Biomedical subjects

M K Reddy

Publications and source records attributed to M K Reddy.

At least 19 recordsLinked to original sources

PubMed2006

Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity.

Abiotic stress-mediated gene expression is regulated via different transcription factors of which drought-responsive element-binding (DREB) proteins play an important role. There are two types of DREBs. Presently, the function of DREB1 type protein is well studied; however, much less information is available for DREB2. In this study, a cDNA with an open reading frame of 332 amino acids, encoding the transcription activation factor DREB2A, was cloned from Pennisetum glaucum, a stress tolerant food grain crop. Phylogenetic tree revealed that PgDREB2A is more close to DREBs isolated from monocots, though it forms an independent branch. The PgDREB2A transcript was up-regulated in response to drought within 1 h of the treatment, whereas the induction was delayed in response to cold and salinity stress. However, during cold stress, the transcript was induced more as compared to drought and salinity. The recombinant PgDREB2A protein having a molecular mass of 36.6 kDa was purified using Ni-NTA affinity chromatography. Gel mobility shift assays using the purified protein and two cis elements of rd29A (responsive to dehydration 29A) gene promoter of Arabidopsis revealed that PgDREB2A binds to drought-responsive element (DRE) ACCGAC and not to GCCGAC. PgDREB2A is a phosphoprotein, which has not been reported earlier. The phosphorylation of PgDREB2A in vitro by P. glaucum total cell extract occurred at threonine residue(s). The phosphorylated PgDREB2A did not bind to the DREs. The present data indicate that stress induction of genes could occur via post-translational modification by phosphorylation of DREB2A.

Acclimatization↗

PubMed2006

Structural and functional analysis of a salt stress inducible gene encoding voltage dependent anion channel (VDAC) from pearl millet (Pennisetum glaucum).

We have cloned and characterized a gene encoding voltage-dependent anion channel from Pennisetum glaucum (PgVDAC). PgVDAC was identified while isolating genes that were differentially up-regulated following salt stress. The genomic organization of PgVDAC clone was well conserved compared to other plant VDAC genes in terms of number of introns, their position and phasing, however, the primary amino acid sequence of voltage dependent anion channel (VDAC) proteins did not show much conservation with other plant VDACs but their secondary and tertiary structures are well conserved as predicted by in silico structural and CD spectra analyses and results show it to be a typical membrane-spanning beta-barrel leading to the formation of pore in the membrane. The heterologous expression of PgVDAC protein in yeast strain lacking the endogenous mitochondrial VDAC gene could not functionally complement it as was also previously observed for the potato VDAC. Using real-time quantitative PCR analysis it was found that transcript expression profile of PgVDAC was quantitatively and kinetically up-regulated in response to salinity, desiccation, cold and exogenous application of salicylic acid (SA); however, there was no effect of exogenous application of abscisic acid (ABA) on its expression. Constitutive over-expression of PgVDAC appears to be deleterious in transgenic rice plant; however, low level of up-regulation imparted salinity stress adaptive response. A search for a more suitable inducible transgene system is currently under way to understand PgVDAC expression levels in plant development and its role in stress adaptation.

Amino Acid Sequence↗

PubMed2006

Role of DREB transcription factors in abiotic and biotic stress tolerance in plants.

Abiotic and biotic stresses negatively influence survival, biomass production and crop yield. Being multigenic as well as a quantitative trait, it is a challenge to understand the molecular basis of abiotic stress tolerance and to manipulate it as compared to biotic stresses. Lately, some transcription factor(s) that regulate the expression of several genes related to stress have been discovered. One such class of the transcription factors is DREB/CBF that binds to drought responsive cis-acting elements. DREBs belong to ERF family of transcription factors consisting of two subclasses, i.e. DREB1/CBF and DREB2 that are induced by cold and dehydration, respectively. The DREBs are apparently involved in biotic stress signaling pathway. It has been possible to engineer stress tolerance in transgenic plants by manipulating the expression of DREBs. This opens an excellent opportunity to develop stress tolerant crops in future. This review intends to focus on the structure, role of DREBs in plant stress signaling and the present status of their deployment in developing stress tolerant transgenic plants.

Abscisic Acid↗

PubMed2006

A novel isoform of ATPase c subunit from pearl millet that is differentially regulated in response to salinity and calcium.

