Project description:Backgropund:In a major paradigm shift in the last decade, the knowledge about a whole class of non-coding RNAs known as miRNAs has emerged, which have proved these to be important regulators of a wide range of cellular processes by the way of modulation of gene expression. It is reported that some of these miRNAs are modified by addition or deletion of nucleotides at their ends, after biogenesis. However, the biogenesis and functions of these modifications are not well studied in eukaryotes, especially in plants. In this study, we examined the miRNA modifications in different tissues of the various plants, namely rice, tomato and Arabidopsis and identified some common features of such modifications. Results:We have analyzed different aspects of miRNA modifications in plants. To achieve this end, we developed a PERL script to find the modifications in the sequences using small RNA deep sequencing data. The modification occurs in both mature and passenger (star) strands, as well as at both the 5' and 3' ends of miRNAs. Interestingly, we found a position-specific nucleotide biased modification, as evident by increased number of modification at the 5' end with the presence of Cytosine (nucleotide 'C') at the 3âend of the miRNA sequence. The level of modifications is not strictly dependent on the abundance of miRNA. Our study showed that the modification events are independent of plant species, tissue and physiological conditions. Our analysis also indicates that the RNAi enzyme, namely, the RNA dependent RNA polymerase 6 (RDR6) may not have any role in Arabidopsis miRNA modifications. Some of these modified miRNAs are bound to AGO1, suggesting their possible roles in biological processes. Conclusions:This is a first report that reveals that 5' nucleotide additions are preferred for mature miRNA sequences with 3â terminal âCâ nucleotide. Our analysis also indicates that the miRNAs modifications involving addition of nucleotides to the 5â or 3â end are independent of RDR6 activity and are not restricted by plant species, physiological conditions and tissue types. The results also indicate that such modifications might be important for biological processes. small RNA profiles of Healthy and ToLCV infected tomato leaves were generated by deep sequencing using Illumina GAII.
Project description:Backgropund:In a major paradigm shift in the last decade, the knowledge about a whole class of non-coding RNAs known as miRNAs has emerged, which have proved these to be important regulators of a wide range of cellular processes by the way of modulation of gene expression. It is reported that some of these miRNAs are modified by addition or deletion of nucleotides at their ends, after biogenesis. However, the biogenesis and functions of these modifications are not well studied in eukaryotes, especially in plants. In this study, we examined the miRNA modifications in different tissues of the various plants, namely rice, tomato and Arabidopsis and identified some common features of such modifications. Results:We have analyzed different aspects of miRNA modifications in plants. To achieve this end, we developed a PERL script to find the modifications in the sequences using small RNA deep sequencing data. The modification occurs in both mature and passenger (star) strands, as well as at both the 5' and 3' ends of miRNAs. Interestingly, we found a position-specific nucleotide biased modification, as evident by increased number of modification at the 5' end with the presence of Cytosine (nucleotide 'C') at the 3âend of the miRNA sequence. The level of modifications is not strictly dependent on the abundance of miRNA. Our study showed that the modification events are independent of plant species, tissue and physiological conditions. Our analysis also indicates that the RNAi enzyme, namely, the RNA dependent RNA polymerase 6 (RDR6) may not have any role in Arabidopsis miRNA modifications. Some of these modified miRNAs are bound to AGO1, suggesting their possible roles in biological processes. Conclusions:This is a first report that reveals that 5' nucleotide additions are preferred for mature miRNA sequences with 3â terminal âCâ nucleotide. Our analysis also indicates that the miRNAs modifications involving addition of nucleotides to the 5â or 3â end are independent of RDR6 activity and are not restricted by plant species, physiological conditions and tissue types. The results also indicate that such modifications might be important for biological processes. small RNA profiles of wild type and RDR6 (-) of Arabidopsis plants were generated using deep sequencing data.
