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

Abstract

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.

Keywords: antinutritional; specialized metabolism; steroidal glycoalkaloids; structural diversity; tomato.

Fig. 1.

The biosynthetic pathway for SGAs in tomato, cultivated eggplant, and other Solanum species. (A) In the tomato plant, the conversion of dehydrotomatidine to tomatidine was previously predicted to be a single-step reaction driven by a hypothetical hydrogenase enzyme (2, 13). SI Appendix, Fig. S3, provides a more detailed SGA pathway schematic. (B) In cultivated eggplant, solasodine, an unsaturated aglycone is glycosylated by STEROL ALKALOID GLYCOSYL TRANSFERASEs (SGTs) to produce unsaturated α-solasonine and α-solamargine SGAs (Left). Cultivated eggplant varieties likely lack a GAME25-like enzyme and therefore do not produce saturated SGAs. Some wild Solanum species (e.g., S. dulcamara) produce a saturated soladulcidine aglycone from solasodine and further glycosylate soladulcidine aglycone to soladulcine A and β-soladulcine (saturated SGAs) (Right). This suggests the presence of GAME25 homologs in S. dulcamara (wild Solanum relative) and other Solanum species producing saturated SGAs starting from solasodine.

Fig. 2.

GAME25 silencing in tomato leaves shifts the SGA pathway to the unsaturated dehydrotomatine branch. SGA levels in leaves of wild-type (nontransformed) and three independent GAME25-RNAi transgenic tomato lines (#2, #3, and #4), as determined by LC–MS. The values represent the means of three biological replicates ±SE (per genotype). Asterisks indicate significant changes from wild-type samples calculated by a Student’s t test (*P value < 0.05; **P value < 0.01; ***P value < 0.001).

Fig. 3.

Green and red stage fruit of GAME25-silenced tomato lines display substantially altered SGA metabolism. (A and B) Levels of (A) saturated α-tomatine– and (B) unsaturated dehydrotomatine-derived SGAs in green fruit of the GAME25-silenced tomato lines. (C and D) Levels of the typical (C) saturated SGAs (esculeoside A and derivatives) and (D) unsaturated SGAs (dehydroesculeoside A and derivatives) in GAME25-silenced red stage fruit compared with wild-type red fruit. The values represent means of three biological replicates ±SE (per genotype). Lines #2, #3, and #4 are three independent GAME25i lines. Asterisks indicate significant changes compared with wild-type samples, calculated by a Student’s t test (*P value < 0.05; **P value < 0.01; ***P value < 0.001). LC–MS was used for targeted SGA profiling.

Fig. 4.

Overexpression of tomato GAME25 results in accumulation of new saturated SGAs and steroidal saponins in cultivated eggplant. (A) Comparison of SGA (Upper) and steroidal saponin (Lower) profile of wild-type (WT, nontransformed) and GAME25-overexpressing transgenic eggplant line #E1 (GAME25-ox). (B) Structures of detected SGAs and saponins. Chemical structures were putatively assigned by calculating elemental compositions from the accurate mass and interpretation of mass fragmentation patterns. Loss of water from steroidal saponins in positive ionization mode is typical for furostanol-type compounds. Presence or absence of a double bond at the C-5,6 position in SGAs and saponins is marked in red. (C) Comparison of mass fragmentation of steroidal SA and steroidal saponin aglycones. (Upper) Overlays of mass spectra of saturated SA aglycones (red) and unsaturated SA aglycones (black). (Lower) Overlays of mass spectra of saturated steroidal saponin aglycones (red) and unsaturated steroidal saponin aglycones (black). Characteristic fragment structures are depicted. The fragments following the loss of the side chain of SGAs or saponins were identical: m/z 253.19 and 271.21 (in blue) for unsaturated compounds and m/z 255.21 and 273.22 (in red) for saturated compounds, respectively. For simplicity, only #E1 is shown here as the representative transgenic line. EIC, extracted ion chromatogram; m/z, mass to charge; Gal, galactosyl; GAME25-ox, GAME25 overexpression (#E1); Glu, glucosyl; Hex, hexosyl; M, molecular mass; Rha, rhamnosyl; WT, wild type. Metabolite analysis was done by LC–MS. Lines #E1 and #E2 are two independent transgenic GAME25-Ox lines (SI Appendix, Fig. S11). Line #E2 showed a similar LC–MS profile as that observed for #E1.

Fig. 5.

Activity of recombinant tomato GAME25 produced in insect cells. (A) Overlay of extracted ion chromatograms of m/z 412.32 Da, [M + H⁺]⁺ (mass of the GAME25 reaction product), and the control reaction obtained with dehydrotomatidine as a substrate. (B) Mass spectra and structures of the detected product (Upper) and substrate (Lower) of the GAME25 enzymatic reaction with dehydrotomatidine as substrate. (C) Overlay of extracted ion chromatograms of m/z 396.32 Da, [M + H⁺]⁺ (mass of the GAME25 reaction product), and the control reaction with solanidine as substrate. (D) Mass fragmentation spectrum of the GAME25 enzymatic reaction product (with dehydrotomatidine as substrate), including the interpretation of the detected mass fragments. The fragmentation pattern corresponds to the tomatid-4-en-3-one (proposed structure of the GAME25 product). (E) Chromatograms of the GAME25 enzymatic reaction (Upper), control reaction (Middle), both with solanidine as substrate, and the solanid-4-en-3-one authentic standard (Lower). The newly formed product (at retention time 23.2 min) coeluted with the solanid-4-en-3-one commercial authentic standard. Comparison of MS-MS spectra between the newly formed product and authentic standard solanid-4-en-3-one was similar and is provided in SI Appendix, Fig. S17. Thus, this newly formed GAME25 product was assigned as solanid-4-en-3-one. (F) Mass spectra and structures of the detected product (Upper) and substrate (Lower) of the GAME25 enzymatic reaction with solanidine as substrate. Analysis of enzyme assay reactions was carried out by LC–MS. The control reaction was performed using protein extracts from nontransfected Sf9 insect cell microsomes. EIC, extracted ion chromatogram; m/z, mass to charge; STD, metabolite standard; TIC, total ion chromatogram.

Fig. 6.

GAME25 enzymes play a key role in the formation of steroidal specialized metabolites in a proposed sequence of three reactions. A proposed three-step reaction sequence for the conversion of dehydrotomatidine to tomatidine in tomato, solanidine to demissidine in wild potatoes (e.g., S. chacoense), and solasodine to soladulcidine in certain Solanum species (e.g., S. dulcamara). Given our results, we propose a three-step reaction for the conversion of unsaturated steroidal saponin aglycone to saturated steroidal saponin aglycone. GAME25, a 3β-hydroxysteroid dehydrogenase/isomerase, performs the first step in this reaction sequence, producing 3-oxo-Δ^5,4 steroidal alkaloid/saponin aglycone derivatives from the respective unsaturated steroidal alkaloid/saponin aglycone substrates, which are further converted to saturated products by successive actions of putative 5-reductases and aldo-keto reductases, respectively. This multistep conversion partly resembles steroid metabolism in species such as Digitalis spp. that produce cardiac glycosides (cardenolides). Dashed arrows indicate multistep reactions.