Steroidal Glycoalkaloids Biosynthesis Research Articles

Incorporation of nitrogen in antinutritional Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

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.

Allelic variation in the autotetraploid potato: genes involved in starch and steroidal glycoalkaloid metabolism as a case study

BackgroundTuber starch and steroidal glycoalkaloid (SGA)-related traits have been consistently prioritized in potato breeding, while allelic variation pattern of genes that underlie these traits is less explored.ResultsHere, we focused on the genes involved in two important metabolic pathways in the potato: starch metabolism and SGA biosynthesis. We identified 119 genes consisting of 81 involved in starch metabolism and 38 in the biosynthesis of steroidal glycoalkaloids, and discovered 96,166 allelic variants among 2,169 gene haplotypes in six autotetraploid potato genomes. Comparative analyses revealed an uneven distribution of allelic variants among gene haplotypes and that the vast majority of deleterious mutations in these genes are retained in heterozygous state in the autotetraploid potato genomes. Leveraging full-length cDNA sequencing data, we find that approximately 70% of haplotypes of the 119 genes are transcribable. Population genetic analyses identify starch and SGA biosynthetic genes that are potentially conserved or diverged between potato varieties with varying starch or SGA content.ConclusionsThese results deepen the understanding of haplotypic diversity within functionally important genes in autotetraploid genomes and may facilitate functional characterization of genes or haplotypes contributing to traits related to starch and SGA in potato.

The transcription factor StMYB113 regulates light-induced greening by modulating steroidal glycoalkaloid biosynthesis in potatoes (Solanum tuberosum L.)

During harvesting, storage, transportation, and processing, potato (Solanum tuberosum L.) tubers undergo greening after exposure to light, leading to the accumulation of toxic glycoside alkaloids, resulting in quality deterioration and economic losses. However, the underlying mechanisms are unclear. This study compared the transcriptome and proteome differences among four potato cultivars during the light-induced greening process, identifying 3,751 unique proteins (high confidence; ≥91.7%). The levels of enzymes involved in steroidal glycoalkaloid biosynthesis varied among the cultivars. In addition, coexpression network analysis of the transcriptomic data identified the transcription factor MYB113 (Soltu.DM.10G020780.1) as a potential positive regulator of steroidal glycoalkaloid biosynthesis. The dual-luciferase assay revealed that StMYB113 could bind to the promoters of steroidal glycoalkaloid biosynthesis-related genes and activate them. The transgenic lines overexpressing Solanum tuberosum L. Myb domain protein (StMYB113) exhibited greater mRNA abundance of these genes and elevated levels of steroidal glycoalkaloids. This study provided a theoretical basis for exploring the impact of light on the synthesis of solanine in potatoes.

GGE Biplot-Based Transcriptional Analysis of 7 Genes Involved in Steroidal Glycoalkaloid Biosynthesis in Potato (Solanum tuberosum L.)

Steroidal glycoalkaloids (SGAs) are secondary metabolites that are closely associated with the sensory and processing qualities of potato tubers. GGE biplots are a widely used tool for analyzing crop breeding analysis. This study aimed to investigate the effect of light on SGA biosynthesis by employing GGE biplots to analyze the transcriptional gene expression of seven genes involved in the SGA biosynthesis pathway. Tubers of five different potato genotypes were incubated for 6, 12, and 24 h under red light. The expression levels of the seven genes were measured using qRT-PCR for analysis. Further analysis of the data was performed using GGE biplots. Our results indicated significantly higher expression levels for Pvs1, Sgt1, and Sgt3 genes than those of the remaining tested genes. Across the three red light illumination durations, Sgt3 showed high and stable expression, although it showed less stability across the different genotypes. Interestingly, the expression patterns of the seven genes were extremely similar for the 12 h and 24 h treatments. It was found that at least 6 h of red light illumination was required for optimal gene expression in all five genotypes, particularly in the genotype Zhuangshu-3 (DXY) after 24 h of treatment. Additionally, significant expression of the seven genes was observed in the L-6 genotype after 12 and 6 h of red light illumination. These results highlight that GGE biplots are an appropriate tool for analyzing and illustrating the differential expression profiles of the seven key genes involved in SGA biosynthesis in potato tubers. This study provides valuable insights into the biosynthesis and metabolism of SGAs in potatoes. Moreover, it demonstrates the potential application of GGE biplots in crop breeding and other research fields.

Comparative transcriptome analysis of different tissues of Solanum khasianum reveals candidate genes involved in steroidal glycoalkaloid biosynthesis.