Project description:Backgropund:In a major paradigm shift in the last decade, the knowledge about a whole class of non-coding RNAs known as miRNAs has emerged, which have proved these to be important regulators of a wide range of cellular processes by the way of modulation of gene expression. It is reported that some of these miRNAs are modified by addition or deletion of nucleotides at their ends, after biogenesis. However, the biogenesis and functions of these modifications are not well studied in eukaryotes, especially in plants. In this study, we examined the miRNA modifications in different tissues of the various plants, namely rice, tomato and Arabidopsis and identified some common features of such modifications. Results:We have analyzed different aspects of miRNA modifications in plants. To achieve this end, we developed a PERL script to find the modifications in the sequences using small RNA deep sequencing data. The modification occurs in both mature and passenger (star) strands, as well as at both the 5' and 3' ends of miRNAs. Interestingly, we found a position-specific nucleotide biased modification, as evident by increased number of modification at the 5' end with the presence of Cytosine (nucleotide 'C') at the 3âend of the miRNA sequence. The level of modifications is not strictly dependent on the abundance of miRNA. Our study showed that the modification events are independent of plant species, tissue and physiological conditions. Our analysis also indicates that the RNAi enzyme, namely, the RNA dependent RNA polymerase 6 (RDR6) may not have any role in Arabidopsis miRNA modifications. Some of these modified miRNAs are bound to AGO1, suggesting their possible roles in biological processes. Conclusions:This is a first report that reveals that 5' nucleotide additions are preferred for mature miRNA sequences with 3â terminal âCâ nucleotide. Our analysis also indicates that the miRNAs modifications involving addition of nucleotides to the 5â or 3â end are independent of RDR6 activity and are not restricted by plant species, physiological conditions and tissue types. The results also indicate that such modifications might be important for biological processes. Total 14 samples were analyzed, each in a pair of control and heat stress.
Project description:Gene-to-gene coexpression analysis is a powerful approach to infer function of uncharacterized genes. To perform non-targeted coexpression analysis of tomato genes, we collected a developmental gene expression dataset using various tissues of tomato plant. Expression data are collected from 24 different tissue types including root, hypocotyl, cotyledon, leaf at different stages, and fruit tissues at 4 different ripening stages from 4 different Solanum lycopersicum cultivars. Fruits were separated to the flesh and the peel. These two tissue types indeed showed remarkably different gene expression profiles. We also collected data from 4 different ripening stages (mature green, yellow, orange, and red) to detail the changes during ripening. By using this gene expression dataset, we calculated pair-wise Pearsonâs correlation coefficients, and performed network-based coexpression analysis. The analysis generated a number of coexpression modules, some of which showed an enrichment of genes associated with specific functional categories. This result will be useful in inferring functions of uncharacterized tomato genes, and in prioritizing genes for further experimental analysis. We used Affymetrix GeneChip Tomato genome Arrays to detail the global gene expression change using 24 different tomato tissue types (67 hybridizations). We collected gene expression data from 24 different tomato tissue types using 67 hybridizations. Root, hypocotyl, cotyledon, and leaf were sampled from 3-week-old or 5-weekâold plant of Solanum lycopersicum cultivar Micro-Tom. Fruit tissues were sampled from S. lycopersicum cultivars Micro-Tom, Anthocyanin fruit (Aft, LA1996), Line27859, and Momotaro 8 (Takii, Japan). From Micro-Tom fruit, the peel and the flesh were separately sampled from 4 different ripening stages: mature green (MG, approximately 30 day after anthesis), yellow (Y, approximately 35 days after anthesis), orange (O, approximately 38-40 days after anthesis), and red (R, approximately 45-48 days after anthesis). From fruits of Aft and Line27859, the peel and the flesh were sampled at mature green (MG, approximately 40 days after anthesis) and red (R, approximately 50-55 days after anthesis) stages. From Momotaro 8, the peel and the flesh were sampled at red (R, 50- approximately 50-55 days after anthesis) stages. For each tissue type, 2-4 biological replicates were made in RNA preparation.
Project description:Photosynthesis is affected by water deficiency (WD) stress, and nitric oxide (NO) is a free radical that participates in the photosynthesis process. Previous studies have suggested that NO regulates the excitation energy distribution of photosynthesis under WD stress. Here, quantitative phosphoproteomic profiling was conducted using isobaric tags for relative and absolute quantitation. Differentially phosphorylated protein species (DEPs) were identified in leaves of NO or polyethylene glycol (PEG)-treated wheat seedlings (D) and in control seedlings, 2,257 unique phosphorylated peptides and 2,416 phosphorylation sites were identified from 1,396 unique phosphoproteins. Of these, 96 DEPs displayed significant changes (≥ 1.50-fold, p < 0.01). These DEPs are involved in photosynthesis and signal transduction, etc. Furthermore, phosphorylation of several DEPs were up-regulated by both D and NO treatments, but down-regulated only in NO treatment. These differences affected the chlorophyll A-B binding protein, chloroplast post-illumination chlorophyll fluorescence increase protein, and SNT7, implying that NO indirectly regulated the absorption and transport of light energy in photosynthesis in response to WD stress. The significant difference of chlorophyll (Chl) content, Chl a fluorescence transient, photosynthesis index, and trapping and transport of light energy further indicated that exogenous NO under D stress enhanced the primary reaction of photosynthesis compared to D treatment. A putative pathway is proposed to elucidate NO regulation of the primary reaction of photosynthesis under WD.
