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

Prashant D. Sonawanea, Uwe Heiniga, Sayantan Pandaa, Netta Segal Gilboab, Meital Yonab, S. Pradeep Kumarc, Noam Alkanc, Tamar Ungerb, Samuel Bocobzaa, Margarita Plinera, Sergey Malitskya, Maria Tkacheva, Sagit Meira, Ilana Rogacheva, and Asaph Aharonia,1

aDepartment of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel; bStructural Proteomics Unit, Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 7610001, Israel; and cDepartment of Postharvest Science of Fresh Produce, Agricultural Research Organization, Volcani Center, Rishon LeZion 7505101, Israel

Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved May 4, 2018 (received for review March 26, 2018) Thousands of specialized, steroidal metabolites are found in a wide As with SGAs, steroidal saponins can be either saturated (e.g., spectrum of plants. These include the steroidal glycoalkaloids sarasapogenin) or unsaturated (e.g., diosgenin) in the C-5,6 posi- (SGAs), produced primarily by most species of the genus Solanum, tion (6) (SI Appendix,Fig.S2). and metabolites belonging to the steroidal saponins class that are Cholesterol serves as the precursor for the biosynthesis of widespread throughout the plant kingdom. SGAs play a protective SGAs (8). Recent studies in the tomato and potato plants role in plants and have potent activity in mammals, including anti- reported on GLYCOALKALOID METABOLISM (GAME) nutritional effects in humans. The presence or absence of the double genes in the core SGA biosynthesis pathway (7, 9–12). The SGA bond at the C-5,6 position (unsaturated and saturated, respectively) biosynthetic pathway can be divided into two main parts. In the creates vast structural diversity within this metabolite class and de- first part, several GAME form unsaturated SA agly- termines the degree of SGA toxicity. For many years, the elimination cones from cholesterol (9, 12). The second part results in the of the double bond from unsaturated SGAs was presumed to occur generation of glycosylated SAs (i.e., SGAs) through the action of through a single hydrogenation step. In contrast to this prior assump- different UDP-glycosyltransferases (7, 10). tion, here, we show that the tomato GLYCOALKALOID METABOLISM25 Dehydrotomatidine (tomatidenol), solanidine, and solasodine (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first are the first unsaturated SA aglycones formed in the SGA of three prospective reactions required to reduce the C-5,6 double pathway of the cultivated tomato, potato, and eggplant, re- bond in dehydrotomatidine to form tomatidine. The recombinant spectively (Fig. 1 and SI Appendix, Fig. S1). These are then β GAME25 displayed 3 -hydroxysteroid dehydrogenase/ further glycosylated to produce diverse unsaturated SGAs (e.g., Δ5,4 activity not only on diverse steroidal alkaloid agly- dehydrotomatine in tomato, α-chaconine and α-solanine in cul- cone substrates but also on steroidal saponin aglycones. Notably, α α GAME25 tivated potato, and -solamargine and -solasonine in cultivated down-regulation rerouted the entire tomato SGA reper- eggplant) (Fig. 1 and SI Appendix, Fig. S1). In the tomato plant, toire toward the dehydro-SGAs branch rather than forming the dehydrotomatidine is further hydrogenated at the C-5,6 position typically abundant saturated α-tomatine derivatives. Overexpress- ing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expres- Significance sion in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates Plants synthesize a vast repertoire of steroidal specialized how a single scaffold modification of steroidal metabolites in metabolites. These include the well-known class of antinutri- plants results in extensive structural diversity and modulation of tional steroidal glycoalkaloids (SGAs), which act as defensive toxicity. chemicals in the Solanaceae, and the pharmacologically im- portant and widespread steroidal saponins. Here, we uncover steroidal glycoalkaloids | specialized metabolism | structural diversity | an elusive enzymatic step that acts on unsaturated steroidal antinutritional | tomato metabolites. We find that GLYCOALKALOID METABOLISM25 (GAME25) acts at a key branch point in the biosynthesis path- ways of steroidal specialized metabolites. The activity of teroidal glycoalkaloids (SGAs) are nitrogen-containing spe- GAME25 not only affects the enormous diversity of SGAs and cialized metabolites present in numerous members of the S steroidal saponins, which are produced in hundreds of plant Solanaceae family. Some well-known representatives of this class species, but also modulates the molecules’ toxic effects. This include α-tomatine and dehydrotomatine in the tomato (Sola- work helps explain the extensive structural diversity in spe- num lycopersicum), α-chaconine and α-solanine in the cultivated cialized metabolism through a relatively simple chemical potato (Solanum tuberosum), and α-solamargine and α-solasonine modification in a single metabolite backbone. in the cultivated eggplant (Solanum melongena)(Fig.1andSI

Appendix, Fig. S1). SGAs play a protective role against a wide Author contributions: P.D.S. and A.A. designed research; P.D.S. and M.P. performed re- range of plant pathogens and predators, including bacteria, fungi, search; N.S.G., M.Y., and T.U. performed protein expression and purification; N.S.G., M.Y., PLANT BIOLOGY oomycetes, viruses, insects, and animals (1–4). While beneficial S.P.K., N.A., and T.U. contributed new reagents/analytic tools; P.D.S., U.H., S.P., S.B., for the plant species that produce them, SGAs are considered S. Malitsky, M.T., S. Meir, and I.R. analyzed data; and P.D.S., U.H., and A.A. wrote the paper. antinutritional and toxic to humans (5–7). SGAs are known for The authors declare no conflict of interest. their enormous structural diversity, mainly based on the structural This article is a PNAS Direct Submission. variations of the steroidal alkaloid (SA) aglycone, which is either Published under the PNAS license. unsaturated (presence of C-5,6 double bond) or saturated (ab- 1To whom correspondence should be addressed. Email: [email protected]. sence of C-5,6 double bond) (Fig. 1 and SI Appendix,Fig.S1). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. addition to SGAs, many plants, including Solanum species and 1073/pnas.1804835115/-/DCSupplemental. monocots, also produce cholesterol-derived steroidal saponins (6). Published online May 21, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1804835115 PNAS | vol. 115 | no. 23 | E5419–E5428 Downloaded by guest on September 25, 2021 A Hydrogenase ? GAME6/8/11/4/12 GAME25

