Tirthartha Chattopadhyay

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Nano-pesticide formulation based on fluorescent organic photoresponsive nanoparticles: for controlled release of 2,4-D and real time monitoring of morphological changes induced by 2,4-D in plant systems

In recent times, nano-pesticide formulations have gained great popularity since they enable effective usage of smaller quantities of the pesticides without creating much damage to the environment. The benefits of a nano-pesticide formulation can be further expanded by adding to its arsenal both tracking ability and precise control over the pesticide release. Recently, fluorescent photoresponsive nanocarriers have gained considerable momentum in the field of drug delivery since they can act as both a “fluorophore” for cell luminescence imaging and a “phototrigger” for regulated drug release by external light stimuli. Hence, we report for the first time a nano-pesticide formulation based on fluorescent photoresponsive organic nanoparticles of perylene-3-ylmethanol for regulated release of pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Further, the fluorescent nature of the photoresponsive organic nanoparticles was used to study the morphological changes induced by 2,4-D inside the plant system using confocal imaging studies. Additionally, the fluorescent colour change by photoresponsive organic nanoparticles before and after photorelease was exploited for real time monitoring of 2,4-D release inside the plants. Bioassay experiments revealed that nano-pesticide, Pe-2,4-D efficiently delivered 2,4-D inside the plant tissues improving its herbicidal activity. Such photoresponsive multifunctional nanocarriers with good fluorescence, cellular uptake property and efficient photoregulated release ability will be of great benefit in the construction of nano-pesticide formulations.

Gene Cloning, Recombinant Expression and Purification of a Germin-Like Protein from Tomato (Solanum lycopersicum L.)

ABSTRACT Germin-like proteins (GLPs) are plant glycoproteins belonging to the “cupin” superfamily. These structurally similar but functionally diverse proteins are reportedly involved in conferring biotic and/or abiotic stress tolerance on different plant species. Here we report the molecular cloning and recombinant expression of an uncharacterized gene encoding a GLP belonging to GER 2 subfamily of tomato. Analyses revealed the presence of germin motifs along with the conserved amino acid residues involved in metal ion (Mn2+) binding. Homology-based modeling revealed the characteristic β jelly-roll structure of the protein. Following optimization of recombinant expression, a simple strategy for purification of the recombinant protein in the presence of detergent was optimized. Keeping in mind the high level of resistance of GLPs against detergents, these results could be useful in identifying functional activity of GLP. Moreover, the recombinant protein will be useful in raising antibodies for further applications for gene expression profiling at protein level.

Spatio-temporal regulation of the OsHFP gene promoter establishes the involvement of this protein in rice anther development.

Anther development leading to pollen maturation, anther dehiscence and pollen dispersal depends upon the precise timing of programmed cell death (PCD) in specified anther tissues. The PCD necessitates a properly tuned transcriptional regulation of some crucial genes. However, the detailed genetic regulation of this PCD in rice anther is yet to be deciphered. Recently, we have established that the OsHFP, a structurally novel hemopexin fold protein of rice is a flower-specific heme binding protein, and plays a role in chlorophyll degradation. Here, we report the spatio-temporal transcriptional regulation of the OsHFP gene, which is proposed to be involved in anther PCD. The OsHFP was immunodetected in rice anthers, and OsHFP-related proteins were also found to be present in anthers of other monocot (lily) and dicot (tobacco) plant species. Unique cis-acting elements, possibly involved in the activation and anther-specificity of the OsHFP promoter were identified based upon in silico prediction and in planta expression profiling of the reporter gene driven by the OsHFP promoter (2051 bp) and its two deleted versions (1057 bp and 437 bp). The temporal regulation of the OsHFP promoter in different developmental stages of tobacco anther implies the physiological function of this protein in anther PCD.

Hairy Root Culture for In Vitro Production of Secondary Metabolites: A Promising Biotechnological Approach

Hairy root culture (HRC)-based in vitro production system has become a promising biotechnological approach in recent years. The hairy root formation is the result of Agrobacterium rhizogenes-induced pathogenesis in plants, characterized by high growth rate, growth in hormone-free media besides genetic stability. These roots can imitate intact plants in the production of secondary metabolites and also amenable to upscaling in the bioreactor. HRCs are being harnessed as one of the methods of choice in tissue culture for high yield of valuable secondary metabolites of medicinal and other commercial importance. Many secondary metabolites, which were earlier extracted from wild plants from their natural habitat, are now being produced using plant cell cultures including HRCs. This book chapter is focussed mainly on the development of hairy root culture in different medicinal plants and its application, challenges, and prospects in the production of valuable secondary metabolites.

Proposed physiological mode of action of rice hemopexin fold protein OsHFP: linking heme-binding with plant cell death

The biosynthetic pathway of tetrapyrrole is under stringent regulation in living systems. Heme, one of the products of this branched pathway is well known to regulate the pathway through feedback inhibition. Since the accumulation of intermediates of tetrapyrrole biosynthetic pathway is found to be cytotoxic and inducer of plant programmed cell death through retrograde signalling, certain role of heme in regulating plant cell death is quite logical. In the present study, we report the in vivo heme-binding property of OsHFP, a novel hemopexin fold protein from the rice (Oryza sativa) plant, and document the effect of heme–OsHFP interaction on oxidative stress responses in bacterial and plant systems. The bound heme is retained by the OsHFP even in presence of SDS, and the amount of bound heme to OsHFP increases upon exogenous application of heme biosynthetic precursor in Escherichia coli. In heme-replete condition, the recombinant OsHFP alters the oxidative stress response in E. coli. Ectopic expression of the OsHFP generates oxidative stress in tobacco leaves, which is visualized through the induction of superoxide dismutase activity and localized accumulation of H2O2. On the basis of the findings from the present study and the available literature, we propose a possible mode of action of OsHFP in regulating cell death in plant system, which might signify the yet-to-be-deciphered physiological role of hemopexin fold proteins in plants.