Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Vitis vinifera tissues (including leaves, flowers and berries). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Vitis vinifera tissues (including leaves, flowers and berries). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from leaves flowers and berries of Vitis vinifera. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Gabriele Di Gaspero for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Bud endodormancy induction response of two genotypes (Seyval, a hybrid white wine grape and Vitis riparia, PI588259, a native North American grape species) was compared under long (15 h) and short (13 h) photoperiods. Proteins were extracted from both genotypes for all time points and experimental conditions. The proteins were separaed by 2D-PAGE, trypsin digested, and the peptides identified with a MALDI-TOF-TOF mass spectrometer. A master gel was made and mapped with all proteins from both genotypes. The proteins were identified by matching the peptide sequences against the 8X Vitis vinifera grape genome in NCBI. This study was funded by NSF grant DBI064755 and is the result of a collaboration between Dr. Anne Fennell at South Dakota State University and Dr. Grant R. Cramer at the University of Nevada, Reno.
Project description:Damage-associated molecular patterns (DAMPs) are endogenous molecules that can activate the plant innate immunity. DAMPs can derive from the plant cell wall, which is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides. Fragments of pectin, called oligogalacturonides (OG), can be released after wounding or by pathogen-encoded cell wall degrading enzymes (CWDEs) such as polygalacturonases (PGs). OG are known to induce innate immune responses, including the activation of mitogen-activated protein kinases (MAPKs), production of H2O2, defense gene activation, and callose deposition. Thus, we hypothesized that xyloglucans (Xh), derived from the plant cell wall hemicellulose, could also act as an endogenous elicitor and trigger a signaling cascade similar to OG. Our results indicate that purified Xh elicit MAPK activation and immune gene expression in grapevine (Vitis vinifera) and Arabidopsis (Arabidopsis thaliana) to trigger induced resistance against necrotrophic (Botrytis cinerea) or biotrophic (Hyaloperonospora arabidopsidis) pathogens. Xh also induce resveratrol production in grapevine cell suspension and callose deposition in Arabidopsis which depends on the callose synthase PMR4. In addition, we characterized some signaling components of Xh-induced immunity using Arabidopsis mutants. Our data suggest that Xh-induced resistance against B. cinerea is dependent on the phytoalexin, salicylate, jasmonate, and ethylene pathways.
Project description:MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample.
Project description:Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (Nc). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.
Project description:In this study, we used a cross-species network approach to uncover nitrogen (N)-regulated network modules conserved across a model and a crop species. By translating gene network knowledge from the data-rich model Arabidopsis (Arabidopsis thaliana, ecotype Columbia-0) to a crop, rice (Oryza sativa spp. japonica (Nipponbare)), we identified evolutionarily conserved N-regulatory modules as targets for translational studies to improve N use efficiency in transgenic plants.
Project description:Grape (Vitis vinifera L.) is a fruit tree with high salt tolerance and high nutritional value, medicinal value, and economic value. Suberin in roots is characterized by long-chain fatty acids and is thought to be related to the salt tolerance of grape. The key enzyme in the fatty acid elongation process is β-ketoacyl-CoA synthase (KCS). The function and the regulatory mechanism of VvKCS in response to salt stress in grape are unclear. In this study, VvKCS was isolated from V. vinifera L. A real-time quantitative polymerase chain reaction analysis showed that salt stress enhanced VvKCS transcription levels in grapes. Overexpression of VvKCS increased the tolerance to salt stress in Arabidopsis during the germination and seedling stages. The improved salt tolerance was the result of the combined contributions of multiple mechanisms including the regulation of expression of ion transporters and channels, accumulation of osmotic regulating substances, and maintenance of membrane stability. The results of this study are valuable information on plant salt tolerance and provide a theoretical basis for the molecular mechanism of grape salt tolerance.
Project description:Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific (‘terroir’) differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue.
Project description:Drought stress dramatically affects the growth and development of grapevine (Vitis vinifera) in many areas of the world, and identifying the mechanisms responsible for drought stress responses is therefore of great interest. The transcription factor, VlbZIP30 has been reported to enhance drought resistance in grapevine, however, the regulatory mechanisms mediated by VlbZIP30 remain poorly understood. Here, we performed chromatin immunoprecipitation (ChIP)-seq and RNA-seq analyses to identify the direct VlbZIP30 target genes at a genome-wide scale. The ChIP-seq analysis determined that VlbZIP30 binds to DNA sequences containing an ACGTG core motif, termed a G-box. By combining the ChIP-seq and RNA-seq results, we identified 48 VlbZIP30-induced target genes, related mainly to the gene ontology (GO) terms: nucleic acid-templated transcription, plant hormone and abiotic stimulus. Through ChIP-qPCR analysis, we confirmed that VlbZIP30 binds directly to the promoters of four of the target genes (VvNAC26, VvDHN1, VvGRAS17 and VvVQ6) containing a G-box motif. In addition, overexpression of VlbZIP30 led to less H2O2 accumulation compared with the wild type under drought conditions in both Arabidopsis thaliana and grapevine, indicating a role for VlbZIP30 in reactive oxygen species (ROS) scavenging. In summary, we report that VlbZIP30 promotes the activity of ROS scavenging by directly regulating the expression of downstream target genes, thereby conferring drought resistance. We also reveal that VlbZIP30 directly regulates the expression of a number of target genes (such as VvDHN1, VvGRAS17 and VvVQ6) to improve plant drought resistance.