Cholesterol Dehydrotomatidine Tomatidine (tomatidenol)

GAME1/17/18/2 GAME1/17/18/2

Dehydrotomatine α-tomatine

Dehydroesculeoside A or Esculeoside A or Fig. 1. The biosynthetic pathway for SGAs in to- Dehydro-lycoperoside H/G lycoperoside G/F mato, cultivated eggplant, and other Solanum spe- cies. (A) In the tomato plant, the conversion of B Hydrogenase ? dehydrotomatidine to tomatidine was previously GAME6/8/11/4/12 GAME25 predicted to be a single-step reaction driven by a hypothetical hydrogenase enzyme (2, 13). SI Appen- dix, Fig. S3, provides a more detailed SGA pathway Cholesterol Solasodine Soladulcidine schematic. (B) In cultivated eggplant, solasodine, an unsaturated aglycone is glycosylated by STEROL GTs? ALKALOID GLYCOSYL (SGTs) to produce SGT1 SGT2 unsaturated α-solasonine and α-solamargine SGAs (Left). Cultivated eggplant varieties likely lack a GAME25- β-soladulcine 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 agly- Soladulcine A cone to soladulcine A and β-soladulcine (saturated SGAs) (Right). This suggests the presence of GAME25 α-solasonine α-solamargine S. dulcamara homologs in S. dulcamara (wild Solanum relative) and (wild Solanum species) other Solanum species producing saturated SGAs S. melongena (cultivated eggplant) starting from solasodine.

to form the saturated tomatidine aglycone (Fig. 1A). Tomatidine Therefore, as with SGAs, unsaturated and saturated aglycone also undergoes glycosylation to produce the saturated α-toma- forms of steroidal saponins determine the degree of structural tine (Fig. 1A). The major tomato SGAs (i.e., α-tomatine and diversity within this metabolite class. dehydrotomatine) accumulate predominantly in green tissues Notably, the unsaturated/saturated SA aglycone and steroidal (9). As the tomato fruit matures and reaches the ripe, red stage, saponin aglycone pairs differ only in their structures by the pres- α-tomatine and dehydrotomatine are mostly converted to diverse ence or absence of the double bond at the C-5,6 position (Fig. 1 saturated and unsaturated SGA derivatives and finally to escu- and SI Appendix, Figs. S1 and S2). However, the biosynthetic leosides and dehydroesculeosides, respectively (Fig. 1 and SI pathway responsible for the formation of saturated steroidal al- Appendix, Fig. S3 for detailed tomato SGA pathway). Therefore, kaloid and steroidal saponin aglycones from their unsaturated dehydrotomatidine and tomatidine are the main SA aglycones forms in Solanaceae or in any other plant family remains to be responsible for the hundreds of SGA derivatives generated in the identified. For decades, it has been hypothesized that the con- tomato plant (Fig. 1 and SI Appendix, Fig. S3). The cultivated version of dehydrotomatidine to tomatidine in the tomato plant, eggplant and potato, on the other hand, do not produce satu- and solanidine to demissidine in wild potato species, occurs by rated SGAs because the hydrogenation step at C-5,6 position elimination of the C-5,6 double bond through a single reaction does not occur (Fig. 1B and SI Appendix, Fig. S1). However, catalyzed by hypothetical hydrogenase enzyme (2, 13–15). several wild potato species (e.g., S. demissum, S. chacoense, and In this study, we present results suggesting that formation of S. commersonii) do produce saturated demissidine and its gly- saturated steroidal specialized metabolites from unsaturated ste- cosylated form, demissine, from unsaturated solanidine (SI Ap- roidal aglycone takes place in multiple steps rather than a single pendix, Fig. S1). Moreover, some wild Solanum species (e.g., step. We discovered that GLYCOALKALOID METABOLISM25 S. dulcamara) produce mostly saturated soladulcidine aglycone- (GAME25), a member of the short-chain dehydrogenase/reductase derived SGAs (e.g., soladulcine A and β-soladulcine) from un- (SDR) gene family, is involved in the first among these multiple saturated solasodine (Fig. 1B). steps, specifically, the conversion of dehydrotomatidine to toma- A main step in steroidal saponin biosynthesis is formation of tidine. Silencing of GAME25 in the tomato plant diverted SGA an unsaturated steroidal saponin aglycone (SI Appendix, Fig. S1). metabolism within the leaves and during fruit development toward The aglycone of steroidal saponins is either spirostanol (closed unsaturated dehydrotomatine-derived SGAs rather than the usual F-ring) or furostanol (open F-ring) (6). Both saponin aglycones formation of saturated α-tomatine–derived SGAs. In vitro, GAME25 undergo either glycosylation to form unsaturated saponin gly- exhibited a 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase cosides (e.g., dioscin) or hydrogenation at the C-5,6 position to activity on various unsaturated steroidal alkaloid and saponin form saturated saponin aglycones (e.g., sarasapogenin) and their aglycone substrates, but not on their glycosylated forms. Fur- corresponding glycosides (e.g., parillin) (SI Appendix, Fig. S2). thermore, overexpression of tomato GAME25 in cultivated

