Short-chain dehydrogenase/reductase governs steroidal ...Short-chain dehydrogenase/reductase governs...

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Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum Prashant D. Sonawane a , Uwe Heinig a , Sayantan Panda a , Netta Segal Gilboa b , Meital Yona b , S. Pradeep Kumar c , Noam Alkan c , Tamar Unger b , Samuel Bocobza a , Margarita Pliner a , Sergey Malitsky a , Maria Tkachev a , Sagit Meir a , Ilana Rogachev a , and Asaph Aharoni a,1 a Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel; b Structural Proteomics Unit, Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 7610001, Israel; and c Department 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 spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including anti- nutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and de- termines 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 assump- tion, 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 agly- cone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA reper- toire toward the dehydro-SGAs branch rather than forming the 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- sion 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. steroidal glycoalkaloids | specialized metabolism | structural diversity | antinutritional | tomato S teroidal glycoalkaloids (SGAs) are nitrogen-containing spe- cialized metabolites present in numerous members of the Solanaceae family. Some well-known representatives of this class include α-tomatine and dehydrotomatine in the tomato (Sola- num lycopersicum), α-chaconine and α-solanine in the cultivated potato (Solanum tuberosum), and α-solamargine and α-solasonine in the cultivated eggplant (Solanum melongena) (Fig. 1 and SI Appendix, Fig. S1). SGAs play a protective role against a wide range of plant pathogens and predators, including bacteria, fungi, oomycetes, viruses, insects, and animals (14). While beneficial for the plant species that produce them, SGAs are considered antinutritional and toxic to humans (57). SGAs are known for their enormous structural diversity, mainly based on the structural variations of the steroidal alkaloid (SA) aglycone, which is either unsaturated (presence of C-5,6 double bond) or saturated (ab- sence of C-5,6 double bond) (Fig. 1 and SI Appendix, Fig. S1). In addition to SGAs, many plants, including Solanum species and monocots, also produce cholesterol-derived steroidal saponins (6). As with SGAs, steroidal saponins can be either saturated (e.g., sarasapogenin) or unsaturated (e.g., diosgenin) in the C-5,6 posi- tion (6) (SI Appendix, Fig. S2). Cholesterol serves as the precursor for the biosynthesis of SGAs (8). Recent studies in the tomato and potato plants reported on GLYCOALKALOID METABOLISM (GAME) genes in the core SGA biosynthesis pathway (7, 912). The SGA biosynthetic pathway can be divided into two main parts. In the first part, several GAME enzymes form unsaturated SA agly- cones from cholesterol (9, 12). The second part results in the generation of glycosylated SAs (i.e., SGAs) through the action of different UDP-glycosyltransferases (7, 10). Dehydrotomatidine (tomatidenol), solanidine, and solasodine are the first unsaturated SA aglycones formed in the SGA pathway of the cultivated tomato, potato, and eggplant, re- spectively (Fig. 1 and SI Appendix, Fig. S1). These are then further glycosylated to produce diverse unsaturated SGAs (e.g., dehydrotomatine in tomato, α-chaconine and α-solanine in cul- tivated potato, and α-solamargine and α-solasonine in cultivated eggplant) (Fig. 1 and SI Appendix, Fig. S1). In the tomato plant, dehydrotomatidine is further hydrogenated at the C-5,6 position Significance Plants synthesize a vast repertoire of steroidal specialized metabolites. These include the well-known class of antinutri- tional steroidal glycoalkaloids (SGAs), which act as defensive chemicals in the Solanaceae, and the pharmacologically im- portant and widespread steroidal saponins. Here, we uncover an elusive enzymatic step that acts on unsaturated steroidal 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 GAME25 not only affects the enormous diversity of SGAs and steroidal saponins, which are produced in hundreds of plant species, but also modulates the moleculestoxic effects. This work helps explain the extensive structural diversity in spe- cialized metabolism through a relatively simple chemical modification in a single metabolite backbone. Author contributions: P.D.S. and A.A. designed research; P.D.S. and M.P. performed re- search; N.S.G., M.Y., and T.U. performed protein expression and purification; N.S.G., M.Y., S.P.K., N.A., and T.U. contributed new reagents/analytic tools; P.D.S., U.H., S.P., S.B., S. Malitsky, M.T., S. Meir, and I.R. analyzed data; and P.D.S., U.H., and A.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Published under the PNAS license. 1 To whom correspondence should be addressed. Email: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1804835115/-/DCSupplemental. Published online May 21, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1804835115 PNAS | vol. 115 | no. 23 | E5419E5428 PLANT BIOLOGY PNAS PLUS Downloaded by guest on July 1, 2020

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Page 1: Short-chain dehydrogenase/reductase governs steroidal ...Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus

Short-chain dehydrogenase/reductase governssteroidal specialized metabolites structural diversityand toxicity in the genus SolanumPrashant 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 LifeSciences Core Facilities, Weizmann Institute of Science, Rehovot 7610001, Israel; and cDepartment of Postharvest Science of Fresh Produce, AgriculturalResearch 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 widespectrum 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 arewidespread throughout the plant kingdom. SGAs play a protectiverole in plants and have potent activity in mammals, including anti-nutritional effects in humans. The presence or absence of the doublebond at the C-5,6 position (unsaturated and saturated, respectively)creates vast structural diversity within this metabolite class and de-termines the degree of SGA toxicity. For many years, the eliminationof the double bond from unsaturated SGAs was presumed to occurthrough a single hydrogenation step. In contrast to this prior assump-tion, here, we show that the tomato GLYCOALKALOIDMETABOLISM25(GAME25), a short-chain dehydrogenase/reductase, catalyzes the firstof three prospective reactions required to reduce the C-5,6 doublebond in dehydrotomatidine to form tomatidine. The recombinantGAME25 enzyme displayed 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid agly-cone substrates but also on steroidal saponin aglycones. Notably,GAME25 down-regulation rerouted the entire tomato SGA reper-toire toward the dehydro-SGAs branch rather than forming thetypically abundant saturated α-tomatine derivatives. Overexpress-ing the tomato GAME25 in the tomato plant resulted in significantaccumulation of α-tomatine in ripe fruit, while heterologous expres-sion in cultivated eggplant generated saturated SGAs and atypicalsaturated steroidal saponin glycosides. This study demonstrateshow a single scaffold modification of steroidal metabolites inplants results in extensive structural diversity and modulation ofproduct toxicity.

steroidal glycoalkaloids | specialized metabolism | structural diversity |antinutritional | tomato

Steroidal glycoalkaloids (SGAs) are nitrogen-containing spe-cialized metabolites present in numerous members of the

Solanaceae family. Some well-known representatives of this classinclude α-tomatine and dehydrotomatine in the tomato (Sola-num lycopersicum), α-chaconine and α-solanine in the cultivatedpotato (Solanum tuberosum), and α-solamargine and α-solasoninein the cultivated eggplant (Solanum melongena) (Fig. 1 and SIAppendix, Fig. S1). SGAs play a protective role against a widerange of plant pathogens and predators, including bacteria, fungi,oomycetes, viruses, insects, and animals (1–4). While beneficialfor the plant species that produce them, SGAs are consideredantinutritional and toxic to humans (5–7). SGAs are known fortheir enormous structural diversity, mainly based on the structuralvariations of the steroidal alkaloid (SA) aglycone, which is eitherunsaturated (presence of C-5,6 double bond) or saturated (ab-sence of C-5,6 double bond) (Fig. 1 and SI Appendix, Fig. S1). Inaddition to SGAs, many plants, including Solanum species andmonocots, also produce cholesterol-derived steroidal saponins (6).

