Computational protocol: Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

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Protocol publication

[…] Arabidopsis thaliana ROPs, ROP-GEFs, ROP-GAPs, ROP-GDIs, RBOHs, and PLDα were used as BLASTP queries against grape, rice, and poplar sequences in the Ensembl Plants () and in the apple genome () to retrieve putative orthologues. Sequences were aligned by CLUSTALX (), the presence for conserved domains was checked, and rooted phylogenetic trees were generated by the neighbour-joining method (). [...] Total RNA was extracted, reverse transcribed, and used for real-time qPCR experiments as described by . Selective primers were constructed on divergent putative 3’-untranslated regions (UTRs) determined by sequence alignments and poly(A)-tail prediction (HCpolyA; ). Primers (Supplementary Table S1, available at JXB online) were designed with Primer3 () and tested with PRaTo (). Data were elaborated with DataAssist (Applied Biosystems, Monza, Italy) and normalized to Md_8283:1:a () using the method. General good-practice guidelines for RT-qPCR (; ) were adopted and primers efficiencies (Supplementary Table S1) were calculated and considered for differentially expressed genes as described by . The choice of RT-qPCR for gene expression analyses was based on suggestions reported by . All analyses were carried out on three independent biological replicates for each time point and experimental condition. [...] RNA samples were processed using TruSeq (Illumina, San Diego, CA, USA) by a third-party service (IGA Technologies Services, Udine, Italy). Raw data were aligned on the Malus×domestica coding sequence (http://www.phytozome.net/apple.php; release 196) and processed using CLC Bio Genomics Workbench software (CLC Bio, Denmark). Hierarchical clusters and heatmaps were generated using R () with the package gplots (http://CRAN.R-project.org/package=gplots) from RNA-seq data normalized on data at harvest before transformation into logarithmic values. Data were filtered for genes with at least five counts on at least three samples and normalized with the full quantile method (EDASeq; ). Differentially expressed genes were obtained by modelling the count with a negative binomial distribution (edgeR; ). P values were adjusted to control the false discovery rate (FDR) (). For gene co-expression, the Pearson correlation coefficient was calculated adopting a cut-off of 0.95 and applied to a dataset of 21 independent RNA-seq experiments (three biological replicates obtained at harvest, at 1 and 6 months of cold storage in control conditions, or after 1-MCP or DPA treatment). Gene Ontology Enrichment Analysis was carried out with BiNGO () with FDR-adjusted P values (Hypergeometric test). […]

Pipeline specifications

Software tools Primer3, PRaTo, geWorkbench, gplots, EDASeq, edgeR, BiNGO
Databases Phytozome
Applications RNA-seq analysis, qPCR
Organisms Malus domestica, Arabidopsis thaliana
Chemicals Hydrogen Peroxide, NADP, Oxygen