Molecular dissection of genes and promoters involved in glycyrrhizin biosynthesis revealed phytohormone induced modulation in Glycyrrhiza glabra L.

The study reports cloning and characterization of complete biosynthetic gene cluster committed to glycyrrhizin biosynthesis along with their corresponding promoter regions from Glycyrrhiza glabra. The identified genes namely, ß-amyrin synthase, ß-amyrin-11-oxidase, 11-oxo-beta-amyrin 30-oxidase and UDP-dependent glucosyltransferase, were hetrologously expressed in Nicotiana benthamiana for functional validation. The phyto-hormone, naphthalene acetic acid was shown to prompt maximum up regulation (1.3-14.0 folds) of all the genes, followed by gibberellic acid (0.001-10.0 folds) and abscisic acid (0.2-7.7 folds) treatments. The promoter-GUS fusion constructs infiltrated leaves of the identified genes exhibited enhanced promoter activity of ß-amyrin synthase (3.9 & 3.0 folds) and 11-oxo-beta-amyrin 30-oxidase (3.6 & 3.2 folds) under the GA3 and NAA treatments, respectively as compared to their respective untreated controls. The transcriptional control of the three phytohormones studied could be correlated to the cis-responsive elements present in the upstream regions of the individual genes. The study provided an insight into the intricate interaction between hormone-responsive motifs with the corresponding co-expression of the glycyrrhizin biosynthetic pathway genes. The study will help in understanding the phytohormones-mediated regulation of glycyrrhizin biosynthesis and its modulation in the plant.

Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.

Genetic enhancement for semi-dwarf and bacterial blight resistance with enhanced grain quality characteristics in traditional Basmati rice through marker-assisted selection.

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC2F2 was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.

Terpenoid and flavonoid spectrum of in vitro cultures of Glycyrrhiza glabra revealed high chemical heterogeneity: platform to understand biosynthesis.

Simultaneous qualitative and quantitative assessment of eight flavonoids and two terpenoids were performed in fourteen in vitro raised morphogenic cultures of Glycyrrhiza glabra. Our study revealed that the spectrum and production of ten compounds, under investigation, were higher in organized tissue than the undifferentiated mass, however, aerial portions of the in vitro raised plants (leaf and stem) were found to be devoid of therapeutically relevant triterpenoid, glycyrrhizin. A correlation was observed between cell maturation, morphological differentiation and glycyrrhizin accumulation. Mature stolons (4 months) were characterized by the maximum accumulation of glycyrrhizin (8.60 µg/mg) in in vitro plantlets. The cytotoxic effect of the extracts evaluated against a panel of human cancer cell lines (in vitro) indicated that the pancreatic cell line (MIA-PaCa-2) were sensitive to all the fourteen extracts investigated. To the best of our knowledge this is the first comprehensive report relating plant growth regulators to metabolite spectrum and cytotoxic assessment in in vitro raised G. glabra cultures. Overall, our findings demonstrated that the metabolite spectrum of in vitro raised morphogenetic lines, under different stages of maturation, might offer a platform to understand the regulatory aspects of the concerned metabolite pathway and their consequent role in differentiation.

Multi-elemental profiling and chemo-metric validation revealed nutritional qualities of Zingiber officinale.

Ginger rhizome is a valued food, spice and an important ingredient of traditional systems of medicine of India, China and Japan. An Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) based multi-elemental profiling was performed to assess the quantitative complement of elements, nutritional quality and toxicity of 46 ginger germplasms, collected from the north western Himalayan India. The abundance of eighteen elements quantified in the acid digested rhizomes was observed to be K>Mg>Fe>Ca>Na>Mn>Zn>Ba>Cu>Cr>Ni>Pb>Co>Se>As>Be>Cd. Toxic element, Hg was not detected in any of the investigated samples. Chemometric analyses showed positive correlation among most of the elements. No negative correlation was observed in any of the metals under investigation. UPGMA based clustering analysis of the quantitative data grouped all the 46 samples into three major clusters, displaying 88% similarity in their metal composition, while eighteen metals investigated grouped into two major clusters. Quantitatively, all the elements analyzed were below the permissible limits laid down by World Health Organization. The results were further validated by cluster analysis (CA) and principal component analysis (PCA) to understand the ionome of the ginger rhizome. The study suggested raw ginger to be a good source of beneficial elements/minerals like Mg, Ca, Mn, Fe, Cu and Zn and will provide platform for understanding the functional and physiological status of ginger rhizome.