As with SGAs, steroidal saponins can be either saturated (e.g.,sarasapogenin) or unsaturated (e.g., diosgenin) in the C-5,6 posi-tion (6) (SI Appendix, Fig. S2).Cholesterol serves as the precursor for the biosynthesis of

SGAs (8). Recent studies in the tomato and potato plantsreported on GLYCOALKALOID METABOLISM (GAME)genes in the core SGA biosynthesis pathway (7, 9–12). The SGAbiosynthetic pathway can be divided into two main parts. In thefirst part, several GAME enzymes form unsaturated SA agly-cones from cholesterol (9, 12). The second part results in thegeneration of glycosylated SAs (i.e., SGAs) through the action ofdifferent UDP-glycosyltransferases (7, 10).Dehydrotomatidine (tomatidenol), solanidine, and solasodine

are the first unsaturated SA aglycones formed in the SGApathway of the cultivated tomato, potato, and eggplant, re-spectively (Fig. 1 and SI Appendix, Fig. S1). These are thenfurther glycosylated to produce diverse unsaturated SGAs (e.g.,dehydrotomatine in tomato, α-chaconine and α-solanine in cul-tivated potato, and α-solamargine and α-solasonine in cultivatedeggplant) (Fig. 1 and SI Appendix, Fig. S1). In the tomato plant,dehydrotomatidine is further hydrogenated at the C-5,6 position

Significance

Plants synthesize a vast repertoire of steroidal specializedmetabolites. These include the well-known class of antinutri-tional steroidal glycoalkaloids (SGAs), which act as defensivechemicals in the Solanaceae, and the pharmacologically im-portant and widespread steroidal saponins. Here, we uncoveran elusive enzymatic step that acts on unsaturated steroidalmetabolites. We find that GLYCOALKALOID METABOLISM25(GAME25) acts at a key branch point in the biosynthesis path-ways of steroidal specialized metabolites. The activity ofGAME25 not only affects the enormous diversity of SGAs andsteroidal saponins, which are produced in hundreds of plantspecies, but also modulates the molecules’ toxic effects. Thiswork helps explain the extensive structural diversity in spe-cialized metabolism through a relatively simple chemicalmodification in a single metabolite backbone.

Author contributions: P.D.S. and A.A. designed research; P.D.S. and M.P. performed re-search; N.S.G., M.Y., and T.U. performed protein expression and purification; N.S.G., M.Y.,S.P.K., N.A., and T.U. contributed new reagents/analytic tools; P.D.S., U.H., S.P., S.B.,S. Malitsky, M.T., S. Meir, and I.R. analyzed data; and P.D.S., U.H., and A.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1804835115/-/DCSupplemental.

Published online May 21, 2018.

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to form the saturated tomatidine aglycone (Fig. 1A). Tomatidinealso undergoes glycosylation to produce the saturated α-toma-tine (Fig. 1A). The major tomato SGAs (i.e., α-tomatine anddehydrotomatine) accumulate predominantly in green tissues(9). As the tomato fruit matures and reaches the ripe, red stage,α-tomatine and dehydrotomatine are mostly converted to diversesaturated and unsaturated SGA derivatives and finally to escu-leosides and dehydroesculeosides, respectively (Fig. 1 and SIAppendix, Fig. S3 for detailed tomato SGA pathway). Therefore,dehydrotomatidine and tomatidine are the main SA aglyconesresponsible for the hundreds of SGA derivatives generated in thetomato plant (Fig. 1 and SI Appendix, Fig. S3). The cultivatedeggplant and potato, on the other hand, do not produce satu-rated SGAs because the hydrogenation step at C-5,6 positiondoes not occur (Fig. 1B and SI Appendix, Fig. S1). However,several wild potato species (e.g., S. demissum, S. chacoense, andS. commersonii) do produce saturated demissidine and its gly-cosylated form, demissine, from unsaturated solanidine (SI Ap-pendix, Fig. S1). Moreover, some wild Solanum species (e.g.,S. dulcamara) produce mostly saturated soladulcidine aglycone-derived SGAs (e.g., soladulcine A and β-soladulcine) from un-saturated solasodine (Fig. 1B).A main step in steroidal saponin biosynthesis is formation of

an unsaturated steroidal saponin aglycone (SI Appendix, Fig. S1).The aglycone of steroidal saponins is either spirostanol (closedF-ring) or furostanol (open F-ring) (6). Both saponin aglyconesundergo either glycosylation to form unsaturated saponin gly-cosides (e.g., dioscin) or hydrogenation at the C-5,6 position toform saturated saponin aglycones (e.g., sarasapogenin) and theircorresponding glycosides (e.g., parillin) (SI Appendix, Fig. S2).

Therefore, as with SGAs, unsaturated and saturated aglyconeforms of steroidal saponins determine the degree of structuraldiversity within this metabolite class.Notably, the unsaturated/saturated SA aglycone and steroidal

saponin aglycone pairs differ only in their structures by the pres-ence or absence of the double bond at the C-5,6 position (Fig. 1and SI Appendix, Figs. S1 and S2). However, the biosyntheticpathway responsible for the formation of saturated steroidal al-kaloid and steroidal saponin aglycones from their unsaturatedforms in Solanaceae or in any other plant family remains to beidentified. For decades, it has been hypothesized that the con-version of dehydrotomatidine to tomatidine in the tomato plant,and solanidine to demissidine in wild potato species, occurs byelimination of the C-5,6 double bond through a single reactioncatalyzed by hypothetical hydrogenase enzyme (2, 13–15).In this study, we present results suggesting that formation of

saturated steroidal specialized metabolites from unsaturated ste-roidal aglycone takes place in multiple steps rather than a singlestep. We discovered that GLYCOALKALOID METABOLISM25(GAME25), a member of the short-chain dehydrogenase/reductase(SDR) gene family, is involved in the first among these multiplesteps, specifically, the conversion of dehydrotomatidine to toma-tidine. Silencing of GAME25 in the tomato plant diverted SGAmetabolism within the leaves and during fruit development towardunsaturated dehydrotomatine-derived SGAs rather than the usualformation of saturated α-tomatine–derived SGAs. In vitro, GAME25exhibited a 3β-hydroxysteroid dehydrogenase/Δ5,4 isomeraseactivity on various unsaturated steroidal alkaloid and saponinaglycone substrates, but not on their glycosylated forms. Fur-thermore, overexpression of tomato GAME25 in cultivated

GAME6/8/11/4/12

Cholesterol Solasodine

α-solasonine α-solamargine

SGT1 SGT2

Soladulcidine

S. dulcamara (wild Solanum species)

Hydrogenase ?GAME25

GTs?

Soladulcine A

β-soladulcine

B

S. melongena (cultivated eggplant)

Cholesterol Tomatidine

Dehydrotomatine α-tomatine

Esculeoside A or lycoperoside G/F

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

Dehydrotomatidine(tomatidenol)

Hydrogenase ? GAME25GAME6/8/11/4/12

Dehydroesculeoside A or Dehydro-lycoperoside H/G

A

Fig. 1. The biosynthetic pathway for SGAs in to-mato, cultivated eggplant, and other Solanum spe-cies. (A) In the tomato plant, the conversion ofdehydrotomatidine to tomatidine was previouslypredicted to be a single-step reaction driven by ahypothetical hydrogenase enzyme (2, 13). SI Appen-dix, Fig. S3, provides a more detailed SGA pathwayschematic. (B) In cultivated eggplant, solasodine,an unsaturated aglycone is glycosylated by STEROLALKALOID GLYCOSYL TRANSFERASEs (SGTs) to produceunsaturated α-solasonine and α-solamargine SGAs(Left). Cultivated eggplant varieties likely lack a GAME25-like enzyme and therefore do not produce saturatedSGAs. Some wild Solanum species (e.g., S. dulcamara)produce a saturated soladulcidine aglycone fromsolasodine and further glycosylate soladulcidine agly-cone to soladulcine A and β-soladulcine (saturatedSGAs) (Right). This suggests the presence of GAME25homologs in S. dulcamara (wild Solanum relative) andother Solanum species producing saturated SGAsstarting from solasodine.

