Nandula Raghuram

Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats

The small intestine exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. We therefore investigated the occurrence of oxidative stress in the small intestine during diabetes by measuring the extent of oxidative damage as well as the status of the antioxidant defense system. Significant increases in lipid peroxidation (four-fold) as measured by TBARS and protein oxidation (38%) as measured by protein carbonyl content were observed after 6 weeks of diabetes. A distinct elevation in the activities of catalase (123.9%) and superoxide dismutase (71.9%) and a decline in the activity of glutathione peroxidase (67.7%) were also observed. The steady state mRNA levels of these enzymes measured by RT-PCR were, however, unchanged suggesting the absence of transcriptional control. In contrast, no changes in the levels of protein and non-protein thiols as well as the activities of glutathione reductase and glutathione-S-transferase were detected. Interestingly, decreases in the activities of xanthine oxidase (XO; 25.7%) and xanthine dehydrogenase (XDH; 42.6%) indicate that they do not contribute significantly to oxidative damage. The results thus reveal the occurrence of oxidative stress in the small intestine during diabetes and suggest its possible involvement in some of the accompanying functional alterations.

Oxidative stress and gene expression of antioxidant enzymes in the renal cortex of streptozotocin- induced diabetic rats

The present study was aimed at addressing the effect of hyperglycemia on antioxidant enzymes. The expression of catalase, superoxide dismutase and glutathione peroxidase, the three primary scavenger enzymes involved in detoxifying reactive oxygen species has been evaluated in the renal cortex of rats after 6 weeks of streptozotocin-induced diabetes. Lipid peroxidation and protein oxidation in the renal cortical homogenate were first performed to confirm a state of oxidative stress. The enzyme assays showed significant and varied alterations in catalase, superoxide dismutase and glutathione peroxidase activities. An opposing response of catalase and glutathione peroxidase activities to diabetes was observed. RT-PCR analysis was used to ascertain whether steady-state transcription levels were altered. While an increase in glutathione peroxidase and Cu-Zn superoxide dismutase mRNA parallels the increase in the activities of the enzymes, an increase in catalase gene expression in contrast to a decrease in enzyme activity suggests a role for post-translational modification in altering the activity of this enzyme.

Evidence for some common signal transduction events for opposite regulation of nitrate reductase and phytochrome-I gene expression by light.

We have explored the possible involvement of the phosphoinositide (PI) cycle and protein kinase C (PKC) in the phytochrome (Pfr)-mediated light signal transduction pathway using nitrate reductase (NR) and phytochrome-I (PhyI) genes as model systems. We have shown earlier that phorbol myristate acetate (PMA) completely replaces the red light effect in stimulating nitrate reductase activity and transcript levels in maize. In this paper, we present detailed evidence to show that PMA mimics the red light effect and follows similar kinetics to enhance NR steady-state transcript accumulation in a nitrate-dependent manner. We also show that PMA inhibits phyI steady-state transcript accumulation in a manner similar to red light, indicating that a PKC-type enzyme(s) may be involved in mediating the light effect in both cases. Serotonin or 5-hydroxytryptamine (5-HT), a stimulator of PI turnover, was also found to mimic the red light effect in enhancing NR transcript levels and inhibiting phyI transcript accumulation, indicating the role of the PI cycle in generating second messengers for regulating the two genes. These results indicate that phytochrome-mediated light regulation of NR and phyI gene expression may involve certain common steps in the signal transduction pathway such as the PI cycle and protein phosphorylation by a PKC-type enzyme.

Rapid production of ethanol in high concentration by immobilized cells of Saccharomyces cerevisiae through soya flour supplementation

SummaryEthanol concentration and fermentation productivities were substantially increased when soya extract was added to the fermentation medium using immobilized cells of a locally isolated strain of S. cerevisiae. Very high concentrations, 152 and 162 g/l of ethanol, were obtained from a medium containing 300 and 350 g/l sugars respectively by supplementing the medium with soya extract. The fermentation time was also reduced by more than 50%.

Improving Plant Nitrogen-Use Efficiency

Nitrogen (N) is a crucial nutrient for plant development, and N-use efficiency (NUE) at the plant level is its ability to maximize its output with minimal input of N fertilizers. NUE is a major issue in agriculture, not only to save fertilizer costs, but also to minimize N pollution due to unutilized fertilizers and associated health hazards and climate change. Most biotechnological efforts to enhance NUE currently rely on plant nitrate uptake and assimilation, or internal remobilization of secondary N metabolites and related pathways. Though research efforts are also underway to transfer the N 2 -fixing ability from microorganisms to crop plants, they are yet to mature into viable technologies and are not elaborated here. This article summarizes a variety of ways to measure NUE, the relevance of biological interventions to improve it and the range of transgenic attempts to manipulate NUE, as well as the patents filed/granted in this field so far. It also documents newer insights and options emerging from recent functional genomics and systems biology studies on organism-wide nitrate response and coordination of C and N metabolic pathways. It concludes with the socioeconomic implications of NUE from the global and regional (Asian) perspectives.

