Rakesh Kumar Shukla

Long Term Transcript Accumulation during the Development of Dehydration Adaptation in Cicer arietinum

Cool season crops face intermittent drought. Exposure to drought and other abiotic stresses is known to increase tolerance of the plants against subsequent exposure to such stresses. Storage of environmental signals is also proposed. Preexposure to a dehydration shock improved adaptive response during subsequent dehydration treatment in a cool season crop chickpea (Cicer arietinum). We have identified 101 dehydration-inducible transcripts of chickpea by repetitive rounds of cDNA subtraction; differential DNA-array hybridization followed by northern-blot analysis and analyzed their responses to exogenous application of abscisic acid (ABA). Steady-state expression levels of the dehydration-induced transcripts were monitored during the recovery period between 2 consecutive dehydration stresses. Seven of them maintained more than 3-fold of expression after 24 h and more than 2-fold of expression level even at 72 h after the removal of stress. Noticeably, all of them were inducible by exogenous ABA treatment. When the seedlings were subjected to recover similarly after an exposure to exogenous ABA, the steady-state abundances of 6 of them followed totally different kinetics returning to basal level expression within 24 h. This observation indicated a correlation between the longer period of abundance of those transcripts in the recovery period and improved adaptation of the plants to subsequent dehydration stress and suggested that both ABA-dependent and -independent mechanisms are involved in the maintenance of the messages from the previous stress experience.

Expression of CAP2, an APETALA2-Family Transcription Factor from Chickpea, Enhances Growth and Tolerance to Dehydration and Salt Stress in Transgenic Tobacco

The APETALA2 (AP2) domain defines a large family of DNA-binding proteins that play important roles in plant morphology, development, and stress response. We describe isolation and characterization of a gene (CAP2) from chickpea (Cicer arietinum) encoding a novel AP2-family transcription factor. Recombinant CAP2 protein bound specifically to C-repeat/dehydration-responsive element in gel-shift assay and transactivated reporter genes in yeast (Saccharomyces cerevisiae) one-hybrid assay. CAP2 appeared to be a single/low copy intronless gene, and the protein product localized in the nucleus. Transcript level of CAP2 increased by dehydration and by treatment with sodium chloride, abscisic acid, and auxin, but not by treatment with low temperature, salicylic acid, and jasmonic acid. The 35S promoter-driven expression of CAP2 in tobacco (Nicotiana tabacum) caused drastic increase in the leaf cell size, and, thereby, in leaf surface area and number of lateral roots. Transgenic plants demonstrated more tolerance to dehydration and salt stress than the wild-type plants. Transgenic plants expressed higher steady-state transcript levels of abiotic stress-response genes NtERD10B and NtERD10C and auxin-response genes IAA4.2 and IAA2.5. Taken together, our results indicated a mutual interrelation between plant growth-development and abiotic stress-response pathways and a probable involvement of CAP2 in both the signaling pathways.

WRKY Transcription Factors: Molecular Regulation and Stress Responses in Plants

Plants in their natural habitat have to face multiple stresses simultaneously. Evolutionary adaptation of developmental, physiological, and biochemical parameters give advantage over a single window of stress but not multiple. On the other hand transcription factors like WRKY can regulate diverse responses through a complicated network of genes. So molecular orchestration of WRKYs in plant may provide the most anticipated outcome of simultaneous multiple responses. Activation or repression through W-box and W-box like sequences is regulated at transcriptional, translational, and domain level. Because of the tight regulation involved in specific recognition and binding of WRKYs to downstream promoters, they have become promising candidate for crop improvement. Epigenetic, retrograde and proteasome mediated regulation enable WRKYs to attain the dynamic cellular homeostatic reprograming. Overexpression of several WRKYs face the paradox of having several beneficial affects but with some unwanted traits. These overexpression-associated undesirable phenotypes need to be identified and removed for proper growth, development and yeild. Taken together, we have highlighted the diverse regulation and multiple stress response of WRKYs in plants along with the future prospects in this field of research.