Vacuolar ATPases help in maintaining the pH of the vacuoles and thereby play a crucial role in the functioning of vacuolar sodium-proton antiporter. Though the various subunits that make V(1) and V(0) sector have been reported in plants their regulation is not understood completely. We have cloned three different isoforms of vacuolar ATPase subunit c (VHA-c) from Pennisetum glaucum with homologies among themselves varying from 38% to approximately 73% at the nucleic acid level. Using real-time PCR approach we have shown that the three isoforms are regulated in a tissue-specific manner under salinity stress. While isoform III is constitutively expressed in roots and shoots and does not respond to stress, isoform I is upregulated under stress. Isoform II is expressed mainly in roots; however, under salinity stress its expression is downregulated in roots and upregulated in shoots. Tissue specific expression under salinity stress of isoform II was also seen after exogenous application of calcium. This study for the first time shows the presence of three isoforms of PgVHA-c and their differential regulation during plant development, and also under abiotic stress.

Abscisic Acid↗

PubMed2005

Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc-spiked soils.

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.

Cadmium↗

PubMed2005

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione.

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.

Chromatography, High Pressure Liquid↗

PubMed2005

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress.

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.

Antioxidants↗

PubMed2005

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes.

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.

Base Sequence↗

PubMed2005

Indoor air quality at Salarjung Museum, Hyderabad, India.

Deterioration of art objects at Salarjung Museum has been noticed such as blackening of white and pink pigments of Indian miniature paintings and other objects like pigments, paints, varnishes, coatings, silver ware, zari works, textiles, which are displayed in museum galleries. The cause of deterioration of the artifacts is attributed to air pollution. The outdoor air pollution levels with respect to suspended particulate matter, sulphur dioxide, oxides of nitrogen, ammonia, aldehydes and oxidants are observed to be high when compared with background environment and ambient air quality standards for sensitive areas. The indoor air quality levels in terms of various parameters including temperature and relative humidity (RH) observed to be more than the threshold limits. The climatic conditions coupled with polluted indoor air are the main causes for the deterioration of art objects. Hence remedial measures are suggested to avoid further deterioration of objects.

Air Pollutants↗

PubMed2004

A pea chloroplast translation elongation factor that is regulated by abiotic factors.

We report the cloning and characterization of both the cDNA (tufA) and genomic clones encoding for a chloroplast translation elongation factor (EF-Tu) from pea. The analysis of the deduced amino acids of the cDNA clone reveals the presence of putative transit peptide sequence and four GTP binding domains and two EF-Tu signature motifs in the mature polypeptide region. Using in vivo immunostaining followed by confocal microscopy pea EF-Tu was localized to chloroplast. The steady state transcript level of pea tufA was high in leaves and not detectable in roots. The expression of this gene is stimulated by light. The differential expression of this gene in response to various abiotic stresses showed that it is down-regulated in response to salinity and ABA and up-regulated in response to low temperature and salicylic acid treatment. These results indicate that regulation of pea tufA may have an important role in plant adaptation to environmental stresses.

Base Sequence↗

PubMed2004

Air quality status of Visakhapatnam (India)--indices basis.

Industrial development in Visakhapatnam is conspicuous to urban agglomeration and the city is located in a topographical bowl formed by two-hill ranges. A major portion of the city is within the bowl area wherein most of the industrial and commercial activities are existing and lies within a distance of 10 km from the shore of the Bay of Bengal. Due to the peculiar geographic location of city, wind movement is either eastern or western and is engulfed within the hill ranges. Hence, there is a possibility of buildup of air pollution levels within the city. Due to gravity of prevailing situation, air quality status of Visakhapatnam on indices basis is analyzed using a non-linear equation for variable parameters i.e. Suspended particulate matter (SPM). Sulfur dioxide (SO2) and Oxides of nitrogen (NO(x)), which are main criteria pollutants in India. For current analysis seasonal air quality data is used, which indicates SPM values in winter at most of the sites and in summer at few sites are exceeding the prescribed standards. Calculated indices reveal that, in winter as well as in summer, most of the locations experienced poor or bad air quality, which is mainly due to higher concentration of SPM and certain extent of SO2 values. Application of Oak Ridge Air Quality Index (ORAQI) type equations (non-linear) are helpful for air quality management plan in the region on long-term basis and it has been also observed that there are certain lapses of weightage assignment for individual pollutant in application.

Air Pollutants↗

PubMed2004

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

Agglutination↗

PubMed2004

Polycyclic aromatic hydrocarbons in the ambient air of Jeedimetla Industrial Development Area-Hyderabad.

