GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato

Abstract

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

Figure 1.

Diversity of SAs in Tomato and Its Correspondence with GAME1 Expression. (A) Diversity of SAs in tomato. Hierarchical clustering of SAs obtained by UPLC-qTOF-MS analysis. Yellow frames enclose several metabolite clusters discussed in the text, whereas white arrows indicate the possible conversion of green tissue–associated metabolites into RR tissue–associated metabolites during fruit ripening. Numbers within parentheses correspond to SAs in Supplemental Table 1 online. Relative levels of five additional putative SAs that were identified in the course of this study could not be measured. These substances are listed in Supplemental Table 1 online. (B) GAME1 expression in tomato tissues, measured by quantitative real-time PCR. The statistical significance of the gene expression data for each examined tissue is presented in Figure 2A. For (A) and (B), the color index refers to the relative levels of a particular metabolite or the GAME1 transcript across the different tissues examined; the highest level is defined as 100% (n = 3). Br, breaker; IG, immature green; M, mature (fully expanded); Y, young.

Figure 2.

Expression of GAME1 in the Wild Type and Fruit of the rin and nor Mutants and Tomatidine-Galactosyltransferase Activity in Wild-Type Tomato Tissues. (A) Quantitative real-time PCR relative expression analyses of GAME1 transcripts in 21 tissues of wild-type tomato (cv MicroTom). Different lettering above the bars denotes significant differences in gene expression as calculated by a Student’s t test (P < 0.05; n = 3). (B) GAME1 expression is elevated in 1-MCP–treated fruit (cv Ailsa Craig) in the MG and Or developmental stages compared with untreated control fruit. Student’s t test results for significance (P < 0.05; n = 3) are indicated by an asterisk. (C) GAME1 expression in tomato fruit of the rin and nor ripening mutants (cv Ailsa Craig) is higher than in fruit of the wild-type (WT) plants at the Or and RR fruit stages. Different lettering above the bars denotes significant differences in GAME1 relative expression levels as calculated by a Student’s t test (P < 0.05; n = 3). (D) Tomatidine-galactosyltransferase activity in five tissues of tomato (cv MicroTom). Activity was measured in freeze-dried samples and is expressed on a dry weight (DW) basis. Different lettering above the bars denotes significant differences in activity as calculated by a Student’s t test (P < 0.05; n = 3). In all panels, the bars represent se. Br, breaker; IG, immature green; ML, mature (fully expanded) leaves; YL, young leaves.

Figure 3.

The Recombinant GAME1 Catalyzes the Galactosylation of Tomatidine. Enzyme activity assays of the recombinant GAME1 produced in E. coli cells. The chromatograms (total ion count in UPLC-qTOF-MS) display analysis of the GAME1 recombinant protein incubated for 1 h with 0.25 mM tomatidine in the presence (top) or absence (bottom) of 8 mM UDP-Gal (UDP-gal). The m/z of eluting compounds is indicated. Arrows point to tomatidenol (dehydrotomatidine) (right) and dehydrotomatine plus Gal (left).

Figure 4.

GAME1-Silenced Tomato Plants Display Severe Morphological Phenotypes. (A) and (B) Three-week-old (3w) wild-type (WT) and GAME1i (SPh) tomato plants (cv MicroTom), respectively. (C) and (D) Six-week-old (6w) GAME1i SPh tomato plants (cv MicroTom). (E) and (F) Four-month-old (4m) wild-type and GAME1i (SPh) tomato plants (cv MicroTom), respectively. (G) to (K) MG and RR wild-type tomato fruit (cv MicroTom; [G] and [H]) compared with fruit in the corresponding stages of the MPh GAME1i plants [(I) and (J)] and to the SPh GAME1i RR stage fruit (K). (L) Suberized stem of a GAME1i plant (cv Ailsa Craig).

Figure 5.

Altering GAME1 Expression Has a Major Effect on in Planta Tomatidine-Galactosyltransferase Activity and on Levels of α-Tomatine and Tomatidine. (A) Tomatidine-galactosyltransferase activity in fully expanded leaves (cv MicroTom, 4-week-old plants). Activity was measured in extracts derived from fresh samples and is expressed on a fresh weight (FW) basis. Different lettering above the bars denotes significant differences in tomatidine-galactosyltransferase activity levels as calculated by a Student’s t test (P < 0.05; n = 3). WT, wild type. (B) GAME1i plants exhibit reduced levels of α-tomatine and accumulation of its precursor tomatidine. Their absolute concentrations were measured in fully expanded leaves of the wild type (4-week-old plants) and compared with those of SPh GAME1i plants of similar age (using UPLC-qTOF-MS). Significant differences according to a Student’s t test (P < 0.05; n = 3) are indicated by an asterisk. In (A) and (B), the bars represent se.

Figure 6.

Changes to SA Levels in MG Fruit of GAME1-Silenced Plants. Metabolites that are abundant at the immature green and MG stages are marked with a green background, those abundant at the breaker and orange stages with an orange background, and those abundant at the RR stage are marked with a red background. Metabolites placed on a white background were not detected in fruit tissues. In the squares, elevated and reduced metabolite levels in fruit of GAME1-silenced plants are marked with red and blue colors, respectively, while a lack of color represents no change in metabolite levels. The numbering of the isomers of a particular metabolite is according to the putative SA numbers in Supplemental Table 1 online. Dashed arrows represent multiple biosynthetic reactions, solid arrows represent a single biosynthetic reaction, and possible points of glycosylation activity of GAME1 are encircled. Acetoxy-hydroxy-methoxy-dehydrotomatine (8), acetoxytomatine + deoxyhexose (14), dehydrotomatidine tetrahexoside (19), di-dehydrotomatine (26), hydroxy-dehydrotomatidine trihexoside + deoxyhexose (33), three isomers of hydroxy-dehydrotomatidine trihexoside (34, 35, and 36), lycoperoside G/F or esculeoside A + hexose - pentose (57), tomatidine dihexoside + pentose + deoxyhexose (68), α-tomatine + C4H6O3 (70), and tri-hydroxy-dehydrotomatine (71) were not included in this scheme.

Figure 7.

GAME1-Silenced Plants Are Altered in Their Sterol Profile and Plastid Morphology. (A) Absolute concentrations of 10 detected phytosterols in fully expanded leaves of 4-week-old GAME1i plants compared with wild-type (WT) plants of a similar age were measured using GC-MS (see Methods). In (A), the bars represent se. Sterols that were found to be significantly altered by a Student’s t test (P < 0.05; for the wild type, n = 5; for GAME1i, n = 6) are marked with an asterisk. The proposed pathway of sterol metabolism in plants and changes in leaves of GAME1-silenced plants are shown in Supplemental Figure 7B online. (B) and (D) TEM images of plastids in cells of mature leaves of the wild-type. In (B), starch grains (sg), plastoglobuli (pg), and grana (gr) are indicated with arrows. (C) and (E) TEM images of plastids in cells of mature leaves of a SPh GAME1i plant.