Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes.

Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

Regulation of lipin1 by nutritional status, adiponectin, sex and pituitary function in rat white adipose tissue.

Lipin1 is a member of the lipin protein family that plays an important role in the regulation of lipid metabolism. The endogenous role of lipin1 was demonstrated by the fact that mutations in lipin1 caused lipodystrophy and metabolic disorders. The aim of this study was to assess the influence of nutritional status, pregnancy, insulin-sensitizers and pituitary hormones on lipin1 mRNA levels in adipose tissue of rats. Lipin1 gene expression was induced in conditions of hypoleptinemia (fasting) and leptin resistance (high fat diet), whereas it was decreased by high circulating leptin levels (leptin administration, pregnancy) and in leptin-deficient mice. Lipin1 mRNA levels were also decreased in adiponectin-deficient mice. Lipin1 mRNA levels are influenced by age in female rats, with peak expression at 25th day of life and decreasing thereafter. Consistently, ovariectomy increased lipin1 expression indicating that estrogens modulate lipin1. Finally, lipin1 was also regulated by pituitary hormones, since its expression was modified by thyroid status and growth hormone deficiency. Our observations indicate that: a) gWAT lipin1 mRNA levels are regulated by nutritional status, and leptin plays an important role in this regard, b) lipin1 is modulated by adiponectin, c) lipin1 is influenced by age and sex, and d) alterations in pituitary function modify lipin1 mRNA levels. To dissect the complicated interactions between key regulators of lipid metabolism like lipin1, may be important for the development of new therapies for the treatment and prevention of obesity and its associated disorders.

Linkage analysis of a rare alkaloid present in a tetraploid potato with Solanum chacoense background.

The potato genotype ND4382-19 has Solanum chacoense Bitt. in its genetic background. Foliar alkaloid analysis of it and its progeny ND5873 (ND4382-19 × Chipeta) by gas chromatography-mass spectrometry (GC-MS) showed that, in addition to the expected alkaloids (solanidine, leptinidine, and acetyl-leptinidine), there was an aglycone of another rare alkaloid. Its molecular mass and some of the m/z fragment ions were similar to leptinidine, but the major fragment ion was the m/z 150 peak of solanidine. This fragmentation pattern suggested that this alkaloid is a solanidine-based compound with mass equal to leptinidine. Leptinidine differs from solanidine by an extra -OH group, but the GC-MS fragmentation pattern of the rare compound indicated hydroxylation at a different position than the C-23 of leptinidine. The exact chemical structure is still unknown, and further analysis, such as NMR will be necessary to determine the structure. Segregation analysis of ND5873 (ND4382-19 × Chipeta) showed that presence of this rare compound segregated in a 1:1 ratio, indicating that a single gene controlled its synthesis and/or accumulation in foliar tissue. Analysis with AFLP and microsatellite markers indicated that the locus-controlling presence of this alkaloid resided on potato chromosome I, with the nearest flanking AFLP markers 0.6 and 9.4 cM apart. This rare alkaloid was present in the foliage and not detected in potato tubers. Its presence in leaves did not affect resistance/susceptibility to Colorado potato beetle.

Technology of compact MAb and its application for medicinal plant breeding named as missile type molecular breeding.

Single chain fragment-variable (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the ScFv solamargine (As)-scFv gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of the parent monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 µg/ml. The expressed anti-solamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology that can produce a high yield of secondary metabolites.

A QTL that confers resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) in tetraploid potato populations segregating for leptine.

Genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) from Solanum chacoense has been incorporated in the tetraploid potato selection, ND4382-19, which is highly resistant and contains moderate level of foliar leptines. We recently reported using ND4382-19 progeny, population ND5873 (ND4382-19 x Chipeta), to map two genes that segregated as complementary epistatic genes that allow accumulation of leptinidine (Lep) and acetyl-leptinidine (AL) on chromosomes 2 and 8, respectively. We describe here the characterization of a second half-sib population NDG116 (ND4382-19 x N142-72). In this population, solasodine from parent N142-72, which has Solanum berthaultii in its background, was predominant over solanidine-based alkaloids. Concentrations of solanidine, leptinidine, and acetyl-leptinidine were 15-, 5-, and 14-fold lower than in the ND5873 population. Nevertheless, Lep and AL mapped to the same locations on chromosomes 2 and 8 of parent ND4382-19, respectively. The two populations were evaluated for resistance to Leptinotarsa in field assays, and by detached leaf assay for population NDG116. In both families, QTL analysis identified a major QTL from ND4382-19 on the distal end of chromosome 2, close to the Lep locus. The contribution of this QTL to resistance ranged from 11 to 34% for ND5873 at four field sites. Contribution to resistance from the linkage group that contains the gene AL for the accumulation of leptine was not detected. In family NDG116, the same chromosome 2 QTL was detected for field and detached leaf assays, explaining 26 and 12% of the variance for defoliation and larval development, respectively. These data may indicate another resistance mechanism besides leptine in the Leptinotarsa resistance observed in these populations.

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants.

We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Transformation of Solanum dulcamara protoplasts and regeneration of transgenic plants.

We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) protoplasts. Under these conditions, a very high transformation efficiency was obtained, with relative transformation frequency being up to 12.4% and absolute transformation frequency 2.4 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.