Lisbeth Jonsson

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

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.

Microarray analysis of the interaction between the aphid Rhopalosiphum padi and host plants reveals both differences and similarities between susceptible and partially resistant barley lines.

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins.

Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol

Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.

Sterol composition and synthesis in potato tuber discs in relation to glycoalkaloid synthesis

Abstract The synthesis of sterols and sterol precursors was analysed in potato tuber discs, which accumulate glycoalkaloids, and in discs where this accumulation was inhibited by the addition of ethephon or tridemorph. In the 4,4-dimethylsterol fraction and the 4α-methylsterol fraction, only compounds with a nonalkylated side-chain were found. The 4-desmethylsterols synthesized de novo were, in tridemorph-treated discs, pollinastanol and 5α-cholest-8-en-3β-ol; in ethephon-treated discs, isofucosterol; and in control discs, isofucosterol and cholesterol. The cholesterol concentration decreased concurrently with the accumulation of glycoalkaloids. The results show that cholesterol synthesis is stimulated in potato discs and indicate that cholesterol is a precursor of glycoalkaloids in potato.

Characterization of UDP-glucose:solanidine glucosyltransferase and UDP-galactose:solanidine galactosyltransferase from potato tuber

Abstract Upon differential fractionation of potato tuber homogenate, the soluble enzym uridine-5′-diphospho-glucose(UDP-glucose):solanidine glucosyltransferase remained for the main part in the supernatants, whereas the membrane-bound UDP-glucose:β-sitosterol glucosyltransferase activity was found both in a 13 000-g and 100 000-g pellet. The-soluble fraction glycosylated not only solanidine, but also the steroidal alkaloids solasodine and tomatidine, using either UDP-glucose or UDP-galactose as sugar donor. The K m -values for the sugar donors in the solanidine glycosylating reactions were similar: UDP-glucose, 22 μM and UDP-galactose, 29 μM. UDP-galactose was a competitive inhibitor in the solanidine glucosylating reaction ( K i 1.1 mM), whereas UDP-glucose showed non-competitive inhibition in the galactosylation reaction ( K i 21 μM). After separations on gelfiltration, ion-exchange or chromatofocusing columns,glucosylating and galactosylating activities were recovered in the same fractions, but with loss of most of the galactosyltransferase activity in the two latter types of separation. The apparent molecular weight of the solanidine glycosylating enzyme(s) was 40 000 and the isoelectric point 4.8.

Sterol composition and growth of transgenic tobacco plants expressing type-1 and type-2 sterol methyltransferases

Abstract. Transgenic tobacco (Nicotiana tabacum L.) plants with altered sterol composition were generated by transformation with plant cDNAs encoding type-1 and type-2 sterol methyltransferases (SMTs; EC 2.1.1.41). For both SMT1 and SMT2 transformants, the transformation was associated with a reduction in the level of cholesterol, a non-alkylated sterol. In SMT1 transformants a corresponding increase of alkylated sterols, mainly 24-methyl cholesterol, was observed. On the other hand, in SMT2 transformants the level of 24-methyl cholesterol was reduced, whereas the level of sitosterol was raised. No appreciable alteration of total sterol content was observed for either genotype. The general phenotype of transformants was similar to that of controls, although SMT2 transformants displayed a reduced height at anthesis. The results show that plant sterol composition can be altered by transformation with an SMT1 cDNA without adverse effects on growth and development, and provide evidence, in planta, that SMT1 acts at the initial step in sterol alkylation.

Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or beta-1,3-glucanase transcript abundance

The effects of infestation by the bird cherry-oat aphid (BCA), (Rhopalosiphum padi L) and the Russian wheat aphid (RWA) (Diuraphis noxia Mordvilko) on callose deposition and transcription of genes related to callose accumulation were investigated in barley (Hordeum vulgare L. cv. Clipper). The BCA, which gives no visible symptoms, induced very limited callose deposition, even after 14 days of infestation. In contrast, RWA, which causes chlorosis, white and yellow streaking and leaf rolling, induced callose accumulation already after 24 h in longitudinal leaf veins. The deposition was pronounced after 72 h, progressing during 7 and 14 days of infestation. In RWA-infested source leaves, callose was also induced in longitudinal veins basipetal to the aphid-infested tissue, whereas in sink leaves, more callose deposition was found above the feeding sites. Eight putative callose synthase genes were identified in a database search, of which seven were expressed in the leaves, but with similar transcript accumulation in control and aphid-infested tissue. Five out of 12 examined beta-1,3-glucanases were expressed in the leaves. All five were upregulated in RWA-infested tissue, but only two in BCA-infested tissue, and to a lesser extent than by RWA. The results suggest that callose accumulation may be partly responsible for the symptoms resulting from RWA infestation and that a callose-inducing signal may be transported in the phloem. Furthermore, it is concluded that the absence of callose deposition in BCA-infested leaves is not because of a stronger upregulation of callose-degrading beta-1,3-glucanases in this tissue, as compared to RWA-infested leaves.

Glycoalkaloid and calystegine levels in table potato cultivars subjected to wounding, light, and heat treatments.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (α-chaconine and α-solanine) and calystegines (A₃, B₂, and B₄) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Regulation of Glycoalkaloid Accumulation in Potato Tuber Discs

Summary The level of glycoalkaloids present in freshly cut potato tuber discs started to increase after 24 hours of incubation. This accumulation was inhibited by the sterol synthesis inhibitor, tridemorph, and was thus due to synthesis de novo . Concomitant to the accumulation of glycoalkaloids, there was an increase in the specific activity of a glycoalkaloid-specific enzyme, UDP-glucose:solanidine glucosyltransferase (solanidine-GT, EC 2.4.1). Time-course studies of enzyme activities in the discs showed that solanidine-GT activity increased at a low rate during the first 11 hours after slicing and at the highest rate between 11 and 16 hours. Other sterol-metabolizing enzymes exhibited different time-course curves:S-adenosyl-L-methionine:cycloartenol methyltransferase (cycloartenol-MT, EC 2.1.1.41) activity increased 5 hours after slicing and peaked at 11 hours. UDP-glucose:sterol glucosyltransferase (sterol-GT, EC 2.4.1) activity was not enhanced as a result of wounding. The accumulation of glycoalkaloids was not affected by light exposure or addition of abscisic acid, gibberellic acid or (2-chloroethyl)trimethylammonium chloride to the discs. Inhibition of ethylene synthesis or action gave a higher content of glycoalkaloids than in control discs, whereas discs incubated at high levels of ethylene had a very low glycoalkaloid content and also a lower activity of solanidine-GT than control discs.

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.