Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites.

Overexpression of Camellia sinensis thaumatin-like protein, CsTLP in potato confers enhanced resistance to Macrophomina phaseolina and Phytophthora infestans infection.

Thaumatin-like proteins (TLPs), a class of pathogenesis related proteins are induced in response to pathogens and exhibit antifungal property when overexpressed in transgenic plants. In the present study, we have raised transgenic potato plants overexpressing TLP gene of Camellia sinensis (CsTLP). Fungal resistance assays of transgenic potato elucidated the potential role of CsTLP in imparting tolerance to fungal pathogens, Macrophomina phaseolina (necrotrophic) and Phytophthora infestans (hemi-biotrophic). Transgenic tubers with higher resistance to M. phaseolina, showed a concomitant and significant increase in transcripts of StPAL, StLOX, and StTLP genes involved in phenylpropanoid, lipoxygenase, and general defense response pathway, respectively after infection. Importantly, leaves of CsTLP transgenic lines inoculated with P. infestans spores under in vitro conditions also showed a resistant phenotype. The resistant phenotype recorded for the two important fungal pathogens by CsTLP transgenic potato plants is remarkable, since no effective control methods and no resistant cv. against M. phaseolina has been identified so far in potato.

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze].

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound.

BACKGROUND: Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. RESULTS: When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide), respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. CONCLUSIONS: We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

Genetic structure of Indian valerian (Valeriana jatamansi) populations in western Himalaya revealed by AFLP.

Valeriana jatamansi Jones is a natural tetraploid species indigenous to the Indian Himalaya. To assess its genetic diversity and population structure, we analyzed six natural populations from the western Himalayan region using amplified fragment length polymorphism. An analysis of molecular variance found that 93% of the genetic variation of V. jatamansi was within populations and 7% among populations. The correlation between genetic and geographic distances (r = 0.14) was not significant. Though the populations are well separated, the lack of distinct genetic variation between populations may be due to either recent rapid fragmentation from a wide and continuous area resulting in genetically similar populations or wide dispersal of seed by wind, since the follicles are feathery. Polyploidy may be the reason for the lack of genetic impoverishment due to fragmentation.

Superoxide dismutase: an industrial perspective.

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.

Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston].

BACKGROUND: Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. RESULTS: A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. CONCLUSION: A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher expression of genes of PP pathway was a general feature for higher shikonins biosynthesis.

Online HPLC-DPPH method for antioxidant activity of Picrorhiza kurroa Royle ex Benth. and characterization of kutkoside by ultra-performance LC-electrospray ionization quadrupole time-of-flight mass spectrometry.

Picrorhiza kurroa Royle ex Benth., is widely used in the Indian systems of medicine for the treatment of various liver ailments. Since, the role of oxidative stress in the pathogenesis of liver injury has become generally recognized, in present study the free radical scavenging effect of P. kurroa was assessed by on-line HPLC-DPPH and colorimetric DPPH methods. The comparative study on antioxidant activity of P. kurroa extracts by both methods revealed that colorimetric method showed very less free radical scavenging effect while HPLC-DPPH method showed high activity. Further, the kutkoside, an important ingredient of a potent hepatoprotective formulation "kutkin/picroliv" was investigated for its chemical composition by ultra-performance liquid chromatography coupled with diode array detection/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-QTOF-MS). Kutkoside was considered to be a single compound and reported as picroside-II or kutkoside, however, present investigation illustrated that kutkoside is a mixture of iridoid glycosides namely, picroside II, picroside IV and 6-ferulloylcatalpol.

Qualitative and quantitative analysis of anthraquinone derivatives in rhizomes of tissue culture-raised Rheum emodi Wall. plants.

This paper presents quantification of five anthraquinone derivatives (emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion) in rhizomes of hardened micro-propagated Rheum emodi plants using high-performance liquid chromatography (HPLC). Aseptic shoot cultures were raised using rhizome buds. Shoot multiplication occurred in both agar gelled and liquid Murashige and Skoog (MS) medium supplemented with 10.0 microM 6-benzylaminopurine (BAP) and 5.0 microM indole-3-butyric acid (IBA). Rooted plantlets obtained on plant growth regulator (PGR)-free medium were transferred to soil with 92% survival. HPLC analysis revealed the presence of five anthraquinone derivatives: emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion in rhizomes of tissue culture-raised plants. Only emodin glycoside (1) and chrysophanol glycoside (2) were present in 6-month-old hardened tissue cultured plants. In addition, the other three derivatives (emodin (3), chrysophanol (4) and physcion (5)) were also detected after 9 months.

Simultaneous densitometric determination of shikonin, acetylshikonin, and beta-acetoxyisovaleryl-shikonin in ultrasonic-assisted extracts of four Arnebia species using reversed-phase thin layer chromatography.

A simple, precise, and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin (1) together with its derivatives acetylshikonin (2), and beta-acetoxyisovalerylshikonin (3) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP-18 F(254S )TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100-600 ng for shikonin and acetylshikonin, and 100-1800 ng for beta-acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1-3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.