Polycyclic aromatic hydrocarbons (PAHs) have received increased attention in recent years in air pollution studies, as some of these compounds are highly carcinogenic or mutagenic. PAHs are mainly associated with respirable suspended particulate matter (RSPM) (diameter <10 m in size). Hence, it is important to determine the concentration of PAHs adsorbed on RSPM to know the extent of human exposure to these potentially carcinogenic substances. The study was carried out at 10 locations in and around the Jeedimetla industrial development area, Hyderabad during April 2000. Higher concentrations of B(a)A and B(b)F were observed at Nandanagar, Py and B(a)P at Kukatpally while, F1 concentrations at Allwyn Colony. These variations in the concentration can be attributed to their source and climatic conditions during sampling.

Adsorption↗

PubMed2003

Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance.

The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of gly I as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of gly I+II together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the gly II gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na+ and K+ in old leaves and negligible accumulation of Na+ in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.

Blotting, Southern↗

PubMed2003

Isolation and characterization of a phospholipase C delta isoform from pea that is regulated by light in a tissue specific manner.

Phosphoinositide-specific phospholipases C (PLCs) play an important role in many cellular responses and are involved in the production of secondary messengers. We report the cloning and characterization of a cDNA encoding a PLC-delta from Pisum sativum (PsPLC). The amino acid sequence deduced from the cDNA sequence showed 75-80% identity to other plant PLCs and contained the characteristic X, Y and C2 domains. The genomic PLC clone from pea was also characterized and found to contain eight introns. The protein was expressed in Escherichia coli, but the recombinant product did not show any phosphoinositide (PI)- or phosphatidylinositol-4, 5-bisphosphate (PIP2)-specific activity, despite having all known residues required for such activity, and in spite of the fact that its C2 domain was shown to bind calcium. Under similar in vitro assay conditions the recombinant tobacco PLC used as a control showed calcium-dependent PI- and PIP2-specific activity. Though PsPLC did not show enzyme activity in vitro, and may represent an inactive form of PLC, such as those reported in some mammalian systems, analysis of the transcription of PsPLC showed that the gene is expressed in all pea tissues, and is regulated by light in a tissue-specific manner. Roots showed higher expression of PsPLC than shoots. A putative PsPLC promoter region (792 bp) was also cloned and found to contain root-specific and light-responsive cis elements, suggesting that this form of PLC may be involved in important functions in plants.

Amino Acid Sequence↗

PubMed2003

Light-mediated regulation defines a minimal promoter region of TOP2.

Light signaling has been demonstrated to be an important factor for plant growth and development; however, its role in the regulation of DNA replication and cell cycle has just started to be unraveled. In this work, we have demonstrated that the TOP2 promoter of Pisum sativum (pea) is activated by a broad spectrum of light including far-red light (FR), red light (RL) and blue light (BL). Deletion analyses of the TOP2 promoter in transformed plants, Arabidopsis thaliana and Nicotiana tobaccum (tobacco), define a minimal promoter region that is induced by RL, FR and BL, and is essential and sufficient for light-mediated activation. The minimal promoter of TOP2 follows the phytochrome- mediated low-fluence response similar to complex light regulated promoters. DNA-protein interaction studies reveal the presence of a DNA binding activity specific to a 106 bp region of the minimal promoter that is crucial for light-mediated activation. These results altogether indicate a direct involvement of light signaling in the regulation of expression of TOP2, one of the components of the DNA replication/cell cycle machinery.

Arabidopsis↗

PubMed2003

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Amino Acid Sequence↗

PubMed2003

Major inorganic ion composition of atmospheric aerosols at the coastal site in Visakhapatnam City--India.

Atmospheric aerosols were sampled at a coastal site and analyzed for H+, NH+4, Ca++, Mg++, Na+, SO4- -, HCO(-3), Cl- and No3-. The analysis demonstrated that these ions are the main constituents of the aerosols sampled. The correlation coefficients are calculated for each possible pair of anions and cations. Ionic balance in the ambient aerosols indicate that the main compounds existed are in the order of sodium chloride; bicarbonates of calcium and magnesium; sulphates of sodium, magnesium and calcium; and nitrates of sodium, magnesium and calcium. The existence of cations has exceeded the anions which indicate that ionic composition of the aerosols are not very much determined by background marine nature but may also be due to transport of polluted air masses from the surrounding areas.

Aerosols↗