Pravej Alam

Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L.

Artemisinin is effective against both chloroquine-resistant and -sensitive strains of Plasmodium species. However, the low yield of artemisinin from cultivated and wild plants is a serious limitation to the commercialization of this drug. Optimization of artemisinin yield either in vivo or in vitro is therefore highly desirable. To this end, we have overexpressed the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthusroseus L. in Artemisia annua L. and analyzed its influence on artemisinin content. PCR and Southern blot analyses revealed that the transgenic plants showed stable integration of the foreign hmgr gene. The reverse transcriptase-PCR results suggested that the hmgr was expressed at the transcriptional level in transgenic lines of Artemisia annua L., while the high-performance liquid chromatography analysis showed that artemisinin content was significantly increased in a number of the transgenic lines. Artemisinin content in one of the A. annua transgenic lines was 38.9% higher than that in non-transgenic plants, and HMGR enzyme activity in transgenic A. annua L. was also higher than that in the non-transgenic lines.

Modulation of artemisinin biosynthesis by elicitors, inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.

Fusarium solani, P1, a new endophytic podophyllotoxin-producing fungus from roots of Podophyllum hexandrum

Podophyllotoxin, a well-known naturally occurring aryl tetralin lignan produced by few plant species is used as precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophos phosphate. The availability of this lignan is limited due to the scarce occurrence of its natural sources. Further, synthetic approaches for its production are still commercially unacceptable. This paper reports the synthesis of podophyllotoxin by an endophytic fungus Fusarium solani isolated from the roots of Podophyllum hexandrum. The presence of podophyllotoxin in fungal biomass was confirmed and quantified by HPLC and mass spectrometry. The fungus is able to produce 29.0 µg/g podophyllotoxin on dry weight basis.

Influence of Agrobacterium rhizogenes on induction of hairy roots for enhanced production of artemisinin in Artemisia annua L. plants

Artemisinin production from plant tissue cultures and induction of hairy roots in vitro have been considered to be a promising alternative, which offer a high degree of genetic stability, grow rapidly and produce the higher spectrum of secondary metabolites than wild type plants. Hairy root cultures developed from infection of different explants of in vitro germinated Artemisia annua L. plant with Agrobacterium rhizogenes LBA 9402 strain were selected on the basis of high artemisinin content and growth. Integration of the TL-DNA ( rol gene) region of the pRi plasmid was confirmed by polymerase chain reaction (PCR) analysis of the gene located in this region. The effect of different environmental factors like temperature, pH, cultivation media and carbon source on growth and artemisinin production were studied in shake flask cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 390 μg L -1 day -1 was obtained in hairy root cultures. Keywords: Agrobacterium , Artemisia annua L., artemisinin, hairy root cultures

Enhanced artemisinin accumulation and metabolic profiling of transgenic Artemisia annua L. plants over-expressing by rate-limiting enzymes from isoprenoid pathway

Artemisinin, a natural product isolated from aerial parts of Artemisia annua L. plant, is a potent antimalarial drug against drug-resistant malaria. In recent times, the demand (101–119 MT) for artemisinin is exponentially increasing with the increased incidence of drug-resistant malaria throughout the world, especially African and Asian continents. However, the commercial production of artemisinin-based combination therapies has limitation because of the presence of low concentration of artemisinin in plants. Therefore, transgenic lines of A. annua L. plants over-expressing both HMG-Co A reductase (hmgr) and amorpha-4, 11-diene synthase (ads) genes were developed to enhance the content of artemisinin. The selected transgenic lines (TR4, TR5, and TR7) were found to accumulate higher artemisinin (0.97–1.2%) as compared to the non-transgenic plants (0.63%). The secondary metabolite profiles of these lines were also investigated employing gas chromatography mass spectrometry, which revealed a clear difference in these metabolites in transgenic and non-transgenic lines of A. annua L. at different growth and developmental stages. The major metabolites reported in these lines at pre-flowering stage were related to essential oil and chlorophyll biosynthesis (71.33% in TR5 transgenic lines vs. 61.70% in non-transgenic line). Based on these results, we concluded that over-expression of both hmgr and ads genes in A. annua L. plants results not only increase in artemisinin content, but also enhances synthesis of other isoprenoid including essential oil. It is also evident from this study that the novel artemisinin-rich varieties of A. annua L. could be developed by suppressing essential oil biosynthesis, so that more carbon could preferentially be diverted from mevalonate pathway to artemisinin biosynthesis.

