Mehar Hasan Asif

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.

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.

De Novo Assembly, Functional Annotation and Comparative Analysis of Withania somnifera Leaf and Root Transcriptomes to Identify Putative Genes Involved in the Withanolides Biosynthesis

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

Differential expression and alternative splicing of rice sulphate transporter family members regulate sulphur status during plant growth, development and stress conditions

Sulphur, an essential nutrient required for plant growth and development, is mainly taken up by the plants as inorganic sulphate from the soil and assimilated into the sulphur reductive pathway. The uptake and transport of sulphate in plants is carried out by transporters encoded by the sulphate transporter gene family. Plant sulphate transporters have been classified with respect to their protein sequences, kinetic properties and tissue-specific localization in Arabidopsis. Though sulphate transporter genes from few other plants have also been characterized, no detailed study with respect to the structure and expression of this family from rice has been carried out. Here, we present genome-wide identification, structural and expression analyses of the rice sulphate transporter gene family. Our analysis using microarray data and MPSS database suggests that 14 rice sulphate transporters are differentially expressed during growth and development in various tissues and during biotic and abiotic stresses. Our analysis also suggests differential accumulation of splice variants of OsSultr1;1 and OsSultr4;1 transcripts during these processes. Apart from known spliced variants, we report an unusual alternative splicing of OsSultr1;1 transcript related to sulphur supply in growth medium and during stress response. Taken together, our study suggests that differential expression and alternative splicing of members of the sulphate transporter family plays an important role in regulating cellular sulphur status required for growth and development and during stress conditions. These findings significantly advance our understanding of the posttranscriptional regulatory mechanisms operating to regulate sulphur demand by the plant.

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.

Prolyl-4-hydroxylase (AtP4H1) mediates and mimics low oxygen response in Arabidopsis thaliana

Proline hydroxylation is an important phenomenon of a living cell. Prolyl-4-hydroxylases (P4H) responsible for this process have been characterized from animals, and one of its forms, HIF-P4H, is regarded as an oxygen sensor. In plants, P4H has been partially characterized from few species, and one of the Arabidopsis P4H (AtP4H1) has been shown to hydroxylate proline-rich peptides in vitro. In order to study its function in planta, we have overexpressed AtP4H1 in Arabidopsis. The AtP4H1oexp plants showed hypoxia-in-normoxia phenotype with strict requirement for carbon source for its growth, increased root hair, absence of trichome, and reduction in seed size. Genome-wide expression analyses suggest that expression of several genes related to hypoxia as well as plant growth and development are upregulated in AtP4H1oexp lines. Based on our studies on AtP4H1oexp lines, we speculate a direct role of AtP4H1 in hypoxia stress and in different stages of plant growth and development.

Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family from banana suggest involvement of specific members in different stages of fruit ripening

Mitogen-activated protein kinases (MAPKs) are important components of the tripartite mitogen-activated protein kinase signaling cascade and play an important role in plant growth and development. Although members of the MAPK gene family have been identified in model plants, little information is available regarding this gene family in fruit crops. In this study, we carried out a computational analysis using the Musa Genome database to identify members of the MAPK gene family in banana, an economically important crop and the most popular fruit worldwide. Our analysis identified 25 members of the MAP kinase (MAPK or MPK) gene family. Phylogenetic analyses of MPKs in Arabidopsis, Oryza, and Populus have classified these MPKs into four subgroups. The presence of conserved domains in the deduced amino acid sequences, phylogeny, and genomic organization strongly support their identity as members of the MPK gene family. Expression analysis during ethylene-induced banana fruit ripening suggests the involvement of several MPKs in the ethylene signal transduction pathway that are necessary for banana fruit ripening. Analysis of the cis-regulatory elements in the promoter regions and the involvement of the identified MPKs in various cellular processes, as analyzed using Pathway Studio, suggest a role for the banana MPK gene family in diverse functions related to growth, development, and the stress response. This report is the first concerning the identification of members of a gene family and the elucidation of their role in various processes using the Musa Genome database.

Transcriptome analysis of ripe and unripe fruit tissue of banana identifies major metabolic networks involved in fruit ripening process

BackgroundBanana is one of the most important crop plants grown in the tropics and sub-tropics. It is a climacteric fruit and undergoes ethylene dependent ripening. Once ripening is initiated, it proceeds at a fast rate making postharvest life short, which can result in heavy economic losses. During the fruit ripening process a number of physiological and biochemical changes take place and thousands of genes from various metabolic pathways are recruited to produce a ripe and edible fruit. To better understand the underlying mechanism of ripening, we undertook a study to evaluate global changes in the transcriptome of the fruit during the ripening process.ResultsWe sequenced the transcriptomes of the unripe and ripe stages of banana (Musa accuminata; Dwarf Cavendish) fruit. The transcriptomes were sequenced using a 454 GSFLX-Titanium platform that resulted in more than 7,00,000 high quality (HQ) reads. The assembly of the reads resulted in 19,410 contigs and 92,823 singletons. A large number of the differentially expressed genes identified were linked to ripening dependent processes including ethylene biosynthesis, perception and signalling, cell wall degradation and production of aromatic volatiles. In the banana fruit transcriptomes, we found transcripts included in 120 pathways described in the KEGG database for rice. The members of the expansin and xyloglucan transglycosylase/hydrolase (XTH) gene families were highly up-regulated during ripening, which suggests that they might play important roles in the softening of the fruit. Several genes involved in the synthesis of aromatic volatiles and members of transcription factor families previously reported to be involved in ripening were also identified.ConclusionsA large number of differentially regulated genes were identified during banana fruit ripening. Many of these are associated with cell wall degradation and synthesis of aromatic volatiles. A large number of differentially expressed genes did not align with any of the databases and might be novel genes in banana. These genes can be good candidates for future studies to establish their role in banana fruit ripening. The datasets developed in this study will help in developing strategies to manipulate banana fruit ripening and reduce post harvest losses.

Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera.

Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.

Differential transcriptional expression following thidiazuron-induced callus differentiation developmental shifts in rice.

Very little is known about molecular events associated with callus differentiation in indica rice. The genes expressed differentially during shoot meristem initiation were identified on genomic arrays applied to efficiently regenerating rice calli. A thidiazuron (TDZ; N-phenyl-N-thiadiazol-1,2,3-5,ylurea)-dependent regeneration protocol was developed for efficient embryogenesis in indica rice. The regenerating embryogenic calli induced by TDZ for 10 days showed transcriptional modulation of a number of genes associated with photosynthesis, hormone metabolism, plant development, signal transduction, light response, and plant defense. Eighteen candidate miRNAs were predicted to target the genes expressed differentially in the embryogenic calli grown in TDZ-containing medium. The majority of the photosynthesis-related genes up-regulated in differentiating calli were not expressed or were down-regulated in developing seeds and inflorescences. Most of the genes down-regulated in differentiating calli were up-regulated in developing seeds. The transcriptome of differentiating callus most closely resembled that of the germinating whole seed.