E5420 | www.pnas.org/cgi/doi/10.1073/pnas.1804835115 Sonawane et al. Downloaded by guest on September 25, 2021 eggplant resulted in the formation of saturated SGAs and atypi- Appendix,Fig.S8A;seeSI Appendix,Fig.S3for the detailed PNAS PLUS cally saturated steroidal saponins. Taken together, GAME25 is a tomato SGA pathway). Conversely, we observed considerable in- key enzyme in SGA and steroidal saponin metabolism, medi- creases in dehydrotomatine (∼4- to 6-fold), dehydrotomatine iso- ating a significant portion of the structural diversity of these mer 1 (∼9- to 11-fold), and dehydrotomatidine +4hexose(∼6- to natural product classes in Solanum as well as in saponin-producing 9-fold) levels compared with wild-type leaves (Fig. 2 and SI Ap- plant species. pendix,Fig.S8A). We noted reduction in α-tomatine and its downstream metabolite levels, yet, accumulation of dehydrotomatine Results and its isomers in GAME25i lines suggested that either (i) Expression of GAME25 Correlates with the Accumulation of Typical GAME25 is involved in α-tomatine biosynthesis directly from Green Tissue Steroidal Glycoalkaloids. A recent report by Cárdenas dehydrotomatine glycoside or (ii) the enzyme mediates toma- et al. (11) demonstrated that the GAME9 AP2-type transcrip- tidine biosynthesis from dehydrotomatidine (i.e., tomatidenol; tion factor is associated with the regulation of SGA biosynthesis Fig. 1). We found no accumulation of dehydrotomatidine in in the tomato and potato plants. Transcriptome analysis revealed GAME25i lines, but this SA aglycone appeared to be converted 27 genes that were up- or down-regulated in GAME9 over- to its glycosylated derivatives (e.g., dehydrotomatine and expression and silenced tomato lines, respectively. This con- dehyrotomatidine +4 hexoses) that did accumulate in leaves β cise gene set included a putative 3- -HYDROXYSTEROID (Fig. 2 and SI Appendix,Fig.S3). Rather than acting on gly- DEHYDROGENASE, a member of the SDR gene family (termed cosylated substrates (e.g., dehydrotomatine), the above findings here GAME25, Solyc01g073640). GAME25 displayed expression position GAME25 activity before the dehydrotomatine glyco- predominantly in flower buds, young leaves, and in the immature sylation steps, possibly in the conversion of dehydrotomatidine to green stage (skin and flesh) of fruit development (SI Appendix,Fig. tomatidine. S4). This expression pattern highly resembled the profile of tomato α SGAs (e.g., -tomatine and dehydrotomatine), which accumulate in GAME25 Silencing Results in Gradual Loss of Saturated SGAs Within the green tissues of the plant (9). Furthermore, the reduced tran- the Developing and Ripening Tomato Fruit. We compared the SGA script levels of GAME25 during later stages of fruit development profile of wild-type and GAME25i tomato fruit through different α correlated with a reduction in -tomatine and dehydrotomatine stages of fruit development and ripening. During the transition content during fruit maturation (SI Appendix,Figs.S3andS4). The from green to red fruit, α-tomatine is typically converted to GAME25 expression pattern during tomato fruit maturation was saturated SGAs (esculeosides and lycoperosides), while dehy- similartothatobservedinwildtomatoaccessions(SI Appendix,Fig. drotomatine is converted to dehydroesculeosides and dehy- A S5 ). The known function of some SDR family members in spe- drolycoperosides (unsaturated minor SGAs) (see SI Appendix, cialized metabolism (16, 17) and the expression profile results showed Fig. S3 for the detailed tomato SGA pathway). As found in here suggest that GAME25 might be involved in SGA metabolism in leaves (Fig. 2), GAME25i green fruit displayed a drastic re- the tomato plant. duction in α-tomatine (∼15- to 25-fold), hydroxytomatine (∼100- SDRs represent one of the largest and most diverse NAD(P) fold), and further α-tomatine–derived downstream SGA levels (H)-dependent enzyme superfamilies that have evolved in plants compared with wild-type green fruit (Fig. 3A and SI Appendix, and were recently categorized into 49 subfamilies (16). The α 259-aa GAME25 protein sequence shows the characteristics of a Fig. S8B). Thus, due to GAME25 silencing, -tomatine and its classical SDR family member, containing the TGxxxGxG downstream saturated SGA intermediates were severely affected in green fruit tissue. In contrast, various unsaturated SGAs including and the YxxxK catalytic motif (17, 18) (SI Appendix, ∼ Fig. S5B). Phylogenetic analysis showed that GAME25 homologs dehydrotomatine ( 10- to 12-fold) and hydroxy-dehydrotomatine of certain Solanaceae species (i.e., tomato, potato, and Solanum pennellii) formed a subclade distinct from other plant SDRs (SI Appendix,Fig.S6). The closest subclade to the GAME25 pro- teins in the phylogenic tree contained the 3β-hydroxysteroid dehydrogenase homologs from tomato and Solanum pennellii (3-βHSD, ∼90% amino acid identity with GAME25 subclade proteins), the function of which is unknown. Phylogenetic analysis also showed no homolog for the GAME25 protein in eggplant or capsicum (SI Appendix,Fig.S6). Moreover, GAME25 proteins were clearly separated from the Digitalis lanata 3-βHSD protein (∼75% amino acid identity with the GAME25 proteins), which is involved in the removal of the C-5,6 double bond from steroid derivatives during progesterone and cardenolide biosynthesis (SI Appendix,Fig.S6). The clear separation of the GAME25 subclade suggested a unique catalytic activity of these enzymes that is most likely different from the closely related 3-βHSD subclade mem- bers (SI Appendix,Fig.S6and Dataset S1).

Tomato SGA Metabolism Is Rerouted from the Native, Predominantly Saturated α-Tomatine Branch to the Unsaturated Dehydrotomatine PLANT BIOLOGY Branch in GAME25-Silenced Leaves. To determine the role of GAME25 in SGA metabolism, we silenced GAME25 in the to- mato plant (i.e., GAME25i lines). GAME25 transcript levels were significantly reduced in GAME25i plant leaves and fruit at Fig. 2. GAME25 silencing in tomato leaves shifts the SGA pathway to the unsaturated dehydrotomatine branch. SGA levels in leaves of wild-type three developmental stages (green, breaker, and red ripe fruit) (nontransformed) and three independent GAME25-RNAi transgenic to- (t test, *P value < 0.05) (SI Appendix,Fig.S7). Notably, GAME25i – α ∼ mato lines (#2, #3, and #4), as determined by LC MS. The values represent leaves showed a substantial decline in -tomatine ( 2.5- to 3-fold), the means of three biological replicates ±SE (per genotype). Asterisks in- hydroxytomatine (∼6- to 10-fold), and acetoxytomatine (∼2- to dicate significant changes from wild-type samples calculated by a Student’s 3.5-fold) levels compared with wild-type leaves (Fig. 2 and SI t test (*P value < 0.05; **P value < 0.01; ***P value < 0.001).

