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Ramaiah M.,Purdue University | Jain A.,National Research Center on Plant Biotechnology | Raghothama K.G.,Purdue University
Plant Physiology | Year: 2014

Inorganic phosphate (Pi) availability is a major factor determining growth and consequently the productivity of crops. However, it is one of the least available macronutrients due to its high fixation in the rhizospheres. To overcome this constraint, plants have developed adaptive responses to better acquire, utilize, and recycle Pi. Molecular determinants of these adaptive mechanisms include transcription factors (TFs) that play a major role in transcriptional control, thereby regulating genome-scale networks. In this study, we have characterized the biological role of Arabidopsis thaliana Ethylene Response Factor070 (AtERF070), a Pi starvationinduced TF belonging to the APETALA2/ETHYLENE RESPONSE FACTOR family of TFs in Arabidopsis (Arabidopsis thaliana). It is localized to the nucleus and induced specifically in Pi-deprived roots and shoots. RNA interference-mediated suppression of AtERF070 led to augmented lateral root development resulting in higher Pi accumulation, whereas there were reductions in both primary root length and lateral root number in 12-d-old transgenic seedlings overexpressing AtERF070. When the overexpressing lines were grown to maturity under greenhouse conditions, they revealed a stunted bushy appearance that could be rescued by gibberellic acid application. Furthermore, a number of Pi starvation-responsive genes were modulated in AtERF070-overexpressing and RNA interference lines, thereby suggesting a potential role for this TF in maintaining Pi homeostasis. © 2014 American Society of Plant Biologists. All rights reserved.

Chakraborty K.,Indian Agriculture Research Institute | Sairam R.K.,Indian Agriculture Research Institute | Bhattacharya R.C.,National Research Center on Plant Biotechnology
Plant Physiology and Biochemistry | Year: 2012

The objective of the present study was to examine the role of SOS pathway in salinity stress tolerance in Brassica spp. An experiment was conducted in pot culture with 4 Brassica genotypes, i.e., CS 52 and CS 54, Varuna and T 9 subjected to two levels of salinity treatments along with a control, viz., 1.65 (S 0), 4.50 (S 1) and 6.76 (S 2) dS m -1. Salinity treatment significantly decreased relative water content (RWC), membrane stability index (MSI) and chlorophyll (Chl) content in leaves and potassium (K) content in leaf, stem and root of all the genotypes. The decline in RWC, MSI, Chl and K content was significantly less in CS 52 and CS 54 as compared to Varuna and T 9. In contrast, the sodium (Na) content increased under salinity stress in all the plant parts in all the genotypes, however, the increase was less in CS 52 and CS 54, which also showed higher K/Na ratio, and thus more favourable cellular environment. Gene expression studies revealed the existence of a more efficient salt overly sensitive pathway composed of SOS1, SOS2, SOS3 and vacuolar Na +/H + antiporter in CS 52 and CS 54 compared to Varuna and T 9. Sequence analyses of partial cDNAs showed the conserved nature of these genes, and their intra and intergenic relatedness. It is thus concluded that existence of an efficient SOS pathway, resulting in higher K/Na ratio, could be one of the major factor determining salinity stress tolerance of Brassica juncea genotypes CS 52 and CS 54. © 2011 Elsevier Masson SAS.

Ramaiah M.,Purdue University | Jain A.,Purdue University | Jain A.,National Research Center on Plant Biotechnology | Baldwin J.C.,Purdue University | And 2 more authors.
Plant Physiology | Year: 2011