Direct rhizogenesis, in vitro stolon proliferation and high-throughput regeneration of plantlets in Glycyrrhiza glabra.

Direct rhizogenesis from leaf explants and establishment of an in vitro stolon culture system and subsequent plant regeneration for Glycyrrhiza glabra have been described. MS liquid medium supplemented with 0.01 mg l-1 of NAA was most effective for stolon proliferation. Extensive proliferation of stolon and shoot regeneration was achieved on medium containing 3 % sucrose with 0.01 mg l-1 NAA. Stolons with nodes showing growth was transferred under light for plantlet regeneration in the same medium. This paper is the first report in G. glabra describing a complete regeneration procedure via in vitro stolon proliferation along with quantitative data for glycyrrhizin and genetic fidelity of plant regenerated in vitro there from. In vitro stolon proliferation described here would be an efficient way for regeneration of plants for functional genomics studies and better understanding of glycyrrhizin (GA) metabolism.

Chromolithic method development, validation and system suitability analysis of ultra-sound assisted extraction of glycyrrhizic acid and glycyrrhetinic acid from Glycyrrhiza glabra.

An ultrasound-assisted extraction and chromolithic LC method was developed for simultaneous determination of glycyrrhizic acid (GA) and glycyrrhetinic acid (GL) from the root extract of Glycyrrhizza glabra using RPLC-PDA. The developed method was validated according to the International Conference on Harmonisation. The method exhibited good linearity (r2 > 0.9989) with high precision and achieved good accuracies between 97.5 to 101.3% of quantitative results. The method is more sensitive and faster (resolved within ten minutes) than the earlier developed methods using normal LC columns.

Composition of a monoterpenoid-rich essential oil from the rhizome of Zingiber officinale from north western Himalayas.

The chemical composition of the essential oil from the rhizome of ginger (Zingiber officinale Roscoe), collected from Nahan, Himachal Pradesh, India, was determined by gas chromatography and GC-MS. Fifty-one compounds, representing 95.1% of the oil, were identified. The oil was characterized by relatively large amounts of the monoterpenoids 1,8-cineole (10.9%), linalool (4.8%), borneol (5.6%), alpha-terpineol (3.6%), neral (8.1%), geraniol (14.5%), geranial (9.5%), trans-dimethoxy citral (5.0%) and geranyl acetate (6.3%). Five compounds, namely trans-linalool oxide, trans-linalool oxide acetate, (Z)-dimethoxycitral, (E)-dimethoxy citral and epi-zingiberenol are reported for the first time in oil of ginger.

RP-thin layer chromatographic method for the quantification of three gingerol homologs of ultrasonic-assisted fresh rhizome extracts of Zingiber officinale collected from North Western Himalayas.

A rapid and sensitive RP high-performance thin-layer chromatographic (RP-HPTLC) methodology was developed and validated for the quantitative estimation of gingerols in methanolic extract of fresh ginger rhizome. The samples were chromatographed on RP-TLC glass plates pre-coated with RP-18 60F(254) as the stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with ternary-solvent system consisting of acetonitrile-water-formic acid (7:2:1 v/v/v) at room temperature (25+/-2 degrees C) and plates were scanned at 500 nm. The system was found to give compact spots for gingerols (R(f) values of 6-gingerol 0.73+/-0.04, 8-gingerol 0.59+/-0.08 and 10-gingerol 0.36+/-0.05). Linearity was found to be in the range of 140-840 ng/spot for 6-gingerol, 168-1008 ng/spot for 8-gingerol and 136-816 ng/spot for 10-gingerol with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed linear relationship (r(2)) and ranged from 0.9992 to 0.9937 for 6-, 8- and 10-gingerol. The method was used for routine analyses and to obtain relative amounts of the gingerols in the fresh rhizomes of the ginger cultivated in different locations of Uttarakhand and Himachal Pradesh of North Western Himalayas (India).