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eggplant resulted in the formation of saturated SGAs and atypi-cally saturated steroidal saponins. Taken together, GAME25 is akey enzyme in SGA and steroidal saponin metabolism, medi-ating a significant portion of the structural diversity of thesenatural product classes in Solanum as well as in saponin-producingplant species.

ResultsExpression of GAME25 Correlates with the Accumulation of TypicalGreen Tissue Steroidal Glycoalkaloids. A recent report by Cárdenaset al. (11) demonstrated that the GAME9 AP2-type transcrip-tion factor is associated with the regulation of SGA biosynthesisin the tomato and potato plants. Transcriptome analysis revealed27 genes that were up- or down-regulated in GAME9 over-expression and silenced tomato lines, respectively. This con-cise gene set included a putative 3-β-HYDROXYSTEROIDDEHYDROGENASE, a member of the SDR gene family (termedhere GAME25, Solyc01g073640). GAME25 displayed expressionpredominantly in flower buds, young leaves, and in the immaturegreen stage (skin and flesh) of fruit development (SI Appendix, Fig.S4). This expression pattern highly resembled the profile of tomatoSGAs (e.g., α-tomatine and dehydrotomatine), which accumulate inthe green tissues of the plant (9). Furthermore, the reduced tran-script levels of GAME25 during later stages of fruit developmentcorrelated with a reduction in α-tomatine and dehydrotomatinecontent during fruit maturation (SI Appendix, Figs. S3 and S4). TheGAME25 expression pattern during tomato fruit maturation wassimilar to that observed in wild tomato accessions (SI Appendix, Fig.S5A). The known function of some SDR family members in spe-cialized metabolism (16, 17) and the expression profile results showedhere suggest that GAME25 might be involved in SGA metabolism inthe tomato plant.SDRs represent one of the largest and most diverse NAD(P)

(H)-dependent enzyme superfamilies that have evolved in plantsand were recently categorized into 49 subfamilies (16). The259-aa GAME25 protein sequence shows the characteristics of aclassical SDR family member, containing the TGxxxGxG cofactorbinding site and the YxxxK catalytic motif (17, 18) (SI Appendix,Fig. S5B). Phylogenetic analysis showed that GAME25 homologsof certain Solanaceae species (i.e., tomato, potato, and Solanumpennellii) formed a subclade distinct from other plant SDRs (SIAppendix, Fig. S6). The closest subclade to the GAME25 pro-teins in the phylogenic tree contained the 3β-hydroxysteroiddehydrogenase homologs from tomato and Solanum pennellii(3-βHSD, ∼90% amino acid identity with GAME25 subcladeproteins), the function of which is unknown. Phylogenetic analysisalso showed no homolog for the GAME25 protein in eggplant orcapsicum (SI Appendix, Fig. S6). Moreover, GAME25 proteinswere clearly separated from the Digitalis lanata 3-βHSD protein(∼75% amino acid identity with the GAME25 proteins), which isinvolved in the removal of the C-5,6 double bond from steroidderivatives during progesterone and cardenolide biosynthesis (SIAppendix, Fig. S6). The clear separation of the GAME25 subcladesuggested a unique catalytic activity of these enzymes that is mostlikely different from the closely related 3-βHSD subclade mem-bers (SI Appendix, Fig. S6 and Dataset S1).

Tomato SGA Metabolism Is Rerouted from the Native, PredominantlySaturated α-Tomatine Branch to the Unsaturated DehydrotomatineBranch in GAME25-Silenced Leaves. To determine the role ofGAME25 in SGA metabolism, we silenced GAME25 in the to-mato plant (i.e., GAME25i lines). GAME25 transcript levelswere significantly reduced in GAME25i plant leaves and fruit atthree developmental stages (green, breaker, and red ripe fruit)(t test, *P value < 0.05) (SI Appendix, Fig. S7). Notably, GAME25ileaves showed a substantial decline in α-tomatine (∼2.5- to 3-fold),hydroxytomatine (∼6- to 10-fold), and acetoxytomatine (∼2- to3.5-fold) levels compared with wild-type leaves (Fig. 2 and SI

Appendix, Fig. S8A; see SI Appendix, Fig. S3 for the detailedtomato SGA pathway). Conversely, we observed considerable in-creases in dehydrotomatine (∼4- to 6-fold), dehydrotomatine iso-mer 1 (∼9- to 11-fold), and dehydrotomatidine +4 hexose (∼6- to9-fold) levels compared with wild-type leaves (Fig. 2 and SI Ap-pendix, Fig. S8A). We noted reduction in α-tomatine and itsdownstreammetabolite levels, yet, accumulation of dehydrotomatineand its isomers in GAME25i lines suggested that either (i)GAME25 is involved in α-tomatine biosynthesis directly fromdehydrotomatine glycoside or (ii) the enzyme mediates toma-tidine biosynthesis from dehydrotomatidine (i.e., tomatidenol;Fig. 1). We found no accumulation of dehydrotomatidine inGAME25i lines, but this SA aglycone appeared to be convertedto its glycosylated derivatives (e.g., dehydrotomatine anddehyrotomatidine +4 hexoses) that did accumulate in leaves(Fig. 2 and SI Appendix, Fig. S3). Rather than acting on gly-cosylated substrates (e.g., dehydrotomatine), the above findingsposition GAME25 activity before the dehydrotomatine glyco-sylation steps, possibly in the conversion of dehydrotomatidine totomatidine.

GAME25 Silencing Results in Gradual Loss of Saturated SGAs Withinthe Developing and Ripening Tomato Fruit. We compared the SGAprofile of wild-type and GAME25i tomato fruit through differentstages of fruit development and ripening. During the transitionfrom green to red fruit, α-tomatine is typically converted tosaturated SGAs (esculeosides and lycoperosides), while dehy-drotomatine is converted to dehydroesculeosides and dehy-drolycoperosides (unsaturated minor SGAs) (see SI Appendix,Fig. S3 for the detailed tomato SGA pathway). As found inleaves (Fig. 2), GAME25i green fruit displayed a drastic re-duction in α-tomatine (∼15- to 25-fold), hydroxytomatine (∼100-fold), and further α-tomatine–derived downstream SGA levelscompared with wild-type green fruit (Fig. 3A and SI Appendix,Fig. S8B). Thus, due to GAME25 silencing, α-tomatine and itsdownstream saturated SGA intermediates were severely affectedin green fruit tissue. In contrast, various unsaturated SGAs includingdehydrotomatine (∼10- to 12-fold) and hydroxy-dehydrotomatine

Fig. 2. GAME25 silencing in tomato leaves shifts the SGA pathway to theunsaturated dehydrotomatine branch. SGA levels in leaves of wild-type(nontransformed) and three independent GAME25-RNAi transgenic to-mato lines (#2, #3, and #4), as determined by LC–MS. The values representthe means of three biological replicates ±SE (per genotype). Asterisks in-dicate significant changes from wild-type samples calculated by a Student’st test (*P value < 0.05; **P value < 0.01; ***P value < 0.001).