Phosphate (Pi) deficiency is one of the leading causes of loss in crop productivity. Plants respond to Pi deficiency by increasing Pi acquisition and remobilization involving organic and inorganic Pi transporters. Here, we report the functional characterization of a putative organic Pi transporter, Glycerol-3-phosphate permease (G3Pp) family, comprising five members (AtG3Pp1 to -5) in Arabidopsis (Arabidopsis thaliana). AtG3Pp1 and AtG3Pp2 showed 24-and 3-fold induction, respectively, in the roots of Pi-deprived seedlings, whereas Pi deficiency-mediated induction of AtG3Pp3 and -4 was evident in both roots and shoots. Furthermore, promoter-b-glucuronidase (GUS) fusion transgenics were generated for AtG3Pp2 to -5 for elucidation of their in planta role in Pi homeostasis. During Pi starvation, there was a strong expression of the reporter gene driven by AtG3Pp4 promoter in the roots, shoots, anthers, and siliques, whereas GUS expression was specific either to the roots (AtG3Pp3) or to stamens and siliques (AtG3Pp5) in other promoter-GUS fusion transgenics. Quantification of reporter gene activities further substantiated differential responses of AtG3Pp family members to Pi deprivation. A distinct pattern of reporter gene expression exhibited by AtG3Pp3 and AtG3Pp5 during early stages of germination also substantiated their potential roles during seedling ontogeny. Furthermore, an AtG3Pp4 knockdown mutant exhibited accentuated total lateral root lengths under +phosphorus and 2phosphorus conditions compared with the wild type. Several Pi starvation-induced genes involved in root development and/or Pi homeostasis were up-regulated in the mutant. A 9-fold induction of AtG3Pp3 in the mutant provided some evidence for a lack of functional redundancy in the gene family. These results thus reflect differential roles of members of the G3Pp family in the maintenance of Pi homeostasis. © 2011 American Society of Plant Biologists. All Rights Reserved.

Mehrotra S.,National Research Center on Plant Biotechnology | Goyal V.,National Research Center on Plant Biotechnology | Goyal V.,Bioseed Research India Pvt. Ltd.
Gene | Year: 2013

With the advent of transgenic technology, it has become possible to mobilize and express foreign genes into plants and to design crop varieties with better agronomic attributes and adaptability to challenging environmental conditions. Recent advances in transgenic technology have led to concerns about safety of transgenic crops to human and animal health and environment. Biosafety focuses on preventing, minimizing and eliminating risks associated with the research, production, and use of transgenic crops. Food biosafety involves studies of substantial equivalence related to compositional analysis, toxicity and allergenicity. Environmental biosafety involves glasshouse and field trials and study of unintended effects on non-target organisms. Transgenics are characterized at phenotypic and molecular levels for understanding the location of transgene insertion site, ploidy level, copy number, integrated vector sequences, protein expression and stability of the transgene. Various techniques employed for transgene characterization include flow cytometry, southern, northern and western analyses, real-time (qRT) PCR, competitive PCR, FISH, fiber-FISH, DNA micro-arrays, mRNA profiling, 2DE-MS, iTRAQ, FT-MS, NMR, GC-MS, CE-MS and biosensor-based approaches. Evaluation of transgene expression involves the application of integrated phenomics, transcriptomics, proteomics and metabolomics approaches. However, the relevance and application of these approaches may vary in different cases. The elaborate analysis of transgenic crops will facilitate the safety assessment and commercialization of transgenics and lead to global food security for the future. © 2012 Elsevier B.V.

Jain A.,National Research Center on Plant Biotechnology | Nagarajan V.K.,Purdue University | Raghothama K.G.,Purdue University
Cellular and Molecular Life Sciences | Year: 2012

Phosphorus (P), an essential macronutrient required for plant growth and development, is often limiting in natural and agro-climatic environments. To cope with heterogeneous or low phosphate (Pi) availability, plants have evolved an array of adaptive responses facilitating optimal acquisition and distribution of Pi. The root system plays a pivotal role in Pi-deficiency-mediated adaptive responses that are regulated by a complex interplay of systemic and local Pi sensing. Cross-talk with sugar, phytohormones, and other nutrient signaling pathways further highlight the intricacies involved in maintaining Pi homeostasis. Transcriptional regulation of Pi-starvation responses is particularly intriguing and involves a host of transcription factors (TFs). Although PHR1 of Arabidopsis is an extensively studied MYB TF regulating subset of Pistarvation responses, it is not induced during Pi deprivation. Genome-wide analyses of Arabidopsis have shown that low Pi stress triggers spatiotemporal expression of several genes encoding different TFs. Functional characterization of some of these TFs reveals their diverse roles in regulating root system architecture, and acquisition and utilization of Pi. Some of the TFs are also involved in phytohormone-mediated root responses to Pi starvation. The biological roles of these TFs in transcriptional regulation of Pi homeostasis in model plants Arabidopsis thaliana and Oryza sativa are presented in this review. © Springer Basel AG 2012.

