Project description:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.
Project description:Polyphenolic-enriched extract of Prunus domestica skin obtained using Pressurized Liquid Extraction (PLE) in methanol and purified using an Amberlite column
Project description:Purpose: The State of Rio Grande do Sul is the largest producer of peaches from Brazil. However, it still has low values of productivity when compared to other States. One of the problems associated to it this is the occurrence of drainage soils problems, which can suffer flooding situations potentially hampering the development and productivity such culture. For studies to assist in the selection of flood tolerant genotypes, it is essential to understand the physiological and molecular changes of the plants in situations of oxygen deprivation. Using Illumina Hiseq2500 we performed transcriptome analysis of leaves from ‘Capdeboscq’ (Prunus persica) and ‘Julior’ (Prunus insititia x Prunus domestica) rootstocks under flooding for 48 hours. Methods: The mRNA of Prunus spp. plants cv. Capdeboscq e Julior was generated using deep sequencing, in triplicate, using Illumina Hi-Seq 2500, for the following treatments:I) control: plants received irrigation daily until field capacity; and II) plants exposed to flood stress, maintaining a water level of approximately 3 cm above the ground. The sequence reads that passed quality filters were analyzed at the transcript level using this method: Mapping using STAR and identification of differentially expressed genes (DEGs) was performed with the edgeR (false discovery rates - FDRs of <0.05). RT–qPCR validation was performed using SYBR Green assays. Results: Flooding stress causes important high transcriptional changes in the ‘Capdeboscq’ compared to 'Julior' and this is mainly due to their sensitivity/tolerance levels. ‘Capdeboscq’ had photosynthesis as the most affected physiological process at the molecular level, showing a large number of down-regulated enriched GOs, even though it activated cellular signaling pathways under flooding. 'Julior' was more efficient in defense responses, which include the activation of flavonoid biosynthesis pathways. Conclusions: The analysis of two Prunus spp. rootstocks contrasting to the level of tolerance / sensitivity provide new insights into the process of plant flood stress tolerance.
Project description:In this study, we used vascular specific promoters and a translating ribosome affinity purification strategy to identify phloem associated translatomes in Prunus domestica L. Three different promoter:FLAG-RPL18 lines were used. These included two phloem specific promoters (pSUC2 and pSULTR2;2), as well as the more ubiquitously expressed cauliflower mosaic virus 35S promoter (p35S). Immunopurification of ribosome-mRNA complexes was accomplished by the method described in Reynoso et al. (Plant Functional Genomics: Methods and Protocols, 185-207; 2015). The dataset includes samples from plum leaves taken at 2, 4 and 6 weeks post vernalization.