Ectopic Expression of Cold Responsive LlaCIPK Gene Enhances Cold Stress Tolerance in Nicotiana tabacum.

Low-temperature stress severely affects the growth, development, and geographical distribution of various crop plants, resulting in significant economic loss to producers. In a quest to identify cold-regulated genes, we constructed a cDNA suppression subtractive library from a high altitude adapted ecotype of Lepidium. We cloned a cold-induced gene LlaCIPK from the subtracted cDNA library which gave homology to Arabidopsis CIPK15 gene. The predicted 3D structure of LlaCIPK protein also showed homology with Arabidopsis CIPK protein. Quantitative real-time PCR analysis in Lepidium seedlings exposed to 6 h of cold stress shows a 3-fold increase in the expression of LlaCIPK transcript. The expression of LlaCIPK was also differentially regulated by ethylene, CaCl2, ABA, and SA treatments. Ethylene and CaCl2 treatments up regulated LlaCIPK expression, whereas ABA and SA treatments down regulated the LlaCIPK expression. Transgenic plants overexpressing LlaCIPK gene under constitutive promoter show an increased level of proline and cell membrane stability. Taken together, our results suggest that the LlaCIPK contributes to the cold-response pathway in Lepidium plants.

Overexpression of Ran gene from Lepidium latifolium L. (LlaRan) renders transgenic tobacco plants hypersensitive to cold stress.

Ran is a multifunctional small GTPase involved in important cellular activities like nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope formation, etc., but is also known to be differentially expressed in response to abiotic stress, particularly low temperature. We have over-expressed Lepidium latifolium (Fam. Brassicaceae) Ran gene in tobacco to study the response of the plants to cold stress (24 h; 4 °C). Transformation of the tobacco plants was verified using PCR targeting Ran gene and co-transformed selectable marker gene nptII. Segregation in Mendelian ratios was validated in five transgenic lines by germination of T1 and T2 seeds on moist filter papers containing 150 mg/l kanamycin. Higher levels of electrolyte leakage and lipid peroxidation pointed towards hypersensitivity of plants. Similarly, lesser proline accumulation compared to wild types also indicated susceptibility of plants to death under chilling conditions. Specific activity of antioxidant enzymes superoxide dismutase and glutathione reductase was also measured under stressed and control conditions. A variation was observed across the different lines, and four out of five lines showed lesser specific activity compared to wild type plants, thus indicating reduced capability of scavenging free radicals. In totality, a strong evidence on induced hypersensitivity to cold stress has been collected which may further be helpful in designing appropriate strategies for engineering crop plants for survival under cold stress conditions.

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5' and 3' UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.

Analysis of Jatropha curcas transcriptome for oil enhancement and genic markers.

Oil-rich seeds of Jatropha curcas are being focussed as a source of bio-diesel. However, prior to its industrial use, a lot of crop improvement efforts are required in Jatropha. Availability of a large number of EST sequences of Jatropha in public domain allow identification of candidate genes for several agronomic characters including oil content in seeds. Here, we have analysed 42,477 ESTs of Jatropha spanning 22.9 Mbp for microsatellites and fatty acid metabolism related sequences. Unigene sequences were built using CAP 3 programme resulted in 12,358 contigs and 5,730 singlets. Nearly, 8 % unigenes showed presence of microsatellites, slightly over-represented compared to their occurrence in ESTs. Most of the microsatellites were either di- or tri-nucleotide repeats, while other categories of tetra-, penta- and hexa-nucleotide repeats together constituted ~4 % of total microsatellites. Assessment of functional relevance of unigenes was carried out using Blast2GO using its default settings. The overall sequence similarity level against sequences in 'nr' database was >80 %. A total of 931 sequences that participated in any of the pathways related to fatty acid or lipid metabolism were found at GO level 6. Among these, GO terms "Fatty acid metabolic process" and "Fatty acid biosynthetic process" were most over-represented. Overall, our work has due relevance in identifying molecular markers for the candidate genes for oil content in Jatropha seeds, and will prove to be an important reference for further studies for identification of trait specific markers in Jatropha.

