynthetic genes can be organized in non-homologous gene clusters, such as in Avena spp., Cucurbitaceae and Solanum spp., or scattered along plant chromosomes as in Barbarea vulgaris. Paralogous genes organized as tandem repeats reflect the extended gene duplication activities in the evolutionary history of the triterpenoid saponin pathways, as seen in B. vulgaris. We review and discuss examples of convergent and divergent evolution in triterpenoid biosynthesis, and the apparent mechanisms occurring in plants that drive their increasing structural diversity within and across species. Using B. vulgaris' saponins as examples, we discuss the impact a single structural modification can have on the structure of a triterpenoid and how this affect its biological properties. These examples provide insight into how plants continuously evolve their specialized metabolome, opening the way to study uncharacterized triterpenoid biosynthetic pathways.

Summary Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin‐based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C‐23 to hederagenin. In vitro‐produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin‐based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.