Sonawane et al. PNAS | vol. 115 | no. 23 | E5421 Downloaded by guest on September 25, 2021 Fig. 3. Green and red stage fruit of GAME25-si- lenced 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)sat- urated SGAs (esculeoside A and derivatives) and (D) unsaturated SGAs (dehydroesculeoside A and deriva- tives) 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.

(∼25-fold) were increased in GAME25i compared with the wild- red fruit) of transgenic tomato lines was significantly higher than type tomato green fruit (Fig. 3B and SI Appendix, Fig. S8B). in wild-type tomato plants (t test, **P value < 0.01) (SI Appendix, These data suggest that GAME25 silencing in the green tomato Fig. S10A). Leaves from GAME25-Ox lines showed higher levels fruit results in redirection of biosynthesis toward unsaturated of α-tomatine (∼1.5-fold), α-tomatine (isomer 2) (∼1.5-fold), and dehydro-SGAs (see SI Appendix, Fig. S3 for detailed tomato acetoxytomatine (∼1.8-fold), with simultaneous reduction of SGA pathway). dehydrotomatine (∼1.5-fold), compared with wild-type leaves (SI The metabolic shift from the α-tomatine–derived saturated Appendix, Fig. S10 B and C). GAME25-Ox green tomato fruit SGA branch to the unsaturated dehydro-SGAs pathway further displayed reduction in dehydrotomatine levels (∼1.5-fold), whereas continued in the GAME25i breaker fruit stage. SGA metabolites no change in α-tomatine content was observed in the same tissues that accumulated following GAME25 silencing but not in wild- (SI Appendix,Fig.S10D). However, we detected increases in ace- type fruit included hydroxy-dehydrotomatine (∼20- to 30-fold), toxytomatine (∼1.9-fold) and acetoxy-hydroxytomatine (∼4- to 7- acetoxy-hydroxy-dehydrotomatine (∼20- to 25-fold), as well as fold) (α-tomatine–derived SGAs) in comparison with wild-type dehydroesculeoside A (∼20- to 25-fold) and its derivatives (SI green fruit (SI Appendix,Fig.S10D;seeSI Appendix,Fig.S3for Appendix, Figs. S8C and S9A). α-Tomatine (∼20- to 50-fold) and the tomato SGA pathway). Analysis of red fruit from the GAME25- its downstream SGAs were almost absent in the GAME25i Ox lines showed accumulation of α-tomatine (∼4- to 6-fold) and its breaker fruit (SI Appendix, Figs. S8C and S9B). In parallel, the downstream saturated SGAs [e.g., acetoxytomatine (∼5- to 7-fold), drastic reduction in α-tomatine levels at the green fruit stage in and acetoxy-hydroxytomatine (∼2- to 3-fold)] compared with wild- GAME25i lines resulted in a severe decline of esculeoside A and type red fruit (SI Appendix,Fig.S10E). Furthermore, GAME25 lycoperoside (∼20- to 25-fold) levels in the red ripe fruit stage overexpressing red tomato fruit did not show any change in levels of compared with levels in wild-type green fruit (Fig. 3C). More- esculeoside A (acetoxy-hydroxytomatine–derived major SGA), over, α-tomatine–derived saturated SGAs were not detected in compared with wild-type red fruit. GAME25-silenced red ripe fruit (Fig. 3C and see SI Appendix, Fig. S3 for the detailed tomato SGA pathway). Compared with Tomato GAME25 Overexpression in Cultivated Eggplant (S. melongena) wild-type green fruit, we did observe a buildup of dehydroto- Results in Newly Produced Saturated SGAs and Steroidal Saponins. matine in GAME25i green fruit that resulted in massive accu- Unlike in the tomato plant, saturated SGAs are normally absent mulation of dehydroesculeoside A (∼20- to 25-fold) and its in cultivated eggplant, suggesting the absence of GAME25 activity derivatives in red ripe fruit (Fig. 3D and SI Appendix, Fig. S8D). in this species. This is further supported by the absence of a These data provide additional evidence regarding the role of GAME25 homolog in cultivated eggplant (SI Appendix,Fig.S6). GAME25 in the conversion of dehydrotomatidine to tomatidine, In cultivated eggplant, α-solasonine, α-solamargine, and malonyl- which we hypothesize to be a bifurcating step between the sat- solamargine are the major unsaturated SGAs (with a C-5,6 double urated (α-tomatine) and unsaturated (dehydrotomatine) SGA bond) (19) derived from the solasodine aglycone (Fig. 4A, Upper). biosynthesis pathway. Moreover, cultivated eggplant also produces unsaturated furostanol- type steroidal saponin glycosides from the unsaturated furostanol- Accumulation of Saturated α-Tomatine and Its Downstream SGAs Due type saponin aglycone (Fig. 4A, Lower). To investigate the impact to GAME25 Overexpression in the Tomato Plant. To further examine of tomato GAME25 activity in cultivated eggplant, we generated the role of GAME25 in SGA biosynthesis, we generated trans- transgenic eggplant lines overexpressing the tomato GAME25 genic tomato lines overexpressing the GAME25 gene (GAME25- gene (SI Appendix,Fig.S11). Specifically, we wanted to assess Ox). GAME25 expression in leaves and fruit tissues (green and whether GAME25 can shift SGA metabolism from predominantly

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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- PLANT BIOLOGY 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 com- pounds. 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.