Fruits and leaves of Solanum khasianum C. B. Clarke have long been used as a common Chinese herbal medicine. Steroidal glycoalkaloids (SGAs), the main active ingredient in S. khasianum, exhibit various pharmacological effects. However, genes involved in the SGA biosynthetic pathway in S. khasianum have not yet been identified. Genes encoding potential key SGA biosynthesis enzymes were identified through comprehensive RNA sequencing analysis (RNA-seq) of S. khasianum leaves, stems, and fruits. A total of 123,704 unigenes were obtained, of which 109,775 (88.74%) were annotated in seven public databases. Among these, 54 unigenes potentially involved in SGA biosynthesis were identified. Additionally, 23,636 differentially expressed genes were identified by comparing gene expression levels among the fruits, stems, and leaves of S. khasianum. The structural characteristics and phylogenetic relationship of cycloartenol synthase involved in SGA biosynthesis were further analyzed. Solasodine constituent was detected by high-performance liquid chromatography. This is the first study to report the comparative transcriptome analysis of different tissues of S. khasianum that identifies valuable genes potentially involved in SGA biosynthesis in this species.

SlERF.H6 mediates the orchestration of ethylene and gibberellin signaling that suppresses bitter-SGA biosynthesis in tomato.

Steroidal glycoalkaloids (SGAs) constitute a characteristic class of antinutritional metabolites that are found in certain Solanum species. Despite the considerable studies on SGA biosynthesis, the mechanisms of crosstalk between hormone signaling pathways that regulate SGA content still remain to be elucidated. Here, we performed a metabolic genome-wide association study (mGWAS) based on the levels of SGA metabolites and identified SlERF.H6 as a negative regulator of bitter-SGA biosynthesis. SlERF.H6 repressed the expression of SGA biosynthetic glycoalkaloid metabolism (GAME) genes and caused a subsequent decrease in the abundance of bitter SGAs. Furthermore, SlERF.H6 were shown to act downstream of GAME9, a regulator of SGA biosynthesis in tomato. We also uncovered the interplay between ethylene and gibberellin (GA) signaling in regulating SGA biosynthesis. SlERF.H6, acting as a downstream component in ethylene signaling, modulated GA content by inhibiting SlGA2ox12 expression. Increasing levels of endogenous GA12 and GA53 in SlERF.H6-OE could inhibit of GA on SGA biosynthesis. Additionally, 1-aminocyclopropane-1-carboxylic acid (ACC) treatment decreased the stability of SlERF.H6, weakening its inhibition on GAME genes and SlGA2ox12, and caused bitter-SGA accumulation. Our findings reveal a key role of SlERF.H6 in the regulation of SGA biosynthesis through the coordinated ethylene-gibberellin signaling.

Downregulation of a cluster of genes encoding nitrate transporter 1/peptide transporter family proteins in tomato with a mutated JRE4 transcription factor.

A group of anti-nutritional specialized metabolites called steroidal glycoalkaloids (SGAs) are produced in Solanum species such as tomato, potato, and eggplant. The transcription factor JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) regulates many SGA biosynthesis genes in tomato and potato. Here we report that the expression of a cluster of genes encoding nitrate transporter 1/peptide transporter family (NPF) members is downregulated in the jre4-1 loss-of-function tomato mutant, which has a low-SGA phenotype compared to the wild type. NPFs are a large family of plant membrane transporters that transport a wide range of substrates, including specialized metabolites. We found that the JRE4-regulated NPF genes are induced by the defense-related phytohormone jasmonate. Conversely, jasmonate-mediated induction of gene expression was attenuated by ethylene treatment of the leaves. The co-regulation of the NPF genes with SGA biosynthesis genes by JRE4 suggests that NPF transporters are involved in the SGA pathway.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

The SlDOG1 Affect Biosynthesis of Steroidal Glycoalkaloids by Regulating GAME Expression in Tomato.

Steroidal alkaloids (SAs) and steroidal glycoalkaloids (SGAs) are common constituents of plant species belonging to the Solanaceae family. However, the molecular mechanism regulating the formation of SAs and SGAs remains unknown. Here, genome-wide association mapping was used to elucidate SA and SGA regulation in tomatoes: a SlGAME5-like glycosyltransferase (Solyc10g085240) and the transcription factor SlDOG1 (Solyc10g085210) were significantly associated with steroidal alkaloid composition. In this study, it was found that rSlGAME5-like can catalyze a variety of substrates for glycosidation and can catalyze SA and flavonol pathways to form O-glucoside and O-galactoside in vitro. The overexpression of SlGAME5-like promoted α-tomatine, hydroxytomatine, and flavonol glycoside accumulation in tomatoes. Furthermore, assessments of natural variation combined with functional analyses identified SlDOG1 as a major determinant of tomato SGA content, which also promoted SA and SGA accumulation via the regulation of GAME gene expression. This study provides new insights into the regulatory mechanisms underlying SGA production in tomatoes.

