Ruchi Singh

Biology and chemistry of Ginkgo biloba

Ginkgo biloba has been existing on earth since 200 million years and is considered as a living fossil. It is among the most sold medicinal plants in the world. A number of secondary metabolites representing terpenoids, polyphenols, allyl phenols, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids have been isolated from the plant. However, the main bioactive constituents are terpene trilactones and flavonoid glycosides which are considered responsible for the pharmacological activities of its standardized leaf extract. Scattered information is available on the extraction and analysis of these pharmacologically important constituents which have been compiled in the present review.

Physiological and proteomic responses of cotton (Gossypium herbaceum L.) to drought stress.

Cotton genotype RAHS 187 was analyzed for changes in physiology, biochemistry and proteome due to drought stress. The deleterious effect of drought in cotton plants was mainly targeted towards photosynthesis. The gas-exchange parameters of net photosynthesis (A), stomatal conductance (g(s)) and transpiration (E) showed a decreasing trend as the drought intensity increased. The fluorescence parameters of, effective quantum yield of PSII (Φ(PSII)), and electron transport rates (ETR), also showed a declining trend. As the intensity of drought increased, both H(2)O(2) and MDA levels increased indicating oxidative stress. Anthocyanin levels were increased by more than four folds in the droughted plants. Two-dimensional gel electrophoresis detected more than 550 protein spots. Significantly expressed proteins were analyzed by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF. The number of up-regulated spots was found to be 16 while 6 spots were down-regulated. The reasonable implications in drought response of the identified proteins vis-à-vis physiological changes are discussed. Results provide some additional information that can lead to a better understanding of the molecular basis of drought-sensitivity in cotton plants.

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance, Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress. Methodology The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5. Results The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana. Conclusions Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.

Genome wide expression profiling of two accession of G. herbaceum L. in response to drought

BackgroundGenome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of Gossypium herbaceum, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roches GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions.ResultsFourteen accessions of Gossypium herbaceum were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roches GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms responses to various abiotic stresses were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death.ConclusionThe comparative genome-wide gene expression profiling study of two accessions of G.herbaceum under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.

SlERF36, an EAR-motif-containing ERF gene from tomato, alters stomatal density and modulates photosynthesis and growth

The AP2 domain class of transcription factors is a large family of genes with various roles in plant development and adaptation but with very little functional information in plants other than Arabidopsis. Here, the characterization of an EAR motif-containing transcription factor, SlERF36, from tomato that affects stomatal density, conductance, and photosynthesis is described. Heterologous expression of SlERF36 under the CaMV35S promoter in tobacco leads to a 25–35% reduction in stomatal density but without any effect on stomatal size or sensitivity. Reduction in stomatal density leads to a marked reduction in stomatal conductance (42–56%) as well as transpiration and is associated with reduced CO2 assimilation rates, reduction in growth, early flowering, and senescence. A prominent adaptive response of SlERF36 overexpressors is development of constitutively high non-photochemical quenching (NPQ) that might function as a protective measure to prevent damage from high excitation pressure. The high NPQ leads to markedly reduced light utilization and low electron transport rates even at low light intensities. Taken together, these data suggest that SlERF36 exerts a negative control over stomatal density and modulates photosynthesis and plant development through its direct or indirect effects.

Characterizing photoinhibition and photosynthesis in juvenile-red versus mature-green leaves of Jatropha curcas L

The new leaves of Jatropha curcas (L.) appear dark red in colour due to the presence of anthocyanin pigments, these leaves subsequently turn green on maturity. The aim of the study was to characterize the photosynthetic efficiency of the juvenile red and mature green leaves and to understand the possible role of anthocyanin pigment in the juvenile leaves of J.xa0curcas. We studied the localization of anthocyanin pigment, reflectance properties, diurnal gas-exchange performance, carboxylation efficiency and photosynthetic efficiency under different light intensities by investigation of the photochemical and non-photochemical energy dissipation processes related to Photosystem II (PSII) and Photosystem I (PSI), of the juvenile and the mature leaves of J.xa0curcas. The JIP test analysis of chlorophyll a fluorescence transients and the gas-exchange studies revels the low photosynthetic efficiency of red leaves is due to the immaturity of the leaf. The low value of quantum yield of non-photochemical energy dissipation due to acceptor side limitation, Y (NA) under high light in the red leaf, suggests that over-reduction of PSI acceptor side was prevented and it results in the accumulation of oxidized P700, which dissipates excess light energy harmlessly as heat and thereby alleviate photoinhibition of PSI in case of the juvenile red leaves. Further our results of photoinhibition and relaxation on exposure of red and green leaves to monochromatic blue light showed that effective quantum yield of PSII recovers faster and completely under darkness in juvenile red leaves as compared to mature green leaves, supporting the role of anthocyanin pigments in protecting both PSII and PSI in the red leaves.

Photosynthetic performance of Jatropha curcas fruits.