Sonawane et al. PNAS | vol. 115 | no. 23 | E5423 Downloaded by guest on September 25, 2021 unsaturated SGAs to saturated SGAs that are not naturally pre- analysis (Fig. 5 C, E,andF and see SI Appendix,Fig.S17for sent in this plant. In transgenic eggplant leaves, GAME25 over- MS-MS analysis of solanid-4-en-3-one standard and solanid-4-en- expression resulted in reduced levels of the unsaturated SGAs, 3-one product after GAME25 assay). Either the recombinant α-solasonine, α-solamargine, and malonyl-solamargine (Fig. 4A, tomato or potato GAME25 enzymes also successfully con- Upper) as well as of unsaturated furostanol saponin glycosides verted solasodine, the cultivated eggplant aglycone, to the putative (Fig. 4A, Lower). Conversely, we observed major accumulation of solasod-4-en-3-one compound (SI Appendix,Fig.S13E and F β-soladulcine, soladulcine A, and the saturated form of malonyl- for potato GAME25 assay and SI Appendix,Fig.S14for tomato solamargine (Fig. 4A, Upper). Both β-soladulcine and soladulcine GAME25 assay). A (lacking the C-5,6 double bond) are SGAs derived from satu- The tomato and potato recombinant GAME25 enzymes showed rated soladulcidine aglycone and are typically found in S. dulca- no activity on glycosylated SA substrates (i.e., α-tomatine, dehy- mara, a wild Solanum relative (Fig. 1B). Thus, S. dulcamara likely drotomatine, α-solanine, α-chaconine, and α-solamargine). These contains an active GAME25 homolog that mediates the formation results suggest that GAME25 catalyzes the oxidation of the 3β- of the above-mentioned saturated SGAs. Moreover, saturated hydroxyl group (3β-hydroxysteroid dehydrogenase activity) and furostanol-type steroidal saponin glycosides (Fig. 4A, Lower)were the isomerization of the double bond from the C-5,6 to the C- detected in GAME25-overexpressing eggplant lines, which are 4,5 position (3-oxosteroid Δ5,4 isomerase activity) in SA aglycone normally undetectable in cultivated eggplant. substrates to form the 3-oxo-Δ5,4 SA intermediates identified The chemical structures of unsaturated and saturated SGAs as here (tomatid-4-en-3-one, solanid-4-en-3-one, or solasod-4-en-3- well as of the steroidal saponins identified here are shown in Fig. one). Thus, GAME25 possesses a previously uncharacterized 4B. Metabolites were identified based on accurate mass-derived 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity. elemental composition and mass fragmentation pattern. Loss of C-3 sugar moieties in unsaturated SGAs leads to the formation Recombinant Tomato GAME25 Expressed in Escherichia coli Confirms of the fragment ion m/z 414.3 that corresponds to solasodine, an 3β-Hydroxysteroid Dehydrogenase and Δ5,4 Isomerase Activity. The unsaturated steroidal aglycone backbone (Fig. 4C, Upper). Fur- observed 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase ac- ther loss of the E and F ring from the solasodine backbone re- tivity of the recombinant tomato and potato GAME25 enzymes sults in the characteristic fragment ion m/z 271.2, which loses a is rather uncommon as other SDR family enzymes participating water molecule to form fragment m/z 253.19 (Fig. 4C, Upper). in specialized metabolism typically possess only 3β-hydroxyste- Due to the absence of a C-5,6 double bond in saturated SGAs, roid dehydrogenase activity (20–26). To confirm that the Δ5,4 all fragment ions showed a mass shift of plus 2 Da, i.e., m/z 416.3 isomerization observed here was a result of GAME25 activity (soladulcidine, saturated aglycone backbone), m/z 273.2, and m/z and not due to activity of an endogenous enzyme of the insect 255.2 after loss of the E/F rings and dehydration, respectively cell microsomes, we expressed tomato GAME25 in E. coli and (Fig. 4C, Upper). Similarly, unsaturated furostanol-type steroidal purified the enzyme for activity assays (SI Appendix, Fig. S15). saponins showed aglycone fragment ions with a mass of m/z Enzyme assay with the recombinant GAME25 enzyme using + 415.3, 271.2, and 253.19, whereas saturated furostanol-type ste- solanidine as a and NAD as a cofactor resulted in the roidal saponins displayed