Mather Ali Khan

HMG-CoA reductase limits artemisinin biosynthesis and accumulation in Artemisia annua L. plants

In vivo modulation of HMG-CoA reductase (HMGR) activity and its impact on artemisinin biosynthesis as well as accumulation were studied through exogenous supply of labeled HMG-CoA (substrate), labeled MVA (the product), and mevinolin (the competitive inhibitor) using twigs of Artemisia annua L. plants collected at the pre-flowering stage. By increasing the concentration (2–16xa0μM) of HMG-CoA (3-14C), incorporation of labeled carbon into artemisinin was enhanced from 7.5 to 17.3xa0nmol (up to 130%). The incorporation of label (14C) into MVA and artemisinin was inhibited up to 87.5 and 82.9%, respectively, in the presence of 200xa0μM mevinolin in incubation medium containing 12xa0μM HMG-CoA (3-14C). Interestingly, by increasing the concentration of MVA (2-14C) from 2 to 18xa0μM, incorporation of label (14C) into artemisinin was enhanced from 10.5 to 35xa0nmol (up to 233%). When HMG-CoA (3-14C) concentration was increased from 12 to 28xa0μM in the presence of 150xa0μM mevinolin, the inhibitions in the incorporation of label (14C) into MVA and artemisinin were, however, reversed and the labels were found to approach their values in twigs fed with 12xa0μM HMG-CoA (3-14C) without mevinolin. In another experiment, 14.2% inhibition in artemisinin accumulation was observed in twigs in the presence of 175xa0μM fosmidomycin, the competitive inhibitor of 1-deoxy-d-xylulose 5-phosphate reductase (DXR). HMG-CoA reductase activity and artemisinin accumulation were also increased by 18.6 to 24.5% and 30.7 to 38.4%, respectively, after 12xa0h of treatment, when growth hormones IAA (100xa0ppm), GA3 (100xa0ppm) and IAAxa0+xa0GA3 (50xa0+xa050xa0ppm) were sprayed on A. annua plants at the pre-flowering stage. The results obtained in this study, hence, demonstrate that the mevalonate pathway is the major contributor of carbon supply to artemisinin biosynthesis and HMGR limits artemisinin synthesis and its accumulation in A. annua plants.

Moving toward a precise nutrition: preferential loading of seeds with essential nutrients over non-essential toxic elements.

Plants and seeds are the main source of essential nutrients for humans and livestock. Many advances have recently been made in understanding the molecular mechanisms by which plants take up and accumulate micronutrients such as iron, zinc, copper and manganese. Some of these mechanisms, however, also facilitate the accumulation of non-essential toxic elements such as cadmium (Cd) and arsenic (As). In humans, Cd and As intake has been associated with multiple disorders including kidney failure, diabetes, cancer and mental health issues. Recent studies have shown that some transporters can discriminate between essential metals and non-essential elements. Furthermore, sequestration of non-essential elements in roots has been described in several plant species as a key process limiting the translocation of non-essential elements to aboveground edible tissues, including seeds. Increasing the concentration of bioavailable micronutrients (biofortification) in grains while lowering the accumulation of non-essential elements will likely require the concerted action of several transporters. This review discusses the most recent advances on mineral nutrition that could be used to preferentially enrich seeds with micronutrients and also illustrates how precision breeding and transport engineering could be used to enhance the nutritional value of crops by re-routing essential and non-essential elements to separate sink tissues (roots and seeds).

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.

HPTLC Fingerprint Analysis: A Quality Control for Authentication of Herbal Phytochemicals

Authentication and consistent quality are the basic requirement for Indian traditional medicine (TIM), Chinese traditional herbal medicine (TCHM), and their commercial products, regardless of the kind of research conducted to modernize the TIM and TCHM. The complexities of TIM and TCHM challenge the current official quality control mode, for which only a few biochemical markers were selected for identification and quantitative assay. Referring too many unknown factors existed in TIM and TCHM, it is impossible and unnecessary to pinpoint qualitatively and quantitatively every single component contained in the herbal drug. Chromatographic fingerprint is a rational option to meet the need for more effective and powerful quality assessment to TIM and TCHM. The optimized chromatographic fingerprint is not only an alternative analytical tool for authentication, but also an approach to express the various pattern of chemical ingredients distribution in the herbal drugs and preserve such “database” for further multifaced sustainable studies. Analytical separation techniques, for example, high-performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS) were among the most popular methods of choice used for quality control of raw material and finished herbal product. Fingerprint analysis approach using high-performance thin-layer chromatography (HPTLC) has become the most potent tool for quality control of herbal medicines because of its simplicity and reliability. It can serve as a tool for identification, authentication, and quality control of herbal drugs. In this chapter, attempts are being made to expand the use of HPTLC and at the same time create interest among prospective researcher in herbal analysis. The developed method can be used as a quality control tool for rapid authentication from a wide variety of herbal samples. Some examples demonstrated the role of fingerprinting in quality control and assessment.

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

BACKGROUNDnThe 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.nnnRESULTSnOil 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.nnnCONCLUSIONnThe 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.

Inheritance of grain filling duration in spring wheat (Triticum aestivum L. em thell)

To understand the genetic control of grain filling duration (GFD), i.e., the number of days from anthesis to physiological maturity, we studied the F1, F2, BC1 and BC2 generations of six spring wheat crosses from nine varieties/genotypes. Generation mean analysis for gene effects indicated that one or more types of epistasis were significant in all crosses. In each pairing, the F1 and F2 means were either intermediate or closer to the mean of the parent having the longer GFD. Our narrow-sense heritability estimate was reasonably high, at 47.67 (based on diallel analysis). This demonstrated that progress could be made from the selection in these crosses for either long or short GFD. The two early varieties that had identical maturity durations differed in their GFD values, indicating that maturity dates are not good criteria when choosing parents for modifying GFD. To utilize favorable additive × additive effects during this selection, we suggest that a single seed descent (SSD) or bulk popula-tion approach be adopted. In comparison, dominance effects would prove quite useful in hybrid wheat breeding programs.

Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation

Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50xa0μM) and sulfur-sufficient (1,000xa0μM) conditions for 30xa0days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions.

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.

Biomolecular Characteristics of Aspergillus niger Under Cadmium Metal Stress

Contamination of heavy metals poses a significant threat to our different ecosystems and their biodegradation as organic contaminants is not possible as heavy metal ions cannot be mineralized to non-toxic forms. Instead, they can be biomobilized into other compounds. Cadmium is supposed to be the most toxic heavy metal occurring naturally in the environment. Fungal biosorption had become an answer to remove toxic metal ions from wastewaters or soils as they can be grown easily and inexpensively. Aspergillus niger is a biomass waste of citric acid production industry and can be used as a biosorbent for this purpose. The cadmium effect on A. niger ITCC 546 and ITCC 6117 has been investigated. The maximum biomass was observed at 475xa0mM containing medium compared to 500 and 525xa0mM. The higher concentration of Cd showed more inhibition of fungal cells. The protein synthesis was increased at 475xa0mM of Cd ions than in control samples. The total RNA expressed from treated fungal cells at 475xa0mM of Cd ions was in greater quantity than the RNA isolated at 500xa0mM, while the DNA from mycelia grown at 500xa0mM were more sheared and degraded than the DNA of 475xa0mM. The biosorption capability of A. niger ITCC 546 has been found to be much more than that of ITCC 6117.