Phenotypic and biochemical alterations in relation to MT2 gene expression in Plantago ovata Forsk under zinc stress.

Plantago ovata Forsk is an annual herb with immense medicinal importance, the seed and husk of which is used in the treatment of chronic constipation, irritable bowel syndrome, diarrhea since ancient times. Zinc, an essential metal, is required by plants as they form important components of zinc finger proteins and also aid in synthesis of photosynthetic pigments such as chlorophyll. However, in excess amount Zn causes chlorosis of leaf and shoot tissues and generate reactive oxygen species. The present study is aimed at investigating the changes in expression levels of MT2 gene in Plantago ovata under zinc stress. Data show up to 1.66 fold increase in expression of PoMT2 in 1000 µM ZnSO4·7H2O treated sample. Our study also describes alteration of MT2 gene expressions in Plantago ovata as observed through Real time PCR (qPCR) done by [Formula: see text] method. In this study we have observed an upregulation (or induction) in the PoMT2 gene expression level in 500 and 800 µM ZnSO4·7H2O treated samples but found saturation on further increasing the dose to 1000 µM of ZnSO4·7H2O. Determination of the phenotypic and biochemical changes in Plantago ovata due to exposure to zinc stress of concentrations 500, 800 and 1000 µM revealed oxidative stress. The enhanced expression of MT2 gene in Plantago ovata has a correlation with the increased total antioxidant activity and increased DPPH radical scavenging activity.

Selenium biofortification improves bioactive composition and antioxidant status in Plantago ovata Forsk., a medicinal plant.

BACKGROUND: Selenium (Se) is an essential micronutrient for humans, but its deficiency as well as toxicity affects large number of people worldwide. Plantago ovata, a commercially important medicinal plant, is mainly cultivated in western regions of India, where elevated levels of Se have been found in soil. Thus, we evaluated the potential of Se biofortification in P. ovata via phytoremediation and its effect on the bioactive composition. RESULTS: The results showed a significant alteration in various morphological and physiological parameters in a dose-dependent manner. The 10 µM Se dose improved seedling height, biomass and total chlorophyll content. There was a gradual increase in total Se content, with highest accumulation of 457.65 µg/g FW at 500 µM Se treatment. Se positively affected the antioxidative metabolism which was measured from the change in total antioxidant capacity, radical scavenging activity and Metallothionein 2 expression. Increasing levels of Se also affected the PAL activity, total polyphenol and flavonoid content. Caffeic acid, Coumaric acid and Rutin were found to be the most abundant phenolic compounds. CONCLUSIONS: Low levels of selenium (below 50 µM) can successfully improve Se accumulation and elicit production of various polyphenols without hampering plant growth. Thus, Se fortification of P. ovata seedlings via phytoremediation appears to be a feasible and efficient way to enhance its nutraceutical value in dietary products.

Radiation induced alterations in Vigna radiata during in vitro somatic embryogenesis.

PURPOSE: Tissue culture has been exploited to understand molecular aspects of regeneration potential of the plants in normal and in stressed conditions. The present study describes ionizing radiation from (60)Co source as the stress stimulator to assess in vitro development of somatic embryo of Vigna radiata, a protein-rich pulse. MATERIALS AND METHODS: Callus culture was established, using leaves of V. radiata. Somatic embryogenesis was induced by manipulating plant hormones. Calli were exposed to gamma rays. Genomic DNA isolated from gamma-irradiated callus samples were subjected to random amplified polymorphic DNA analysis. A band of molecular weight 1440 bp was used as a probe and Southern hybridization was carried out. To determine alterations in DNA following irradiation, RAPD bands were cloned and sequenced from control and irradiated samples. Embryogenic calli were exposed to gamma irradiation and the effects were assessed immediately and after seven days of exposure. Phenotypic alterations were observed using scanning electron microscopy. RESULTS: Exposed calli revealed altered frequency of somatic embryo formation. Results showed that the 1440 bp molecular weight probe hybridized with bands of low molecular weight. DNA sequences from irradiated samples showed recombination when compared to control. Scanning electron micrography illustrated presence of transient pores on the exposed embryos. BLAST search of the DNA sequences showed partial homology with some sequences from Arabidopsis thaliana. CONCLUSION: The present report might help in designing a breeding program, where both radiation coupled with somatic embryogenesis could be employed to build up the desired variants.

In vitro radiation induced alterations in heavy metals and metallothionein content in Plantago ovata Forsk.

Proton Induced X-ray emission (PIXE) and fluorescence-activated cell sorting (FACS) have been used to study the effects of gamma irradiation on heavy metal accumulation in callus tissue of Plantago ovata-an important cash crop of India. PIXE analysis revealed radiation-induced alteration in trace element profile during developmental stages of the callus of P. ovata. Subsequent experiments showed antagonism between Fe and Cu and also Cu and Zn and synergistic effect between Fe and Zn. FACS analysis showed significant induction of the metallothionein (MT) protein following gamma-irradiation, and maximum induction was noted at the 50-Gy absorbed dose. This indicated a progressive increment of MTs as a measure for protection against gamma-rays, to combat alteration in the homeostasis of heavy metals like Fe, Cu, Zn, and Mn.