Identification of differentially displayed proteins in cerebrospinal fluid of Parkinson's disease patients: a proteomic approach.

BACKGROUND Clinical proteomics has been widely used to identify differentially displayed proteins in blood and cerebrospinal fluid (CSF) to understand the molecular and cellular events leading to Parkinsons disease (PD). The close connection between CSF and the brain offers reliable and reproducible way to assess the majority of changes in the brain proteome profile directly into CSF throughout the course of neurodegeneration. METHODS We identified the differentially displayed proteins in CSF of PD patients as compared with controls using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry. RESULTS Comparative 2-D PAGE electrophoretograms of CSF of PD patients with case controls and/or neurological controls revealed significant differential display of six protein spots. The differentially displayed proteins were identified as serum albumin precursor, serum albumin chain-A, hemoglobin beta fragment, mutant globin, proline rich repeat 14 (PRR 14) and serum transferrin N-terminal lobe. Although the level of hemoglobin beta fragment and mutant globin was attenuated, serum albumin precursor, serum albumin chain-A, PRR 14 and serum transferrin N-terminal lobe were augmented in PD patients as compared with case controls. The level of serum albumin chain-A, PRR 14 and serum transferrin N-terminal lobe was not significantly altered when compared with neurological controls. CONCLUSIONS The results obtained thus suggest that differential display of CSF serum albumin precursor, serum albumin chain-A, PRR 14 and serum transferrin N-terminal lobe could be associated with neuronal dysfunction and hemoglobin/globin with the onset/progression of PD in humans.

Wound Induced Tanscriptional Regulation of Benzylisoquinoline Pathway and Characterization of Wound Inducible PsWRKY Transcription Factor from Papaver somniferum

Wounding is required to be made in the walls of the green seed pod of Opium poppy prior exudation of latex. To withstand this kind of trauma plants regulate expression of some metabolites through an induced transcript level. 167 unique wound-inducible ESTs were identified by a repetitive round of cDNA subtraction after 5 hours of wounding in Papaver somniferum seedlings. Further repetitive reverse northern analysis of these ESTs revealed 80 transcripts showing more than two fold induction, validated through semi-quantitative RT-PCR & real time expression analysis. One of the major classified categories among identified ESTs belonged to benzylisoquinoline transcripts. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to wounding revealed increased accumulation of narcotine and papaverine. Promoter analysis of seven transcripts of BIAs pathway showed the presence of W-box cis-element with the consensus sequence of TGAC, which is the proposed binding site for WRKY type transcription factors. One of the Wound inducible ‘WRKY’ EST isolated from our subtracted library was made full-length and named as ‘PsWRKY’. Bacterially expressed PsWRKY interacted with the W-box element having consensus sequence TTGACT/C present in the promoter region of BIAs biosynthetic pathway genes. PsWRKY further activated the TYDC promoter in yeast and transiently in tobacco BY2 cells. Preferential expression of PsWRKY in straw and capsule and its interaction with consensus W-box element present in BIAs pathway gene transcripts suggest its possible involvement in the wound induced regulation of BIAs pathway.

PsAP2 an AP2/ERF family transcription factor from Papaver somniferum enhances abiotic and biotic stress tolerance in transgenic tobacco

The AP2/ERFs are one of the most important family of transcription factors which regulate multiple responses like stress, metabolism and development in plants. We isolated PsAP2 a novel AP2/ERF from Papaver somniferum which was highly upregulated in response to wounding followed by ethylene, methyl jasmonate and ABA treatment. PsAP2 showed specific binding with both DRE and GCC box elements and it was able to transactivate the reporter genes in yeast. PsAP2 overexpressing transgenic tobacco plants exhibited enhanced tolerance towards both abiotic and biotic stresses . Real time transcript expression analysis showed constitutive upregulation of tobacco Alternative oxidase1a and Myo-inositol-1-phosphate synthase in PsAP2 overexpressing tobacco plants. Further, PsAP2 showed interaction with NtAOX1a promoter in vitro, it also specifically activated the NtAOX1a promoter in yeast and tobacco BY2 cells. The silencing of PsAP2 using VIGS lead to significant reduction in the AOX1 level in P. somniferum. Taken together PsAP2 can directly bind and transcriptionally activate NtAOX1a and its overexpression in tobacco imparted increased tolerance towards both abiotic and biotic stress.

