Surekha Mandal

Bio: Surekha Mandal is an academic researcher from Bose Institute. The author has contributed to research in topic(s): Quenching (fluorescence) & Spodoptera litura. The author has an hindex of 2, co-authored 2 publication(s) receiving 63 citation(s).

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor CHARACTERIZATION, POST-TRANSLATIONAL PROCESSING STUDIES, AND TRANSGENIC EXPRESSION TO DEVELOP INSECT-RESISTANT PLANTS

Abstract: A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank(TM) accession number ) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28-0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.

Dielectric relaxation in a single tryptophan protein.

Abstract: Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single-tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift (∼5 nm at 50% quenching by acrylamide) is striking in a single-tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady-state polarization and time-resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.

Genetic Transformation of Crops for Insect Resistance: Potential and Limitations

Abstract: Transgenic resistance to insects has been demonstrated in plants expressing insecticidal genes such as δ -endotoxins from Bacillus thuringiensis (Bt), protease inhibitors, enzymes, secondary plant metabolites, and plant lectins. While transgenic plants with introduced Bt genes have been deployed in several crops on a global scale, the alternative genes have received considerably less attention. The protease inhibitor and lectin genes largely affect insect growth and development and, in most instances, do not result in insect mortality. The effective concentrations of these proteins are much greater than the Bt toxin proteins. Therefore, the potential of some of the alternative genes can only be realized by deploying them in combination with conventional host plant resistance and Bt genes. Genes conferring resistance to insects can also be deployed as multilines or synthetic varieties. Initial indications from deployment of transgenics with insect resistance in diverse cropping systems in USA, Canada, Arge...

[Proteinase inhibitors and their function in plants: a review].

Abstract: The spread, classification, and properties of plant proteins capable of inhibiting proteinases have been reviewed. Data from the literature on the likely physiological functions of these inhibitors in plants are analyzed.

Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective

Abstract: Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their 'unintended' pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

Multiple functional proteins are produced by cleaving asn-gln bonds of a single precursor by vacuolar processing enzyme

Abstract: Precursor-accumulating vesicles mediate transport of the precursors of seed proteins to protein storage vacuoles in maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97,310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CXXXC motifs, a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Both immunocytochemistry and immunoblots with anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa vicilin-like protein were accumulated in the vacuoles. To identify the mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass spectrometry and peptide sequencing showed that two Cys-rich peptides, three Arg/Glu-rich peptides, and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a pyroglutamate at their NH2 termini. To clarify the cleavage mechanism, in vitro processing of PV100 was performed with purified vacuolar processing enzyme (VPE). Taken together, these results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins. It is likely that the Asn-Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. We also found that the Cys-rich peptide functions as a trypsin inhibitor. Our findings suggested that PV100 is converted into different functional proteins, such as a proteinase inhibitor and a storage protein, in the vacuoles of seed cells.

The use of versatile plant antimicrobial peptides in agribusiness and human health.

Abstract: Plant immune responses involve a wide diversity of physiological reactions that are induced by the recognition of pathogens, such as hypersensitive responses, cell wall modifications, and the synthesis of antimicrobial molecules including antimicrobial peptides (AMPs). These proteinaceous molecules have been widely studied, presenting peculiar characteristics such as conserved domains and a conserved disulfide bond pattern. Currently, many AMP classes with diverse modes of action are known, having been isolated from a large number of organisms. Plant AMPs comprise an interesting source of studies nowadays, and among these there are reports of different classes, including defensins, albumins, cyclotides, snakins and several others. These peptides have been widely used in works that pursue human disease control, including nosocomial infections, as well as for agricultural purposes. In this context, this review will focus on the relevance of the structural-function relations of AMPs derived from plants and their proper use in applications for human health and agribusiness.