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(∼25-fold) were increased in GAME25i compared with the wild-type tomato green fruit (Fig. 3B and SI Appendix, Fig. S8B).These data suggest that GAME25 silencing in the green tomatofruit results in redirection of biosynthesis toward unsaturateddehydro-SGAs (see SI Appendix, Fig. S3 for detailed tomatoSGA pathway).The metabolic shift from the α-tomatine–derived saturated

SGA branch to the unsaturated dehydro-SGAs pathway furthercontinued in the GAME25i breaker fruit stage. SGA metabolitesthat accumulated following GAME25 silencing but not in wild-type fruit included hydroxy-dehydrotomatine (∼20- to 30-fold),acetoxy-hydroxy-dehydrotomatine (∼20- to 25-fold), as well asdehydroesculeoside A (∼20- to 25-fold) and its derivatives (SIAppendix, Figs. S8C and S9A). α-Tomatine (∼20- to 50-fold) andits downstream SGAs were almost absent in the GAME25ibreaker fruit (SI Appendix, Figs. S8C and S9B). In parallel, thedrastic reduction in α-tomatine levels at the green fruit stage inGAME25i lines resulted in a severe decline of esculeoside A andlycoperoside (∼20- to 25-fold) levels in the red ripe fruit stagecompared with levels in wild-type green fruit (Fig. 3C). More-over, α-tomatine–derived saturated SGAs were not detected inGAME25-silenced red ripe fruit (Fig. 3C and see SI Appendix,Fig. S3 for the detailed tomato SGA pathway). Compared withwild-type green fruit, we did observe a buildup of dehydroto-matine in GAME25i green fruit that resulted in massive accu-mulation of dehydroesculeoside A (∼20- to 25-fold) and itsderivatives in red ripe fruit (Fig. 3D and SI Appendix, Fig. S8D).These data provide additional evidence regarding the role ofGAME25 in the conversion of dehydrotomatidine to tomatidine,which we hypothesize to be a bifurcating step between the sat-urated (α-tomatine) and unsaturated (dehydrotomatine) SGAbiosynthesis pathway.

Accumulation of Saturated α-Tomatine and Its Downstream SGAs Dueto GAME25 Overexpression in the Tomato Plant. To further examinethe role of GAME25 in SGA biosynthesis, we generated trans-genic tomato lines overexpressing the GAME25 gene (GAME25-Ox). GAME25 expression in leaves and fruit tissues (green and

red fruit) of transgenic tomato lines was significantly higher thanin wild-type tomato plants (t test, **P value < 0.01) (SI Appendix,Fig. S10A). Leaves from GAME25-Ox lines showed higher levelsof α-tomatine (∼1.5-fold), α-tomatine (isomer 2) (∼1.5-fold), andacetoxytomatine (∼1.8-fold), with simultaneous reduction ofdehydrotomatine (∼1.5-fold), compared with wild-type leaves (SIAppendix, Fig. S10 B and C). GAME25-Ox green tomato fruitdisplayed reduction in dehydrotomatine levels (∼1.5-fold), whereasno change in α-tomatine content was observed in the same tissues(SI Appendix, Fig. S10D). However, we detected increases in ace-toxytomatine (∼1.9-fold) and acetoxy-hydroxytomatine (∼4- to 7-fold) (α-tomatine–derived SGAs) in comparison with wild-typegreen fruit (SI Appendix, Fig. S10D; see SI Appendix, Fig. S3 forthe tomato SGA pathway). Analysis of red fruit from theGAME25-Ox lines showed accumulation of α-tomatine (∼4- to 6-fold) and itsdownstream saturated SGAs [e.g., acetoxytomatine (∼5- to 7-fold),and acetoxy-hydroxytomatine (∼2- to 3-fold)] compared with wild-type red fruit (SI Appendix, Fig. S10E). Furthermore, GAME25overexpressing red tomato fruit did not show any change in levels ofesculeoside A (acetoxy-hydroxytomatine–derived major SGA),compared with wild-type red fruit.

Tomato GAME25 Overexpression in Cultivated Eggplant (S. melongena)Results in Newly Produced Saturated SGAs and Steroidal Saponins.Unlike in the tomato plant, saturated SGAs are normally absentin cultivated eggplant, suggesting the absence of GAME25 activityin this species. This is further supported by the absence of aGAME25 homolog in cultivated eggplant (SI Appendix, Fig. S6).In cultivated eggplant, α-solasonine, α-solamargine, and malonyl-solamargine are the major unsaturated SGAs (with a C-5,6 doublebond) (19) derived from the solasodine aglycone (Fig. 4A, Upper).Moreover, cultivated eggplant also produces unsaturated furostanol-type steroidal saponin glycosides from the unsaturated furostanol-type saponin aglycone (Fig. 4A, Lower). To investigate the impactof tomato GAME25 activity in cultivated eggplant, we generatedtransgenic eggplant lines overexpressing the tomato GAME25gene (SI Appendix, Fig. S11). Specifically, we wanted to assesswhether GAME25 can shift SGA metabolism from predominantly

Fig. 3. Green and red stage fruit of GAME25-si-lenced tomato lines display substantially altered SGAmetabolism. (A and B) Levels of (A) saturatedα-tomatine– and (B) unsaturated dehydrotomatine-derived SGAs in green fruit of the GAME25-silencedtomato 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 comparedwith wild-type red fruit. The values represent meansof three biological replicates ±SE (per genotype).Lines #2, #3, and #4 are three independent GAME25ilines. Asterisks indicate significant changes comparedwith wild-type samples, calculated by a Student’st test (*P value < 0.05; **P value < 0.01; ***P value <0.001). LC–MS was used for targeted SGA profiling.

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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 ofSGA (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 massand 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 steroidalSA and steroidal saponin aglycones. (Upper) Overlays of mass spectra of saturated SA aglycones (red) and unsaturated SA aglycones (black). (Lower) Overlaysof mass spectra of saturated steroidal saponin aglycones (red) and unsaturated steroidal saponin aglycones (black). Characteristic fragment structures aredepicted. 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 compoundsand 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.

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unsaturated SGAs to saturated SGAs that are not naturally pre-sent in this plant. In transgenic eggplant leaves, GAME25 over-expression resulted in reduced levels of the unsaturated SGAs,α-solasonine, α-solamargine, and malonyl-solamargine (Fig. 4A,Upper) as well as of unsaturated furostanol saponin glycosides(Fig. 4A, Lower). Conversely, we observed major accumulation ofβ-soladulcine, soladulcine A, and the saturated form of malonyl-solamargine (Fig. 4A, Upper). Both β-soladulcine and soladulcineA (lacking the C-5,6 double bond) are SGAs derived from satu-rated soladulcidine aglycone and are typically found in S. dulca-mara, a wild Solanum relative (Fig. 1B). Thus, S. dulcamara likelycontains an active GAME25 homolog that mediates the formationof the above-mentioned saturated SGAs. Moreover, saturatedfurostanol-type steroidal saponin glycosides (Fig. 4A, Lower) weredetected in GAME25-overexpressing eggplant lines, which arenormally undetectable in cultivated eggplant.The chemical structures of unsaturated and saturated SGAs as

well as of the steroidal saponins identified here are shown in Fig.4B. Metabolites were identified based on accurate mass-derivedelemental composition and mass fragmentation pattern. Loss ofC-3 sugar moieties in unsaturated SGAs leads to the formationof the fragment ion m/z 414.3 that corresponds to solasodine, anunsaturated steroidal aglycone backbone (Fig. 4C, Upper). Fur-ther loss of the E and F ring from the solasodine backbone re-sults in the characteristic fragment ion m/z 271.2, which loses awater molecule to form fragment m/z 253.19 (Fig. 4C, Upper).Due to the absence of a C-5,6 double bond in saturated SGAs,all fragment ions showed a mass shift of plus 2 Da, i.e., m/z 416.3(soladulcidine, saturated aglycone backbone), m/z 273.2, and m/z255.2 after loss of the E/F rings and dehydration, respectively(Fig. 4C, Upper). Similarly, unsaturated furostanol-type steroidalsaponins showed aglycone fragment ions with a mass of m/z415.3, 271.2, and 253.19, whereas saturated furostanol-type ste-roidal saponins displayed m/z 417.3, 273.2, and 255.2 fragmentions after MS-fragmentation analysis (Fig. 4C, Lower).

