Yogesh Mishra

Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 degrees C), NaCl (6% w/v), carbofuron (0.025 mg ml(-1)), CdCl2 (4 mM), CuCl2 (1mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses.

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 degrees C), NaCl (6% w/v), carbofuron (0.025mgml(-1)), CdCl(2) (4mM), CuCl(2) (1mM), and UV-B (10min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture, mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.

Metabolomic analysis of extreme freezing tolerance in Siberian spruce (Picea obovata)

Siberian spruce (Picea obovata) is one of several boreal conifer species that can survive at extremely low temperatures (ELTs). When fully acclimated, its tissues can survive immersion in liquid nitrogen. Relatively little is known about the biochemical and biophysical strategies of ELT survival. We profiled needle metabolites using gas chromatography coupled with mass spectrometry (GC-MS) to explore the metabolic changes that occur during cold acclimation caused by natural temperature fluctuations. In total, 223 metabolites accumulated and 52 were depleted in fully acclimated needles compared with pre-acclimation needles. The metabolite profiles were found to develop in four distinct phases, which are referred to as pre-acclimation, early acclimation, late acclimation and fully acclimated. Metabolite changes associated with carbohydrate and lipid metabolism were observed, including changes associated with increased raffinose family oligosaccharide synthesis and accumulation, accumulation of sugar acids and sugar alcohols, desaturation of fatty acids, and accumulation of digalactosylglycerol. We also observed the accumulation of protein and nonprotein amino acids and polyamines that may act as compatible solutes or cryoprotectants. These results provide new insight into the mechanisms of freezing tolerance development at the metabolite level and highlight their importance in rapid acclimation to ELT in P. obovata.

Non-Photochemical Quenching Capacity in Arabidopsis thaliana Affects Herbivore Behaviour

Under natural conditions, plants have to cope with numerous stresses, including light-stress and herbivory. This raises intriguing questions regarding possible trade-offs between stress defences and growth. As part of a program designed to address these questions we have compared herbivory defences and damage in wild type Arabidopsis thaliana and two “photoprotection genotypes”, npq4 and oePsbS, which respectively lack and overexpress PsbS (a protein that plays a key role in qE-type non-photochemical quenching). In dual-choice feeding experiments both a specialist (Plutella xylostella) and a generalist (Spodoptera littoralis) insect herbivore preferred plants that expressed PsbS most strongly. In contrast, although both herbivores survived equally well on each of the genotypes, for oviposition female P. xylostella adults preferred plants that expressed PsbS least strongly. However, there were no significant differences between the genotypes in levels of the 10 most prominent glucosinolates; key substances in the Arabidopsis anti-herbivore chemical defence arsenal. After transfer from a growth chamber to the field we detected significant differences in the genotypes’ metabolomic profiles at all tested time points, using GC-MS, but no consistent “metabolic signature” for the lack of PsbS. These findings suggest that the observed differences in herbivore preferences were due to differences in the primary metabolism of the plants rather than their contents of typical “defence compounds”. A potentially significant factor is that superoxide accumulated most rapidly and to the highest levels under high light conditions in npq4 mutants. This could trigger changes in planta that are sensed by herbivores either directly or indirectly, following its dismutation to H2O2.

Heat Pretreatment Alleviates UV‐B Toxicity in the Cyanobacterium Anabaena doliolum: A Proteomic Analysis of Cross Tolerance

This study offers proteomic elucidation of heat pretreatment‐induced alleviation of UV‐B toxicity in Anabaena doliolum. Heat‐pretreated cells exposed to UV‐B showed improved activity of PSI, PSII, whole chain, 14C fixation, ATP and NADPH contents compared to UV‐B alone. Proteomic analysis using two‐dimensional gel electrophoresis (2‐DE), MALDI‐TOF MS/MS and reverse transcription polymerase chain reaction (RT‐PCR) of UV‐B and heat pretreatment followed by UV‐B–treated cells exhibited significant and reproducible alterations in nine proteins homologous to phycocyanin‐α‐chain (PC‐α‐chain), phycoerythrocyanin‐α‐chain (PEC‐α‐chain), hypothetical protein alr0882, phycobilisome core component (PBS‐CC), iron superoxide dismutase (Fe‐SOD), fructose‐1,6‐bisphosphate aldolase (FBA), nucleoside diphosphate kinase (NDPK), phosphoribulokinase (PRK) and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCo) large chain. Except the PEC‐α‐chain, hypothetical protein alr0882 and PBS‐CC, all other proteins showed upregulation at low doses of UV‐B (U2) and significant downregulation at higher doses of UV‐B (U5). The disruption of redox status, signaling, pentose phosphate pathway and Calvin cycle appears to be due to the downregulation of Fe‐SOD, NDPK, FBA, PRK and RuBisCo thereby leading to the death of Anabaena. In contrast to this, the upregulation of all the above proteins in heat‐pretreated cells, harboring different heat shock proteins (HSPs) like 60, 26 and 16.6, followed by UV‐B treatment than only the UV‐B–treated ones suggests a protective role of HSPs in mitigating UV‐B toxicity.