Choudhary M.,National Research Center on Plant Biotechnology | Padaria J.C.,National Research Center on Plant Biotechnology
Physiology and Molecular Biology of Plants | Year: 2015

Pearl millet (Pennisetum glaucum) is an important cereal of traditional farming systems that has the natural ability to withstand various abiotic stresses. The present study aims at the identification and validation of major differentially expressed genes in response to drought stress in P. glaucum by Suppression Subtractive Hybridization (SSH) analysis. Twenty-two days old seedlings of P. glaucum cultivar PPMI741 were subjected to drought stress by treatment of 30 % Polyethylene glycol for different time periods 30 min (T1), 2 h (T2), 4 h (T3), 8 h (T4), 16 h (T5), 24 h (T6) and 48 h (T7) respectively, monitored by examining the RWC of seedlings. Total RNA was isolated to construct drought responsive subtractive cDNA library through SSH, sequenced to identify the differentially expressed genes in response to drought stress and validated by qRT-PCR.745 ESTs were assembled into a collection of 299 unigenes having 52 contigs and 247 singletons. All 745 ESTs were submitted to ENA-EMBL databases (Accession no. HG516611- HG517355). After analysis, 10 differentially expressed genes were validated namely Abscisic stress ripening protein, Ascorbate peroxidase, Inosine-5′-monophosphate dehydrogenase, Putative beta-1, 3-glucanase, Glyoxalase, Rab7, Aspartic proteinase Oryzasin, DnaJ—like protein and Calmodulin—like protein by qRT-PCR. The identified ESTs reveal a major portion of the stress responsive transcriptome that may prove to be a vent to unravel molecular basis underlying tolerance of pearl millet (Pennisetum glaucum) to drought stress. These genes could be utilized for transgenic breeding or transferred to crop plants through marker assisted selection for the development of better drought resistant cultivars having enhanced adaptability to survive harsh environmental conditions. © 2015, Prof. H.S. Srivastava Foundation for Science and Society.

Chakraborty A.,ICAR Central Research Institute for Jute and Allied Fibres CRIJAF | Sarkar D.,ICAR Central Research Institute for Jute and Allied Fibres CRIJAF | Satya P.,ICAR Central Research Institute for Jute and Allied Fibres CRIJAF | Karmakar P.G.,ICAR Central Research Institute for Jute and Allied Fibres CRIJAF | Singh N.K.,National Research Center on Plant Biotechnology
Molecular Genetics and Genomics | Year: 2015

We generated the bast transcriptomes of a deficient lignified phloem fibre mutant and its wild-type jute (Corchorus capsularis) using Illumina paired-end sequencing. A total of 34,163 wild-type and 29,463 mutant unigenes, with average lengths of 1442 and 1136  bp, respectively, were assembled de novo, ~77–79 % of which were functionally annotated. These annotated unigenes were assigned to COG (~37–40 %) and GO (~22–28 %) classifications and mapped to 189 KEGG pathways (~19–21 %). We discovered 38 and 43 isoforms of 16 and 10 genes of the upstream shikimate-aromatic amino acid and downstream monolignol biosynthetic pathways, respectively, rendered their sequence similarities, confirmed the identities of 22 of these candidate gene families by phylogenetic analyses and reconstructed the pathway leading to lignin biosynthesis in jute fibres. We also identified major genes and bast-related transcription factors involved in secondary cell wall (SCW) formation. The quantitative RT-PCRs revealed that phenylalanine ammonia-lyase 1 (CcPAL1) was co-down-regulated with several genes of the upstream shikimate pathway in mutant bast tissues at an early growth stage, although its expression relapsed to the normal level at the later growth stage. However, cinnamyl alcohol dehydrogenase 7 (CcCAD7) was strongly down-regulated in mutant bast tissues irrespective of growth stages. CcCAD7 disruption at an early growth stage was accompanied by co-up-regulation of SCW-specific genes cellulose synthase A7 (CcCesA7) and fasciclin-like arabinogalactan 6 (CcFLA6), which was predicted to be involved in coordinating the S-layers’ deposition in the xylan-type jute fibres. Our results identified CAD as a promising target for developing low-lignin jute fibres using genomics-assisted molecular approaches. © 2015, Springer-Verlag Berlin Heidelberg.

