Prashant Misra

Effect of arsenic on growth, oxidative stress, and antioxidant system in rice seedlings ☆

The physiological, biochemical, and proteomic changes in germinating rice seedlings were investigated under arsenic stress. A marked decrease in germination percentage, shoot, and root elongation as well as plant biomass was observed with arsenic treatments, as compared to control, whereas accumulation of arsenic and malondialdehyde (MDA) in seedlings were increased significantly with increasing arsenic concentration (both AsIII and AsV). The up-regulation of some antioxidant enzyme activities and the isozymes of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), peroxidase (POD, EC 1.11.1.7), and glutathione reductase (GR, 1.6.4.2) substantiated that arsenic accumulation generated oxidative stress, which was more pronounced in As(III) treatment. We also studied the protective effect of reduced glutathione (GSH) and cysteine (Cys) to As(III)/As(V) stressed seedlings. Both GSH and Cys imparted enhanced tolerance to seedlings against arsenic stress. Seedlings growth improved while level of MDA declined significantly when GSH and Cys were supplemented to As(III)/As(V) treatments suggesting GSH and Cys-mediated protection against oxidative stress. The arsenic content was highest in roots of seedlings grown in As(III) in the presence of GSH/Cys. However, in case of As(V) plus GSH or Cys, the arsenic content in seedlings was highest in shoots. The results are suggestive of differential metabolism of As(III) and As(V) in rice.

Comparative transcriptome analysis of arsenate and arsenite stresses in rice seedlings

The effect of arsenic (As) exposure on genome-wide expression was examined in rice (Oryza sativa L., ssp. Indica). A group of defense and stress-responsive genes, transporters, heat-shock proteins, metallothioneins, sulfate-metabolizing proteins, and regulatory genes showed differential expression in rice seedlings challenged with arsenate (AsV) and arsenite (AsIII). AsV stress led to upregulation or downregulation of an additional set of genes in comparison to AsIII. Differential expression of several genes that showed the highest contrast in a microarray analysis was validated by following the quantitative changes in the levels of individual transcripts following challenge with AsV, AsIII, Cd, Cr, and Pb. Most of the selected genes responded to challenge by heavy metals such as arsenic. However, expression of one of the cytochrome P450 genes (Os01g43740) in rice root was induced by AsV but not by other heavy metals. Similarly, one glutaredoxin (Os01g26912) is expressed specifically in the AsIII-treated shoot.

Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMyb12, leads to insect resistance

Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

Transcriptomic and metabolomic shifts in rice roots in response to Cr (VI) stress

BackgroundWidespread use of chromium (Cr) contaminated fields due to careless and inappropriate management practices of effluent discharge, mostly from industries related to metallurgy, electroplating, production of paints and pigments, tanning, and wood preservation elevates its concentration in surface soil and eventually into rice plants and grains. In spite of many previous studies having been conducted on the effects of chromium stress, the precise molecular mechanisms related to both the effects of chromium phytotoxicity, the defense reactions of plants against chromium exposure as well as translocation and accumulation in rice remain poorly understood.ResultsDetailed analysis of genome-wide transcriptome profiling in rice root is reported here, following Cr-plant interaction. Such studies are important for the identification of genes responsible for tolerance, accumulation and defense response in plants with respect to Cr stress. Rice root metabolome analysis was also carried out to relate differential transcriptome data to biological processes affected by Cr (VI) stress in rice. To check whether the Cr-specific motifs were indeed significantly over represented in the promoter regions of Cr-responsive genes, occurrence of these motifs in whole genome sequence was carried out. In the background of whole genome, the lift value for these 14 and 13 motifs was significantly high in the test dataset. Though no functional role has been assigned to any of the motifs, but all of these are present as promoter motifs in the Database of orthologus promoters.ConclusionThese findings clearly suggest that a complex network of regulatory pathways modulates Cr-response of rice. The integrated matrix of both transcriptome and metabolome data after suitable normalization and initial calculations provided us a visual picture of the correlations between components. Predominance of different motifs in the subsets of genes suggests the involvement of motif-specific transcription modulating proteins in Cr stress response of rice.

