Vishal Acharya

Genome-Wide Organization and Expression Profiling of the NAC Transcription Factor Family in Potato (Solanum tuberosum L.)

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

Genome-Wide Identification and Expression Analysis of NBS-Encoding Genes in Malus x domestica and Expansion of NBS Genes Family in Rosaceae

Nucleotide binding site leucine-rich repeats (NBS-LRR) disease resistance proteins play an important role in plant defense against pathogen attack. A number of recent studies have been carried out to identify and characterize NBS-LRR gene families in many important plant species. In this study, we identified NBS-LRR gene family comprising of 1015 NBS-LRRs using highly stringent computational methods. These NBS-LRRs were characterized on the basis of conserved protein motifs, gene duplication events, chromosomal locations, phylogenetic relationships and digital gene expression analysis. Surprisingly, equal distribution of Toll/interleukin-1 receptor (TIR) and coiled coil (CC) (1∶1) was detected in apple while the unequal distribution was reported in majority of all other known plant genome studies. Prediction of gene duplication events intriguingly revealed that not only tandem duplication but also segmental duplication may equally be responsible for the expansion of the apple NBS-LRR gene family. Gene expression profiling using expressed sequence tags database of apple and quantitative real-time PCR (qRT-PCR) revealed the expression of these genes in wide range of tissues and disease conditions, respectively. Taken together, this study will provide a blueprint for future efforts towards improvement of disease resistance in apple.

Comparative phylogenetic analysis and transcriptional profiling of MADS-box gene family identified DAM and FLC-like genes in apple (Malusx domestica).

The MADS-box transcription factors play essential roles in various processes of plant growth and development. In the present study, phylogenetic analysis of 142 apple MADS-box proteins with that of other dicotyledonous species identified six putative Dormancy-Associated MADS-box (DAM) and four putative Flowering Locus C-like (FLC-like) proteins. In order to study the expression of apple MADS-box genes, RNA-seq analysis of 3 apical and 5 spur bud stages during dormancy, 6 flower stages and 7 fruit development stages was performed. The dramatic reduction in expression of two MdDAMs, MdMADS063 and MdMADS125 and two MdFLC-like genes, MdMADS135 and MdMADS136 during dormancy release suggests their role as flowering-repressors in apple. Apple orthologs of Arabidopsis genes, FLOWERING LOCUS T, FRIGIDA, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 and LEAFY exhibit similar expression patterns as reported in Arabidopsis, suggesting functional conservation in floral signal integration and meristem determination pathways. Gene ontology enrichment analysis of predicted targets of DAM revealed their involvement in regulation of reproductive processes and meristematic activities, indicating functional conservation of SVP orthologs (DAM) in apple. This study provides valuable insights into the functions of MADS-box proteins during apple phenology, which may help in devising strategies to improve important traits in apple.

Braving the attitude of altitude: Caragana jubata at work in cold desert of Himalaya

The present work was conducted to understand the basis of adaptation in Caragana jubata in its niche environment at high altitude cold desert of Himalaya. Molecular data showed predominance of genes encoding chaperones and those involved in growth and development at low temperature (LT), a major cue operative at high altitude. Importantly, these genes expressed in C. jubata in its natural habitat. Their homologues in Arabidopsis thaliana, Oryza sativa, and Glycine max did not exhibit similar trend of gene expression at LT. Constitutive expression and a quick up-regulation of the above genes suggested the ability of C. jubata to adjust its cellular machinery to maintain growth and development in its niche. This was reflected in LT50 (the temperature at which 50% injury occurred) and LT mediated photosynthetic acclimatory response. Such molecular and physiological plasticity enables C. jubata to thrive in the high altitude cold desert of Himalayas.

Integrated network analysis and logistic regression modeling identify stage-specific genes in Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is associated with substantial mortality and morbidity but, OSCC can be difficult to detect at its earliest stage due to its molecular complexity and clinical behavior. Therefore, identification of key gene signatures at an early stage will be highly helpful.MethodsThe aim of this study was to identify key genes associated with progression of OSCC stages. Gene expression profiles were classified into cancer stage-related modules, i.e., groups of genes that are significantly related to a clinical stage. For prioritizing the candidate genes, analysis was further restricted to genes with high connectivity and a significant association with a stage. To assess predictive power of these genes, a classification model was also developed and tested by 5-fold cross validation and on an independent dataset.ResultsThe identified genes were enriched for significant processes and functional pathways, and various genes were found to be directly implicated in OSCC. Forward and stepwise, multivariate logistic regression analyses identified 13 key genes whose expression discriminated early- and late-stage OSCC with predictive accuracy (area under curve; AUC) of ~0.81 in a 5-fold cross-validation strategy.ConclusionsThe proposed network-driven integrative analytical approach can identify multiple genes significantly related to an OSCC stage; the classification model that is developed with these genes may help to distinguish cancer stages. The proposed genes and model hold promise for monitoring of OSCC stage progression, and our findings may facilitate cancer detection at an earlier stage, resulting in improved treatment outcomes.

