The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.

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Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, H N -H ␣ coupling constants, 1 H-15 N nuclear Overhauser effects, and 15 N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 M guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.The folding of a protein is conceptually described in terms of a folding funnel (1-6). The narrow end of the funnel represents the folded native state, and the broad end represents the unfolded state consisting of millions of rapidly inter-converting conformers. As a protein folds from an unfolded state, it goes through one or more partially folded intermediates, which need to be characterized for elucidation of its folding pathways. Experimentally, this is a very challenging task. The most common and direct approach relies on kinetic pulse labeling experiments of amide protons coupled with hydrogen exchange at different time points along the folding reaction of the protein (reviewed in Ref. 7). However, as has been pointed out (8), this also has limitations. First, it is limited by the necessity of detectable protection against exchange. Second, any lack of protection does not necessarily imply absence of structure. Third, it biases the interpretation of the structure in intermediates toward the native state. Even so, useful information has been obtained in many protein systems (7). However, this approach is complicated in the case of proteases with autolytic property, because the autolytic reaction interferes with interpretation of the hydrogen exchange data. Therefore, in these sys...

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