Reduction of Cholesterol and Glycoalkaloid Levels in Transgenic Potato Plants by Overexpression of a Type 1 Sterol Methyltransferase cDNA

Abstract

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.Glycoalkaloids are a family of steroidal toxic secondary metabolites present in plants of the Solanaceae family. In cultivated potato (Solanum tuberosum) the main glycoalkaloids, ␣-chaconine and ␣-solanine, are triglycosylated products of the same aglycone, solanidine, but they differ in their sugar moieties (Friedman and McDonald, 1997). The highest glycoalkaloid level in potato plants is found in flowers and sprouts, followed by the leaves, and the lowest amounts are detected in stems and tubers. The amount of glycoalkaloids increases upon wounding and light exposure, something that may render tubers unsuitable for human consumption. Mild clinical symptoms of glycoalkaloid poisoning include abdominal pain, vomiting, and diarrhea, and an upper safe limit in tubers of 200 mg total glycoalkaloids (TGA) kg Ϫ1 fresh weight has been recommended by leading authorities. However, this upper limit is close to levels found in tubers destined for human consumption, and efforts should be made to keep TGA levels low when introducing new varieties on the market (see Valkonen et al., 1996).The biosynthesis of glycoalkaloids in potato is currently not fully understood. Solanidine has been proposed to be synthesized from the key precursor in plant sterol synthesis, cycloartenol, in a biosynthetic route including cholesterol, a sterol lacking alkylations at the C-24 position in the side chain (Heftmann, 1983; Bergenstråhle et al., 1996;Friedman and McDonald, 1997; Fig. 1). Cholesterol is in most plant species only a minor sterol, but is present at relatively high levels, approximately 15% to 20% of total sterols, in Solanaceous plants such as potato and tobacco (Nicotiana tabacum). One of the final reactions in the synthesis of glycoalkaloids is the glucosylation or galactosylation of solanidine to yield ␥-chaconine or ␥-solanine, respectively. A cDNA encoding the solanidine glucosyltransferase (SGT) enzyme has been cloned (Moehs et al., 1997). The SGT mRNA increased after wounding, in line with previous measurements of wound-induced SGT activity and glycoalkaloid levels. However, the ga...

Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol, the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato

“…The presence of the alkyl group at C-24 that originates from this reaction is the structural characteristic differentiating C-24 alkylsterol from cholesterol. Therefore, cholesterol levels in plants are hypothesized to be modulated by SMT; this hypothesis is supported by the lower cholesterol level in transgenic plants that overexpress SMT and the higher cholesterol level in SMT knockout plants (Diener et al, 2000;Schaeffer et al, 2000;Sitbon and Jonsson 2001;Holmberg et al, 2002;Arnqvist et al, 2003). However, the considerable amounts of cholesterol and its derivatives, SGAs, in solanaceous plants have suggested that these plants have a mechanism to produce cholesterol that is distinct from other plants.…”

“…In plants, cycloartenol is the first D 24(25) -sterol. If cycloartenol is reduced to cycloartanol by SSR2, the biosynthetic pathway would be efficiently redirected toward cholesterol (Figure 1) (Diener et al, 2000;Schaeffer et al, 2000;Sitbon and Jonsson 2001;Holmberg et al, 2002;Arnqvist et al, 2003). We conducted in vitro assays to examine whether SSR2 has cycloartenol reductase activity and to compare the substrate specificities of SSR1 and SSR2 (Figure 4).…”

GLYCOALKALOID METABOLISM1 Is Required for Steroidal Alkaloid Glycosylation and Prevention of Phytotoxicity in Tomato

“…Consistent with the inability of sterol treatment to complement plants defective in SMT2 (Carland et al, 2010), our attempts to reverse the GAME1i phenotype by application of b-sitosterol and/or stigmasterol were unsuccessful. The effect of GAME1 silencing on sterol metabolism demonstrates once again the tight link between SAs and the sterol pathway (Arnqvist et al, 2003).…”

“…Alteration of sterol metabolism was also considered as a reason for the morphological changes, since SA and sterol biosynthesis are tightly linked (Arnqvist et al, 2003). Moreover, tomatidine, which accumulates to high levels in GAME1i plants, was reported to mimic the effect of sterol biosynthesis inhibitors (Simons et al, 2006).…”

“…SAs have been extensively investigated for their diverse biological activities and occurrence in important crop plants (e.g., tomato [Solanum lycopersicum], potato [Solanum tuberosum], and eggplant [Solanum melongena]) (Eich, 2008). The synthesis of SAs, which is presumed to start from cholesterol, likely occurs in the cytosol and in most cases involves further glycosylation of the alkamine steroidal skeleton (aglycone) at C-3b to form steroidal glycoalkaloids (SGAs) (Bowles, 2002;Friedman, 2002;Arnqvist et al, 2003;Kalinowska et al, 2005;Bowles et al, 2006).…”