Grover A.,National Research Center on Plant Biotechnology
Critical Reviews in Plant Sciences | Year: 2012

Chitinase proteins are widely distributed across diverse biological systems. Chitinases hydrolyze chitin, chitosan, lipochitooligosaccharides, peptidoglycan, arabinogalactan and glycoproteins containing N-acetylglucosamine. Analyses of genome-wide sequence and microarray expression profilings show that chitinase genes are represented by large families and the individual member genes are expressed in diverse conditions. Chitinase proteins are members in the group of the pathogenesis-related proteins that are strongly induced when host plant cells are challenged by pathogen stress and thus chitinases constitute an important arsenal of plants against fungal pathogens. Transgenic plants have been produced that overexpress chitinases alone or in conjunction with other defense-related proteins. The phenotype analyses of such plants have shown enhanced disease resistance in large number of cases. Apart from defense against pathogen stress, chitinases are implicated in relationships between plant cells and fungi (e.g., mycorrhizae associations) and bacteria (e.g., legume/Rhizobium associations). Chitinases are also involved in plant abiotic stress responses as noted for osmotic, salt, cold, wounding and heavy metal stresses. Chitinases play a role in developmental aspects of plants too (i.e., regulation of plant embryogenesis process). A detailed account of the genetic diversity and functional aspects of plant chitinases is presented in this review. © 2012 Taylor and Francis Group, LLC.

Pattanayak D.,National Research Center on Plant Biotechnology | Solanke A.U.,National Research Center on Plant Biotechnology | Kumar P.A.,National Research Center on Plant Biotechnology
Plant Molecular Biology Reporter | Year: 2013

Small non-coding RNA-mediated gene-silencing pathways, collectively called RNA interference (RNAi), are involved in regulation of endogenous gene expression and plant defence. It is manifested through two broad classes of small non-coding regulatory RNAs, small interfering RNA (siRNA) and microRNA (miRNA). siRNAs, generated from cleavage of long hairpin RNA by RNase III-class endonuclease, Dicer-like, mediate transcriptional or post-transcriptional gene silencing. At transcriptional level, 24-nucleotide (nt)-long-siRNAs guide an effector complex for DNA methylation, which leads to heterochromatinisation of target loci and consequently transcriptional silencing. At post-transcriptional level, a different size class of 21-nt-long siRNAs guides a silencing complex, called RNA-induced silencing complex, for cleavage of target mRNA. cis-acting siRNAs are involved in plant defence against viruses and transposons, and trans-acting siRNAs regulate endogenous genes involved in plant growth. miRNAs are generated from processing of imperfect stem-loop RNA precursors by Dicer-like. They regulate plant growth and adaptive stress responses by either degradation or translational repression of target mRNAs. © 2012 Springer Science+Business Media New York.

Babu S.R.,Indian Agricultural Research Institute | Subrahmanyam B.,Indian Agricultural Research Institute | Srinivasan,National Research Center on Plant Biotechnology | Santha I.M.,Indian Agricultural Research Institute
Journal of Biosciences | Year: 2012

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera. © Indian Academy of Sciences.

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