Sub-acute toxicity of cultured mycelia of himalayan entomogenous fungus Cordyceps sinensis (Berk.) Sacc. in rats.

Oral administration of laboratory cultured mycelia powder of C. sinensis did not show any sign of toxicity as no significant change was observed in organ weight and serological parameters in rats. However, there was a significant increase in food intake, body weight gain and hematological parameters like WBC, RBC, Hb and lymphocytes in treated groups. Histopathology of vital organs also supported the non toxic effect of C. sinensis. The results conclude that laboratory cultured mycelia powder of C. sinensis is safe and non toxic up to 2 g/kg body weight dose.

Semi-quantitative analysis of transcript accumulation in response to drought stress by Lepidium latifolium seedlings.

Cross-amplification of five Arabidopsis abiotic stress-responsive genes (AtPAP, ZFAN, Vn, LC4 and SNS) in Lepidium has been documented in plants raised out of seeds pre-treated with potassium nitrate (KNO 3) for assessment of enhanced drought stress tolerance. cDNA was synthesized from Lepidium plants pre-treated with KNO 3 (0.1% and 0.3%) and exposed to drought conditions (5% and 15% PEG) at seedling stage for 30 d. Transcript accumulation of all the five genes were found suppressed in set of seedlings, which were pre-treated with 0.1% KNO 3 and were exposed to 15% PEG for 30 d. The present study establishes that different pre-treatments may further enhance the survivability of Lepidium plants under conditions of drought stress to different degrees.

Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 µM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.

Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes.

Our containment trials have established cold tolerance in Nicotiana tabacum osmotin (Nt Osm) transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby). Though, the stress tolerance mechanisms have been studied at physio-biochemical levels, molecular mechanisms underlying the tolerant response are still not well studied. Therefore, quantitative transcript expression of Osmotin and other stress responsive genes (CBF1, P5CS and APX) was studied in response to cold (4°C; 2 and 24 h) treatment in the transgenic and wild type tomato plants. The expression analysis revealed differential transcript regulation in the transgenic and wild type plants on the cold exposure. In general, the genes were either earlier induced or the extent of fold change in transcript expression over the respective untreated controls was higher in transgenic than in the wild type plants on cold exposure. The transcript expression data also supported the metabolite analysis on free Proline and ascorbate content. The results thus suggest that constitutive over expression of the Osmotin modulate transcript abundance and functional expression products of the other stress responsive genes thereby, imparting cold tolerance in the transgenic tomato plants.

Influence of nitrogen sources on biomass productivity of microalgae Scenedesmus bijugatus.

The influence of different nitrogen source of varying concentrations on biomass production of green algae Scenedesmus was investigated. The result revealed that there was a significant difference among nitrogen sources in promoting algal biomass growth at lower concentrations of 5 and 10 mM. Nitrate was found to be a preferred form of nitrogen source and potassium (0.32 g/L) and sodium nitrates (0.28 g/L) performed better for biomass growth of Scenedesmus. Among the ammonical forms, urea (0.25 g/L) resulted in almost equal biomass as nitrates, making it an economical substitute for nitrogen source in large scale culturing of algae being commercially available.

Construction of cold induced subtracted cDNA library from Cicer microphyllum and transcript characterization of identified novel wound induced gene.

A forward cold induced subtracted cDNA library was constructed to identify the stress regulated genes in a high altitude cold desert-adapted species Cicer microphyllum, a wild relative of cultivated chickpea, distributed in western and trans-Himalayas. A total of 1,040 clones were obtained from the subtracted cDNA library. These clones were screened for presence of insert with colony PCR. A total of 523 clones were picked by colony PCR among which 300 clones were observed differentially expressed as per dot blot analysis. Differentially expressed clones were sequenced and assembled into clusters based on the presence of overlapping, identical, or similar sequences. A total of 283 ESTs were submitted in gene bank (accession numbers GO241043 to GO241326). BLAST analysis of these ESTs revealed its similarity for regulatory proteins like kinases, metallothionin, and enzymes/proteins with unknown functions. A cDNA encoding wound induced like protein, identified from this cold induced subtraction cDNA library, was full-length cloned using RACE and sequenced (accession number GQ914056). Southern blot of C. microphyllum indicated single copy of the gene in genome. Transcript expression profiling of this gene by quantitative real-time PCR and northern blot confirmed its up-regulation during low temperature stress. Further, in situ RNA hybridization also revealed cold (4°C) induced expression of the gene.