Insect Cells Expressing GAME25 Convert Dehydrotomatidine toTomatid-4-En-3-One. We examined the potential role of GAME25in the conversion of dehydrotomatidine to tomatidine by express-ing either the recombinant tomato or potato enzymes in Sf9 insectcells and testing microsomal fractions for their activity (SI Ap-pendix, Fig. S12). We performed enzymatic assays in the presenceof NAD+ as a cofactor and with dehydrotomatidine, solanidine,and solasodine (unsaturated SA aglycones) as substrates. Surpris-ingly, assays with either enzyme did not result in the formation ofthe expected reaction products, tomatidine, demissidine, or sol-adulcidine (saturated SA aglycones). However, an assay with eachrecombinant GAME25 enzyme (either tomato or potato) anddehydrotomatidine resulted in the formation of a compound withthe mass m/z 412.3 (M + H+) (Fig. 5 A and B for tomatoGAME25 assay and SI Appendix, Fig. S13 A and B for the potatoGAME25 assay). MS-MS fragmentation pattern analysis of thenewly formed compounds (Fig. 5 A, B, and D) showed three majorfragment ions derived from two parallel fragmentation routes. Lossof the carbonyl oxygen and formation of an additional double bondled to a fragment ion with m/z 394.3 (Fig. 5D). Loss of the E and Frings of the steroidal skeleton led to the fragment ion m/z 269.2,which was then dehydrated to form fragment m/z 251.17 (Fig. 5D).The newly formed compound was putatively assigned as tomatid-4-en-3-one (Fig. 5 A and B).Using solanidine as a substrate, the GAME25 enzyme assays

(either with tomato or potato GAME25 enzymes) resulted in theformation of a new product with an apparent molecular ion ofm/z 396.3 (M + H+) (Fig. 5 C, E, and F for the tomato GAME25assay and SI Appendix, Fig. S13 C and D for the potato GAME25assay). We identified the compound as solanid-4-en-3-one bycomparing retention time and mass spectrum to the authentic,commercially available, solanid-4-en-3-one standard and MS-MS

analysis (Fig. 5 C, E, and F and see SI Appendix, Fig. S17 forMS-MS analysis of solanid-4-en-3-one standard and solanid-4-en-3-one product after GAME25 assay). Either the recombinanttomato or potato GAME25 enzymes also successfully con-verted solasodine, the cultivated eggplant aglycone, to the putativesolasod-4-en-3-one compound (SI Appendix, Fig. S13 E and Ffor potato GAME25 assay and SI Appendix, Fig. S14 for tomatoGAME25 assay).The tomato and potato recombinant GAME25 enzymes showed

no activity on glycosylated SA substrates (i.e., α-tomatine, dehy-drotomatine, α-solanine, α-chaconine, and α-solamargine). Theseresults suggest that GAME25 catalyzes the oxidation of the 3β-hydroxyl group (3β-hydroxysteroid dehydrogenase activity) andthe isomerization of the double bond from the C-5,6 to the C-4,5 position (3-oxosteroid Δ5,4 isomerase activity) in SA aglyconesubstrates to form the 3-oxo-Δ5,4 SA intermediates identifiedhere (tomatid-4-en-3-one, solanid-4-en-3-one, or solasod-4-en-3-one). Thus, GAME25 possesses a previously uncharacterized3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity.

Recombinant Tomato GAME25 Expressed in Escherichia coli Confirms3β-Hydroxysteroid Dehydrogenase and Δ5,4 Isomerase Activity. Theobserved 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase ac-tivity of the recombinant tomato and potato GAME25 enzymesis rather uncommon as other SDR family enzymes participatingin specialized metabolism typically possess only 3β-hydroxyste-roid dehydrogenase activity (20–26). To confirm that the Δ5,4

isomerization observed here was a result of GAME25 activityand not due to activity of an endogenous enzyme of the insectcell microsomes, we expressed tomato GAME25 in E. coli andpurified the enzyme for activity assays (SI Appendix, Fig. S15).Enzyme assay with the recombinant GAME25 enzyme usingsolanidine as a substrate and NAD+ as a cofactor resulted in theformation of the same solanid-4-en-3-one product that we ob-served in the insect cell enzyme assay (SI Appendix, Fig. S16A).We confirmed the identity of the product by comparing retentiontime, mass spectrum, and MS-MS fragments with an authenticsolanid-4-en-3-one standard (SI Appendix, Fig. S17). Thus, thisresult provided substantial evidence that the recombinant GAME25enzymes possess both 3β-hydroxysteroid dehydrogenase and Δ5,4

isomerase activities.

The Recombinant Tomato GAME25 Converts Diosgenin, a Spirostanol-Type Saponin Aglycone, to Diosgen-4-En-3-One. Like SGAs, steroidalsaponins display two structural forms, saturated or unsaturatedC-5,6 (SI Appendix, Fig. S2). Our observation that new saturatedfurostanol-type saponins were formed as a result of GAME25overexpression in cultivated eggplant suggested the potential roleof GAME25 in elimination of the C-5,6 double bond, not onlyfrom SA substrates but also from steroidal saponins. To examinethis possibility, we performed assays with the recombinant enzymeand diosgenin [(M + H+, m/z 415.3), a major spirostanol-typesteroidal saponin aglycone produced by Dioscorea species]. In-terestingly, GAME25 activity resulted in the formation of a novelcompound with the molecular ion m/z 413.3 (M + H+), repre-senting oxidation of the 3β-hydroxyl group and isomerization ofthe double bond from the C-5,6 position to the C-4,5 position (SIAppendix, Fig. S18A). While the unsaturated diosgenin substrateproduced three major fragment ions with m/z 415.3, 271.2, and253.2, the newly formed compound (m/z 413.3) showed fragmentions with m/z 413.3, 269.2, and 251.2, respectively (SI Appendix,Fig. S18B) and thus putatively was assigned as diosgen-4-en-3-onebased on mass fragmentation spectra analysis (SI Appendix, Fig.S18). Our results suggest that recombinant GAME25 can catalyzethe oxidation of the 3β-hydroxyl group and the isomerization ofthe double bond from the C-5,6 to the C-4,5 position in steroidalsaponin 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 ofthe GAME25 reaction product), and the control reaction obtained with dehydrotomatidine as a substrate. (B) Mass spectra and structures of the detectedproduct (Upper) and substrate (Lower) of the GAME25 enzymatic reaction with dehydrotomatidine as substrate. (C) Overlay of extracted ion chromatogramsof m/z 396.32 Da, [M + H+]+ (mass of the GAME25 reaction product), and the control reaction with solanidine as substrate. (D) Mass fragmentation spectrumof the GAME25 enzymatic reaction product (with dehydrotomatidine as substrate), including the interpretation of the detected mass fragments. Thefragmentation pattern corresponds to the tomatid-4-en-3-one (proposed structure of the GAME25 product). (E) Chromatograms of the GAME25 enzymaticreaction (Upper), control reaction (Middle), both with solanidine as substrate, and the solanid-4-en-3-one authentic standard (Lower). The newly formedproduct (at retention time 23.2 min) coeluted with the solanid-4-en-3-one commercial authentic standard. Comparison of MS-MS spectra between the newlyformed 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 wasassigned as solanid-4-en-3-one. (F) Mass spectra and structures of the detected product (Upper) and substrate (Lower) of the GAME25 enzymatic reaction withsolanidine as substrate. Analysis of enzyme assay reactions was carried out by LC–MS. The control reaction was performed using protein extracts fromnontransfected Sf9 insect cell microsomes. EIC, extracted ion chromatogram; m/z, mass to charge; STD, metabolite standard; TIC, total ion chromatogram.

