G. S. Shekhawat

Haem oxygenase (HO): an overlooked enzyme of plant metabolism and defence

Haem oxygenase (HO) degrades free haem released from haem proteins with the generation of ferrous iron (Fe2+), biliverdin-IXalpha (BV-IXalpha), and carbon monoxide (CO). The mechanism of haem cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of HO differ substantially. The crystal structure of HO1, a monomeric protein, has been extensively reported in mammals, pathogenic bacteria, and cyanobacteria, but no such reports are available for higher plant HOs except a predicted model for pea HO1. Along with haem degradation, HO performs various cellular processes including iron acquisition/mobilization, phytochrome chromophore synthesis, cell protection, and stomatal regulation. To date, four HO genes (HO1, HO2, HO3, and HO4) have been reported in plants. HO1 has been well explored in cell metabolism; however, the divergent roles of the other three HOs is less known. The transcriptional up-regulation of HO1 in plants responds to many agents, such as light, UV, iron deprivation, reactive oxygen species (ROS), abscisic acid (ABA), and haematin. Recently the HO1/CO system has gained more attention due to its physiological cytoprotective role in plants. This review focuses on the recent advances made in plant HO research involving its role in environmental stresses. Moreover, the review emphasizes physiological, biochemical, and molecular aspects of this enzyme in plants.

Critical review on medicinally potent plant species: Gloriosa superba

Gloriosa superba L. is a perennial climber and is used as an ayurvedic medicinal herb to cure diseases in various parts of Africa and Southeast Asia. The plant was under threatened category due to its imprudent harvesting from wild as it is extensively used by medicinal industries for its colchicine content. It also faces a low seed set problem, but due to its industrial demand it is now under cultivation. The plant is used to cure arthritis, gout, rheumatism, inflammation, ulcers, bleeding piles, skin diseases, leprosy, impotency, snakebites, etc. Various compounds have been isolated from the plant parts mainly tubers and seeds, viz colchicine, colchicoside (its semi-synthetic derivative - thiocolchicoside), superbine, gloriosine, lumicolchicine, 3-demethyl-N-deformyl-N-deacetylcolchicine, 3-demethylcolchicine, N-formyl deacetylcolchicine. In the present review, we have summarized the information concerning the occurrence, botanical description, ethanopharmacology, medicinal uses, biological activities and toxicological studies on this plant.

Cadmium induced oxidative stress and changes in soluble and ionically bound cell wall peroxidase activities in roots of seedling and 3–4 leaf stage plants of Brassica juncea (L.) czern

Metabolic adaptations to heavy metal toxicity in plants are thought to be related with developmental growth stage and the type of metal by which plant is affected. In the present study, changes in ionically bound CWP, soluble peroxidase activity, H2O2 level and Malonaldehyde content in roots of cadmium and copper stressed seedlings and cadmium stressed 3–4 leaf stage plants of Brassica juncea were investigated. Cadmium inhibits root growth and reduces fresh biomass. The reduction in root growth and fresh biomass is correlated with increased lipid peroxidation and reduced tolerance. Treatment with cadmium resulted in an increase in ionically bound CWP activity in roots of seedlings but no significant change in its activity was found in roots of 3–4 leaf stage plants. Increased level of H2O2 in roots of cadmium and copper treated seedlings, show a direct correlation with increased activity of ionically bound CWP. H2O2 level in 3–4 leaf stage plant roots was found to be very low. Soluble peroxidase activity decreased in cadmium (50 and 100xa0μM) treated seedlings but it was ineffective to cause any change in its activity in 3–4 leaf stage plants. Copper treated seedlings showed an increase in ionically bound CWP activity, H2O2 level and MDA content. Ascorbic acid (50xa0mM) pretreated seedlings shows significant decrease in ionically bound CWP activity when exposed to 50xa0μM cadmium. Hence, it is concluded that inhibition of root growth in Brassica juncea seedlings by cadmium, is associated with CWP catalyzed H2O2 dependent reactions which are involved in metabolic adaptations to heavy-metal stress.

Nitric oxide (NO) counteracts cadmium induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H2O2 thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25xa0μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5xa0mM SNP for 6xa0h when exposed to 25xa0μM Cd for 24xa0h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100xa0μM cPTIO pretreatment to roots for 1xa0h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.

In vitro biochemical evaluation of cadmium tolerance mechanism in callus and seedlings of Brassica juncea

