Brewer's spent yeast replacement in carp diet leads to muscle biomass production, recycling, waste management and resource conservation.

Brewer's spent yeast (BSY) is among the most voluminous by-products generated in brewery industry that adds to the waste; however, smart utilization of BSY could lead to edible biomass production besides waste management. To utilize it for biomass production, it is being used in fish feeds; however, its effect on the fish physiology has been scantily studied. The present study investigated the proteomic changes in muscle tissues of carp Labeo rohita fed with BSY-based diet, to understand its impact on muscle physiology and biomass. Six feeds were prepared with different grades of BSY (0, 20, 30, 40, 50, 100% replacement of fishmeal with BSY) and fishes were fed for 90 days. Highest weight gain%, feed conversion efficiency, specific growth rate% were observed in 30% BSY-replaced group and this group was considered for the proteomic study. Comparative shotgun proteomic analysis was carried out by LC-MS/MS and data generated have been deposited in ProteomeXchange Consortium with dataset identifier PXD020093. A total of 62 proteins showed differential abundance; 29 increased and 33 decreased in the 30% BSY-replaced group. Pathway analysis using IPA and Panther tools revealed that the proteins tyrosine protein kinase, PDGFα, PKRCB and Collagen promote muscle growth by inducing the PI3K-AKT pathway. Conversely, the proteins Serine/threonine-protein phosphatase, Phosphatidylinositol 3,4,5-trisphosphate5-phosphatase 2A and Ras-specific guanine- nucleotide-releasing factor inhibit muscle growth indicating that 30% BSY-replaced feed promote muscle growth in a highly controlled manner. Findings suggest that BSY could be recycled for carp feed production in large scale thereby leading to resource conservation, reducing environmental effects.

Proteomic Profiling and Pathway Analysis of Acid Stress-Induced Vasorelaxation of Mesenteric Arteries In Vitro.

Although metabolic acidosis is associated with numerous pathophysiological conditions and its vasorelaxation effects have been well described in different animal and culture models, the molecular mechanisms of acidosis-induced vasorelaxation are not fully understood. Mesenteric artery models have been used extensively to examine the vascular response to various pathophysiological conditions. Our previous studies and several other reports have suggested the vascular responses of goat mesenteric arteries and human arteries to various stimuli, including acidic stress, are highly similar. In this study, to further identify the signaling molecules responsible for altered vasoreactivity in response to acidic pH, we examined the proteomic profile of acid stress-induced vasorelaxation using a goat mesenteric artery model. The vascular proteomes under acidic pH were compared using 2D-GE with 7 cm IPG strips and mini gels, LC-MS/MS, and MALDI TOF MS. The unique proteins identified by mass spectroscopy were actin, transgelin, WD repeat-containing protein 1, desmin, tropomyosin, ATP synthase ß, Hsp27, aldehyde dehydrogenase, pyruvate kinase, and vitamin K epoxide reductase complex subunit 1-like protein. Out of five protein spots identified as actin, three were upregulated > 2-fold. ATP synthase ß was also upregulated (2.14-fold) under acid stress. Other actin-associated proteins upregulated were transgelin, desmin, and WD repeat-containing protein 1. Isometric contraction studies revealed that both receptor-mediated (histamine) and non-receptor-mediated (KCl) vasocontraction were attenuated, whereas acetylcholine-induced vasorelaxation was augmented under acidosis. Overall, the altered vasoreactivity under acidosis observed in the functional studies could possibly be attributed to the increase in expression of actin and ATP synthase ß.

Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.

Draft Genome Sequence of the Plant Growth-Promoting Rhizobacterium Klebsiella sp. Strain KBG6.2, Imparting Salt Tolerance to Rice.

Klebsiella sp. strain KBG6.2 is a potential salt-tolerant, plant growth-promoting rhizobacterium isolated from a rice field in Konark, Odisha, India. Here, we report the whole-genome sequencing of Klebsiella sp. strain KBG6.2, which has a 5.038-Mb genome containing 4,867 predicted protein-coding sequences and 79 RNA genes.

A comparative metabolomics study on anadromous clupeid Tenualosa ilisha for better understanding the influence of habitat on nutritional composition.

INTRODUCTION: Fish inhabiting different aquatic habitats adapts to the environment by metabolomic readjustments. Understanding the combined activities of all the metabolic pathways (metabolome) helps in better understanding the complex interactions between gene and environment. OBJECTIVES: The anadromous migratory Tenualosa ilisha is a high value food fish comprising the dominant fishery of the rivers Padma and Hooghly. The present study aimed at understanding the influence of the two habitats on the nutritional composition of hilsa. METHODS: Metabolite profiling was carried out by GC/MS. De novo assembly of hilsa liver transcriptome was generated under Illumina HiSeq platform and multivariate analysis was employed for correlation and comparison. RESULTS: GC/MS fingerprinting showed C16:0, C18:1, C20:5 and C22:6 to be the predominant fatty acids present in hilsa liver, which were also found to be significantly higher in Hooghly hilsa. Comparative transcriptome analysis revealed that the differentially expressed genes were mainly associated with 'lipid metabolism' and 'amino acid metabolism' pathways. Multivariate analysis between the metabolites amino acid, fatty acid and corresponding gene expression showed that few genes of amino acid metabolism (EZH1, ALAS2 and ALDH4A1) positively correlated with individual amino acids (lysine, glycine and glutamate) in Hooghly hilsa. Similarly, the key genes for LC-PUFA biosynthesis (ELOVL5, FADS2, CPT1) showed positive correlation with individual LC-PUFAs (C18:3, C20:4, C20:5, C22:6), indicating higher LC-PUFA biosynthesis potential in Hooghly hilsa. CONCLUSION: Comparative metabolomic study in hilsa from the two different habitats showed that the habitats influence the nutritional composition as evidenced by high abundance of amino acids lysine, leucine and arginine and LC-PUFAs C18:3, C20:4, C20:5, C22:6 in Hooghly hilsa.

Proteomic and transcriptomic changes in rat liver following oral feeding of formaldehyde.

Formaldehyde (FA), a ubiquitous volatile organic compound present in a wide range of resources, is a hazardous chemical and human carcinogen. Contamination of FA in food, especially perishable commodities like fish and meat, is a major source of exposure, although it is not recommended for use in food and food products owing to its carcinogenicity. Effects of oral feeding of FA have been studied by evaluating general health, haematology and clinical chemistry in rat. Recent studies have shown that FA exposure leads to detrimental cardiovascular effects. It regulates vascular tensions through nitric oxide-cGMP signalling pathway and ion channels in rats. Although FA is an established carcinogen, molecular studies on carcinogenic potential with dose dependency are meagre. In this context, the present study was undertaken to investigate the toxicogenomic and proteomic alterations in liver of rats fed FA through drinking water. By proteomic analysis, 621 proteins/protein-subunits showed differential abundance (proteome data available via ProteomeXchange with identifier PXD010534), whereas 536 differentially-expressed-genes were identified by transcriptome analysis (data available via Sequence Read Archive with identifier SRR7974113). Gene ontology analysis showed that binding, catalysis, signal transduction were affected in formaldehyde-fed rats. Pathway analysis revealed that formaldehyde-exposure activated PI3K-AKT pathway that leads to inhibition of caspase activity thereby assisting cells to survive against apoptosis. Decreased abundance/down-regulation of ANGPT, eNOS, STAT3 proteins/transcripts and increased abundance of EDN1 indicated decrease in angiogenesis and vasodilatation that restricted hepatic cells from becoming tumorigenic; thus, indicating FA could be less toxic and non-tumorigenic at low concentrations.

Heat stress-induced alterations in the expression of genes associated with gonadal integrity of the teleost Puntius sophore.

Temperature plays an important role on reproductive physiology of vertebrates including mammals, fish, and birds. It has varying effects on fish reproduction depending on the species; higher temperatures favor the spring-spawning species, while lower temperatures stimulate reproduction in autumn spawners. To evaluate the impact of high temperature on the reproductive physiology of minnow Puntius sophore, we carried out expression analysis of selected genes associated with gamete quality (hsp60, hsp70, hsp90, hsf1, vtg), pleuripotency (sox2, oct4, nanog), and sex determination (dmrt1) in gonads (ovary and testis) of P. sophore, heat stressed for different time periods (36 °C/7 days or 60 days) using real-time quantitative polymerase chain reaction (RT-qPCR). Expression of most of the hsp, vtg, and pleuripotency marker genes sox-2, oct-4, and nanog genes was downregulated in both ovary and testis of heat-stressed fish. The expression of dmrt-1 was upregulated in testis but downregulated in ovary of the heat-stressed fish which could be a male favoring effect of high temperature in P. sophore. This study suggests that the reproductive physiology and health of the nutrient dense P. sophore would be negatively affected by high temperature stress.

