Expression of CspE by a Psychrotrophic Bacterium Enterobacter ludwigii PAS1, Isolated from Indian Himalayan Soil and In silico Protein Modelling, Prediction of Conserved Residues and Active Sites

Proteome analysis of Enterobacter ludwigii PAS1 provide a powerful set of tool to study the cold shock proteins along with that combination of bioinformatics is useful for interpretation of comparative results from many species. There is a considerable interest in the use of psychrotrophic bacteria for nitrogen fixation, especially at hilly regions, thus better understanding of cold adaptation mechanisms too. The psychrotrophic E. ludwigii PAS1 grown at 30 and 4 °C, isolated from Himalaya soil was undertaken for proteomic responses during optimal and cold shock conditions. Comparative proteomic analyses using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS revealed the presence of Cold shock protein E (CspE). Three-dimensional structure of CspE of E. ludwigii PAS1 divulge the presence of five antiparallel β-sheets forming a β-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding and also reveals the presence of highly conserved nucleic acid-binding motifs RNP1 and RNP2 in Csp family.     

Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Positive correlation between menthol content andin vitro menthol tolerance inMentha arvensis L. cultivars

Menthol is a highly valued monoterpene produced by Japanese mint (Mentha arvensis) as a natural product with wide applications in cosmetics, confectionery, flavours, beverages and therapeutics. Selection of high menthol yielding genotypes is therefore the ultimate objective of all genetic improvement programmes inMentha arvensis. A positive correlation was observed in the present study between menthol content in oils of evaluated genotypes and the level of tolerance to externally supplied menthol of explants of these genotypes in culture medium. The easy use of this relationship as a selectable biochemical marker opens the practical applicability of largescalein vitro screening of the germplasm, clones and breeders' material for selection of elite genotypes.     

High regenerative nature ofMentha arvensis internodes

Media and incubation conditions have been defined for highly efficient regeneration of shoots from internode explants of slow and fast growing cultivars ofMentha arvensis. Internodal segments excised from thein vitro raised shoots were inoculated on the MS medium supplemented with combinations of 5 concentrations of l-napthalene acetic acid (NAA) and 3 concentrations of 6-benzyl amino purine (BAP). The media containing 2 μg ml⁻¹ NAA, 10 Μg ml⁻¹ BAP and 1 μg ml⁻¹ NAA, 5 μg ml⁻¹ BAP proved best for shoot regeneration and growth responses on cv Himalaya and cv Kalka explants, respectively. In 12 weeks time, on average one explant of cv Himalaya produced about 200 shoots and that of cv Kalka produced about 180 shoots. The Himalaya explants required higher concentrations of NAA and BAP for high efficiency proliferation as compared to the Kalka explants. The experiments demonstrated that internodal tissue inMentha arvensis can be induced to obtain direct shoot regenerants with high efficiency. The analysis of the RAPD profiles of 100 regenerated plantlets each of cv Himalaya and Kalka showed more than 99.9% homogeneity in bands with respect to the parents.     

Differentially Expressed Peroxidases from <Emphasis Type="Italic">Artemisia annua</Emphasis> and Their Responses to Various Abiotic Stresses

Artemisia annua is well-known for producing the antimalarial phytomolecule, artemisinin. The role of peroxidases has been hypothesized in artemisinin metabolism owing to the presence of an –O–O– linkage in this sesquiterpene lactone. Earlier, using a microarray, we identified differentially expressed genes, including peroxidases, in plant growth stages having contrasting artemisinin content. Here, three peroxidases—Aa547, having higher expression in low-artemisinin stage, and Aa540 and Aa528, having higher expression in high artemisinin stage, which could be associated with trichomes on the basis of their approximate gene expression pattern inferred from EST counts in UniGene—were selected for full-length cloning, tissue-specific expression profiling, and in silico analyses. The upstream genomic region of Aa547 was cloned and various cis-regulatory elements were identified. All the three candidates were predicted to be class III plant peroxidases. Further, this study aimed to check the responsiveness of the logically selected peroxidase genes to various abiotic stress factors. Taking cues from previous reports and the regulatory elements observed in the Aa547 promoter, hydration, salinity, temperature, salicylic acid, hydrogen peroxide, and methyl jasmonate, were selected to study their effect on the expression of the peroxidase genes through qRT-PCR. The peroxidases were found to be highly sensitive to the various factors but differed in their responses. Broadly, except for responses to high temperature and salicylic acid, the response of Aa547 to various factors was distinct from that of Aa540 and Aa528, which was in line with its distinctness from the other two peroxidases, considering the in planta artemisinin content and predicted structural features.     

Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes

In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml^–1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml^–1 followed by 60 and 70 g ml^–1. In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5×2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml^–1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml^–1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar `Saksham' was released from the selections by CIMAP for superior performance.