Chandrama Prakash Upadhyaya

Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses

Abstractl-Ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing l-gulono-γ-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6–7xa0weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl Viologen (MV), NaCl or mannitol, respectively. The T1 transgenic plants exposed to salt stress (100xa0mM NaCl) survived better with increased shoot and root length when compared to untransformed plants. The elevated level of AsA accumulation in transgenics was directly correlated with their ability to withstand abiotic stresses. These results further demonstrated that the overexpression of GLOase gene enhanced basal levels of AsA in potato tubers and also the transgenics showed better survival under various abiotic stresses.

Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing d-galacturonic acid reductase gene in response to various abiotic stresses

Upregulation of the antioxidant enzyme system in plants provides protection against various abiotic stresses. Transgenic potato plants overexpressing the strawberry d-galacturonicacidreductase (GalUR) gene with enhanced accumulation of ascorbate (AsA) were used to study the antioxidant system involving the ascorbate–glutathione cycle in order to understand the tolerance mechanism in plants in response to various abiotic stresses under in vitro conditions. Transgenic potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed enhanced activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.1.1.1.6) and enzymes of the ascorbate–glutathione cycle such as ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.8.1.7), as well as increased levels of ascorbate, glutathione (GSH) and proline when compared to untransformed tubers. The increased enzyme activities correlated with the mRNA transcript levels in the stressed transgenic tubers. Significant differences in redox status of AsA and GSH were also observed in stressed transgenic potato tubers that showed increased tolerance to abiotic stresses compared to untransformed tubers. This study suggests that the increased accumulation of AsA could upregulate the antioxidant system which imparts improved tolerance against various abiotic stresses in transgenic tubers compared to untransformed tubers.

Dynamic proteomic profile of potato tuber during its in vitro development.

Potato tuberization is a complicated biochemical process, which is dependent on external environmental factors. Tuber development in potato consists of a series of biochemical and morphological processes at the stolon tip. Signal transduction proteins are involved in the source-sink transition during potato tuberization. In the present study, we examined protein profiles under in vitro tuber-inducing conditions using a shotgun proteomic approach involving denaturing gel electrophoresis and liquid chromatography-mass spectrometry. A total of 251 proteins were identified and classified into 9 groups according to distinctive expression patterns during the tuberization stage. Stolon stage-specific proteins were primarily involved in the photosynthetic machinery. Proteins specific to the initial tuber stage included patatin. Proteins specific to the developing tuber stage included 6-fructokinase, phytoalexin-deficient 4-1, metallothionein II-like protein, and malate dehydrogenase. Novel stage-specific proteins identified during in vitro tuberization were ferredoxin-NADP reductase, 34 kDa porin, aquaporin, calmodulin, ripening-regulated protein, and starch synthase. Superoxide dismutase, dehydroascorbate reductase, and catalase I were most abundantly expressed in the stolon; however, the enzyme activities of these proteins were most activated at the initial tuber. The present shotgun proteomic study provides insights into the proteins that show altered expression during in vitro potato tuberization.

Characterization of a cinnamoyl derivative from broccoli (Brassica oleracea L. var. italica) florets

A new intact glucosinolate Cinnamoyl derivative [6-O-trans-(4″- hydroxy cinnamoyl)-4-(methylsulphinyl) butyl glucosinolate] (A) has been isolated from Broccoli (Brassica oleracea L. var. italica) florets. The compound was isolated and characterized by LC, MS-ESI, FTIR, (1)H and (13)C NMR as well as (1)H-(1)H COSY, DEPT 135° spectrometric experiments.