Variation in oil content and fatty acid composition of the seed oil of Acacia species collected from the northwest zone of India

BACKGROUND The oil content and fatty acid composition of the mature seeds of Acacia species collected from natural habitat of the northwest zone of the Indian subcontinent (Rajasthan) were analyzed in order to determine their potential for human or animal consumption. RESULTS Oil content varied between 40 and 102 g kg⁻¹. The highest oil content was obtained in Acacia bivenosa DC. (102 g kg⁻¹) among the nine Acacia species. The fatty acid composition showed higher levels of unsaturated fatty acids, especially linoleic acid (~757.7 g kg⁻¹ in A. bivenosa), oleic acid (~525.0 g kg⁻¹ in A. nubica) and dominant saturated fatty acids were found to be 192.5 g kg⁻¹ palmitic acid and 275.6 g kg⁻¹ stearic acid in A. leucophloea and A. nubica respectively. Seed oils of Acacia species can thus be classified in the linoleic-oleic acid group. Significant variations were observed in oil content and fatty acid composition of Acacia species. CONCLUSION The present study revealed that the seed oil of Acacia species could be a new source of high linoleic-oleic acid-rich edible oil and its full potential should be exploited. The use of oil from Acacia seed is of potential economic benefit to the poor native population of the areas where it is cultivated. The fatty acid composition of Acacia seed oils is very similar to that reported for commercially available edible vegetable oils like soybean, mustard, sunflower, groundnut and olive. Hence the seed oil of Acacia species could be a new source of edible vegetable oil after toxicological studies.

Isolation, characterization and structural studies of amorpha - 4, 11-diene synthase (ADS(3963)) from Artemisia annua L.

: With the escalating prevalence of malaria in recent years, artemisinin demand has placed considerable stress on its production worldwide. At present, the relative low-yield of artemisinin (0.01-1.1 %) in the source plant (Artemisia annua L. plant) has imposed a serious limitation in commercializing the drug. Amorpha-4, 11-diene synthase (ADS) has been reported a key enzyme in enhancing the artemisinin level in Artemisia annua L. An understanding of the structural and functional correlations of Amorpha-4, 11-diene synthase (ADS) may therefore, help in the molecular up-regulation of the enzyme. In this context, an in silico approach was used to study the ADS₃₉₆₃ (3963 bp) gene cloned by us, from high artemisinin (0.7-0.9% dry wt basis) yielding strain of A. annua L. The full-length putative gene of ADS₃₉₆₃ was found to encode a protein consisting of 533 amino acid residues with conserved aspartate rich domain. The isoelectric point (pI) and molecular weight of the protein were 5.25 and 62.2 kDa, respectively. The phylogenetic analysis of ADS genes from various species revealed evolutionary conservation. Homology modeling method was used for prediction of the 3D structure of ADS₃₉₆₃ protein and Autodock 4.0 version was used to study the ligand binding. The predicted 3D model and docking studies may further be used in characterizing the protein in wet laboratory.

Efficient Method for Agrobacterium Mediated Transformation of Artemisia annua L.

Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

Mitigation of sodium chloride toxicity in Solanum lycopersicum L. by supplementation of jasmonic acid and nitric oxide

ABSTRACT We investigated the effects of exogenous application of jasmonic acid (JA) and nitric oxide (NO) on growth, antioxidant metabolism, physio-biochemical attributes and metabolite accumulation, in tomato (Solanum lycopersicum L.) plants exposed to salt stress. Treating the plants with NaCl (200 mM) resulted in considerable growth inhibition in terms of biomass, relative water content, and chlorophyll content, all of which were significantly improved upon application of JA and NO under both normal and NaCl-stress treatments. Salt treatment particularly 200 mM NaCl caused an apparent increase in electrolyte leakage, lipid peroxidation, and hydrogen peroxide production, which were reduced by exogenous application of JA and NO. Salt treatment triggered the induction of antioxidant system by enhancing the activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Application of JA and NO separately as well as in combination caused a significant improvement in activities of SOD, CAT, APX, and GR activities. JA and NO either applied individually or in combination boosted the flavonoid, proline and glycine betaine synthesis under NaCl treatments. In conclusion, the exogenous application of JA and NO protected tomato plants from NaCl-induced damage by up-regulating the antioxidant metabolism, osmolyte synthesis, and metabolite accumulation.

Signal Transduction and Regulatory Networks in Plant-Pathogen Interaction: A Proteomics Perspective

Plant diseases are amongst the major limiting factors of crop production worldwide. They devastate not only food supply, but also the economy of a nation. Depending upon the time of infection and severity of the disease, they can cause average yield loss of about 10–90%. Keeping in view of the global food scarcity, there is, an urgent need to develop crop plants with improved biotic stress tolerance so as to meet the global food demands. A detailed study of the molecular interactions between crops plants and their pathogens would, therefore, be of primary importance for devising new strategies based on plants self defense mechanisms to develop crops with increased disease tolerance for sustainable agricultural production.