Expression patterns of heat shock protein genes in Rita rita from natural riverine habitat as biomarker response against environmental pollution.

River pollution is one of the principal environmental concerns and biomonitoring tools can play an important role in pollution assessment in the riverine environment. Heat shock proteins (Hsps) have been found to be suitable tools for monitoring stress response. In the present study, expression analyses of hsp genes (hsp27, hsp47, hsp60, hsp70, hsc70, and hsp90) and selected hsp-regulatory genes (hsf1, hyou1, ask1, jnk) were carried out by RT-qPCR in catfish Rita rita collected from selected stretches of river Ganga to investigate changes in their expression patterns as biomarker response. Water quality characteristics were measured in terms of physico-chemical characteristics (DO, BOD, COD, pH, conductivity), element profile (arsenic, mercury, cadmium, lead, chromium, zinc, copper) and persistent organic pollutants (POPs; HCH, DDT, aldrin, endosulphan, heptachlor). Water quality index was calculated and sampling sites were categorized as good/medium/bad. Multivariate analysis was carried out taking the water quality parameters and the fold changes in hsp gene expression as variables, which showed that hsp47 and hsp70b correlated well with BOD, an indicator of organic pollution. To identify the organic pollutant(s) which could be influencing the expression of hsps, again multivariate analysis was employed taking concentration of POPs and fold changes of hsps, which showed up-regulation of hsp47 and hsp70b (HSP72i) correlated well with concentrations of aldrin and HCH. Synergistic effects of these POPs could be responsible for the up-regulation of said hsps, although individually present in low concentration; thus, indicating synergistic effect of the POPs on hsp47 and hsp70b up-regulation as biomarker response.

Expression patterns and mutation analysis of p53 in fish Rita rita from polluted riverine environment.

The present study was undertaken to investigate the alterations in gene expression patterns and for mutation analysis of p53 in the riverine catfish Rita rita collected from polluted riverine habitat. The partial p53 gene sequence of Rita rita generated showed a high degree of similarities with the DNA binding domains of fishes, mice and human. Transcriptomic analysis, carried out by quantitative real-time Polymerase Chain Reaction (RT-qPCR), showed significant down-regulation of p53 in fishes collected from most of the polluted stretches. Similar trend in protein abundance was observed by western blot analysis. Down-regulation of p53 was more pronounced in gill than liver. Expression patterns of p53 suggest that exposure to a multitude of contaminants in the natural riverine ecosystem could suppress the expression of p53. Genomic DNA showed a low stained smear pattern upon electrophoresis, with no evidence of DNA fragmentation. For mutation analysis PCR-SSCP followed by sequence analysis was carried out, which identified eight mutations; two at codon level and six missense mutations in the DNA binding domain IV and V. Secondary structure prediction showed that these mutations could lead to impairment of protein structure. Thus, the present study indicated that aquatic pollution has impacted these lower vertebrates which are reflected by the down-regulation of tumor suppressor protein (p53) in majority of the stretches studied.

The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses.

The occurrence of various stresses, as the outcome of global climate change, results in the yield losses of crop plants. Prospecting of genes in stress tolerant plant species may help to protect and improve their agronomic performance. Finger millet (Eleusine coracana L.) is a valuable source of superior genes and alleles for stress tolerance. In this study, we isolated a novel endoplasmic reticulum (ER) membrane tethered bZIP transcription factor from finger millet, EcbZIP17. Transgenic tobacco plants overexpressing this gene showed better vegetative growth and seed yield compared with wild type (WT) plants under optimal growth conditions and confirmed upregulation of brassinosteroid signalling genes. Under various abiotic stresses, such as 250 mM NaCl, 10% PEG6000, 400 mM mannitol, water withdrawal, and heat stress, the transgenic plants showed higher germination rate, biomass, primary and secondary root formation, and recovery rate, compared with WT plants. The transgenic plants exposed to an ER stress inducer resulted in greater leaf diameter and plant height as well as higher expression of the ER stress-responsive genes BiP, PDIL, and CRT1. Overall, our results indicated that EcbZIP17 improves plant growth at optimal conditions through brassinosteroid signalling and provide tolerance to various environmental stresses via ER signalling pathways.

Nutrigenomic studies on hilsa to evaluate flesh quality attributes and genes associated with fatty acid metabolism from the rivers Hooghly and Padma.