Computational Analysis of “-omics” Data to Identify Transcription Factors Regulating Secondary Metabolism in Rauvolfia serpentina

Rauvolfia serpentina has been known to produce therapeutically important indole alkaloids used in treatment of various diseases. Despite its medicinal importance, complete understanding of its secondary metabolism is challenging due to complex interplay among various transcription factors (TFs) and genes. However, weighted co-expression analysis of transcriptome along with integration of metabolomics data has proficiency to elucidate topological properties of complex regulatory interactions in secondary metabolism. We aimed to implement an integrative strategy using “-omics” data to identify TFs of “unknown function” and exemplify their role in regulation of valuable metabolites as well as metabolic traits. A total of 69 TFs were identified through significant thresholds and removal of false positives based on cis-regulatory motif analysis. Network-biology inspired analysis of co-expression network lead to generation of four statistically significant and biologically robust modules. Similar to known regulatory roles of WRKY and AP2-EREBP TF families in Catharanthus roseus, this study presented them to regulate synthesis of alkaloids in R. serpentina as well. Moreover, TFs in module 4 were observed to be regulating connecting steps between primary and secondary metabolic pathways in the synthesis of terpenoid indole alkaloids. Integration of metabolomics data further highlight the significance of module 1 since it was statistically predicted to be involved in synthesis of specialized metabolites, and associated genes may physically clustered on genome. Importantly, putative TFs in module 1 may modulate the major indole alkaloids synthesis in response to various environmental stimuli. The methodology implemented herein may provide a better reference to identify and explore functions of transcriptional regulators.

Genome-wide characterization and expression profiling of TIFY gene family in pigeonpea (Cajanus cajan (L.) Millsp.) under copper stress

Copper is a vital micronutrient for plant growth and development, but in high concentration it leads to the oxidative damage thereby initiating biosynthesis of jasmonic acid (JA). The JA signalling is mediated by various transcription factors (TFs) such as JAZ and MYC2. JAZ TFs are the negative regulators of JA signalling which are degraded in presence of JA. JAZ proteins belong to TIFY family and regulate various biological processes such as plant development, response to phytohormones, biotic and abiotic stresses. In the present study, we identified 18 TIFY family proteins in pigeonpea, which are further classified into TIFY, JAZ and ZML subfamilies. Gene expression of 12 CcTIFY genes in pigeonpea was studied under methyl jasmonate (Me-JA), JA in presence and absence of copper (Cu). Our results showed that the transcript levels of CcTIFY3, CcTIFY4, CcTIFY5, CcTIFY9 and CcTIFY16 were upregulated under Cu stress, indicating their involvement in Cu stress signalling. The presence of defense and stress responsive cis-regulatory elements in the promoter regions of these genes further confirms their involvement in response to Cu stress. Apart from feedback regulation of JAZ proteins, the expression of CcTIFY3 and CcTIFY9 was observed to be upregulated in Me-JA and JA treatment, respectively, which suggest involvement of alternative JA and Me-JA signalling.

Plant STAND P-loop NTPases: a current perspective of genome distribution, evolution, and function

STAND P-loop NTPase is the common weapon used by plant and other organisms from all three kingdoms of life to defend themselves against pathogen invasion. The purpose of this study is to review comprehensively the latest finding of plant STAND P-loop NTPase related to their genomic distribution, evolution, and their mechanism of action. Earlier, the plant STAND P-loop NTPase known to be comprised of only NBS–LRRs/AP–ATPase/NB–ARC ATPase. However, recent finding suggests that genome of early green plants comprised of two types of STAND P-loop NTPases: (1) mammalian NACHT NTPases and (2) NBS–LRRs. Moreover, YchF (unconventional G protein and members of P-loop NTPase) subfamily has been reported to be exceptionally involved in biotic stress (in case of Oryza sativa), thereby a novel member of STAND P-loop NTPase in green plants. The lineage-specific expansion and genome duplication events are responsible for abundance of plant STAND P-loop NTPases; where “moderate tandem and low segmental duplication” trajectory followed in majority of plant species with few exception (equal contribution of tandem and segmental duplication). Since the past decades, systematic research is being investigated into NBS–LRR function supported the direct recognition of pathogen or pathogen effectors by the latest models proposed via ‘integrated decoy’ or ‘sensor domains’ model. Here, we integrate the recently published findings together with the previous literature on the genomic distribution, evolution, and distinct models proposed for functional molecular mechanism of plant STAND P-loop NTPases.

Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat

Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology.

Computational Identification Raises a Riddle for Distribution of Putative NACHT NTPases in the Genome of Early Green Plants

NACHT NTPases and AP-ATPases belongs to STAND (signal transduction ATPases with numerous domain) P-loop NTPase class, which are known to be involved in defense signaling pathways and apoptosis regulation. The AP-ATPases (also known as NB-ARC) and NACHT NTPases are widely spread throughout all kingdoms of life except in plants, where only AP-ATPases have been extensively studied in the scenario of plant defense response against pathogen invasion and in hypersensitive response (HR). In the present study, we have employed a genome-wide survey (using stringent computational analysis) of 67 diverse organisms viz., archaebacteria, cyanobacteria, fungi, animalia and plantae to revisit the evolutionary history of these two STAND P-loop NTPases. This analysis divulged the presence of NACHT NTPases in the early green plants (green algae and the lycophyte) which had not been previously reported. These NACHT NTPases were known to be involved in diverse functional activities such as transcription regulation in addition to the defense signaling cascades depending on the domain association. In Chalmydomonas reinhardtii, a green algae, WD40 repeats found to be at the carboxyl-terminus of NACHT NTPases suggest probable role in apoptosis regulation. Moreover, the genome of Selaginella moellendorffii, an extant lycophyte, intriguingly shows the considerable number of both AP-ATPases and NACHT NTPases in contrast to a large repertoire of AP-ATPases in plants and emerge as an important node in the evolutionary tree of life. The large complement of AP-ATPases overtakes the function of NACHT NTPases and plausible reason behind the absence of the later in the plant lineages. The presence of NACHT NTPases in the early green plants and phyletic patterns results from this study raises a quandary for the distribution of this STAND P-loop NTPase with the apparent horizontal gene transfer from cyanobacteria.