Expression of Arabidopsis MYB transcription factor, AtMYB111 , in tobacco requires light to modulate flavonol content

Flavonoids, due to their pharmacological attributes, have recently become target molecules for metabolic engineering in commonly consumed food crops. Strategies including expression of single genes and gene pyramiding have provided only limited success, due principally to the highly branched and complex biosynthetic pathway of the flavonoids. Transcription factors have been demonstrated as an efficient tool for metabolic engineering of this pathway, but often exhibit variation in heterologous systems relative to that in the homologous system. In the present work, Arabidopsis MYB transcription factor, AtMYB111, has been expressed in tobacco to study whether this can enhance flavonoid biosynthesis in heterologous system. The results suggest that AtMYB111 expression in transgenic tobacco enhances expression of genes of the phenylpropanoid pathway leading to an elevated content of flavonols. However, dark incubation of transgenic and wild type (WT) plants down-regulated both the expression of genes as well as flavonoid content as compared to light grown plants. The study concludes that AtMYB111 can be effectively used in heterologous systems, however, light is required for its action in modulating biosynthetic pathway.

Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity.

Isoflavones, a group of flavonoids, restricted almost exclusively to family Leguminosae are known to exhibit anticancerous and anti-osteoporotic activities in animal systems and have been a target for metabolic engineering in commonly consumed food crops. Earlier efforts based on the expression of legume isoflavone synthase (IFS) genes in nonlegume plant species led to the limited success in terms of isoflavone content in transgenic tissue due to the limitation of substrate for IFS enzyme. In this work to overcome this limitation, the activation of multiple genes of flavonoid pathway using Arabidopsis transcription factor AtMYB12 has been carried out. We developed transgenic tobacco lines constitutively co-expressing AtMYB12 and GmIFS1 (soybean IFS) genes or independently and carried out their phytochemical and molecular analyses. The leaves of co-expressing transgenic lines were found to have elevated flavonol content along with the accumulation of substantial amount of genistein glycoconjugates being at the highest levels that could be engineered in tobacco leaves till date. Oestrogen-deficient (ovariectomized, Ovx) mice fed with leaf extract from transgenic plant co-expressing AtMYB12 and GmIFS1 but not wild-type extract exhibited significant conservation of trabecular microarchitecture, reduced osteoclast number and expression of osteoclastogenic genes, higher total serum antioxidant levels and increased uterine oestrogenicity compared with Ovx mice treated with vehicle (control). The skeletal effect of the transgenic extract was comparable to oestrogen-treated Ovx mice. Together, our results establish an efficient strategy for successful pathway engineering of isoflavones and other flavonoids in crop plants and provide a direct evidence of improved osteoprotective effect of transgenic plant extract.

Heavy metals induce oxidative stress and genome-wide modulation in transcriptome of rice root

Industrial growth, ecological disturbances and agricultural practices have contaminated the soil and water with many harmful compounds, including heavy metals. These heavy metals affect growth and development of plants as well as cause severe human health hazards through food chain contamination. In past, studies have been made to identify biochemical and molecular networks associated with heavy metal toxicity and uptake in plants. Studies suggested that most of the physiological and molecular processes affected by different heavy metals are similar to those affected by other abiotic stresses. To identify common and unique responses by different metals, we have studied biochemical and genome-wide modulation in transcriptome of rice (IR-64 cultivar) root after exposure to cadmium (Cd), arsenate [As(V)], lead (Pb) and chromium [Cr(VI)] in hydroponic condition. We observed that root tissue shows variable responses for antioxidant enzyme system for different heavy metals. Genome-wide expression analysis suggests variable number of genes differentially expressed in root in response to As(V), Cd, Pb and Cr(VI) stresses. In addition to unique genes, each heavy metal modulated expression of a large number of common genes. Study also identified cis-acting regions of the promoters which can be determinants for the modulated expression of the genes in response to different heavy metals. Our study advances understanding related to various processes and networks which might be responsible for heavy metal stresses, accumulation and detoxification.