m/z 417.3, 273.2, and 255.2 fragment formation of the same solanid-4-en-3-one product that we ob- ions after MS-fragmentation analysis (Fig. 4C, Lower). served in the insect cell enzyme assay (SI Appendix, Fig. S16A). Insect Cells Expressing GAME25 Convert Dehydrotomatidine to We confirmed the identity of the product by comparing retention Tomatid-4-En-3-One. We examined the potential role of GAME25 time, mass spectrum, and MS-MS fragments with an authentic in the conversion of dehydrotomatidine to tomatidine by express- solanid-4-en-3-one standard (SI Appendix, Fig. S17). Thus, this ing either the recombinant tomato or potato enzymes in Sf9 insect result provided substantial evidence that the recombinant GAME25 β Δ5,4 cells and testing microsomal fractions for their activity (SI Ap- enzymes possess both 3 -hydroxysteroid dehydrogenase and pendix,Fig.S12). We performed enzymatic assays in the presence isomerase activities. + of NAD as a cofactor and with dehydrotomatidine, solanidine, and solasodine (unsaturated SA aglycones) as substrates. Surpris- The Recombinant Tomato GAME25 Converts Diosgenin, a Spirostanol- ingly, assays with either enzyme did not result in the formation of Type Saponin Aglycone, to Diosgen-4-En-3-One. Like SGAs, steroidal the expected reaction products, tomatidine, demissidine, or sol- saponins display two structural forms, saturated or unsaturated adulcidine (saturated SA aglycones). However, an assay with each C-5,6 (SI Appendix,Fig.S2). Our observation that new saturated recombinant GAME25 enzyme (either tomato or potato) and furostanol-type saponins were formed as a result of GAME25 dehydrotomatidine resulted in the formation of a compound with overexpression in cultivated eggplant suggested the potential role + the mass m/z 412.3 (M + H )(Fig.5A and B for tomato of GAME25 in elimination of the C-5,6 double bond, not only GAME25 assay and SI Appendix,Fig.S13A and B for the potato from SA substrates but also from steroidal saponins. To examine GAME25 assay). MS-MS fragmentation pattern analysis of the this possibility, we performed assays with the recombinant enzyme + + newly formed compounds (Fig. 5 A, B,andD) showed three major and diosgenin [(M H , m/z 415.3), a major spirostanol-type fragment ions derived from two parallel fragmentation routes. Loss steroidal saponin aglycone produced by Dioscorea species]. In- terestingly, GAME25 activity resulted in the formation of a novel of the carbonyl oxygen and formation of an additional double bond + led to a fragment ion with m/z 394.3 (Fig. 5D). Loss of the E and F compound with the molecular ion m/z 413.3 (M + H ), repre- rings of the steroidal skeleton led to the fragment ion m/z 269.2, senting oxidation of the 3β-hydroxyl group and isomerization of which was then dehydrated to form fragment m/z 251.17 (Fig. 5D). the double bond from the C-5,6 position to the C-4,5 position (SI The newly formed compound was putatively assigned as tomatid-4- Appendix,Fig.S18A). While the unsaturated diosgenin substrate en-3-one (Fig. 5 A and B). produced three major fragment ions with m/z 415.3, 271.2, and Using solanidine as a substrate, the GAME25 enzyme assays 253.2, the newly formed compound (m/z 413.3) showed fragment (either with tomato or potato GAME25 enzymes) resulted in the ions with m/z 413.3, 269.2, and 251.2, respectively (SI Appendix, formation of a new product with an apparent molecular ion of Fig. S18B) and thus putatively was assigned as diosgen-4-en-3-one + m/z 396.3 (M + H )(Fig.5C, E,andF for the tomato GAME25 based on mass fragmentation spectra analysis (SI Appendix, Fig. assay and SI Appendix,Fig.S13C and D for the potato GAME25 S18). Our results suggest that recombinant GAME25 can catalyze assay). We identified the compound as solanid-4-en-3-one by the oxidation of the 3β-hydroxyl group and the isomerization of comparing retention time and mass spectrum to the authentic, the double bond from the C-5,6 to the C-4,5 position in steroidal commercially available, solanid-4-en-3-one standard and MS-MS saponin aglycones to form the 3-oxo-Δ5,4 saponin intermediate.

