Salt-stress-responsive chloroplast proteins in <Emphasis Type="Italic">Brassica juncea</Emphasis> genotypes with contrasting salt tolerance and their quantitative PCR analysis

Brassica juncea is mainly cultivated in the arid and semi-arid regions of India where its production is significantly affected by soil salinity. Adequate knowledge of the mechanisms underlying the salt tolerance at sub-cellular levels must aid in developing the salt-tolerant plants. A proper functioning of chloroplasts under salinity conditions is highly desirable to maintain crop productivity. The adaptive molecular mechanisms offered by plants at the chloroplast level to cope with salinity stress must be a prime target in developing the salt-tolerant plants. In the present study, we have analyzed differential expression of chloroplast proteins in two Brassica juncea genotypes, Pusa Agrani (salt-sensitive) and CS-54 (salt-tolerant), under the effect of sodium chloride. The chloroplast proteins were isolated and resolved using 2DE, which facilitated identification and quantification of 12 proteins that differed in expression in the salt-tolerant and salt-sensitive genotypes. The identified proteins were related to a variety of chloroplast-associated molecular processes, including oxygen-evolving process, PS I and PS II functioning, Calvin cycle and redox homeostasis. Expression analysis of genes encoding differentially expressed proteins through real time PCR supported our findings with proteomic analysis. The study indicates that modulating the expression of chloroplast proteins associated with stabilization of photosystems and oxidative defence plays imperative roles in adaptation to salt stress.     

FLEXc: protein flexibility prediction using context-based statistics,predicted structural features,and sequence information

Background

The fluctuation of atoms around their average positions in protein structures provides important information regarding protein dynamics. This flexibility of protein structures is associated with various biological processes. Predicting flexibility of residues from protein sequences is significant for analyzing the dynamic properties of proteins which will be helpful in predicting their functions.

Results

In this paper, an approach of improving the accuracy of protein flexibility prediction is introduced. A neural network method for predicting flexibility in 3 states is implemented. The method incorporates sequence and evolutionary information, context-based scores, predicted secondary structures and solvent accessibility, and amino acid properties. Context-based statistical scores are derived, using the mean-field potentials approach, for describing the different preferences of protein residues in flexibility states taking into consideration their amino acid context.The 7-fold cross validated accuracy reached 61 % when context-based scores and predicted structural states are incorporated in the training process of the flexibility predictor.

Conclusions

Incorporating context-based statistical scores with predicted structural states are important features to improve the performance of predicting protein flexibility, as shown by our computational results. Our prediction method is implemented as web service called “FLEXc” and available online at: http://hpcr.cs.odu.edu/flexc.

     

Identification,cDNA Cloning,and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.     

Physical and chemical mutagens improved Sporotrichum thermophile,strain ST20 for enhanced Phytase activity

ObjectivePhosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi.MethodologyIn current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile.ResultsWe report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production.ConclusionThe current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.     

Characterisation of plant growth‐promoting rhizobacteria from rhizosphere soil of heat‐stressed and unstressed wheat and their use as bio‐inoculant

• High temperature induces several proteins in plants that enhance tolerance to high temperature shock. The fate of proteins synthesised in microbial cells or secreted into culture media by interacting microbes has not been fully elucidated. The present investigation aimed to characterise plant growth‐promoting rhizobacteria (PGPR) isolated from the rhizosphere of wheat genotypes (differing in tolerance to high temperature stress) and evaluate their performance as bioinoculant for use in wheat.
• Four bacterial strains, viz. Pseudomonas brassicacearum, Bacillus thuringiensis, Bacillus cereus strain W6 and Bacillus subtilis, were isolated from the rhizosphere of heat‐stressed and unstressed wheat genotypes. The wheat genotypes were exposed to high temperature stress at 45 °C for 10 days (3 h daily) at pre‐anthesis phase. Isolates were identified on the basis of morphology and biochemical characteristics, 16S rRNA gene sequencing and whole cell protein profiles. Results were further complemented by size exclusion chromatography (SEC) with fast protein liquid chromatography (FPLC) and SDS PAGE of 80% ammonium sulphate precipitates of the cell‐free supernatants.
• Isolates were positive for catalase, oxidases and antimicrobial activity . P. brassicacearum from the rhizosphere of the heat‐tolerant genotype was more efficient in phosphate solubilisation, bacteriocin production, antifungal and antibacterial activity against Helminthosporium sativum, Fusarium moniliforme and Klebsiella pneumonia, respectively. The inoculated seedlings had significantly higher root and shoot fresh weight, enhanced activity of antioxidant enzymes, proline and protein content. Total profiling of the culture with SDS‐PAGE indicated expression of new protein bands in 95 kDa in P. brassicacearum.
• Temperature‐induced changes in PGPR isolates are similar to those in the host plant. P. brassicacearum may be a good candidate for use in biofertiliser production for plants exposed to high temperature stress.

     

Modifying bio-catalytic properties of enzymes for efficient biocatalysis: a review from immobilization strategies viewpoint

Enzyme-based catalysis has become one of the most important disciplines in organic synthesis and plays a noteworthy role in the establishment of many chemical industries, e.g. fine chemicals, food or energy, textiles, agricultural, cosmeceutical, medicinal and pharmaceutical industries. However, pristine enzymes fail to demonstrate requisite functionalities for an industrial setting where extremely specific and stable catalysts are required. Immobilization enhances the catalytic stability and activity of enzymes and trims the overall cost burden of the enzyme. Therefore, it widely endeavours for proficient, sustainable, and environmentally responsive catalytic processes. Amongst several immobilization strategies, e.g. (1) supports-assisted, i.e. physical or covalent coupling and (2) supports-free techniques, i.e. cross-linked enzyme crystals (CLECs) or aggregates are the most promising ones and widely pursued for enzyme immobilization purposes. This perspective review focuses on up-to-date developments in the area of enzyme immobilization and presents their potentialities to upgrade and/or modify enzyme properties. Both types of immobilization strategies, i.e. supports-assisted and supports-free techniques are discussed with particular reference to CLECs or aggregates and protein-coated microcrystals. Also, several useful traits achieved after immobilization are also discussed in the second half of the review.