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The Presence of the C-5,6 Double Bond in SGAs Inhibits Fungal Growthand Pathogenicity. α-Tomatine in green tomato tissues is knownto affect the growth of pathogenic fungi, including Botrytis cinerea,Fusarium oxysporum, and Colletotrichum gloeosporioides (2). Incontrast, the role of unsaturated dehydrotomatine in phyto-pathogenicity has not been previously examined primarily be-cause it is typically produced in small amounts in tomato tissues.As silencing of GAME25 in the tomato plant redirected SGAmetabolism toward formation of dehydro-SGAs, we examined theeffects of dehydro-SGAs on fungal growth and pathogenicity.Analysis of GAME25i leaf extracts showed mycelial growth in-hibition of the pathogenic fungi C. gloeosporioides and B. cinereacompared with wild-type saturated SGA-containing extracts (SIAppendix, Fig. S19 A, B, E, and F). In addition, C. gloeosporioidesand B. cinerea fungal conidia germination was severely reducedupon treatment with GAME25i extracts compared with treatmentwith wild-type extracts (SI Appendix, Fig. S19 C, D, and G–J).

DiscussionGAME25 Is a Key Branch Point Enzyme That Determines the Diversityof SGAs Produced in Solanum Species and Modulates Their Level ofToxicity. The presence or absence of a double bond at the C-5,6 position within the core SGA scaffold is a major source ofstructural diversity among SGAs produced by Solanum species.In tomato, both dehydrotomatidine and tomatidine SA agly-cones are highly toxic to plant cells, and it is therefore likely thatthey undergo glycosylation to prevent self-toxicity (9). Studies inanimal models demonstrated that SGAs lacking the C-5,6 doublebond (e.g., α-tomatine) are much less toxic to animals and hu-mans compared with those SGAs that contain this double bond(e.g., α-chaconine and α-solanine from potato) (2). These priorfindings also suggest that dehydrotomatine is likely a more toxicSGA compared with α-tomatine. Although less toxic to humansand animals, α-tomatine is a highly active molecule involved in arange of host-plant resistance mechanisms in tomato plants (2, 4,5). However, the contribution of dehydrotomatine, typicallyproduced at lower levels in tomato, to plant resistance againstpathogens remains unclear. In the present study, severe growthand conidia germination inhibition of the pathogenic fungi B.cinerea and C. gloeosporioides by extracts enriched with dehydro-derivatives (due to GAME25 silencing) suggest enhanced toxicityof these compounds compared with wild-type samples containingmainly saturated α-tomatine and related metabolites (SI Appendix,Fig. S19). Thus, in Solanum plants, α-tomatine and dehydroto-matine SGAs may act synergistically against pathogens and mighthave coevolved to exert a combined effect against a broad range ofdisease-causing pathogens. As demonstrated here, GAME25 cat-alyzes the first step in the conversion of dehydrotomatidine totomatidine in which the double bond at the C-5,6 position is re-duced. This reaction, of the C-5,6 double bond removal in SAaglycones, is therefore a key branch point that not only determinesthe diversity of SGAs produced in hundreds of Solanum speciesbut also modulates the toxic effects of this metabolite class to theplant itself, other animals, and likely also as a plant defense againstpathogens and herbivores.

Production of Tomatidine from Dehydrotomatidine in the TomatoPlant Involves a Yet-Unknown Plant SDR-Type GAME25 EnzymeActivity. To date, the biosynthesis of dehydrotomatine andα-tomatine was hypothesized to occur through several differentpathways (1, 2, 14). In one scenario, dehydrotomatidine wasproposed to be derived from cholesterol (contains a C-5,6 doublebond), while tomatidine was predicted to be synthesized fromcholestanol (lacking the C-5,6 double bond) (1, 2). Thus, con-version of cholesterol to cholestanol was thought to be re-sponsible for the formation of tomatidine. An alternativesuggested pathway is that the cholesterol-derived teneimine in-termediate (possessing a double bond) is partitioned, leading to

formation of both tomatidine (through the action of a hypotheticalhydrogenase that reduces the double bond) and dehydrotomatidine(1, 2, 14). In a third hypothesis, tomatidine was proposed to bepartly dehydrogenated to form dehydrotomatidine by a hypothet-ical dehydrogenase (1, 2). Finally, the formation of tomatidinefrom dehydrotomatidine was hypothesized as a single-step hy-drogenation reaction (2, 13, 14). In the present study, functionalcharacterization of GAME25 provided strong evidence that theformation of tomatidine from dehydrotomatidine [i.e., the re-duction of the Δ5 (C-5,6 position) bond in the SA aglycones] islikely carried out in multiple steps and that the GAME25 cata-lyzes the first of these. In vitro enzyme assays with the recombi-nant tomato and potato GAME25 enzyme demonstrated theenzyme’s dual activity, namely, oxidation of the 3β-hydroxyl group(3β-hydroxysteroid dehydrogenase activity) and isomerization ofthe double bond from the C-5,6 position to the C-4,5 position(3-oxosteroid Δ5,4 isomerase activity) on both SA and steroidalsaponin aglycones (Fig. 5 and SI Appendix, Figs. S13, S14, and S18).These results also suggest that formation of the saturated steroidalsaponin aglycone (by removal of the C-5,6 double bond) is likelynot a single-step reaction, but rather that GAME25 catalyzes thefirst step, as we observed for SA biosynthesis.In plants, SDR enzymes catalyze NAD(P)(H)-dependent ox-

idation/reduction reactions involving a wide range of primary orspecialized metabolites (16–18, 20, 23–26). Members of thisfamily have been reported to participate in the metabolism ofvarious specialized metabolites including cardiac glycosides (i.e.,cardenolides) in Digitalis spp. (3-βHSD), tropane-like alkaloids(SDR65C), terpenoids (SDR110C, SDR114C), benzylisoquino-line alkaloids in poppy (NOS), oryzalexin diterpenoids in rice(MSI and MI1-3), and phenolics (SDR108E) (20, 23–26). GAME25is a unique plant SDR enzyme that exhibits dual activity, with theability to oxidize the 3β-hydroxyl group (3β-hydroxysteroid de-hydrogenase activity) and isomerization of the double bond from theC-5,6 to the C-4,5 position (3-oxosteroid Δ5,4 isomerase activity) insteroidal substrates. Most, if not all, 3-βHSD enzymes participatingin specialized metabolism in plants (i.e., members of the 3-βHSDand SDR family) merely possess 3β-hydroxysteroid dehydrogenaseactivity and not Δ5,4 isomerase activity (21–25). For example, inDigitalis, the oxidation (of the 3β-hydroxyl group) and isomerization(C-5,6 to the C-4,5 position) steps, required during the conversion ofpregnenolone to progesterone, are carried out successively by twoseparate enzymes, 3-βHSD (3β-hydroxysteroid dehydrogenase) and3-KSI (Δ5-3-ketosteroid isomerase) (20–22). Phylogenetic analysisof SDR family proteins from plants involved in specialized me-tabolism suggested that the GAME25 proteins of the Solanaceaefamily have undergone significant diversification compared withother SDR proteins. Moreover, the dual enzyme activity ofGAME25 proteins reported here suggests that it could haveevolved from the classical monofunctional SDRs. Interestingly, inmammalian steroid hormone metabolism, the conversion ofpregnenolone to progesterone includes a 3-βHSD enzyme that,similar to GAME25, possesses dual dehydrogenase and isomeraseactivities (27, 28).