CAP2 enhances germination of transgenic tobacco seeds at high temperature and promotes heat stress tolerance in yeast

We reported earlier that ectopic expression of CAP2, a single AP2 domain containing transcription activator from chickpea (Cicer arietinum) in tobacco improves growth and development, and tolerance to dehydration and salt stress, of the transgenic plants. Here, we report that, in addition, the CAP2‐transgenic tobacco seeds also exhibit higher germination efficiency at high temperature and show higher expression levels of genes for tobacco heat shock proteins and a heat shock factor. CAP2 was able to activate the 5′‐upstream activating sequence of tobacco heat shock factor. Surprisingly, expression of CAP2 cDNA in Saccharomyces cerevisiae also enhanced heat tolerance, with increased expression of the gene for yeast heat shock factor 1 (Hsf1) and its target, the gene for yeast heat shock protein 104 (Hsp104). Sequence analysis of the Hsf1 promoter revealed the presence of a dehydration‐responsive element/C‐repeat‐like element (DRE/CRE). Recombinant CAP2 protein bound to the DRE/CRE in the Hsf1 promoter in a gel shift assay and transactivated the Hsf1 promoter–His reporter construct. The full‐length CAP2 protein was required to provide thermotolerance in yeast. If these findings are taken together, our results suggest that CAP2 is involved in the heat stress response and provides an example of functioning of a plant transcription factor in yeast, highlighting the strong evolutionary conservation of the stress response mechanism.

Soybean cultivar resistant to Mungbean Yellow Mosaic India Virus infection induces viral RNA degradation earlier than the susceptible cultivar

Yellow mosaic disease caused by whitefly-transmitted bipartite Geminiviruses is one of the major constraints on productivity of a number of pulse crops. We have cloned the bipartite genome of Mungbean Yellow Mosaic India Virus isolated from infected Soybean. We report here that agroinfection of Soybean seedlings with a single uncut recombinant binary plasmid containing tandem dimers of both DNA A and DNA B resulted in 100% infectivity in susceptible varieties. To understand the mechanism of natural resistance in a Soybean variety, we compared the abundance of the viral RNAs in a resistant and a susceptible variety at the early time points after agroinfection. Whilst the resistant variety displayed synthesis but rapid degradation of the early viral RNAs; the degradation in the susceptible variety was delayed resulting in accumulation of those transcripts later in infection. Accumulation of the late viral transcripts and DNA replication were detectable only in the susceptible variety. This indicates that rapid degradation of the early viral transcripts, possibly through siRNA mechanism, is one of the probable mechanisms of natural resistance against geminivirus.

Regulation of Apetala2/Ethylene Response Factors in Plants

Multiple environmental stresses affect growth and development of plants. Plants try to adapt under these unfavorable condition through various evolutionary mechanisms like physiological and biochemical alterations connecting various network of regulatory processes. Transcription factors (TFs) like APETALA2/ETHYLENE RESPONSE FACTORS (AP2/ERFs) are an integral component of these signaling cascades because they regulate expression of a wide variety of down stream target genes related to stress response and development through different mechanism. This downstream regulation of transcript does not always positively or beneficially affect the plant but also they display some developmental defects like senescence and reduced growth under normal condition or sensitivity to stress condition. Therefore, tight auto/cross regulation of these TFs at transcriptional, translational and domain level is crucial to understand. The present manuscript discuss the multiple regulation and advantage of plasticity and specificity of these family of TFs to a wide or single downstream target(s) respectively. We have also discussed the concern which comes with the unwanted associated traits, which could only be averted by further study and exploration of these AP2/ERFs.

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition.