Arun K. Sharma

Plants as bioreactors: Recent developments and emerging opportunities.

Abstract In recent years, the use of plants as bioreactors has emerged as an exciting area of research and significant advances have created new opportunities. The driving forces behind the rapid growth of plant bioreactors include low production cost, product safety and easy scale up. As the yield and concentration of a product is crucial for commercial viability, several strategies have been developed to boost up protein expression in transgenic plants. Augmenting tissue-specific transcription, elevating transcript stability, tissue-specific targeting, translation optimization and sub-cellular accumulation are some of the strategies employed. Various kinds of products that are currently being produced in plants include vaccine antigens, medical diagnostics proteins, industrial and pharmaceutical proteins, nutritional supplements like minerals, vitamins, carbohydrates and biopolymers. A large number of plant-derived recombinant proteins have reached advanced clinical trials. A few of these products have already been introduced in the market.

Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato

Ethylene responsive transcription factors have been shown to be intimately connected to plant development, defense responses and stress signaling pathways and in order to use them for plant improvement, we need to have better understanding of these proteins. In this study, 85 ERF genes have been identified from tomato using raw EST data in various public repositories. Phylogenetic analysis with tomato ERF domains revealed their distribution in all the groups, previously identified in model systems. MEME motif analysis resulted in identification of conserved domains, characteristic to member of each clade, in addition to ERF domain. Expression analysis during vegetative and reproductive stages of development using QPCR and tomato GeneChip® arrays, revealed their tissue-specific/preferential accumulation. In total, 57 genes were found to be differentially expressed during temporal stages of tomato fruit development. The expression analysis of 23 ERF family genes representing each clade in response to seven abiotic stress treatments revealed their differential expression in response to more than one abiotic stress treatments. Results suggest that ERF genes play diverse roles in plant’s life and comprehensive data generated will be helpful in conducting functional genomics studies to understand their precise role during plant development and stress response.

Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice

The short persistence of cecropin B peptide in plants, due to post‐translational degradation, is a serious impediment in its effective utilization for developing bacterial resistance transgenic plants. Two DNA constructs encoding the full‐length precursor of cecropin B peptide and the mature sequence of cecropin B peptide preceded by a signal peptide derived from rice chitinase gene were transformed in rice. The differences in the transcriptional levels in independent transgenic lines showed moderate to high expression of cecropin B gene that correlated well with the differences in cecropin B accumulation observed by Western blot analysis. The development of lesions resulting from infection by Xanthomonas oryzae pv. oryzae was significantly confined in the infected leaflet of transgenic lines, when compared with the control plants.

Role of plant hormones and their interplay in development and ripening of fleshy fruits

Plant hormones have been extensively studied for their roles in the regulation of various aspects of plant development. However, in the last decade important new insights have been made into their action during development and ripening, in both dry and fleshy fruits. Emerging evidence suggests that relative functions of plant hormones are not restricted to a particular stage, and a complex network of more than one plant hormone is involved in controlling various aspects of fruit development. Though some areas are extensively covered, considerable gaps in our knowledge and understanding still exist in the control of hormonal networks and crosstalk between different hormones during fruit expansion, maturation, and various other aspects of ripening. Here, we evaluate the new knowledge on their relative roles during tomato fruit development with a view to understand their mechanism of action in fleshy fruits. For a better understanding, pertinent evidences available on hormonal crosstalk during fruit development in other species are also discussed. We envisage that such detailed knowledge will help design new strategies for effective manipulation of fruit ripening.

Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development.

Auxin response transcription factors have been widely implicated in auxin-mediated responses during various developmental processes ranging from root and shoot development to flower and fruit development in plants. In order to use them for improvement of agronomic traits related to fruit, we need to have better understanding of their role during fruit development. In this study, 17 SlARF genes have been identified from tomato (Solanum lycopersicum), using various publically available tomato EST databases. Phylogenetic analysis of the 23 AtARF and 17 SlARF proteins results in formation of three major classes and a total of 14 sister pairs, including seven SlARF–AtARF, four SlARF–SlARF and three AtARF–AtARF sister pairs, providing insights into various orthologous relationships between AtARFs and SlARFs. Further, search for orthologs of these SlARFs resulted in identification of nine, ten, four and three ARF genes from potato, tobacco, N. benthemiana and pepper, respectively. A phylogenetic analysis of these genes, along with their orthologs from Solanaceae species, suggests the presence of a common set of the ARF genes in this family. Comparison of the expression of these SlARF genes in wild type and rin mutant provides an insight into their role during different stages of flower and fruit development. This study suggests that ARF genes may play diverse role during flower and fruit development. Comprehensive data generated here will provide a platform for identification of ARF genes and elucidation of their function during reproductive development stages in Solanaceae in general and fruit development in tomato, in particular.

