Ashutosh Pandey

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.

Physiological and proteomic responses of cotton (Gossypium herbaceum L.) to drought stress.

Cotton genotype RAHS 187 was analyzed for changes in physiology, biochemistry and proteome due to drought stress. The deleterious effect of drought in cotton plants was mainly targeted towards photosynthesis. The gas-exchange parameters of net photosynthesis (A), stomatal conductance (g(s)) and transpiration (E) showed a decreasing trend as the drought intensity increased. The fluorescence parameters of, effective quantum yield of PSII (Φ(PSII)), and electron transport rates (ETR), also showed a declining trend. As the intensity of drought increased, both H(2)O(2) and MDA levels increased indicating oxidative stress. Anthocyanin levels were increased by more than four folds in the droughted plants. Two-dimensional gel electrophoresis detected more than 550 protein spots. Significantly expressed proteins were analyzed by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF. The number of up-regulated spots was found to be 16 while 6 spots were down-regulated. The reasonable implications in drought response of the identified proteins vis-à-vis physiological changes are discussed. Results provide some additional information that can lead to a better understanding of the molecular basis of drought-sensitivity in cotton plants.

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.

MicroRNA858 Is a Potential Regulator of Phenylpropanoid Pathway and Plant Development

miR858a targets MYB transcription factors involved in flavonoid biosynthesis and plant growth and development. MicroRNAs (miRNAs) are endogenous, noncoding small RNAs that function as critical regulators of gene expression. In plants, miRNAs have shown their potential as regulators of growth, development, signal transduction, and stress tolerance. Although the miRNA-mediated regulation of several processes is known, the involvement of miRNAs in regulating secondary plant product biosynthesis is poorly understood. In this study, we functionally characterized Arabidopsis (Arabidopsis thaliana) miR858a, which putatively targets R2R3-MYB transcription factors involved in flavonoid biosynthesis. Overexpression of miR858a in Arabidopsis led to the down-regulation of several MYB transcription factors regulating flavonoid biosynthesis. In contrast to the robust growth and early flowering of miR858OX plants, reduction of plant growth and delayed flowering were observed in Arabidopsis transgenic lines expressing an artificial miRNA target mimic (MIM858). Genome-wide expression analysis using transgenic lines suggested that miR858a targets a number of regulatory factors that modulate the expression of downstream genes involved in plant development and hormonal and stress responses. Furthermore, higher expression of MYBs in MIM858 lines leads to redirection of the metabolic flux towards the synthesis of flavonoids at the cost of lignin synthesis. Altogether, our study has established the potential role of light-regulated miR858a in flavonoid biosynthesis and 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.

Constitutive expression of Arabidopsis MYB transcription factor, AtMYB11, in tobacco modulates flavonoid biosynthesis in favor of flavonol accumulation

Key messageHeterologous expression ofAtMYB11, a flavonol-specific transcription factor fromArabidopsis, in tobacco modulates flavonoid biosynthesis, however, with a lower efficiency as compared to its paralogsAtMYB12andAtMYB111.AbstractTranscriptional regulation is the most important means for controlling flavonoid biosynthesis under temporal and spatial cues. In Arabidopsis, three functionally redundant MYB transcription factors (AtMYB11, AtMYB111 and AtMYB12) have been characterized as flavonol-specific regulators which positively modulate expression of biosynthetic genes involved in flavonol biosynthesis. Based on expression of AtMYB111 and AtMYB12 in heterologous systems, studies suggest that these transcription factors can be used to develop plants with enhanced flavonol biosynthesis. The potential of AtMYB11 to activate flavonol biosynthesis in a heterologous system has not yet been studied. In this study, the regulatory potential of AtMYB11 has been studied in Nicotiana tabacum by developing transgenic plants constitutively expressing AtMYB11. Our analysis using leaf and petal tissues of the transgenic plants indicates that AtMYB11 enhances flavonol and chlorogenic acid (CGA) biosynthesis in tobacco through up-regulation of the biosynthetic genes. Activation of flavonol biosynthesis in tobacco by AtMYB11 is not as pronounced as with AtMYB12 or AtMYB111. Taken together, these results reveal a differential regulatory mechanism in plants for modulating flavonol biosynthesis. This study demonstrated that AtMYB11 can be strategically used for enhancing the health beneficial flavonols in species other than Arabidopsis.