Jatropha curcas (L.) trees under north Indian conditions (Lucknow) produce fruits in two major flushes, once during autumn-winter (October-December). The leaves at this time are at the senescence stages and already shedding. The second flush of fruit setting occurs during the summer (April-June) after the leaves have formed during spring (March-April). Photosynthetic performance of detached jatropha fruits was studied at three developmental stages, immature, mature and ripe fruits. Studies were made in both winter and summer fruits in response to light, temperature and vapour pressure deficit (VPD) under controlled conditions to assess the influence of these environmental factors on the photosynthetic performance of jatropha fruits. Immature fruits showed high light saturating point of around 2000xa0μmolxa0m(-2)xa0s(-1). High VPD did not show an adverse effect on the fruit A. Stomatal conductance (g(s)) showed an inverse behaviour to increasing VPD, however, transpiration (E) was not restricted by the increasing VPD in both seasons. During winter in absence of leaves on the jatropha tree the fruits along with the bark contributes maximum towards photoassimilation. Dark respiration rates (R(d)) monitored in fruit coat and seeds independently, showed maximum R(d) in seeds of mature fruit and these were about five times more than its fruit coat, reflecting the higher energy requirement of the developing fruit during maximum oil synthesis stage. Photosynthesis and fluorescence parameters studied indicate that young jatropha fruits are photosynthetically as efficient as its leaves and play a paramount role in scavenging the high concentration of CO(2) generated by the fruit during respiration.

Expression of Rice CYP450-Like Gene (Os08g01480) in Arabidopsis Modulates Regulatory Network Leading to Heavy Metal and Other Abiotic Stress Tolerance

Heavy metal (HM) toxicity has become a grave problem in the world since it leads to hazardous effects on living organisms. Transcriptomic/proteomic studies in plants have identified a large number of metal-responsive gene families. Of these, cytochrome-P450 (CYPs) family members are composed of enzymes carrying out detoxification of exogenous molecules. Here, we report a CYP-like protein encoded by Os08g01480 locus in rice that helps the plant to combat HM and other abiotic stresses. To functionally characterize CYP-like gene, cDNA and promoter were isolated from rice to develop Arabidopsis transgenic lines. Heterologous expression of Os08g01480 in Arabidopsis provided significant tolerance towards abiotic stresses. In silico analysis reveals that Os08g01480 might help plants to combat environmental stress via modulating auxin metabolism. Transgenic lines expressing reporter gene under control of Os08g01480 promoter demonstrated differential promoter activity in different tissues during environmental stresses. These studies indicated that differential expression of Os08g01480 might be modulating response of plants towards environmental stresses as well as in different developmental stages.

The EAR motif controls the early flowering and senescence phenotype mediated by over-expression of SlERF36 and is partly responsible for changes in stomatal density and photosynthesis.

The EAR motif is a small seven amino acid motif associated with active repression of several target genes. We had previously identified SlERF36 as an EAR motif containing gene from tomato and shown that its over-expression results in early flowering and senescence and a 25–35% reduction of stomatal density, photosynthesis and stomatal conductance in transgenic tobacco. In order to understand the role of the EAR motif in governing the phenotypes, we have expressed the full-length SlERF36 and a truncated form, lacking the EAR motif under the CaMV35S promoter, in transgenic Arabidopsis. Plants over-expressing the full-length SlERF36 show prominent early flowering under long day as well as short day conditions. The early flowering leads to an earlier onset of senescence in these transgenic plants which in turn reduces vegetative growth, affecting rosette, flower and silique sizes. Stomatal number is reduced by 38–39% while photosynthesis and stomatal conductance decrease by about 30–40%. Transgenic plants over-expressing the truncated version of SlERF36 (lacking the C-terminal EAR motif), show phenotypes largely matching the control with normal flowering and senescence indicating that the early flowering and senescence is governed by the EAR motif. On the other hand, photosynthetic rates and stomatal number were also reduced in plants expressing SlERF36ΔEAR although to a lesser degree compared to the full- length version indicating that these are partly controlled by the EAR motif. These studies show that the major phenotypic changes in plant growth caused by over-expression of SlERF36 are actually mediated by the EAR motif.

Factors affecting podophyllotoxin yield in the ex situ grown Podophyllum hexandrum, an endangered alpine native of the western Himalayas

This study reports an appreciable yield of podophyllotoxin (PDT) in P. hexandrum plants grown ex situ under polyhouse conditions of a temperate locale. The PDT content of below-ground parts was affected by both plant age and growth period. However, only the effect of plant age on PDT content was significant. Thus, the highest amounts of PDT were recorded in the below-ground parts of 2-year-old plants harvested during the late-growth period (LGP). High total soluble sugars in the below-ground parts during the early growth period (EGP) and the highest nitrate and nitrate reductase in the leaves of 2-year-old plants during the peak-growth period (PGP) indicated higher mobilization and assimilation of starch and nitrate. Probably the surplus carbon and nitrogen gained during the PGP were diverted from aerial parts to below-ground parts during the LGP and in turn contributed to the synthesis of higher amounts of PDT. This study shows that commercial cultivation of P. hexandrum is possible under ex situ temperate conditions.