Chromium (VI) - induced stress response in the plant Plantago ovata Forsk in vitro.

BACKGROUND: Plants experience severe physiological stress from heavy metal pollution caused by improper discarding of the industrial wastes. Hexavalent chromium [Cr (VI)] is one of the major heavy metal pollutants in India and is present particularly in some regions where Plantago ovata grows to a great extent. This study was aimed at finding the effects of Cr (VI) on P. ovata and manoeuvres of the plant to combat such heavy metal exposure in vitro. METHODS: Potassium dichromate was used as a source of Cr (VI) to induce the heavy metal stress. Range of Cr (VI) sublethal doses [0 mM (control), 0.1 mM, 0.3 mM, 0.5 mM, 1 mM, 1.5 mM and1.8 mM] was used to observe its effect on the plant. The seeds of the plant were grown on sucrose-agar media with different concentrations of potassium dichromate, and ten-day old seedlings were then harvested and examined. RESULTS: The germination rate reduced below 50% at 1.9 mM Cr (VI) concentration and thus, 0 mM-1.8 mM concentration ranges were found to be suitable for sublethal dose. Morphological changes namely, reduction of the shoot-root length and multiple root development were caused by Cr (VI) in a dose-dependent manner. The plant showed elevated responses against Cr (VI), up to 1.5 mM (10 days treated) in terms of increasing accumulation of secondary metabolites like polyphenols, chlorophyll content (chlorophyll a, b and total chlorophyll), carotenoids and total antioxidant activity. DPPH radical scavenging activity along with malondialdehyde (MDA) content was not significantly elevated with the increase in Cr (VI) concentration indicating that the lipid peroxidation rate within the tissue was low. Phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) gene expressions were upregulated by 1 mM Cr (VI) concentration, which decreased at higher concentrations. The atomic absorption spectroscopy analysis also showed significant accumulation of Cr (VI) in the shoot and root with an increase in the potassium dichromate concentration. CONCLUSION: Cr (VI) reduced the shoot-root length and seed germination in a dose-dependent manner. The plant system tried to combat the Cr (VI) stress by upregulating the stress response genes in the phenylpropanoid pathway along with an increase in polyphenol and antioxidant contents, which were evident from the lowering of lipid peroxidation rate and increase in PAL and PPO gene expressions.

Partial alleviation of oxidative stress induced by gamma irradiation in Vigna radiata by polyamine treatment.

PURPOSE: Environmental changes generate free radicals and reactive oxygen species (ROS) resulting in abiotic stress in plants. This causes alterations in germination, morphology, growth and development ultimately leading to yield loss. Gamma irradiation was used to experimentally induce oxidative damage in an important pulse crop Vigna radiata (L.) Wilczek or mung bean. Our research was aimed towards augmentation of oxidative stress tolerance through treatment with a group of aliphatic amines known as polyamines. MATERIALS AND METHODS: We used sub-lethal doses of gamma irradiation to generate oxidative damage which was evaluated using Nitro blue tetrazolium (NBT) staining, total antioxidant activity, 1, 1-Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay, proline content and lipid peroxidation. Changes in internal free polyamines and messenger ribonucleic acid (mRNA) expression of key rate-limiting S-adenosylmethionine decarboxylase (SAMDC) enzyme in polyamine biosynthetic pathway was studied using real-time polymerase chain reaction (PCR). RESULTS: We observed increased oxidative damage with higher irradiation dose which was partially alleviated by putrescine treatment. Internal levels of putrescine and spermidine increased with 1 mM (50 and 100 Gy) and 2 mM putrescine treatment. Expression of SAMDC also increased with putrescine treatment. CONCLUSION: This study shows that treatment with putrescine can partially alleviate oxidative damage caused by gamma rays.

Cross-adaptation to cadmium stress in Plantago ovata by pre-exposure to low dose of gamma rays: Effects on metallothionein and metal content.

PURPOSE: To investigate the effects of gamma pre-exposure on cadmium accumulation in Plantago ovata seedlings. Metallothionein (MT) localization was also studied following Cadmium (Cd) treatment in P. ovata. MATERIALS AND METHODS: DNA damage was determined by alkaline comet assay. MT gene and protein expression were studied by real-time polymerase chain reaction and flow cytometry, respectively, in root and shoot tissues. Metal accumulation (Cd, zinc [Zn], iron [Fe]) was evaluated by Atomic Absorption Spectroscopy. RESULTS: Cd treatment decreased seed germination rate, biomass and free radical scavenging activity and increased DNA damage in a dose-dependent manner. When P. ovata seeds were pre- exposed to 5 Gy gamma dose (prior to Cd treatment) seed germination rate, biomass and free radical scavenging activity increased significantly. MT genes (PoMT1, PoMT2 and PoMT3) and MT protein expression enhanced when 5 Gy gamma-irradiated seeds were grown in Cd containing medium and Cd accumulation also increased in a dose-dependent manner. CONCLUSIONS: Higher Cd accumulation in P. ovata seedlings may be attributed to the upregulation of PoMT genes in gamma pretreated seedlings. Localization of metallothionein in cytosol and nucleus indicated its positive role against Cd-mediated cytotoxic and genotoxic effects.