Roles of nitrate, nitrite and ammonium ion in phytochrome regulation of nitrate reductase gene expression in maize

The influence of nitrate and its metabolites on the nitrate reductase (NR) gene expression and its relationship with phytochrome (Pfr) regulation of NR in etiolated maize leaves is examined. Nitrate induction and Pfr stimulation are brought about by independent signalling phenomena. Phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), mimicked the effect of red light but could not replace the nitrate requirement for the induction of NR transcript accumulation. This suggests that while PKC‐type enzymes may be involved in mediating the Pfr signal, nitrate may follow an independent signalling mechanism. Experiments with 5‐hydroxytryptamine (5HT) and lithium ions (Li+), which are known to modulate phosphoinositide (PI) turnover, indicated that in addition to generating Pfr‐induced second messengers for PKC activation, PI cycle may also generate other signals which mediate nitrate induction of NR gene expression in the dark. The products of nitrate reduction ie, nitrite and ammonium ion had inhibitory and stimulatory effects respectively, on NR transcript accumulation. They work mainly at the level of nitrate induction.

Transcriptome analysis of Arabidopsis GCR1 mutant reveals its roles in stress, hormones, secondary metabolism and phosphate starvation.

The controversy over the existence or the need for G-protein coupled receptors (GPCRs) in plant G-protein signalling has overshadowed a more fundamental quest for the role of AtGCR1, the most studied and often considered the best candidate for GPCR in plants. Our whole transcriptome microarray analysis of the GCR1-knock-out mutant (gcr1-5) in Arabidopsis thaliana revealed 350 differentially expressed genes spanning all chromosomes. Many of them were hitherto unknown in the context of GCR1 or G-protein signalling, such as in phosphate starvation, storage compound and fatty acid biosynthesis, cell fate, etc. We also found some GCR1-responsive genes/processes that are reported to be regulated by heterotrimeric G-proteins, such as biotic and abiotic stress, hormone response and secondary metabolism. Thus, GCR1 could have G-protein-mediated as well as independent roles and regardless of whether it works as a GPCR, further analysis of the organism-wide role of GCR1 has a significance of its own.

Genomewide computational analysis of nitrate response elements in rice and Arabidopsis

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualifies to be the cis-acting NRE for all the responsive genes. We searched for the specific occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-specific bias. This is the first comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any specificity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-specific and shoot-specific response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants.

Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold

The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides, carbohydrates, receptors and lipids, morphogenesis, flower development, and cell homeostasis. We also mined 63 miRNAs that bind to the stress responsive transcripts identified in this study, indicating their post-transcriptional regulation. Overall, these results indicate the potentially extensive role of RGA1 in the regulation of multiple abiotic stresses in rice for further validation.

G-protein Signaling Components GCR1 and GPA1 Mediate Responses to Multiple Abiotic Stresses in Arabidopsis

G-protein signaling components have been implicated in some individual stress responses in Arabidopsis, but have not been comprehensively evaluated at the genetic and biochemical level. Stress emerged as the largest functional category in our whole transcriptome analyses of knock-out mutants of GCR1 and/or GPA1 in Arabidopsis (Chakraborty et al., 2015a,b). This led us to ask whether G-protein signaling components offer converging points in the plants response to multiple abiotic stresses. In order to test this hypothesis, we carried out detailed analysis of the abiotic stress category in the present study, which revealed 144 differentially expressed genes (DEGs), spanning a wide range of abiotic stresses, including heat, cold, salt, light stress etc. Only 10 of these DEGs are shared by all the three mutants, while the single mutants (GCR1/GPA1) shared more DEGs between themselves than with the double mutant (GCR1-GPA1). RT-qPCR validation of 28 of these genes spanning different stresses revealed identical regulation of the DEGs shared between the mutants. We also validated the effects of cold, heat and salt stresses in all the 3 mutants and WT on % germination, root and shoot length, relative water content, proline content, lipid peroxidation and activities of catalase, ascorbate peroxidase and superoxide dismutase. All the 3 mutants showed evidence of stress tolerance, especially to cold, followed by heat and salt, in terms of all the above parameters. This clearly shows the role of GCR1 and GPA1 in mediating the plants response to multiple abiotic stresses for the first time, especially cold, heat and salt stresses. This also implies a role for classical G-protein signaling pathways in stress sensitivity in the normal plants of Arabidopsis. This is also the first genetic and biochemical evidence of abiotic stress tolerance rendered by knock-out mutation of GCR1 and/or GPA1. This suggests that G-protein signaling pathway could offer novel common targets for the development of tolerance/resistance to multiple abiotic stresses.