Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea. Identification of microRNAs and phased small inferfering RNAs in chickpea (Cicer arietinum) by analyzing small RNA sequencing profiles of leaves and flowers using Illumina GAII.
Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea.
Project description:Chickpea (Cicer arietinum L.) is an important pulse crop grown mainly in the arid and semi-arid regions. Due to its taxonomic proximity with the model legume Medicago truncatula and its ability to grow in arid soil, chickpea has its unique advantage to understand how plant responds to drought stress. This microarray was used for analyze the transcriptomic profiles of unigenes in leaf and root of chickpea seedling under drought stress, respectively. Microarray data showed that 4805 differentially expressed unigenes were either greater than 2 fold up- or less than 0.5 fold down-regulated in at least one of the 5 time points during drought stress. 2003 and 3488 unigenes were time-dependent differentially expressed in root and leaf, respectively. 110 pathways in two tissues were found to respond to drought stress. Compared to control, 88 and 52 unigenes were expressed only in drought-stressed root and leaf, respectively, while nine genes were expressed in both tissues. 2484 function-unknown unigenes were found to be remarkably regulated by drought stress. The expression profiles of these time-dependent differentially expressed unigenes were useful in furthering our knowledge of the drought tolerance mechanism of plant.
Project description:Chickpea (Cicer arietinum L.) seed proteins show a lot of functional properties leading this leg-ume an interesting component for the development of protein-enriched foods. However, both in-depth proteomic investigation and structural characterization of chickpea proteins seed are still lacking. In this paper we report a detailed characterization of chickpea seed protein fraction by means SDS-PAGE, in-gel protein digestion, high-resolution mass spectrometry, and database searching. By this approach twenty SDS gel bands were cut and analysed. While the majority of bands and the identified peptides were related to vicilin and legumin storage proteins, also metabolic functional proteins were detected. Legumins, as expected, were revealed at 45÷65 kDa, as whole subunits with the α- and β-chains linked together by a disulphide bond, but also at lower mass ranges (α- and β-chains migrating alone). Similarly, but not expected, also the vi-cilins were spread along the mass region between 65 and 23 kDa, with some of them identified in several bands. In-depth MS structural characterization allowed to determine that, although chickpea vicilins were always described as proteins lacking of cysteine residues, they contain this amino acid residue. Moreover, similarly to legumins, these storage proteins are firstly syn-thesized as pre-propolypeptides (Mr 50÷80 kDa), that may undergo to proteolytic steps that cut not only the signal peptides but also produce different subunits having lower molecular masses. Overall, about 360 different proteins specific of the Cicer arietinum L. species were identified and characterized, a result that up to date represents the most detailed description of seed’s proteome of this legume.
Project description:Drought is one of the major constraints for crop productivity across the globe. Chickpea (Cicer arietinum L.) is mainly cultivated in the arid and semi-arid tropical regions under rain-fed conditions and drought stress is one of the major constraints, which causes up to 50% yield losses annually. In this study, transcriptomics, proteomics and metabolomics datasets from root tissues of contrasting drought responsive chickpea genotypes, ICC 4958 (drought-tolerant), JG 11 (drought-tolerant); an introgression line, JG 11+ (drought-tolerant) and ICC 1882, (drought-sensitive) under control and stress conditions were generated. The integrated analysis of these multi-omics data revealed complex molecular mechanism underlying drought stress response in chickpea. Transcriptomics integrated with proteomics data identified enhancement of hub proteins encoding isoflavone 4’-O-methyltransferase (Ca_06356), UDP-D-glucose/UDP-D-Galactose 4-epimerase (Ca_15037) and delta-1-pyrroline-5-carboxylate synthesis (Ca_24241). These proteins highlighted the involvement of critical pathways such as antibiotic biosynthesis, galactose metabolism and isoflavonoid biosynthesis in activating drought stress response mechanism. Subsequently, integration of metabolomics data identified six key metabolites (fructose, galactose, glucose, myo-inositol, galactinol and raffinose) that showed enhanced correlation with galactose metabolism. Further, integration of root -omics data together with genomic dataset of the “QTL-hotspot” region harbouring several drought tolerance related traits revealed involvement of candidate genes encoding aldo keto reductase family oxidoreductase (Ca_04551) and leucine rich repeat extensin 2 (Ca_04564). These results from integrated multi-omics approach provided a comprehensive understanding and new insights into the drought stress response mechanism of chickpea.
Project description:Background The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on RocheM-bM-^@M-^Ys 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. Results We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress. Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. Conclusions This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26bp tags by SuperSAGE. SuperSAGE libraries from chickpea roots and nodules were constructed starting from hydro aeroponics-generated material. Control and stressed plants were grown in parallel Sequencing Instrument: First gerenration 454-Machine Place of sequencing: 454 Life Sciences, Branford, CT, USA