Catechin and catechin fractions as biochemical markers to study the diversity of Indian tea (Camellia sinensis (L.) O. Kuntze) germplasm.

The heterogeneous Indian tea germplasm includes 'China', 'Assam', 'Cambod', and their hybrids which were evaluated using biochemical markers viz., total catechin and their fractions, for varietal identification and characterization. Principal component analysis (PCA) of biochemical characters showed that the total catechin and trihydroxylated catechin has higher eigenvalues. The first two principal components (PCs) could differentiate more than 90% of the clones studied. This grouping based on first two principal component matrices differentiated 'China', and their hybrids with 'Assam' and 'Cambod' variety. Morphologically indistinct large-leaved 'Cambod' variety and 'Assam' varieties could not be differentiated using biochemical markers, since both varietal types taxonomically belong to a single species. Clones of 'China' type showed low total catechin content and catechin ratio which are distinctly grouped. The 'China-Assam' and 'China-Cambod' hybrids formed intermediate groups between 'China' PC group and 'Cambod'/'Assam' PC groups, providing evidence for genetic control of catechin ratio variation. Tea clones which are differentially positioned in the PC group could be explained based on the genetic contribution by other varietal type as parents. This biochemical characterization will be a useful tool in the development of quality-tea clones with different proportion of total catechin and their fractions.

An early gene of the flavonoid pathway, flavanone 3-hydroxylase, exhibits a positive relationship with the concentration of catechins in tea (Camellia sinensis).

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan 3-ols or catechins. Apart from being responsible for tea quality, these compounds have medicinal properties. Flavanone 3-hydroxylase (F3H) is an abundant enzyme in tea leaves that catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. We report a full-length cDNA sequence of F3H from tea (CsF3H Accession no. AY641730). CsF3H comprised 1365 bp with an open reading frame of 1107 nt (from 43 to 1149) encoding a polypeptide of 368 amino acids. Expression of CsF3H in an expression vector in Escherichia coli yielded a functional protein with a specific activity of 32 nmol min(-1) mg protein(-1). There was a positive correlation between the concentration of catechins and CsF3H expression in leaves of different developmental stages. CsF3H expression was down-regulated in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in response to wounding. The concentration of catechins paralleled the expression data. Exposure of tea shoots to 50-100 microM catechins led to down-regulation of CsF3H expression suggesting substrate mediated feedback regulation of the gene. The strong correlation between the concentration of catechins and CsF3H expression indicates a critical role of F3H in catechin biosynthesis.

Essential oil composition of three accessions of Dracocephalum heterophyllum Benth. cultivated at Palampur, India.

Seeds of three accessions of Dracocephalum heterophyllum growing wild in high altitude locations in the cold desert regions of trans-Himalaya were collected and propagated under polyhouse conditions at IHBT Palampur to evaluate, compare their growth performance and essential oil composition. Three different chemotypes viz., citronellol-rose oxides type, citronellol type and citronellal-trans rose oxide type were identified. Samples of essential oils were distilled separately from leaves and inflorescence of all the three cultivated accessions and characterised by capillary GC and GC-MS techniques, which showed variation in the volatile constituents. In all the three accessions, citronellol was higher in the inflorescence oils as compared to leaf oils. Rose oxides, which are important high odour value cyclic monoterpene ethers were found in the essential oil samples of leaves as well as in the inflorescence of accession II, whereas these constituents were absent in the leaves of other two accessions. The essential oil from the inflorescence part of accession I was devoid of both cis and trans isomers of rose oxide, while accession III contained only one isomer of trans rose oxide. Under polyhouse conditions, seed germination was higher in accession III, whereas aerial biomass was higher in accession I. In comparison to other accessions, yield of volatile oil was higher in both leaf and inflorescence of accessions I, whereas accession II had higher oil yield from inflorescence. This is the first report of volatile oil composition of three accessions of D. heterophyllum cultivated at Palampur having potential as a new source of high valued essential oil for introduction in the perfumery industry.

High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi.

A high-performance thin layer chromatographic (HPTLC) method for the rapid and simple quantification of the four major anthraquinone derivatives i.e. physcion, chrysophanol, emodin and chrysophanol glycoside in Rheum emodi is described. HPTLC of anthraquinone derivatives was performed on pre-coated RP-18 F254S HPTLC plates. For achieving good separation, the mobile phase of methanol-water-formic acid (80:19:1, v/v/v) was used. The densitometric determination of anthraquinone derivatives was carried out at 445 nm in reflection/absorption mode. The calibration curves were linear in the range of 20-100 ng for physcion, 80-400 ng for chrysophanol and emodin, and 200-1000 ng for chrysophanol glycoside. The method was found to be reproducible and convenient for quantitative analysis of anthraquinone derivatives in the methanolic extract of rhizomes of R. emodi collected from three different locations of Western Himalaya, India.