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+ + 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 PLANT BIOLOGY 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.

Sonawane et al. PNAS | vol. 115 | no. 23 | E5425 Downloaded by guest on September 25, 2021 The Presence of the C-5,6 Double Bond in SGAs Inhibits Fungal Growth formation of both tomatidine (through the action of a hypothetical and Pathogenicity. α-Tomatine in green tomato tissues is known hydrogenase that reduces the double bond) and dehydrotomatidine to affect the growth of pathogenic fungi, including Botrytis cinerea, (1, 2, 14). In a third hypothesis, tomatidine was proposed to be Fusarium oxysporum,andColletotrichum gloeosporioides (2). In partly dehydrogenated to form dehydrotomatidine by a hypothet- contrast, the role of unsaturated dehydrotomatine in phyto- ical dehydrogenase (1, 2). Finally, the formation of tomatidine pathogenicity has not been previously examined primarily be- from dehydrotomatidine was hypothesized as a single-step hy- cause it is typically produced in small amounts in tomato tissues. drogenation reaction (2, 13, 14). In the present study, functional As silencing of GAME25 in the tomato plant redirected SGA characterization of GAME25 provided strong evidence that the metabolism toward formation of dehydro-SGAs, we examined the formation of tomatidine from dehydrotomatidine [i.e., the re- effects of dehydro-SGAs on fungal growth and pathogenicity. duction of the Δ5 (C-5,6 position) bond in the SA aglycones] is Analysis of GAME25i leaf extracts showed mycelial growth in- likely carried out in multiple steps and that the GAME25 cata- hibition of the pathogenic fungi C. gloeosporioides and B. cinerea lyzes the first of these. In vitro enzyme assays with the recombi- compared with wild-type saturated SGA-containing extracts (SI nant tomato and potato GAME25 enzyme demonstrated the Appendix, Fig. S19 A, B, E, and F). In addition, C. gloeosporioides enzyme’s dual activity, namely, oxidation of the 3β-hydroxyl group and B. cinerea fungal conidia germination was severely reduced (3β-hydroxysteroid dehydrogenase activity) and isomerization of upon treatment with GAME25i extracts compared with treatment the double bond from the C-5,6 position to the C-4,5 position with wild-type extracts (SI Appendix,Fig.S19C, D,andG–J). (3-oxosteroid Δ5,4 isomerase activity) on both SA and steroidal saponin aglycones (Fig. 5 and SI Appendix, Figs. S13, S14, and S18). Discussion These results also suggest that formation of the saturated steroidal GAME25 Is a Key Branch Point Enzyme That Determines the Diversity saponin aglycone (by removal of the C-5,6 double bond) is likely of SGAs Produced in Solanum Species and Modulates Their Level of not a single-step reaction, but rather that GAME25 catalyzes the Toxicity. The presence or absence of a double bond at the C- first step, as we observed for SA biosynthesis. 5,6 position within the core SGA scaffold is a major source of In plants, SDR enzymes catalyze NAD(P)(H)-dependent ox- structural diversity among SGAs produced by Solanum species. idation/reduction reactions involving a wide range of primary or In tomato, both dehydrotomatidine and tomatidine SA agly- specialized metabolites (16–18, 20, 23–26). Members of this cones are highly toxic to plant cells, and it is therefore likely that family have been reported to participate in the metabolism of they undergo glycosylation to prevent self-toxicity (9). Studies in various specialized metabolites including cardiac glycosides (i.e., animal models demonstrated that SGAs lacking the C-5,6 double cardenolides) in Digitalis spp. (3-βHSD), tropane-like alkaloids bond (e.g., α-tomatine) are much less toxic to animals and hu- (SDR65C), terpenoids (SDR110C, SDR114C), benzylisoquino- mans compared with those SGAs that contain this double bond line alkaloids in poppy (NOS), oryzalexin diterpenoids in rice (e.g., α-chaconine and α-solanine from potato) (2). These prior (MSI and MI1-3), and phenolics (SDR108E) (20, 23–26). GAME25 findings also suggest that dehydrotomatine is likely a more toxic is a unique plant SDR enzyme that exhibits dual activity, with the SGA compared with α-tomatine. Although less toxic to humans ability to oxidize the 3β-hydroxyl group (3β-hydroxysteroid de- and animals, α-tomatine is a highly active molecule involved in a hydrogenase activity) and isomerization of the double bond from the range of host-plant resistance mechanisms in tomato plants (2, 4, C-5,6 to the C-4,5 position (3-oxosteroid Δ5,4 isomerase activity) in 5). However, the contribution of dehydrotomatine, typically steroidal substrates. Most, if not all, 3-βHSD enzymes participating produced at lower levels in tomato, to plant resistance against in specialized metabolism in plants (i.e., members of the 3-βHSD pathogens remains unclear. In the present study, severe growth and SDR family) merely possess 3β-hydroxysteroid dehydrogenase and conidia germination inhibition of the pathogenic fungi B. activity and not Δ5,4 isomerase activity (21–25). For example, in cinerea and C. gloeosporioides by extracts enriched with dehydro- Digitalis, the oxidation (of the 3β-hydroxyl group) and isomerization derivatives (due to GAME25 silencing) suggest enhanced toxicity (C-5,6 to the C-4,5 position) steps, required during the conversion of of these compounds compared with wild-type samples containing pregnenolone to progesterone, are carried out successively by two mainly saturated α-tomatine and related metabolites (SI Appendix, separate enzymes, 3-βHSD (3β-hydroxysteroid dehydrogenase) and Fig. S19). Thus, in Solanum plants, α-tomatine and dehydroto- 3-KSI (Δ5-3-ketosteroid isomerase) (20–22). Phylogenetic analysis matine SGAs may act synergistically against pathogens and might of SDR family proteins from plants involved in specialized me- have coevolved to exert a combined effect against a broad range of tabolism suggested that the GAME25 proteins of the Solanaceae disease-causing pathogens. As demonstrated here, GAME25 cat- family have undergone significant diversification compared with alyzes the first step in the conversion of dehydrotomatidine to other SDR proteins. Moreover, the dual enzyme activity of tomatidine in which the double bond at the C-5,6 position is re- GAME25 proteins reported here suggests that it could have duced. This reaction, of the C-5,6 double bond removal in SA evolved from the classical monofunctional SDRs. Interestingly, in aglycones, is therefore a key branch point that not only determines mammalian steroid hormone metabolism, the conversion of the diversity of SGAs produced in hundreds of Solanum species pregnenolone to progesterone includes a 3-βHSD enzyme that, but also modulates the toxic effects of this metabolite class to the similar to GAME25, possesses dual dehydrogenase and isomerase plant itself, other animals, and likely also as a plant defense against activities (27, 28). pathogens and herbivores. The Absence of GAME25 Activity in Cultivated Potato and Eggplant Production of Tomatidine from Dehydrotomatidine in the Tomato Underlies the Lack of Saturated SGAs in These Plants. The pathway Plant Involves a Yet-Unknown Plant SDR-Type GAME25 Enzyme from the unsaturated SA aglycone solanidine to the saturated SA Activity. To date, the biosynthesis of dehydrotomatine and aglycone demissidine and its glycosylated form (i.e., demissine) in α-tomatine was hypothesized to occur through several different wild potato species corresponds to the tomato pathway in which pathways (1, 2, 14). In one scenario, dehydrotomatidine was the C-5,6 double bond is eliminated from dehydrotomatidine to- proposed to be derived from cholesterol (contains a C-5,6 double ward tomatidine and glycosylated α-tomatine (SI Appendix,Figs. bond), while tomatidine was predicted to be synthesized from S1 and S3). The domesticated potato does not accumulate satu- cholestanol (lacking the C-5,6 double bond) (1, 2). Thus, con- rated demissidine or demissine SGAs. The presence of a GAME25 version of cholesterol to cholestanol was thought to be re- homolog (SI Appendix,Fig.S6) but the absence of saturated SGAs sponsible for the formation of tomatidine. An alternative in cultivated potato tubers suggests that these SGAs were lost suggested pathway is that the cholesterol-derived teneimine in- during the domestication process, possibly through altered GAME25 termediate (possessing a double bond) is partitioned, leading to gene activity. Moreover, in vitro, the recombinant potato GAME25

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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 un- saturated 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-Δ5,4 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.

