Browsing by Author "Sen, Jayanti"

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Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus
(Wiley-Blackwell, 2007) Kumar, Santosh; Dutta, Ajaswrata; Sinha, Alok Krishna; Sen, Jayanti
Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide 1 primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a ‘three intron’ category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a beta-glucuronidase–green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed.
Downregulation of terpenoid indole alkaloid biosynthetic pathway by low temperature and cloning of a AP2 type C-repeat binding factor (CBF) from Catharanthus roseus (L). G. Don
(Springer, 2007) Dutta, Ajaswrata; Sen, Jayanti; Deswal, Renu
Plants produce secondary metabolites in response to various external signals. Coordinated transcriptional control of biosynthetic genes emerges as a major mechanism dictating the accumulation of secondary metabolites in plant cells. However, information about stress regulation of secondary metabolites and the molecular mechanisms regulating these specialized pathways are poorly understood. Here, we show that terpenoid indole alkaloid (TIA) biosynthetic pathway is differentially regulated in response to different abiotic stresses in Catharanthus roseus, a model medicinal plant producing important anticancer and antihypertensive drugs. Semiquantitative RT-PCR analysis of TIA and related primary pathway genes in response to dehydration, low temperature, salinity, UV-light and wounding revealed their negative regulation in response to low temperature. HPLC analysis further supports the notion that TIA biosynthetic pathway is negatively controlled by low temperature stress.
Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don
(Springer, 2005) Dutta, Ajaswrata; Batra, Jyoti; Pandey-Rai, Sashi; Singh, Digvijay; Kumar, Sushil; Sen, Jayanti
Madagascar periwinkle, Catharanthus roseus (L.) G. Don, a medicinally important plant, produces anticancer dimeric alkaloids, vinblastine and vincristine, in the leaves and accumulates antihypertensive alkaloids, ajmalicine and serpentine, in the roots. This plant grows wild in distant tropical and sub-tropical geographical locations with different agro-climates and shows wide variations in morphological and alkaloid yield-related traits. In order to understand the correlation between the expression of terpenoid indole alkaloid (TIA) pathway genes and accumulation of related alkaloids, six different genetic resources of C. roseus, including the medicinal cultivars Nirmal, Prabal, Dhawal, the mutants gsr-3 and gsr-6, and one horticultural variety, Pacifica blush, were studied. The expression profiles of one early and two late TIA biosynthetic pathway genes, namely, strictosidine synthase, desacetoxyvindoline 4-hydroxylase and deacetyl vindoline 4-O-acetyl transferase were analyzed in these plants. A positive correlation between transcript abundance and accumulation of related alkaloids was observed in the different genetic resources. The potential of these TIA biosynthetic pathway genes for use in screening of high-yielding C. roseus germplasm has been discussed.
Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration
(Springer, 2004) Batra, Jyoti; Dutta, Ajaswrata; Singh, Digvijay; Kumar, Sushil; Sen, Jayanti
Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pat- tern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri TL-DNA and TR-DNA genes, there were only three different categories of independent hairy root clones–C1 (rolA&B+/ags+), C2 (rolA&B-/ags+) and C3 (rolA&B+/ags ). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the TL-DNA and TR-DNA genes (C1) and the TL-DNA gene (C3) alone. Two rolA&B but ags+ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the TL-DNA and TR-DNA genes on growth and alkaloid accumulation in these root clones is discussed
Level of endogenous free amino acids during various stages of culture of Vigna mungo (L.) Hepper - somatic embryogenesis, organogenesis and plant regeneration
(Indian Academy of Sciences, 2002) Sen, Jayanti; Kalia, Sanjeev; Guha-Mukherjee, Sipra
A comparative study on endogenous free amino acid levels was conducted in embryogenic and regenerating tissues vis-à-vis regenerated shoots of Vigna mungo PS1. A number of important amino acids were found to increase during initiation of embryogenesis and regeneration. To study the effects of exogenous application of amino acids in this system, media were supplemented with three amino acids, tryptophan (Trp), serine (Ser) and proline (Pro). We found that in the presence of proline, a stress associated amino acid, there was an enhancement in shoot-bud regeneration as well as in the production of somatic embryos. We also analysed the endogenous levels of amino acids in these tissues. This communication thus deals with the induction of globular embryo by auxin and its promotion by proline in V. mungo.
New evidences about strictosidine synthase (Str) regulation by salinity, cold stress and nitric oxide in Catharanthus roseus
(Springer, 2013) Dutta, Ajaswrata; Sen, Jayanti; Deswal, Renu
Alkaloid production in plants is altered by abiotic stressors, but the mechanism(s) are poorly understood. Present study provides novel evidences about differential regulation of strictosidine synthase (Str), the key gene of terpenoid indole alkaloid (TIA) biosynthetic pathway in response to salinity and low temperature stress in Catharanthus roseus. HPLC analysis of terpene indole alkaloids correlated with differential regulation of Str by low temperature and salinity stress. Administration of exogenous calcium and calcium channel modulator preferentially regulated Str transcript. In addition, administration of kinase and phosphatase inhibitors modulated Str expression. Involvement of nitric oxide (NO) signaling was ascertained by NO donor and nitric oxide synthase (NOS) scavenger treatments. This finding suggests co-ordinated action of calcium, protein kinases, phosphatases and nitric oxide in abiotic stress signaling for TIA biosynthetic pathway in C. roseus.
Transcript profiling of terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in Catharanthus roseus cell cultures
(Springer, 2007) Dutta, Ajaswrata; Singh, Digvijay; Kumar, Sushil; Sen, Jayanti
The understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseus, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseus cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol-4- phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macro- array, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseus comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseus cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseus cell cultures.
Use of a herbicide or lysine and threonine for non-antibiotic selection of transgenic chickpea
(Springer, 2004) Tewari-Singh, N.; Sen, Jayanti; Kiesecker, H.; Reddy, V. S.; Jacobsen, H.J.; Guha-Mukherjee, S.
A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea (Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 M thidiazuron (TDZ) and 2 mM lysine and 2 mM threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l-1 PPT. Increasing the concentrations of PPT to 15 mg l-1 reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.