DRE-binding transcription factor gene (LlaDREB1b) is regulated by various abiotic stresses in Lepidium latifolium L.

We have isolated and in silico characterized a cold-induced LlaDREB1b encoding a putative DRE-binding transcription factor from Lepidium latifolium. Its cDNA (JN214345) sequence (998 bp) consisted of a 642 bp ORF, 168 and 188 bp of 5' and 3' UTR regions, respectively, encoding a protein of 213 aa with deduced molecular mass 23.85 kDa and pI of 4.63. In silico and phylogenetic analysis further suggested that the protein showed features of a typical member of the AP2/EREBP family of DNA-binding proteins. Southern blot analysis indicated that multiple copies of the gene could be present in the genome. Its transcripts were abundant in leaves, petiole and stem, but scarce in roots and could strongly be induced by cold treatment (4 °C), weakly by drought and salt stress, and did not respond to ABA treatment. Thus, LlaDREB1b is a potential candidate for abiotic stress-tolerance engineering in crop plants upon its further functional validation.

Cold tolerance in thiourea primed capsicum seedlings is associated with transcript regulation of stress responsive genes.

Benefits of seed priming in seedling establishment and tolerance to subsequent stress exposure are well reported. However, the molecular mechanisms underlying the priming mediated benefits are not much discovered. Results of our earlier experiments established that thiourea (TU) seed priming imparts cold tolerance to capsicum seedlings. Therefore, to understand molecular mechanisms underlying priming mediated cold stress tolerance, quantitative transcript expression of stress responsive genes involved in transcript regulation (CaCBF1A, CaCBF1B, Zinc Finger protein, CaWRKY30), osmotic adjustment (PROX1, P5CS, Osmotin), antioxidant defence (CAT2, APX, GST, GR1, Cu/Zn SOD, Mn SOD, Fe SOD), signaling (Annexin), movement of solutes and water (CaPIP1), and metabolite biosynthesis through phenylpropanoid pathway (CAH) was studied in response to cold (4 °C; 4 and 24 h) stress in seedlings grown from the TU primed, hydroprimed and unsoaked seeds. The transcript expression of CaWRKY30, PROX1, Osmotin, Cu/Zn SOD and CAH genes was either higher or induced earlier on cold exposure in thiourea priming than that of hydroprimed and unsoaked over the respective unstressed controls. The results thus suggest that the TU priming modulate expression of these genes thereby imparting cold tolerance in capsicum seedlings.

Isolation and characterization of cold responsive NAC gene from Lepidium latifolium.

Cold stress is one of the major limiting factor in crop productivity. Plants growing in colder regions acclimatize to severe conditions owing to the presence of 'cold stress tolerant genes'. Isolation and functional characterization of these genes are important before their exploitation in modern agricultural practices. Here, we have cloned full length NAC gene (1,388 bp) from Lepidium latifolium (LlaNAC). This gene belongs to NAP sub-group which also includes ANAC056 of Arabidopsis thaliana, nearest relative of LlaNAC. Upstream analysis and microarray data analysis of ANAC056 suggested that LlaNAC might also be ABA-regulated. However, quantitative transcript expression analysis revealed that LlaNAC transcript upregulated by cold stress and downregulated in response to varying concentrations of abscisic acid, salicylic acid, calcium chloride and ethylene. There is also a possibility that the gene may be getting regulated by a pathway whose components are still unknown. Any further investigations to understand the mechanism of regulation of LlaNAC gene expression are likely to find immense importance in plant biotechnology and crop improvement.