enzyme shows 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase reductase (5β-POR), (iv) which is then converted to pregnanolone activity on diverse unsaturated steroidal metabolites (SI Appendix, by a 3βHSD enzyme (Fig. 6) (20–22, 27, 28). Thus, the conversion Fig. S13). Therefore, even if GAME25 is considered active in of pregnenolone to pregnanolone resembles the formation of potato (in vivo), additional enzymes required for the formation of tomatidine from dehydrotomatidine in tomato, or demissidine from saturated SA aglycone might be absent or inactive in the domes- solanidine in wild potato plants, in which the C-5,6 double bond ticated potato. In the case of the cultivated eggplant, it is likely that is also removed (Fig. 6). Assays assessing recombinant GAME25 GAME25 activity is the single factor that is responsible for the lack activity clearly showed that GAME25 is not sufficient to catalyze of saturated SGA production, as overexpression of tomato GAME25 the entire Δ5 reduction in unsaturated SA aglycones and that resulted in accumulation of saturated SGAs (Fig. 4). In addition additional enzymes are required to eliminate the C-5,6 double to saturated SGAs, novel saturated steroidal saponins were formed bond and form saturated products. The additional enzymatic steps in GAME25-Ox eggplant transgenic lines, underscoring GAME25’s required after GAME25 activity resemble the Digitalis (iii)and crucial role in steroidal saponin biosynthesis in Solanum plants (Fig. (iv) reactions described above. Thus, we expected that the Digitalis 4). Thus, GAME25-like enzymes might be involved in eliminating 5β-POR reductase homolog in tomato (Solyc10g049620, termed the C-5,6 double bond from unsaturated steroidal saponin aglycone here GAME35) might act downstream to the pair of reactions substrates in numerous plant species, including those outside the catalyzed by GAME25. In this case, Digitalis 5β-POR would cat- Solanaceae family. alyze reduction of various 3-oxo-Δ5,4 SA aglycone intermediates (GAME25 enzyme products; e.g., solanid-4-en-3-one). Enzyme The Pathway to Saturated Steroidal Specialized Metabolites in assays with the purified recombinant GAME35 protein (homolog Solanum and Other Species. During cardenolide biosynthesis in of the Digitalis 5β-POR) showed that it is not active on the solanid- Digitalis species, pregnenolone is converted to pregnanolone. 4-en-3-one substrate (SI Appendix, Figs. S15 and S16B). Thus, a PLANT BIOLOGY Both pregnenolone and pregnanolone are steroid derivatives different reductase enzyme is likely required to carry out this that differ only by the presence or absence of the double bond at second removal of C-4,5 bond reaction. the C-5,6 position (Fig. 6). The conversion of pregnenolone to Based on the intermediates produced by GAME25 and the pregnanolone (removal of C-5,6 double bond) occurs in the four further requirement of analogous enzymatic reactions, we propose following steps: (i) oxidation of (3β-hydroxyl group) pregneno- that the conversion of dehydrotomatidine to tomatidine, and like- lone by the 3βHSD enzyme, followed by (ii) isomerization of the wise the conversion of other SAs, requires a three-step reaction double bond from the C-5,6 to the C-4,5 position by the 3-KSI sequence with GAME25 catalyzing the first step, converting dehy- enzyme to form progesterone; (iii) conversion of progesterone to drotomatidine to tomatid-4-en-3-one (Fig. 6). Tomatid-4-en-3-one 5β-pregnan-3,20-dione (removal of C-4,5 bond) by 5β-progesterone is subsequently reduced to tomatidine by the successive action of a

Sonawane et al. PNAS | vol. 115 | no. 23 | E5427 Downloaded by guest on September 25, 2021 putative 5-reductase and an aldo-keto reductase, which remain to evidence suggests its relevance in determining the level of toxicity be identified (Fig. 6). We also predict a similar three-step conver- of these molecules to humans. SGAs, primarily the unsaturated sion in wild potato species producing saturated demissidine from ones prevalent in potato tubers, are renowned antinutritionals, and the solanidine aglycone (Fig. 6). In addition, tomato GAME25 their levels in the cultivated potato are tightly regulated. The current overexpression in cultivated eggplant generated saturated SGAs work has implications for commercial farming. Together with the naturally produced by certain Solanum species (e.g., S. dulcamara). previously reported structural and regulatory genes, overexpression Based on these findings, we anticipate similar reactions in Solanum of GAME25 could be a valuable strategy to reduce the levels of species that produce a saturated soladulcidine aglycone from sol- these substances in commercial potato varieties. asodine (e.g., S. dulcamara; Fig. 6). Our in vivo and in vitro results support a multireaction sequence in the biosynthesis of saturated Materials and Methods steroidal saponins (Fig. 6). Hence, evolution of structural diversity Plant Extract Preparation and Targeted Profiling of Steroidal Metabolites. in steroidal alkaloids and saponins by elimination of the C- Preparation of plant extracts and the profiling of steroidal metabolites in 5,6 double bond and its underlying enzymatic base is likely con- various tomato (leaves, green fruit, breaker fruit, and red fruit) and eggplant served in a wide range of plant families that are rich in steroidal leaf tissue were performed as described previously (9, 11). Detailed liquid specialized metabolites. chromatography–mass spectrometry (LC–MS) methods are provided in SI Characterization of GAME25 activity in the SGAs biosynthesis Appendix, SI Materials and Methods. pathway is a significant step toward resolving the entire core SGA pathway in Solanaceae species. This work further contributes to Protein Expression and in Vitro Enzyme Assay. The detailed steps for tomato/ the understanding of how a large portion of structural diversity in potato GAME25 and tomato GAME35 protein expression and recombinant SGAs and steroidal saponin-producing species is generated. protein enzyme assay are in SI Appendix, SI Materials and Methods. Nevertheless, the enzymes completing the elimination of the C-5,6 double bond succeeding GAME25 still remain to be identified. Fungal Inhibition Assay. B. cinerea (B05.10) and C. gloeosporioides (Cg14) fungal inhibition activity of the GAME25i and wild-type methanolic extracts The dramatic shift from saturated to unsaturated SGAs in GAME25- was determined by the disk diffusion method (9). Details of the fungal in- silenced tomato plants, including the dominance of dehydro- hibition assay are in SI Appendix, SI Materials and Methods. esculeosides in ripening fruit, make this genetic material an excellent resource for future investigation. Further genetic and biochemical ACKNOWLEDGMENTS. We thank the Adelis Foundation; the Leona M. and analyses will enable linking the structural information on steroidal Harry B. Helmsley Charitable Trust; the Jeanne and Joseph Nissim Foundation specialized metabolites to the potency of these molecules with for Life Sciences; the Tom and Sondra Rykoff Family Foundation Research; and respect to plant pathogens and herbivores. Ripe fruit accumulating the Raymond Burton Plant Genome Research Fund for supporting the A.A. α-tomatine (instead of the typical esculeosides) as a result of GAME25 laboratory activity. The work also was supported by Israel Science Foundation Grant 1805/15 and European Research Council (SAMIT-FP7) personal grants (to overexpression will also be of value for carrying out similar in- A.A.). The research in the A.A. laboratory was supported by the European teraction studies. The presence of the double bond at the C-5,6 Union Seventh Framework Program FP7/2007–2013 Grant 613692–TriForC. A.A. position is not merely an issue of structural variation as ample is the incumbent of the Peter J. Cohn Professorial Chair.

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