The Absence of GAME25 Activity in Cultivated Potato and EggplantUnderlies the Lack of Saturated SGAs in These Plants. The pathwayfrom the unsaturated SA aglycone solanidine to the saturated SAaglycone demissidine and its glycosylated form (i.e., demissine) inwild potato species corresponds to the tomato pathway in whichthe C-5,6 double bond is eliminated from dehydrotomatidine to-ward tomatidine and glycosylated α-tomatine (SI Appendix, Figs.S1 and S3). The domesticated potato does not accumulate satu-rated demissidine or demissine SGAs. The presence of a GAME25homolog (SI Appendix, Fig. S6) but the absence of saturated SGAsin cultivated potato tubers suggests that these SGAs were lostduring the domestication process, possibly through alteredGAME25gene activity. Moreover, in vitro, the recombinant potato GAME25

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enzyme shows 3β-hydroxysteroid dehydrogenase/Δ5,4 isomeraseactivity on diverse unsaturated steroidal metabolites (SI Appendix,Fig. S13). Therefore, even if GAME25 is considered active inpotato (in vivo), additional enzymes required for the formation ofsaturated SA aglycone might be absent or inactive in the domes-ticated potato. In the case of the cultivated eggplant, it is likely thatGAME25 activity is the single factor that is responsible for the lackof saturated SGA production, as overexpression of tomatoGAME25resulted in accumulation of saturated SGAs (Fig. 4). In additionto saturated SGAs, novel saturated steroidal saponins were formedin GAME25-Ox eggplant transgenic lines, underscoring GAME25’scrucial role in steroidal saponin biosynthesis in Solanum plants (Fig.4). Thus, GAME25-like enzymes might be involved in eliminatingthe C-5,6 double bond from unsaturated steroidal saponin aglyconesubstrates in numerous plant species, including those outside theSolanaceae family.

The Pathway to Saturated Steroidal Specialized Metabolites inSolanum and Other Species. During cardenolide biosynthesis inDigitalis species, pregnenolone is converted to pregnanolone.Both pregnenolone and pregnanolone are steroid derivativesthat differ only by the presence or absence of the double bond atthe C-5,6 position (Fig. 6). The conversion of pregnenolone topregnanolone (removal of C-5,6 double bond) occurs in the fourfollowing steps: (i) oxidation of (3β-hydroxyl group) pregneno-lone by the 3βHSD enzyme, followed by (ii) isomerization of thedouble bond from the C-5,6 to the C-4,5 position by the 3-KSIenzyme to form progesterone; (iii) conversion of progesterone to5β-pregnan-3,20-dione (removal of C-4,5 bond) by 5β-progesterone

reductase (5β-POR), (iv) which is then converted to pregnanoloneby a 3βHSD enzyme (Fig. 6) (20–22, 27, 28). Thus, the conversionof pregnenolone to pregnanolone resembles the formation oftomatidine from dehydrotomatidine in tomato, or demissidine fromsolanidine in wild potato plants, in which the C-5,6 double bondis also removed (Fig. 6). Assays assessing recombinant GAME25activity clearly showed that GAME25 is not sufficient to catalyzethe entire Δ5 reduction in unsaturated SA aglycones and thatadditional enzymes are required to eliminate the C-5,6 doublebond and form saturated products. The additional enzymatic stepsrequired after GAME25 activity resemble the Digitalis (iii) and(iv) reactions described above. Thus, we expected that the Digitalis5β-POR reductase homolog in tomato (Solyc10g049620, termedhere GAME35) might act downstream to the pair of reactionscatalyzed by GAME25. In this case, Digitalis 5β-POR would cat-alyze reduction of various 3-oxo-Δ5,4 SA aglycone intermediates(GAME25 enzyme products; e.g., solanid-4-en-3-one). Enzymeassays with the purified recombinant GAME35 protein (homologof theDigitalis 5β-POR) showed that it is not active on the solanid-4-en-3-one substrate (SI Appendix, Figs. S15 and S16B). Thus, adifferent reductase enzyme is likely required to carry out thissecond removal of C-4,5 bond reaction.Based on the intermediates produced by GAME25 and the

further requirement of analogous enzymatic reactions, we proposethat the conversion of dehydrotomatidine to tomatidine, and like-wise the conversion of other SAs, requires a three-step reactionsequence with GAME25 catalyzing the first step, converting dehy-drotomatidine to tomatid-4-en-3-one (Fig. 6). Tomatid-4-en-3-oneis subsequently reduced to tomatidine by the successive action of a

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), andsolasodine 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 stepin this reaction sequence, producing 3-oxo-Δ5,4 steroidal alkaloid/saponin aglycone derivatives from the respective unsaturated steroidal alkaloid/saponinaglycone 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 arrowsindicate multistep reactions.

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putative 5-reductase and an aldo-keto reductase, which remain tobe identified (Fig. 6). We also predict a similar three-step conver-sion in wild potato species producing saturated demissidine fromthe solanidine aglycone (Fig. 6). In addition, tomato GAME25overexpression in cultivated eggplant generated saturated SGAsnaturally produced by certain Solanum species (e.g., S. dulcamara).Based on these findings, we anticipate similar reactions in Solanumspecies that produce a saturated soladulcidine aglycone from sol-asodine (e.g., S. dulcamara; Fig. 6). Our in vivo and in vitro resultssupport a multireaction sequence in the biosynthesis of saturatedsteroidal saponins (Fig. 6). Hence, evolution of structural diversityin steroidal alkaloids and saponins by elimination of the C-5,6 double bond and its underlying enzymatic base is likely con-served in a wide range of plant families that are rich in steroidalspecialized metabolites.Characterization of GAME25 activity in the SGAs biosynthesis

pathway is a significant step toward resolving the entire core SGApathway in Solanaceae species. This work further contributes tothe understanding of how a large portion of structural diversity inSGAs and steroidal saponin-producing species is generated.Nevertheless, the enzymes completing the elimination of the C-5,6double bond succeeding GAME25 still remain to be identified.The dramatic shift from saturated to unsaturated SGAs inGAME25-silenced tomato plants, including the dominance of dehydro-esculeosides in ripening fruit, make this genetic material an excellentresource for future investigation. Further genetic and biochemicalanalyses will enable linking the structural information on steroidalspecialized metabolites to the potency of these molecules withrespect to plant pathogens and herbivores. Ripe fruit accumulatingα-tomatine (instead of the typical esculeosides) as a result ofGAME25overexpression will also be of value for carrying out similar in-teraction studies. The presence of the double bond at the C-5,6position is not merely an issue of structural variation as ample

evidence suggests its relevance in determining the level of toxicityof these molecules to humans. SGAs, primarily the unsaturatedones prevalent in potato tubers, are renowned antinutritionals, andtheir levels in the cultivated potato are tightly regulated. The currentwork has implications for commercial farming. Together with thepreviously reported structural and regulatory genes, overexpressionof GAME25 could be a valuable strategy to reduce the levels ofthese substances in commercial potato varieties.

