Manish Kumar Patel

The SbMT-2 Gene from a Halophyte Confers Abiotic Stress Tolerance and Modulates ROS Scavenging in Transgenic Tobacco

Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13) showed significantly enhanced salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA) analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2 −; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed) condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5’-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.

Physio-Biochemical Composition and Untargeted Metabolomics of Cumin (Cuminum cyminum L.) Make It Promising Functional Food and Help in Mitigating Salinity Stress.

Cumin is an annual, aromatic, herbaceous, medicinal, spice plant, most widely used as a food additive and flavoring agent in different cuisines. The study is intended to comprehensively analyse physiological parameters, biochemical composition and metabolites under salinity stress. Seed germination index, rate of seed emergence, rate of seed germination, mean germination time, plant biomass, total chlorophyll and carotenoid contents decreased concomitantly with salinity. In contrast, total antioxidant activity, H2O2, proline and MDA contents increased concurrently with stress treatments. Total phenolic and flavonoid contents were decreased initially about 1.4-fold at 50 mM, and thereafter increased about 1.2-fold at 100 mM NaCl stress. Relative water content remained unchanged up to 50 mM NaCl stress, and thereafter decreased significantly. About 2.8-fold electrolyte leakage was found in 50 mM, which increases further 4-fold at 100 mM NaCl stress. Saturated fatty acids (FAs) increased gradually with salinity, whereas unsaturation index and degree of unsaturation change arbitrarily along with the percent quantity of unsaturated FAs. Total lipid and fatty acid composition were significantly influenced by salinity stress. A total of 45 differentially expressed metabolites were identified, including luteolin, salvianolic acid, kaempferol and quercetin, which are phenolic, flavonoid or alkaloids in nature and contain antioxidant activities. Additionally, metabolites with bioactivity such as anticancerous (docetaxel) and antimicrobial (megalomicin) properties were also identified. The study evidenced that plant shoots are a rich source of metabolites, essential amino acids, phenolic compounds and fatty acids, which unveil the medicinal potential of this plant, and also provide useful insight about metabolic responses under salinity stress.

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

Non-targeted Metabolite Profiling and Scavenging Activity Unveil the Nutraceutical Potential of Psyllium (Plantago ovata Forsk)

Non-targeted metabolomics implies that psyllium (Plantago ovata) is a rich source of natural antioxidants, PUFAs (ω-3 and ω-6 fatty acids) and essential and sulfur-rich amino acids, as recommended by the FAO for human health. Psyllium contains phenolics and flavonoids that possess reducing capacity and reactive oxygen species (ROS) scavenging activities. In leaves, seeds, and husks, about 76, 78, 58% polyunsaturated, 21, 15, 20% saturated, and 3, 7, 22% monounsaturated fatty acids were found, respectively. A range of FAs (C12 to C24) was detected in psyllium and among different plant parts, a high content of the nutritive indicators ω-3 alpha-linolenic acid (57%) and ω-6 linoleic acid (18%) was detected in leaves. Similarly, total content of phenolics and the essential amino acid valine were also detected utmost in leaves followed by sulfur-rich amino acids and flavonoids. In total, 36 different metabolites were identified in psyllium, out of which 26 (13 each) metabolites were detected in leaves and seeds, whereas the remaining 10 were found in the husk. Most of the metabolites are natural antioxidants, phenolics, flavonoids, or alkaloids and can be used as nutrient supplements. Moreover, these metabolites have been reported to have several pharmaceutical applications, including anti-cancer activity. Natural plant ROS scavengers, saponins, were also detected. Based on metabolomic data, the probable presence of a flavonoid biosynthesis pathway was inferred, which provides useful insight for metabolic engineering in the future. Non-targeted metabolomics, antioxidants and scavenging activities reveal the nutraceutical potential of the plant and also suggest that psyllium leaves can be used as a green salad as a dietary supplement to daily food.

Cloning and functional characterization of the Na+/H+ antiporter (NHX1) gene promoter from an extreme halophyte Salicornia brachiata

Salinity is one of the major abiotic stresses which affect plant growth and productivity by imposing dual stress, ionic and osmotic stress, on plants. Halophytes which are adapted to complete their life cycle in saline soil keep the transcript expression of stress-responsive genes constitutively higher in the optimum growth environments, which can be further increased by several folds under stress conditions. The transcript expression of SbNHX1 gene, cloned from a leafless succulent halophyte Salicornia brachiata, was up-regulated under salinity stress, but its transcriptional regulation has not been studied so far. In the present study, a 1727 bp putative promoter (upstream to translation start site) of the SbNHX1 gene was cloned using a genome walking method. The bioinformatics analysis identified important stress-responsive cis-regulatory motifs, GT1, MBS, LTR and ARE, in addition to two leaf-specific enhancer motifs. The GUS expression analysis of stable transgenic tobacco plants, transformed with a transcriptional fusion of GUS with the full SbNHX1 promoter (NP1) or any of its five deletion fragments (NP2 to NP6), showed that the deletion of two enhancer motifs resulted in the sudden decrease in GUS expression in leaves but not in the stem or root tissues. In contrast, under salinity stress, the higher induction of GUS expression observed in NP1 and NP2 was correlated by the presence of salt-inducible GT1- and MBS-motifs which is distributed only in NP1 and NP2 deletion promoter fragments. Finally, we concluded that the SbNHX1 promoter has a 624 bp (-1727 to -1103 bp) regulatory region which contains the two leaf-specific enhancer motifs and salinity stress-inducible GT-1 and MBS motifs. We suggest the SbNHX1 gene promoter and fragments as a candidate alternative promoter/s for crop engineering for better stress tolerance, which can be amended according to the desired level of expression needed.

Physicochemical characterization, antioxidant and anti-proliferative activities of a polysaccharide extracted from psyllium (P. ovata) leaves

Psyllium leaves polysaccharides were physicochemically characterized and evaluated for antioxidant, free-radical scavenging and anticancer activities. The polysaccharide comprised of arabinose, rhamnose, xylose, galactose, and glucose. FTIR and NMR study exhibited the presence of key functional groups whereas morphological study revealed that crude polysaccharides are aggregates of irregular shape, non-porous, and smooth surface, while the purified products are porous and fibrous in nature. Extracted polysaccharides were thermally stable and exhibited the characteristic diffraction curve with a higher viscosity and size distribution. Biochemical analyses demonstrated the higher antioxidant and scavenging activities. In vitro anticancer assay showed the maximum inhibitory effect about 33% on Huh7 and about 30% on HeLa cell lines. The study confirmed the presence of high antioxidant and free-radical scavenging activities with a potential anticancer activity. The polysaccharide could be developed as a potential natural antioxidant and anticancer agents after comprehensive studies on experimental animal models.