Preparation of Stroma, Thylakoid Membrane, and Lumen Fractions from Arabidopsis thaliana Chloroplasts for Proteomic Analysis

For many studies regarding important chloroplast processes such as oxygenic photosynthesis, fractionation of the total chloroplast proteome is a necessary first step. Here, we describe a method for isolating the stromal, the thylakoid membrane, and the thylakoid lumen subchloroplast fractions from Arabidopsis thaliana leaf material. All three fractions can be isolated sequentially from the same plant material in a single day preparation. The isolated fractions are suitable for various proteomic analyses such as simple mapping studies or for more complex experiments such as differential expression analysis using two-dimensional difference gel electrophoresis (2D-DIGE) or mass spectrometry (MS)-based techniques. Besides this, the obtained fractions can also be used for many other purposes such as immunological assays, enzymatic activity assays, and studies of protein complexes by native-polyacrylamide gel electrophoresis (native-PAGE).

Proteomic evaluation of the non-survival of Anabaena doliolum (Cyanophyta) at elevated temperatures

This paper presents proteomic evidence for the non-survival of Anabaena doliolum at elevated temperatures (43°C, 48°C, 53°C and 58°C), when photosystem II (PSII), carbon fixation, ATP and NADPH contents were significantly decreased. A total of 215, 200, 261, 229 and 99 spots were clearly visible in two-dimensional gel electrophoresis (2DE) gels of the cyanobacterium from the control and those subjected to 1 h treatment at the above temperatures, respectively. Proteomic analysis using 2DE, MALDI-TOF MS/MS and reverse transcription polymerase chain reaction of Anabaena exposed to the above temperatures displayed significant and reproducible alterations in 13 proteins homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1, 6-bisphosphate aldolase (FBA), fructose-1, 6-bisphosphatase (FBPase), keto-acid reductoisomerase, phycocyanin-α-chain, peroxiredoxin, ATP synthase-β-chain, RNA binding protein, nucleoside diphosphate kinase (NDPK), GroES, phycoerythrocyanin-α-chain, AhpC/TSA family and phycobilisome rod-core linker (PBS-RCL) protein. Except Prx, GroES and ATP synthase-β-chain, other metabolic and oxidative stress proteins were down-regulated at temperatures over 48°C. The down-regulation of FBA, GAPDH, and FBPase beyond 48°C possibly disrupted glycolysis, the pentose phosphate pathway and the Calvin cycle, thereby leading to the death of Anabaena above 48°C. Notwithstanding the above, an appreciable down-regulation of NDPK (involved in protein phosphorylation) and AhpC/TSA (specific scavenger of reactive sulphur species), may lead to loss of signalling and accumulation of reactive sulphur species, respectively, which contribute further to temperature stress. Appreciable induction of heat-shock proteins (HSPs), required for maintenance of metabolic proteins during thermal stress, as well as metabolic proteins up to 48°C and their down-regulation at 53°C and 58°C suggested a close interlinking of these proteins. However, down-regulation of HSP60, HSP26 and HSP 16.6 appears to be responsible for cell death.

Copper pretreatment augments ultraviolet B toxicity in the cyanobacterium Anabaena doliolum: a proteomic analysis of cell death

This study provides first-hand proteomic characterisation of Cu-pretreatment-induced augmentation of ultraviolet B toxicity in the cyanobacterium Anabaena doliolum Bharadwaja. Of the three treatments (i.e. Cu, UV-B and Cu + UV-B) tested, the UV-B treatment of Cu-pretreated Anabaena produced a greater inhibition of oxygen evolution, 14C fixation, ATP and NADPH contents than UV-B alone. Proteomic analysis using two-dimensional gel electrophoresis (2DE), MALDI-TOF MS/MS and reverse transcription polymerase chain reaction (RT-PCR) of Cu, UV-B, and Cu + UV-B treated Anabaena exhibited significant and reproducible alterations in 12 proteins. Of these, manganese superoxide dismutase (Mn-SOD), iron superoxide dismutase (Fe-SOD) and peroxiredoxin (PER) are antioxidative enzymes; ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), phosphoribulokinase (PRK), flavodoxin (Flv), plastocyanin (PLC), phosphoglycerate kinase (PGK), phycocyanin (PC) and phycoerythrocyanin α-chain (PC α-chain) are linked with photosynthesis and respiration; and DnaK and nucleoside diphosphate kinase (NDPK) are associated with cellular processes and light signalling, respectively. However, when subjected to a high dose of UV-B, Cu-pretreated Anabaena depicted a severe down-regulation of DnaK, NDPK and Flv, probably because of inevitable oxidative stress. Thus, the augmentation of UV-B toxicity by Cu can be attributed to the down-regulation of DnaK, NDPK and Flv.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120

Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry 1prx) as the template.