In vitro grown callus and seedlings of Brassica juncea were treated with equimolar concentrations of cadmium and compared for their respective tolerance to cadmium. Calli cultures were grown on Murashige and Skoog medium supplemented with α 6-benzyl aminopurine (200xa0µg L−1, naphthalene acetic acid 200xa0µg L−1) and 2,4-dichloro-phenoxy acetic acid (65xa0µg L−1) while the seedlings grown on Hoaglands nutrient solution have been carried out. Cellular homeostasis and detoxification to cadmium in B. juncea were studied by analyzing the growth in terms of fresh weight and dry weight, lipid peroxidation, proline accumulation, and antioxidative enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)). At 200xa0µM cadmium, callus and seedlings showed 73.61% and 74.76% reduction in tolerance, respectively. A significant increase in malondialdehyde (MDA) content was found in both calli and seedlings; however, the amount of MDA content was more in seedlings. Proline content increased on lower concentration of cadmium (up to 50xa0µM), and it further decreased (up to 200xa0µM). But the accumulation of proline was higher in callus cultures. The overall activity of antioxidative enzymes (SOD, CAT, and APX) was found to be higher in callus in comparison to seedlings of B. juncea. Callus and seedlings showed a significant (Pu2009≤u20090.5) increase in SOD activity in a concentration-dependent manner up to 50xa0µM cadmium concentration but decreased further. APX activity increased significantly at low cadmium levels but CAT activity decreased significantly throughout on increasing cadmium concentrations from 5 to 200xa0µM, respectively. Hence, it was observed that callus of B. juncea was more tolerant in comparison to seedlings exposed to equimolar concentrations of cadmium. Thus, from the present studies, it is concluded that calli were more tolerant toward cadmium-induced oxidative stress. Hence, it is suitable material for the study of cadmium tolerance mechanisms and for the manipulations within them for better understanding of cadmium detoxification strategies in B. juncea.

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9xa0μM) and naphthalene acetic acid (10.7xa0μM). The optimal conditions for biomass growth in suspension cultures were found to be 10xa0gxa0l−1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27xa0μM), benzyl aminopurine (0.27xa0μM) and ascorbic acid (0.06xa0μM), 1.0× NH4NO3 (24.7xa0mM), 3.0× KNO3 (56.4xa0mM), 3.0× MgSO4 (4.5xa0mM) and 3.0× KH2PO4 (3.75xa0mM), in 150xa0ml Erlenmeyer flask with 50xa0ml media and incubated in dark at 110xa0rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26xa0day−1, doubling time of 26.35xa0h and cell viability of 75xa0%, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.

Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of Anethum graveolens

In vitro regeneration protocol for Anethum graveolens (Apiaceae) was developed using leaf explants. MS basal medium used in experiments was augmented with various hormones for caulogenic and rhizogenic response. The optimum callus induction (100%) was obtained by leaf explants on MS media fortified with BA (0.5xa0mgxa0l−1) singly and in combination with NAA (0.1 and 0.2xa0mgxa0l−1). BA at 0.5xa0mgxa0l−1, KN at 1.0xa0mgxa0l−1 and NAA at 0.1xa0mgxa0l−1 induced highest number of multiple shoots (10.0xa0±xa00.25) per explant and they also showed in vitro flowering within 3xa0weeks of culture. Influence of adenine sulfate on regeneration frequency of callus was evaluated. The highest frequency of rooting (100%) with 6.0xa0±xa00.25 roots per explants was obtained in one-fourth strength MS medium supplemented with 1/4 MSxa0+xa0IBA 0.5xa0mgxa0l−1 within 4xa0weeks of transfer to the rooting medium. In vitro flowering (35%) was obtained with MS fortified with BA alone and also in combination with KN and NAA (5.3xa0±xa00.42 flowers per explants). In vitro flowering response was tested with different carbohydrates (fructose, glucose, mannose and sorbitol) and optimized. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species.

Molecular characterization of two distinct monopartite begomoviruses infecting tomato in india

BackgroundTomato leaf curl viruses, which are the members of the genus Begomovirus, have emerged as devastating pathogens worldwide causing huge economic losses and threatening production of crops like cassava, cotton, grain legumes and vegetables. Even though the ToLCV isolates from Northern India have been shown to possess bipartite genome (designated as DNA A and DNA B), those from Australia, Taiwan and Southern India have a single genomic component (DNA A). We describe here the genetic diversity of two isolates of monopartite Tomato leaf curl virus infecting tomato in two extreme regions (North and South) of Indian subcontinent.ResultsThe rolling circle amplification (RCA) products obtained from symptomatic samples were digested, cloned and sequenced. The complete DNA sequence of two Tomato leaf curl virus isolates identified as ToLCV-CTM (India, New Delhi, 2005) and ToLCVK3/K5 (India, Kerala, 2008) are reported here. These isolates had the characteristic features of Begomovirus genome organization with six conserved open reading frames (ORFs). The ToLCV-K3 and ToLCV-K5 isolates may be the strains of the same virus since they show sequence homology of 97% over their entire genome. This, according to the guidelines established by the ICTV Geminiviridae Study-Group is higher than threshold (92%) for delineation of different viral variants and hence single, average value has been assigned for all their analyses presented here. The ToLCV-CTM and ToLCV-K3/K5 viruses were found to be monopartite, as neither DNA-B component nor betasatellite associated with begomovirus species, were detected. The complete nucleotide sequence of DNA-A genome of CTM exhibited highest sequence homology (88%) to Croton yellow vein mosaic virus (AJ507777), and of isolates K3/K5 (88.5%) to Tomato leaf curl Pakistan virus (DQ116884). This is less than the threshold value for demarcation of species in the genus Begomovirus.ConclusionK3/K5 and CTM are considered to be novel isolates of Tomato leaf curl virus. Sequence analyses and phylogenetic study indicate that these two ToLCV isolates might have evolved by recombination between viruses related to two or more viral ancestors. The existence of different ToLCV isolates having high genome diversity in India poses a threat to the tomato production in the Asian continent.