Evaluation of antioxidant activity of two important memory enhancing medicinal plants Baccopa monnieri and Centella asiatica.

BACKGROUND: Free radicals or highly reactive oxygen species are capable of inducing oxidative damage to human body. Antioxidants are the compounds which terminate the attack of reactive species and reduce the risk of diseases. Both Baccopa monnieri and Centella asiatica are used in treatment of brain disorders in humans and have almost similar effects. OBJECTIVE: The study was conducted to determine the antioxidant properties of two well-known memory enhancer medicinal plants Baccopa monnieri and Centella asiatica. RESULTS: The antioxidant activity of these two medicinal plants was evaluated by measuring reducing ability, free radical scavenging activity by DPPH and hydrogen peroxide methods. The antioxidants compounds like ascorbic acid, total phenols and tannins were also evaluated in these plants. Baccopa monnieri and Centella asiatica exhibited significant differences (P<0.05) in their antioxidant values. The methanolic extract of whole leaf powder of Baccopa monnieri exhibited significantly higher antioxidant activity than the Centella asiatica. The antioxidant components viz. ascorbic acid, total phenols and tannins were also found in a higher concentration in Baccopa monnieri as compared to Centella asiatica. CONCLUSION: It can be concluded from the study that regular use of Baccopa monnieri as a supplement could be more helpful compared to Centella asiatica in treatment of neurological disorders caused by free radical damage.

Two new for science species of genus Cordyceps fr. (Ascomycetes) from Indian Himalaya.

Two new species for science, Cordyceps kurijimeaensis and C. nirtolii (Clavicipitaceae, Ascomycetes), collected from the forests of the Munsyari Region of the Himalayan hills (Uttarakhand State, India) are described and illustrated. C. kurijimeaensis has the same host, larvae of Hepialus armoricanus, as does Cordyceps (=Ophiocordyceps) sinensis, and C. nirtolii was found on Melanotus communis. Morphology and other diagnostic characteristics show a close relationship between C. kurijimeaensis and C. nirtolii. ITS sequences show a close phylogenetic relationship with members of Clavicipitaceae clade C, strongly supporting the classification of these as new species.

Female plants of Hippophae salicifolia D. Don are more responsive to cold stress than male plants.

Hippophae salicifolia D. Don (Seabuckthorn) grows in stressful environment of high altitude under conditions of low temperature and low availability of water. We have studied gender based differences in physiochemical response to cold stress in male and female plants of Seabuckthorn. After 24 h of cold stress about 32 and 66 % higher electrolyte leakage (EL) was recorded in male and female plants respectively. Relative water content (RWC) at the end of 24 h stress was higher in male plants (~64 %) compared to female plants (~60 %). Proline content in leaf samples of cold stressed male and female plants also increased upon cold stress. After 24 h about 2.7 fold higher amount of proline was assessed in male and female in comparison to control plants. Similarly, about two fold increase in the specific activities of superoxide dismutase (SOD) and catalase was also observed upon cold stress in male and female plants. These findings have important inferences for community of molecular biologists exploring seabuckthorn genome for agronomically important genes.

Induced ectopic expression of At-CBF1 in marker-free transgenic tomatoes confers enhanced chilling tolerance.

In an attempt to improve chilling stress tolerance, an Arabidopsis C-repeat binding factor 1 (At-CBF1) gene driven by the inducible promoter RD29A was co-transferred into tomato var. Shalimar. Marker (NPTII)-free transgenic were obtained in T(1) generation because of unlinked integration of CBF1 and NPTII genes. Reverse transcription-polymerase chain reaction confirmed the expression of CBF1 in T(1) transgenic lines. Study of expression pattern in T(1) transgenic line showed a gradual increase with increasing chilling stress period and also confirmed the reversibility of expression on removal of stress. The transgenic plants exhibited no morphological and agronomical differences as compared to non-transformed plants. When young transgenic plants were exposed to chilling stress (4°C) for 3 days, increased survival (50%) was observed in transgenic lines than non-transformed plants (10%). Transgenic plants subjected to the chilling stress showed a significant decrease in membrane injury index and lipid peroxidation and also increased significantly free proline content in the leaf tissues as compared to non-transformed plants. Thus, these findings indicate that marker-free transgenic tomato plants expressing Arabidopsis CBF1 gene provided protection and conferred cold tolerance to transgenic tomato without any phenotypic variation.