The Indian shad hilsa (Tenualosa ilisha), a commercially important food fish rich in oils, enjoys high consumer preference in the South Asian countries owing to its unique flavour and culinary properties. The present study was undertaken with the primary objective of determining the flesh quality attributes of hilsa in terms of nutritive value (gross chemical composition, amino acid, fatty acid and mineral composition), pH, water holding capacity (WHC) and expression of genes associated with fatty acid metabolism and flesh quality. Additionally, comparative studies on the flesh quality attributes in hilsa from two distributaries of river Ganga i.e. Hooghly and Padma were also carried out. A high WHC (>80%) suggested juicy and tender nature of hilsa meat. The protein content was 18-21% in hilsa from both the rivers and essential amino acid lysine, valine and functional amino acids leucine and arginine were significantly higher in Hooghly hilsa (P<0.05). The predominance of umami taste amino acids, glutamic acid and aspartic acid and sweet taste amino acids, serine, glycine and alanine in hilsa from both the rivers could be the contributing factors to its unique flavour. The fat content in hilsa from river Hooghly and Padma were found to be 9.94 and 7.84%, respectively. The concentration of flavouring fatty acids like saturated fatty acids (SFA) (myristic acid) and omega (ω)-3 polyunsaturated fatty acids (PUFAs) (linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) were significantly higher in Hooghly hilsa (P<0.05). Among the genes associated with fatty acid metabolism studied, expression of cluster of differentiation (CD36), acetyl CoA oxidase (ACO), fatty acid synthase (FAS), peroxisome proliferator activated receptor beta (PPARß), peroxisome proliferator activated receptor gamma (PPARγ) and desaturase were significantly higher in Padma hilsa (P<0.05), and the change was <2 fold. Comparative gene expression profiling of flesh quality genes (actin, GAPDH, LDH, TPI) showed similar levels of expression in hilsa from both the rivers (P<0.05). The nutrigenomic information generated on various flesh quality attributes of hilsa has enriched the knowledgebase. Further, from comparative nutrient analysis on hilsa from river Hooghly and Padma, it was observed that Hooghly hilsa is superior in terms of oil content, ω-3 PUFAs EPA and DHA and essential amino acids; however, the expression profile of genes associated with flesh quality were found to be similar. Thus, within the scope of the present study, Hooghly hilsa (medium size category, 500-700g size) was found to be nutritionally superior.

Overexpression of plastidic maize NADP-malate dehydrogenase (ZmNADP-MDH) in Arabidopsis thaliana confers tolerance to salt stress.

The plastidic C4 Zea mays NADP-malate dehydrogenase (ZmNADP-MDH), responsible for catalysis of oxaloacetate to malate, was overexpressed in Arabidopsis thaliana to assess its impact on photosynthesis and tolerance to salinity stress. Different transgenic lines were produced having ~3-6-fold higher MDH protein abundance and NADP-MDH enzyme activity than vector control. The overexpressors had similar chlorophyll, carotenoid, and protein content as that of vector control. Their photosynthetic electron transport rates, carbon assimilation rate, and consequently fresh weight and dry weight were almost similar. However, these overexpressors were tolerant to salt stress (150 mM NaCl). In saline environment, the Fv/Fm ratio, yield of photosystem II, chlorophyll, and protein content were higher in ZmNADP-MDH overexpressor than vector control. Under identical conditions, the generation of reactive oxygen species (H2O2) and production of malondialdehyde, a membrane lipid peroxidation product, were lower in overexpressors. In stress environment, the structural distortion of granal organization and swelling of thylakoids were less pronounced in ZmNADP-MDH overexpressing plants as compared to the vector control. Chloroplastic NADP-MDH in consort with cytosolic and mitochondrial NAD-MDH plays an important role in exporting reducing power (NADPH) and exchange of metabolites between different cellular compartments that maintain the redox homeostasis of the cell via malate valve present in chloroplast envelope membrane. The tolerance of NADP-MDH overexpressors to salt stress could be due to operation of an efficient malate valve that plays a major role in maintaining the cellular redox environment.

Isolation and expression analysis of EcbZIP17 from different finger millet genotypes shows conserved nature of the gene.

Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we report isolation and characterization of EcbZIP17, a group B bZIP transcription factor from a climate smart cereal, finger millet (Eleusine coracana L.). The genomic sequence of EcbZIP17 is 2662 bp long encompassing two exons and one intron with ORF of 1722 bp and peptide length of 573 aa. This gene is homologous to AtbZIP17 (Arabidopsis), ZmbZIP17 (maize) and OsbZIP60 (rice) which play a key role in endoplasmic reticulum (ER) stress pathway. In silico analysis confirmed the presence of basic leucine zipper (bZIP) and transmembrane (TM) domains in the EcbZIP17 protein. Allele mining of this gene in 16 different genotypes by Sanger sequencing revealed no variation in nucleotide sequence, including the 618 bp long intron. Expression analysis of EcbZIP17 under heat stress exhibited similar pattern of expression in all the genotypes across time intervals with highest upregulation after 4 h. The present study established the conserved nature of EcbZIP17 at nucleotide and expression level.

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress.

BACKGROUND: High ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (ßactin), ß-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder. RESULTS: In ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis. CONCLUSIONS: In conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita.

Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water and food leads to arsenicosis, a major public health problem in many parts of the world. Early detection of arsenic toxicity would greatly benefit patients; however, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D proteome profiles, 14 unique spots were identified by MALDI-TOF/TOF MS and/or LC-MS/MS which included Apolipoprotein-A1 (Apo-A1) (6 spots), α-2 macroglobulin-like protein (A2ML) (2 spots), transferrin (TF) (3 spots) and warm-temperature acclimation related 65kDa protein (Wap65). The proteome data are available via ProteomeXchange with identifier PXD003404. Highly abundant protein spots identified in plasma from arsenic-exposed fish i.e. Apo-A1 (>10-fold), A2ML (7-fold) and Wap65 (>2-fold) indicate liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure.

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of nrf-2-mediated antioxidative enzymes and hsps in Puntius sophore.

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.

hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for surviving in the hot spring run-off aquatic ecosystem.

Changes in the expression of a number of hsp genes in minnow Puntius sophore collected from a hot spring run-off (Atri hot spring in Odisha, India; 20o09'N 85°18'E, 36-38 °C) were investigated to study the upper thermal acclimation response under heat stress, using same species from aquaculture ponds (water temperature 27 °C) as control. Expression of hsp genes was analyzed in both groups using RT-qPCR, which showed up-regulation of hsp90 (2.1-fold) and hsp47 (2.5-fold) in hot spring run-off fishes, whereas there was no alteration in expression of other hsps. As the fish inhabit the hot spring run-off area for very long duration, they could have adapted to the environment. To test this hypothesis, fishes collected from hot spring run-off were divided into two groups; one was heat-shocked at 41 °C/24 h, and the other was acclimatized at 27 °C/24 h. Up-regulation of all the hsps (except hsp78) was observed in the heat-shocked fishes, whereas expression of all hsps was found to be down-regulated to the basal level in fishes maintained at 27 °C/24 h. Pathway analysis showed that the expressions of all the hsps except hsp90 are regulated by the transcription factor heat shock factor 1 (Hsf1). This study showed that hsp90 and hsp47 play an important role in Puntius sophore for surviving in the high-temperature environment of the hot spring run-off. Additionally, we show that plasticity in hsp gene expression is not lost in the hot spring run-off population.

Proteomic changes in the liver of Channa striatus in response to high temperature stress.

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were exposed to 36°C for 4 days and liver proteome changes were analyzed using gel- based proteomics, i.e. 2DE, MALDI-TOF/TOF-MS, and validation by transcript analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular retinol binding protein (CRBP), glutathione-S-transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring runoff at 36-38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight-, 2.5-, 2.4-, and 2.45-fold, respectively, in the hot spring runoff fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Thus, while short-term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.

Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of 35S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress.

Quantitative real-time polymerase chain reaction is the most advanced method of quantifying gene expression studies; however, the significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. In the present study, expression analysis of six different constitutively expressed genes viz. 18S ribosomal RNA, glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (ßactin), ribosomal binding protein L13, tubulin and TATA-box-binding protein (tbp) were carried out to test their efficacy as reference genes in three different tissues, namely liver, gill and muscle of murrel Channa striatus exposed to high temperature for variable time periods. The stability and suitability of the genes were determined by using bioinformatic tools: GeNorm, NormFinder and BestKeeper. Based on the results, tub/ßactin could be used as the reference genes for liver and gill tissues and ßactin/gapdh could be the reference genes for muscle tissues in Channa striatus under both short- and long-term thermal stress.