Development of AtMYB12 -expressing transgenic tobacco callus culture for production of rutin with biopesticidal potential

Flavonoids synthesized by the phenylpropanoid pathway participate in a number of physiological and biochemical processes in plants. Flavonols, among flavonoids, are considered as health-protective components in functional foods and they protect plants against certain insect pests. There have been efforts to develop strategies for the enhanced production of flavonols in plants, but limited success was achieved due to complex regulation and poor substrate availability. In the present study, we have developed and optimized method for callus cultures for transgenic tobacco line expressing a flavonol-specific transcription factor, AtMYB12, with an objective to use callus as an alternative source of rutin. Transgenic callus displayed enhanced expression of genes related to biosynthetic pathway leading to increased accumulation of flavonols, especially rutin. At each time point of callus growth, the rutin content of transgenic callus was several folds higher than that of wild-type tobacco callus. Supplementation of semi-synthetic diet with extract from transgenic callus as well as purified rutin led to mortality and growth reduction in the Spodopteralitura and Helicoverpaarmigera larvae. This study suggests the biotechnological potential of AtMYB12-expressing callus cultures for the production of rutin, which can be used for biopesticide formulations against insect pests.Key messageTobacco callus cultures expressing AtMYB12 accumulate enhanced content of rutin and can be used as a potential alternative source of rutin as well as biopesticides against insect pests.

Prolyl-4-hydroxylase (AtP4H1) mediates and mimics low oxygen response in Arabidopsis thaliana

Proline hydroxylation is an important phenomenon of a living cell. Prolyl-4-hydroxylases (P4H) responsible for this process have been characterized from animals, and one of its forms, HIF-P4H, is regarded as an oxygen sensor. In plants, P4H has been partially characterized from few species, and one of the Arabidopsis P4H (AtP4H1) has been shown to hydroxylate proline-rich peptides in vitro. In order to study its function in planta, we have overexpressed AtP4H1 in Arabidopsis. The AtP4H1oexp plants showed hypoxia-in-normoxia phenotype with strict requirement for carbon source for its growth, increased root hair, absence of trichome, and reduction in seed size. Genome-wide expression analyses suggest that expression of several genes related to hypoxia as well as plant growth and development are upregulated in AtP4H1oexp lines. Based on our studies on AtP4H1oexp lines, we speculate a direct role of AtP4H1 in hypoxia stress and in different stages of plant growth and development.

AtMYB12 expression in tomato leads to large scale differential modulation in transcriptome and flavonoid content in leaf and fruit tissues.

Plants synthesize secondary metabolites, including flavonoids, which play important role during various stresses for their survival. These metabolites are also considered as health-protective components in functional foods. Flavonols, one of the important groups of flavonoids, apart from performing several roles in plants have been recognized as potent phytoceuticals for human health. Tomato fruits are deficient in this group of flavonoids and have been an important target for enhancing the accumulation of flavonols through genetic manipulations. In the present study, AtMYB12 transcription factor of the Arabidopsis has been expressed under constitutive promoter in tomato. Transgenic tomato lines exhibited enhanced accumulation of flavonols and chlorogenic acid (CGA) in leaf and fruit accompanied with elevated expression of phenylpropanoid pathway genes involved in flavonol biosynthesis. In addition, global gene expression analysis in leaf and fruit suggested that AtMYB12 modulates number of molecular processes including aromatic amino acid biosynthesis, phytohormone signaling and stress responses. Besides this, a differential modulation of the genes in fruits and leaves is reported in this study. Taken together, results demonstrate that modulation of primary carbon metabolism and other pathways by AtMYB12 in tomato may lead to sufficient substrate supply for enhanced content of phenolics in general and flavonols in particular.