Project description:Salt stress causes the quality change and significant yield loss of tomato. However, the resources of salt-resistant tomato were still deficient and the mechanisms of tomato resistance to salt stress were still unclear. In this study, the proteomic profiles of two salt-tolerant and salt-sensitive tomato cultivars were investigated to deciphered the salt-resistance mechanism of tomato and provide novel resources for tomato breeding. We found that there is an over-abundant proteins relevant to Nitrate and amino acids metabolisms in the Salt-tolerant cultivars. The significant increase in expression of proteins involved in Brassinolides and GABA biosynthesis were verified in salt-tolerant cultivars, strengthening the salt resistance of tomato. Meanwhile, salt-tolerant cultivars with higher abundance and activity of antioxidant-related proteins have more advantages in dealing with reactive oxygen species caused by salt stress. And the salt-tolerant cultivars had higher photosynthetic activity based on overexpression of proteins functioned in chloroplast, guaranteeing the sufficient nutrient for plant growth under salt stress. Furthermore, three key proteins were identified as important salt-resistant resources for breeding salt-tolerant cultivars, including Sterol side chain reductase, gamma aminobutyrate transaminase and Starch synthase. Our results provided series valuable strategies for salt-tolerant cultivars which can be used in future
Project description:A comparative study ware made to know the abiotic stress tolerance machanism between tolerant and susceptible plants at flowering stage. The tolerance in response to abiotic stresses are sum of expression of thousands of genes at a particular stage. Tomato plants were exposed to drought and heat stress for RNA extraction and hybridization on Affymetrix microarrays. To study the molecular mechanism of abiotic stress tolerance to increase the tolerance in tomato plants, transcripts of tolerant and susceptible plants at flowering stage were compared in response to heat and water stress.
Project description:Tomato pericarp tissues at three different stages (green, breaker and ripening) of fruit development from both the above genotypes were collected using a sterile surgical blade and flash frozen in liquid nitrogen. High quality RNA was extracted from the frozen pericarp tissue using TRI Reagent (Ambion, INC. USA) and pooled at least from three independent samples and treated with DNase-I (QIAGEN GmbH, Germany). RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5ug of total RNA from each sample with three biological replications were reverse-transcribed to double stranded cDNA using the GeneChipR One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipR IVT Labelling Kit (Affymetrix, CA, USA). 15 ug of cRNA samples was fragmented and was hybridized for 16 hours at 45C to the Affymetrix tomato GeneChip Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the GenechipR Fluidics Station 450, the arrays were scanned by the GenechipR 3000 Scanner. The chip images were scanned and extracted using default settings and the .CEL files were produced by using the Affymetrix GeneChip Operating Software (GCOS 1.2). Samples that passed through various quality checks (data not shown) were normalized with RMA algorithm using GeneSpring GX 10 (Agilent Technologies Inc., Santa Clara, CA, USA). Probes showing twofold up- or down-regulation (in Log2 transformed values) compared to corresponding control condition were taken only into consideration for further analysis
Project description:A comparative study ware made to know the Early Blight resistance mechanism between resistant and susceptible plants at vegetative stage. Transcriptional changes during compatible and resistant reaction between A. solani and tomato genotypes have been identified using Affymetrix tomato gene chips. Results were confirmed by reverse transcription-polymerase chain reaction with the samples collected after 24hrs of inoculation from inoculated and control susceptible and resistant genotypes. The number of up and down regulated genes in three different combinations of treatments i.e. Early Blight Resistant Treated and Control (EBTT and EBTC) and Early Blight Suceptible Treated and Control (EBST and EBSC) has observed and compared. In EBST vs. EBSC condition, 3478 genes are up regulated and 2663 are down regulated. Similarly when analysis is done between EBST and EBRT, 2475 genes are up regulated and 3662 are down regulated. Total 3442 genes are up regulated and 2695 genes are found to be down regulated in resistant line of tomato for early blight when the analysis is done for EBRT Vs. EBRC. These genes, that are induced in the resistant genotype EC-520061(Solanum habrochaites , wild) of tomato, after Alternaria solani infection, have been classified in to following categories, response to stress, cellular component, Metabolic activity, regulation of transcription, regulation of translation, and few were unknown. Data from this study are a resource for understanding the mechanism, which works for resistance against pathogen of early blight in tomato, which is critical and will be useful in improving this trait through transgenic approach or gene silencing. Tomato plants were exposed to A. solani spores and after 24 hrs (at the stage of penetration of tissues with fungal hyphae) samples were collected from both susceptible and resistant genotypes for RNA extraction and hybridization on Affymetrix chips. To study the molecular mechanism of biotic stress resistance, transcripts of both the genotypes were compared in response to A. solani.