Shrikant Mantri

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.

Genome wide expression profiling of two accession of G. herbaceum L. in response to drought

BackgroundGenome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of Gossypium herbaceum, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roches GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions.ResultsFourteen accessions of Gossypium herbaceum were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roches GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms responses to various abiotic stresses were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death.ConclusionThe comparative genome-wide gene expression profiling study of two accessions of G.herbaceum under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.

Development and characterization of genomic and expressed SSRs for levant cotton ( Gossypium herbaceum L.)

Four microsatellite-enriched genomic libraries for CA(15), GA(15), AAG(8) and ATG(8) repeats and transcriptome sequences of five cDNA libraries of Gossypium herbaceum were explored to develop simple sequence repeat (SSR) markers. A total of 428 unique clones from repeat enriched genomic libraries were mined for 584 genomic SSRs (gSSRs). In addition, 99,780 unigenes from transcriptome sequencing were explored for 8,900 SSR containing sequences with 12,471 expressed SSRs. The present study adds 1,970 expressed SSRs and 263 gSSRs to the public domain for the use of genetic studies of cotton. When 150 gSSRs and 50 expressed SSRs were tested on a panel of four species of cotton, 68 gSSRs and 12 expressed SSRs revealed polymorphism. These 200 SSRs were further deployed on 15 genotypes of levant cotton for the genetic diversity assessment. This is the first report on the successful use of repeat enriched genomic library and expressed sequence database for microsatellite markers development in G. herbaceum.

Targeting MicroRNAs in Prevention and Treatment of Neurodegenerative Disorders

Preclinical Research

Tissue specific transcript profiling of wheat phosphate transporter genes and its association with phosphate allocation in grains.

Approaches enabling efficient phosphorus utilization in crops are of great importance. In cereal crop like wheat, utilization of inorganic phosphate (Pi) is high and mature grains are the major sink for Pi utilization and storage. Research that addresses the importance of the Pi homeostasis in developing grains is limited. In an attempt to understand the Pi homeostasis in developing wheat grains, we identified twelve new phosphate transporters (PHT), these are phyologentically well distributed along with the members reported from Arabidopsis and rice. Enhanced expression of PHT1-subfamily genes was observed in roots subjected to the Pi starvation suggesting their active role in Pi homeostasis. Differential expression patterns of all the PHT genes during grain filling stages suggested their importance in the filial tissues. Additionally, high accumulation of Pi and total P in aleurone correlates well with the expression of TaPHTs and other phosphate starvation related genes. Tissue specific transcript accumulation of TaPHT1.1, TaPHT1.2, TaPHT1.4 in aleurone; TaPHT3.1 in embryo and TaPHT4.2 in the endosperm was observed. Furthermore, their transcript abundance was affected in low phytate wheat grains. Altogether, this study helps in expanding the knowledge and prioritize the candidate wheat Pi-transporters to modulate the Pi homeostasis in cereal grains.

Isomalto-oligosaccharides, a prebiotic, functionally augment green tea effects against high fat diet-induced metabolic alterations via preventing gut dysbacteriosis in mice

&NA; High fat diet (HFD)‐induced alterations in gut microbiota and resultant ‘leaky gut’ phenomenon promotes metabolic endotoxemia, ectopic fat deposition, and low‐grade systemic inflammation. Here we evaluated the effects of a combination of green tea extract (GTE) with isomalto‐oligosaccharide (IMOs) on HFD‐induced alterations in mice. Male Swiss albino mice were fed with HFD (58% fat kcal) for 12 weeks. Systemic adiposity, gut derangement parameters and V3‐V4 region based 16S rRNA metagenomic sequencing, ectopic fat deposition, liver metabolome analysis, systemic and tissue inflammation, and energy homeostasis markers along with gene expression analysis in multiple tissues were done in mice supplemented with GTE, IMOs or their combination. The combination of GTE and IMOs effectively prevented HFD‐induced adiposity and lipid accumulation in liver and muscle while normalizing fasting blood glucose, insulin, glucagon, and leptin levels. Co‐administration of GTE with IMOs effectively modulated liver metabolome associated with lipid metabolism. It also prevented leaky gut phenotype and HFD‐induced increase in circulating lipopolysaccharides and pro‐inflammatory cytokines (e.g. resistin, TNF‐&agr;, and IL‐1&bgr;) and reduction in anti‐inflammatory cytokines (e.g. adiponectin and IL‐6). Gene expression analysis across multiple tissues further supported these functional outcomes. Most importantly, this combination improved beneficial gut microbiota (Lactobacillus sp., Bifidobacteria, Akkermansia muciniphila, Roseburia spp.) abundances, restored Firmicutes/Bacteriodetes and improved Prevotella/Bacteroides proportions. In particular, a combination of these two agents has shown improved beneficial effects on multiple parameters studied. Data presented herein suggests that strategically chosen food components might be highly effective in the prevention of HFD‐induced alterations and may further be developed as functional foods. Graphical abstract Figure. No caption available. Preventive effects of co‐administration of GTE with IMOs against HFD‐induced alterations in mice. Administration of GTE with IMOs to HFD‐fed mice prevented diet induced pathologies across multiple organ system. Its supplementation prevented adipose tissue mass building and prevented systemic obesity. It also prevented HFD‐induced alteration in insulin, glucagon and leptin levels. In liver, GTE with IMOs most effectively prevented HFD‐induced lipid and glucose metabolism aberrations, inflammation (via NF‐kB pathway activation) and metabolites patterns. Systemically, it prevented HFD‐induced increase in pro‐inflammatory cytokines and reduction in anti‐inflammatory adipokines. Its supplementation also prevented HFD‐induced impairment in glucose tolerance and in turn improved insulin sensitivity. It also prevented HFD‐induced gut microbial dysbiosis along with structural damages and SCFAs production. A reduced lipopolysaccharide production further assisted towards improved insulin sensitivity and reduced systemic inflammation.

Genome-wide identification and expression characterization of ABCC-MRP transporters in hexaploid wheat

The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots (TaABCC2, TaABCC3, and TaABCC11 and TaABCC12), stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds (TaABCC6, TaABCC8, TaABCC12, TaABCC13, and TaABCC17) implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily.

A T9G Mutation in the Prototype TATA-Box TCACTATATATAG Determines Nucleosome Formation and Synergy with Upstream Activator Sequences in Plant Promoters

We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-box TCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterized mutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhanced transcription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not light dependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box, suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed.

Comparative transcriptional profiling of two wheat genotypes, with contrasting levels of minerals in grains, shows expression differences during grain filling.

Wheat is one of the most important cereal crops in the world. To identify the candidate genes for mineral accumulation, it is important to examine differential transcriptome between wheat genotypes, with contrasting levels of minerals in grains. A transcriptional comparison of developing grains was carried out between two wheat genotypes- Triticum aestivum Cv. WL711 (low grain mineral), and T. aestivum L. IITR26 (high grain mineral), using Affymetrix GeneChip Wheat Genome Array. The study identified a total of 580 probe sets as differentially expressed (with log2 fold change of ≥2 at p≤0.01) between the two genotypes, during grain filling. Transcripts with significant differences in induction or repression between the two genotypes included genes related to metal homeostasis, metal tolerance, lignin and flavonoid biosynthesis, amino acid and protein transport, vacuolar-sorting receptor, aquaporins, and stress responses. Meta-analysis revealed spatial and temporal signatures of a majority of the differentially regulated transcripts.

PmiRExAt: plant miRNA expression atlas database and web applications

High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database. Database URL: http://pmirexat.nabi.res.in.