Ionizing radiation induced changes in phenotype, photosynthetic pigments and free polyamine levels in Vigna radiata (L.) Wilczek.

Effects of gamma rays on the free polyamine (PA) levels were studied in Vigna radiata (L.) Wilczek. Seeds exposed to different doses of gamma rays were checked for damage on phenotype, germination frequency and alteration in photosynthetic pigments. Free polyamine levels were estimated from seeds irradiated in dry and water imbibed conditions. Polyamine levels of seedlings grown from irradiated seeds, and irradiated seedlings from unexposed seeds were also measured. Damage caused by gamma irradiation resulted in decrease in final germination percentage and seedling height. Photosynthetic pigments decreased in a dose dependent manner as marker of stress. Polyamines decreased in irradiated dry seeds and in seedlings grown from irradiated seeds. Radiation stress induced increase in free polyamines was seen in irradiated imbibed seeds and irradiated seedlings. Response of polyamines towards gamma rays is dependent on the stage of the life cycle of the plant.

Alterations in transcriptome and proteome on metallothioneins following oxidative stress induced by sublethal doses of cadmium and gamma rays in Plantago ovata.

PURPOSE: To study the oxidative stress-induced changes by sublethal doses of cadmium chloride (CdCl2) and gamma irradiation, two redox-inducing agents, on metallothionein (MT) gene and protein expression in Plantago ovata Forsk (P. ovata). MATERIALS AND METHODS: Chlorophyll content was estimated to study the stress response in P. ovata seedlings following exposure to gamma irradiation and CdCl2. Lipid peroxidation and proline content, two oxidative stress markers, were also studied. The level of metallothionein gene and protein expression was further investigated using polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), immunocytochemistry and flow cytometry. RESULTS: Three MT genes of P. ovata namely PoMT 1, PoMT 2 and PoMT 3 were isolated, sequenced and characterized and their expressions were found to be altered in the case of both oxidative stresses in a dose-dependent and tissue-specific manner. The results were in agreement with the observations from immunocytochemistry and FACS analysis. CONCLUSIONS: The results suggest that both gamma irradiation and CdCl2 alter redox balance in P. ovata. The metallothionein gene may play an important role in metal tolerance and stress balance. It is conjectured that the stress-mediated imbalance is maintained by altered MT gene and protein expression.

Formulation of nutrient medium for in vitro somatic embryo induction in Plantago ovata forsk.

A nutrient medium has been formulated by altering the macro- and micro-elemental concentration in the culture medium for in vitro somatic embryo induction of economically important medicinal plant Plantago ovata Forsk .A comparison was made between induced embryos with normal embryos (produced in Murashige and Skoog (MS) medium) to observe frequency of embryo induction and also to determine regeneration efficiency. In the present investigation, three different media have been formulated. Among them, FM3 (formulated media, treatment 3) was the most suitable for increasing the frequency of somatic embryo production and regeneration of P. ovata Forsk. Better result was obtained using formulated medium than with MS medium.

Analysis of trace elements during different developmental stages of somatic embryogenesis in Plantago ovata Forssk using energy dispersive X-ray fluorescence.

Energy dispersive X-ray fluorescence (ED-XRF) technique has been used for the determination of trace element profile during different developmental stages of somatic embryogenic callus of an economically important medicinal plant, Plantago ovata Forssk. Somatic embryogenesis is a plant tissue culture-based technique, which is used for plant regeneration and crop improvement. In the present investigation, elemental content was analysed using ED-XRF technique during different developmental stages and also determine the effect of additives--casein hydrolysate and coconut water on the trace elemental profile of embryogenic callus tissue of P. ovata. Subsequent experiments showed significant alteration in the concentration of K, Ca, Mn, Fe, Zn, Cu, Br, and Sr in both the embryogenic and non-embryogenic callus. Higher K, Ca, Fe, Cu, and Zn accumulation was in embryogenic tissue stage compared to other stages, suggesting these elements are crucial for successful embryogenesis. The results suggest that this information could be useful for formulating a media for in vitro embryo induction of P. ovata.

PIXE analysis of trace elements in relation to chlorophyll concentration in Plantago ovata Forsk.

Plantago ovata Forsk - an economically important medicinal plant - was analyzed for trace elements and chlorophyll in a study of the effects of gamma radiation on physiological responses of the seedlings. Proton-induced X-ray emission (PIXE) technique was used to quantify trace elements in unirradiated and gamma-irradiated plants at the seedling stage. The experiments revealed radiation-induced changes in the trace element and chlorophyll concentrations.