Breaking seed dormancy in Hippophae salicifolia, a high value medicinal plant.

Seed dormancy is an important limiting factor in exploitation of an economically important species to its fullest. Hippophae salicifolia D. Don (seabuckthorn), a rich source of medicinal metabolites shows both exogenous and endogenous dormancy. Evidently, we recorded a high seed viability (94 %) but poor germination (22 %) of untreated seeds. We applied different pre-sowing seed priming treatments including NaCl (50, 100, 200, 500 mM), KNO3 (0.1, 0.2, 0.3 %), Thiourea (1, 2, 3 %), GA3 (100, 250, 500 mg/L), Sulphuric acid (98 %) and cold (4 °C) and warm water (65 °C) stratifications to explore improvements in its germination percentage, if any. We found KNO3 (0.1 %) and Thiourea (1 %) treatments to be superior to other methods for enhancement of mean seed germination percentage of H. salicifolia. Considering the practical applicability and cost effectiveness, these treatments can be applied to overcome seed dormancy and recommended for mass multiplication through seeds of H. salicifolia.

Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses.

Cicer microphyllum, a wild relative of cultivated chickpea, is a high altitude cold desert-adapted species distributed in western and trans-Himalayas. A complementary DNA (cDNA) encoding metallothionein-like protein has been identified from a cold-induced subtraction cDNA library from C. microphyllum. The sequence of the cloned metallothionein gene from C. microphyllum (GQ900702) contains 240-bp-long open reading frame and encodes predicted 79-amino acid protein of 7.9 kDa. Sequence analysis identified the motifs characteristic of type II metallothionein and designated as CmMet-2. Southern hybridization confirms a single copy of the CmMet-2 gene in C. microphyllum genome. In situ hybridization indicated spatial transcript regulation of CmMet-2 in root and aerial parts and also confirmed through real-time PCR-based quantitative transcript analysis. The data revealed a significantly low level of transcript in the aerial parts than the roots. Quantitative analysis using real-time PCR assay revealed induction of transcript in all parts of plants in response to cold stress at 4°C. The transcript abundance was found to increase exponentially with time course from 6 to 24 h after exposure. Further, regulation of transcript accumulation in response to abscisic acid application, polyethylene glycol (100 µM)-induced osmotic stress, or ZnSO(4) (1 µM) foliar spray indicated by Northern hybridization suggests the involvement of CmMet-2 in multiple stress response.

Shilajit: A panacea for high-altitude problems.

High altitude problems like hypoxia, acute mountain sickness, high altitude cerebral edema, pulmonary edema, insomnia, tiredness, lethargy, lack of appetite, body pain, dementia, and depression may occur when a person or a soldier residing in a lower altitude ascends to high-altitude areas. These problems arise due to low atmospheric pressure, severe cold, high intensity of solar radiation, high wind velocity, and very high fluctuation of day and night temperatures in these regions. These problems may escalate rapidly and may sometimes become life-threatening. Shilajit is a herbomineral drug which is pale-brown to blackish-brown, is composed of a gummy exudate that oozes from the rocks of the Himalayas in the summer months. It contains humus, organic plant materials, and fulvic acid as the main carrier molecules. It actively takes part in the transportation of nutrients into deep tissues and helps to overcome tiredness, lethargy, and chronic fatigue. Shilajit improves the ability to handle high altitudinal stresses and stimulates the immune system. Thus, Shilajit can be given as a supplement to people ascending to high-altitude areas so that it can act as a "health rejuvenator" and help to overcome high-altitude related problems.