DEVELOPMENT AND OPTIMIZATION OF HPLC-PDA-MS-MS METHOD FOR SIMULTANEOUS QUANTIFICATION OF THREE CLASSES OF FLAVONOIDS IN LEGUME SEEDS, VEGETABLES, FRUITS, AND MEDICINAL PLANTS

Certain plants of the daily human diet are an important source of beneficially healthy flavonoids, possessing significant pharmacological activities against various diseases. Flavonoids display an array of chemical diversity based on relative positioning of benzopyrano moiety and aromatic ring. Due to structural diversity of these molecules, simultaneous quantification of these molecules in plant materials becomes difficult. In the present study, a rapid, simple, and sensitive, High Performance Liquid Chromatography-Photo Diode Array Mass Spectrometry (HPLC-PDA-MS-MS) method was developed for the simultaneous determination of three different classes of flavonoids,namely, isoflavonoids, flavanones, and flavonols in legume seeds, vegetables, fruits, and medicinal plants. The method was evaluated for validation parameters, such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, selectivity, and compound stability. The developed method was applied on extracts of different plant samples for quantification of the flavonoids daidzein, genistein, naringenin, quercetin, kaempferol, and rutin. Method developed in this study can be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations. Supplemental materials are available for this article. Go to the publishers online edition of the Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Genetically engineered flavonol enriched tomato fruit modulates chondrogenesis to increase bone length in growing animals

Externally visible body and longitudinal bone growth is a result of proliferation of chondrocytes. In growth disorder, there is delay in the age associated increase in height. The present study evaluates the effect of extract from transgenic tomato fruit expressing AtMYB12 transcription factor on bone health including longitudinal growth. Constitutive expression of AtMYB12 in tomato led to a significantly enhanced biosynthesis of flavonoids in general and the flavonol biosynthesis in particular. Pre-pubertal ovary intact BALB/c mice received daily oral administration of vehicle and ethanolic extract of wild type (WT-TOM) and transgenic AtMYB12-tomato (MYB12-TOM) fruits for six weeks. Animal fed with MYB12-TOM showed no inflammation in hepatic tissues and normal sinusoidal Kupffer cell morphology. MYB12-TOM extract significantly increased tibial and femoral growth and subsequently improved the bone length as compared to vehicle and WT-TOM. Histomorphometry exhibited significantly wider distal femoral and proximal tibial growth plate, increased number and size of hypertrophic chondrocytes in MYB12-TOM which corroborated with micro-CT and expression of BMP-2 and COL-10, marker genes for hypertrophic cells. We conclude that metabolic reprogramming of tomato by AtMYB12 has the potential to improve longitudinal bone growth thus helping in achievement of greater peak bone mass during adolescence.

Genome-wide analysis of the AP2/ERF family in Musa species reveals divergence and neofunctionalisation during evolution

AP2/ERF domain containing transcription factor super family is one of the important regulators in the plant kingdom. The involvement of AP2/ERF family members has been elucidated in various processes associated with plant growth, development as well as in response to hormones, biotic and abiotic stresses. In this study, we carried out genome-wide analysis to identify members of AP2/ERF family in Musa acuminata (A genome) and Musa balbisiana (B genome) and changes leading to neofunctionalisation of genes. Analysis identified 265 and 318 AP2/ERF encoding genes in M. acuminata and M. balbisiana respectively which were further classified into ERF, DREB, AP2, RAV and Soloist groups. Comparative analysis indicated that AP2/ERF family has undergone duplication, loss and divergence during evolution and speciation of the Musa A and B genomes. We identified nine genes which are up-regulated during fruit ripening and might be components of the regulatory machinery operating during ethylene-dependent ripening in banana. Tissue-specific expression analysis of the genes suggests that different regulatory mechanisms might be involved in peel and pulp ripening process through recruiting specific ERFs in these tissues. Analysis also suggests that MaRAV-6 and MaERF026 have structurally diverged from their M. balbisiana counterparts and have attained new functions during ripening.