Expression of cholera toxin B subunit in transgenic tomato plants

Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. The latter binds to GM1-ganglioside receptors as a pentamer (∼55 kDa). Tomato plants were transformed with the gene encoding cholera toxin B subunit (ctxB) along with an endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter via Agrobacterium-mediated transformation. PCR and Southern analysis confirmed the presence of the ctxB gene in transformed tomato plants. Northern analysis showed the presence of the ctxB-specific transcript. Immunoblot assays of the plant-derived protein extract showed the presence of cholera toxin subunit B (CTB) with mobility similar to purified CTB from V. cholerae. Both tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04% of total soluble protein, respectively. The GM1-ELISA showed that the plant-derived CTB bound specifically to GM1-ganglioside receptor, suggesting that it retained its native pentameric form. This study forms a basis for exploring the utility of CTB to develop tomato-based edible vaccines against cholera.

Granulocyte colony-stimulating factor in severe alcoholic hepatitis: a randomized pilot study.

OBJECTIVES:Severe alcoholic hepatitis has high short-term mortality. The aim of this study was to test the hypothesis that treatment of patients with alcoholic hepatitis with granulocyte colony-stimulating factor (G-CSF) might mobilize bone marrow–derived stem cells and promote hepatic regeneration and thus improve survival.METHODS:Forty-six patients with severe alcoholic hepatitis were prospectively randomized in an open study to standard medical therapy (SMT) plus G-CSF (group A; n=23) at a dose of 5 μg/kg subcutaneously every 12 h for 5 consecutive days or to SMT alone (group B; n=23) at a tertiary care center. We assessed the mobilization of CD34+ cells on day 6, Child-Turcotte-Pugh (CTP), model for end-stage liver disease (MELD), and modified Maddrey’s discriminant function (mDF) scores, and survival until day 90.RESULTS:There was a statistically significant increase in the number of CD34+ cells in peripheral blood in group A as compared with group B (P=0.019) after 5 days of G-GSF therapy. There was a significant reduction in median Δ change% in CTP, MELD, and mDF at 1, 2, and 3 months in group A as compared with group B (P<0.05). There was marked improvement in survival in group A as compared with group B (78.3% vs. 30.4%; P=0.001) at 90 days.CONCLUSIONS:G-CSF is safe and effective in the mobilization of hematopoietic stem cells and improves liver function as well as survival in patients with severe alcoholic hepatitis.

Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum)

In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.

Signal transduction during cold stress in plants

Cold stress signal transduction is a complex process. Many physiological changes like tissue break down and senescence occur due to cold stress. Low temperature is initially perceived by plasma membrane either due to change in membrane fluidity or with the help of sensors like Ca2+ permeable channels, histidine kinases, receptor kinases and phospholipases. Subsequently, cytoskeleton reorganization and cytosolic Ca2+ influx takes place. Increase in cytosolic Ca2+ is sensed by CDPKs, phosphatase and MAPKs, which transduce the signals to switch on transcriptional cascades. Photosynthetic apparatus have also been thought to be responsible for low temperature perception and signal transduction. Many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. However, the importance of CBF independent pathways in cold acclimation is supported by few Arabidopsis mutants’ studies. Cold stress signaling has certain pathways common with other abiotic and biotic stress signaling which suggest cross-talks among these. Most of the economically important crops are sensitive to low temperature, but very few studies are available on cold susceptible crop plants. Therefore, it is necessary to understand signal transducing components from model plants and utilize that knowledge to improve survival of cold sensitive crop plants at low temperature.

A simple and efficient Agrobacterium-mediated procedure for transformation of tomato

We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and β-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.