A RING membrane-anchor E3 ubiquitin ligase gene is co-expressed with steroidal glycoalkaloid biosynthesis genes in tomato.

RING membrane-anchor (RMA) E3 ubiquitin ligases are involved in endoplasmic reticulum (ER)-associated protein degradation, which mediates the regulated destruction of ER-resident enzymes in various organisms. We determined that the transcription factor JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) co-regulates the expression of the RMA-type ligase gene SlRMA1, but not its homolog SlRMA2, with steroidal glycoalkaloid biosynthesis genes in tomato, perhaps to prevent the overaccumulation of these metabolites.

A Jasmonate-Responsive ERF Transcription Factor Regulates Steroidal Glycoalkaloid Biosynthesis Genes in Eggplant

Steroidal glycoalkaloids (SGAs) are a class of cholesterol-derived anti-nutritional defense compound that are produced in species of the genus Solanum, such as tomato (S. lycopersicum), potato (S. tuberosum), and eggplant (S. melongena). However, the regulation of defense-related metabolites in eggplant remains underexplored. In tomato and potato, the JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) transcription factor positively regulates a large number of genes involved in SGA biosynthesis. Here, we report that the overexpression of eggplant JRE4 (SmJRE4) induces numerous metabolic genes involved in SGA biosynthesis in leaves. We demonstrate the jasmonate-dependent induction of SmJRE4 and its downstream metabolic genes and show that ethylene treatment attenuates this induction. Our findings thus provide molecular insights into SGA biosynthesis and its regulation in this major crop.

Identification and Characterization of the Tomato UGT Gene Family and Effects of GAME 17 Overexpression on Plants and Growth and Development under High-CO2 Conditions

Steroidal glycoalkaloids (SGAs), the nitrogen-containing compounds produced primarily by Liliaceae and Solanaceae species, are toxic to animals and humans and have putative roles in defense against pests. UDP-glycosyltransferases (UGTs) catalyze the final glycosylation steps of SGA biosynthesis. Although previously published studies focused on the effect of UGT proteins on SGA biosynthesis, research to understand the effects of constitutive overexpression of UGTs on plant phenotype and fruit development is limited. The constitutive overexpression of a UGT encoding gene, GAME 17, may provide an alternative method to study the role of UGTs on the fruit development. In this study, we have identified 162 SlUGT proteins in tomato that are classified into t 23 groups. Gene structure and motif analyses have demonstrated that all SlUGTs have similar intron/exon distribution and motif compositions. RNA-seq data analysis has shown that SlUGTs exhibit differential expression patterns in different organs or different stages of fruit development. When the constitutive promoter 35S is used to control the expression of GAME 17, we have observed significant differences in growth parameters (i.e., plant height, leaf length, leaf width, internode length, and stem diameter) between WT and transgenic plants under high-CO2 conditions, and slight differences in growth parameters between WT and transgenic plants have beendetected. In addition, the contents of glucose, fructose, and soluble sugar of transgenic plants are significantly higher than those of WT plants. The increases in glucose, fructose, and soluble sugar in transgenic tomato fruits at three developmental stages under high-CO2 conditions are significantly higher than under natural conditions. This study provides additional evidence that the GAME 17 gene plays an important role in controlling plant phenotype and sugar homeostasis, especially in environments with high concentration of CO2.

The basic helix-loop-helix transcription factors MYC1 and MYC2 have a dual role in the regulation of constitutive and stress-inducible specialized metabolism in tomato.

Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.

Multi-Omics Analysis Reveals That SlERF.D6 Synergistically Regulates SGAs and Fruit Development.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules that contribute to the pathogen defense in tomato but are toxic and considered to be antinutritional compounds to humans. APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors (TFs) play an indispensable role in various biological processes, such as plant growth and development, fruit ripening, biotic and abiotic stresses responses, and SGA biosynthesis. In this study, we identified 176 AP2/ERF genes that were domesticated or improved SlAP2/ERF in the tomato variome (Solanum lycopersicum) within either domestication or improvement sweeps, respectively. According to the RNA-sequencing data, 93 of the ERF genes with high transcriptional level (Transcripts Per Million, TPM > 1) belong to six clusters. Weighted gene co-expression network analysis (WGCNA) and metabolite-based genome-wide association study (mGWAS) analyses revealed that the expression level of the Solyc04g071770 (SlERF.D6) gene in the cluster six gradually increased as the fruit matured. Transient transformation verified that the overexpression of SlERF.D6 significantly promoted fruit ripening and regulated the expression of multiple genes in the SGA synthesis pathway, thereby affecting the SGA content of the fruit. Virus-induced gene silencing (VIGS) showed that the silencing of SlERF.D6 delayed fruit ripening and influenced the content of SGAs. Our data provide new insights into AP2/ERF TFs in tomato, offer a candidate TF for fruit development and steroidal glycoalkaloids, and provide new resources for tomato breeding and improvement.