Materials and MethodsPlant Extract Preparation and Targeted Profiling of Steroidal Metabolites.Preparation of plant extracts and the profiling of steroidal metabolites invarious tomato (leaves, green fruit, breaker fruit, and red fruit) and eggplantleaf tissue were performed as described previously (9, 11). Detailed liquidchromatography–mass spectrometry (LC–MS) methods are provided in SIAppendix, SI Materials and Methods.

Protein Expression and in Vitro Enzyme Assay. The detailed steps for tomato/potato GAME25 and tomato GAME35 protein expression and recombinantprotein enzyme assay are in SI Appendix, SI Materials and Methods.

Fungal Inhibition Assay. B. cinerea (B05.10) and C. gloeosporioides (Cg14)fungal inhibition activity of the GAME25i and wild-type methanolic extractswas determined by the disk diffusion method (9). Details of the fungal in-hibition assay are in SI Appendix, SI Materials and Methods.

ACKNOWLEDGMENTS. We thank the Adelis Foundation; the Leona M. andHarry B. Helmsley Charitable Trust; the Jeanne and Joseph Nissim Foundationfor Life Sciences; the Tom and Sondra Rykoff Family Foundation Research; andthe Raymond Burton Plant Genome Research Fund for supporting the A.A.laboratory activity. The work also was supported by Israel Science FoundationGrant 1805/15 and European Research Council (SAMIT-FP7) personal grants (toA.A.). The research in the A.A. laboratory was supported by the EuropeanUnion Seventh Framework Program FP7/2007–2013 Grant 613692–TriForC. A.A.is the incumbent of the Peter J. Cohn Professorial Chair.

1. Friedman M, McDonald G, Filadelfi-keszi M (1997) Potato glycoalkaloids: Chemistryanalysis safety and plant physiology. Crit Rev Plant Sci 16:55–132.

2. Friedman M (2002) Tomato glycoalkaloids: Role in the plant and in the diet. J AgricFood Chem 50:5751–5780.

3. Friedman M (2006) Potato glycoalkaloids and metabolites: Roles in the plant and inthe diet. J Agric Food Chem 54:8655–8681.

4. Milner SE, et al. (2011) Bioactivities of glycoalkaloids and their aglycones from Solanumspecies. J Agric Food Chem 59:3454–3484.

5. Roddick JG (1996) Steroidal glycoalkaloids: Nature and consequences of bioactivity.Adv Exp Med Biol 404:277–295.

6. Eich E (2008) Solanaceae and Convolvulaceae–Specialized Metabolites: BiosynthesisChemotaxonomy Biological and Economic Significance: A Handbook (Springer, Berlin).

7. Itkin M, et al. (2013) Biosynthesis of antinutritional alkaloids in solanaceous crops ismediated by clustered genes. Science 341:175–179.

8. Sonawane PD, et al. (2016) Plant cholesterol biosynthetic pathway overlaps withphytosterol metabolism. Nat Plants 3:16205.

9. Itkin M, et al. (2011) GLYCOALKALOID METABOLISM1 is required for steroidal alka-loid glycosylation and prevention of phytotoxicity in tomato. Plant Cell 23:4507–4525.

10. Cárdenas PD, et al. (2015) The bitter side of the nightshades: Genomics drives dis-covery in Solanaceae steroidal alkaloid metabolism. Phytochemistry 113:24–32.

11. Cárdenas PD, et al. (2016) GAME9 regulates the biosynthesis of steroidal alkaloids andupstream isoprenoids in the plant mevalonate pathway. Nat Commun 7:10654.

12. Umemoto N, et al. (2016) Two cytochrome P450 monooxygenases catalyze early hy-droxylation steps in the potato steroid glycoalkaloid biosynthetic pathway. PlantPhysiol 171:2458–2467.

13. Friedman M, Levin CE (1998) Dehydrotomatine content in tomatoes. J Agric FoodChem 46:4571–4576.

14. Laurila J, Laakso I, Valkonen JP, Hiltunen R, Pehu E (1996) Formation of parental-typeand novel glycoalkaloids in somatic hybrids between Solanumbrevidens and S. tuberosum.Plant Sci 118:145–155.

15. Ginzberg I, Tokuhisa JG, Veilleux RE (2009) Potato steroidal glycoalkaloids: Bio-synthesis and genetic manipulation. Potato Res 52:1–15.

16. Moummou H, Kallberg Y, Tonfack LB, Persson B, van der Rest B (2012) The plantshort-chain dehydrogenase (SDR) superfamily: Genome-wide inventory and di-versification patterns. BMC Plant Biol 12:219.

17. Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) Medium- and short-chaindehydrogenase/reductase gene and protein families: The SDR superfamily: Functionaland structural diversity within a family of metabolic and regulatory enzymes. Cell MolLife Sci 65:3895–3906.

18. Kallberg Y, Oppermann U, Jörnvall H, Persson B (2002) Short-chain dehydrogenases/reductases (SDRs). Eur J Biochem 269:4409–4417.

19. Wu SB, Meyer RS, Whitaker BD, Litt A, Kennelly EJ (2013) A new liquid chromatography-mass spectrometry-based strategy to integrate chemistry, morphology, and evolution ofeggplant (Solanum) species. J Chromatogr A 1314:154–172.

20. Herl V, Frankenstein J, Meitinger N, Müller-Uri F, Kreis W (2007) Δ 5-3β-hydroxyste-roid dehydrogenase (3 β HSD) from Digitalis lanata. Heterologous expression andcharacterisation of the recombinant enzyme. Planta Med 73:704–710.

21. Meitinger N, Geiger D, Augusto TW, Maia de Pádua R, Kreis W (2015) Purification ofΔ(5)-3-ketosteroid isomerase from Digitalis lanata. Phytochemistry 109:6–13.

22. Meitinger N, et al. (2016) The catalytic mechanism of the 3-ketosteroid isomerase ofDigitalis lanata involves an intramolecular proton transfer and the activity is not as-sociated with the 3β-hydroxysteroid dehydrogenase activity. Tetrahedron Lett 57:1567–1571.

23. Chen X, Facchini PJ (2014) Short-chain dehydrogenase/reductase catalyzing the finalstep of noscapine biosynthesis is localized to laticifers in opium poppy. Plant J 77:173–184.

24. Kitaoka N, Wu Y, Zi J, Peters RJ (2016) Investigating inducible short-chain alcoholdehydrogenases/reductases clarifies rice oryzalexin biosynthesis. Plant J 88:271–279.

25. Ringer KL, Davis EM, Croteau R (2005) Monoterpene metabolism. Cloning, expression,and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermintand spearmint. Plant Physiol 137:863–872.

26. Okamoto S, et al. (2011) A short-chain dehydrogenase involved in terpene metabo-lism from Zingiber zerumbet. FEBS J 278:2892–2900.

27. Gavidia I, Tarrío R, Rodríguez-Trelles F, Pérez-Bermúdez P, Seitz HU (2007) Plantprogesterone 5β-reductase is not homologous to the animal enzyme. Molecularevolutionary characterization of P5betaR from Digitalis purpurea. Phytochemistry 68:853–864.

28. Herl V, Fischer G, Müller-Uri F, Kreis W (2006) Molecular cloning and heterologousexpression of progesterone 5β-reductase from Digitalis lanata Ehrh. Phytochemistry67:225–231.

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