Colorado potato beetle exploits frass-associated bacteria to suppress defense responses in potato plants.

Colorado potato beetle (CPB; Leptinotarsa decemlineata) is a destructive quarantine pest that develops broad physiological adaptations to potato plants. During feeding, CPB deposits a copious amount of wet frass onto the surface of leaves and stems that remains in place for long periods. Insect behaviors such as feeding, crawling and oviposition are able to mediate plant defenses. However, the specific role of CPB defecation-associated cues in manipulating plant defenses remains unclear. CPB larval frass significantly suppressed potato polyphenol oxidase activity and enhanced larval growth on treated potato plants. The incorporation of antibiotics into larval frass triggered higher jasmonic acid (JA)-regulated defense responses in potato plants compared with antibiotic-free frass. Four bacterial symbionts belonging to the genera Acinetobacter, Citrobacter, Enterobacter and Pantoea were isolated from larval frass and suppressed plant defenses. After reinoculation of these bacteria into axenic larvae, Acinetobacter and Citrobacter were found to be highly abundant in the frass, whereas Enterobacter and Pantoea were less abundant probably due to the negative effect of potato steroidal glycoalkaloids (SGA) such as α-solanine. Furthermore, direct application of Acinetobacter and Citrobacter to wounded potato plants significantly inhibited the expression of genes associated with the JA-mediated defense signaling pathway and SGA biosynthesis. Our findings demonstrate that CPB exploits frass-associated bacteria as a deceptive strategy of plant defense suppression, adding an interesting dimension to our understanding of how CPB successfully specializes on potato plants. © 2022 Society of Chemical Industry.

Multiomic Analysis Reveals Core Regulatory Mechanisms underlying Steroidal Glycoalkaloid Metabolism in Potato Tubers.

Steroidal glycoalkaloids (SGAs) present in germinated potato tubers are toxic; however, the mechanisms underlying SGA metabolism are poorly understood. Therefore, integrated transcriptome, metabolome, and hormone analyses were performed in this study to identify and characterize the key regulatory genes, metabolites, and phytohormones related to glycoalkaloid regulation. Based on transcriptome sequencing of bud eyes of germinated and dormant potato tubers, a total of 6260 differentially expressed genes were identified, which were mainly responsible for phytohormone signal transduction, carbohydrate metabolism, and secondary metabolite biosynthesis. Two TCP14 genes were identified as the core transcription factors that potentially regulate SGA synthesis. Metabolite analysis indicated that 149 significantly different metabolites were detected, and they were enriched in metabolic and biosynthetic pathways of secondary metabolites. In these pathways, the α-solanine content was increased and the expression of genes related to glycoalkaloid biosynthesis was upregulated. Levels of gibberellin and jasmonic acid were increased, whereas that of abscisic acid was decreased. This study lays a foundation for investigating the biosynthesis and regulation of SGAs and provides the reference for the production and consumption of potato tubers.

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Genome-wide characterization of 2-oxoglutarate and Fe(II)-dependent dioxygenase family genes in tomato during growth cycle and their roles in metabolism

Background2-Oxoglutarate and Fe(II)-dependent dioxygenases (2ODDs) belong to the 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily and are involved in various vital metabolic pathways of plants at different developmental stages. These proteins have been extensively investigated in multiple model organisms. However, these enzymes have not been systematically analyzed in tomato. In addition, type I flavone synthase (FNSI) belongs to the 2ODD family and contributes to the biosynthesis of flavones, but this protein has not been characterized in tomato.ResultsA total of 131 2ODDs from tomato were identified and divided into seven clades by phylogenetic classification. The Sl2ODDs in the same clade showed similar intron/exon distributions and conserved motifs. The Sl2ODDs were unevenly distributed across the 12 chromosomes, with different expression patterns among major tissues and at different developmental stages of the tomato growth cycle. We characterized several Sl2ODDs and their expression patterns involved in various metabolic pathways, such as gibberellin biosynthesis and catabolism, ethylene biosynthesis, steroidal glycoalkaloid biosynthesis, and flavonoid metabolism. We found that the Sl2ODD expression patterns were consistent with their functions during the tomato growth cycle. These results indicated the significance of Sl2ODDs in tomato growth and metabolism. Based on this genome-wide analysis of Sl2ODDs, we screened six potential FNSI genes using a phylogenetic tree and coexpression analysis. However, none of them exhibited FNSI activity.ConclusionsOur study provided a comprehensive understanding of the tomato 2ODD family and demonstrated the significant roles of these family members in plant metabolism. We also suggest that no FNSI genes in tomato contribute to the biosynthesis of flavones.