RNA isolation and microarray scanning
Root tissue from 15-day-old plants cultivated under hydroponic
conditions was recollected at 48 hours after initiating the
treatments. At least 12 plants per line and treatment were pooled to
perform 3 biological replicates. Leaves were immersed in liquid
nitrogen, homogenized to a fine powder, and stored at -80º C. The total
RNA of about 100 mg of leaf powder for each biological replicate was
extracted using the Maxwell® plant RNA kit (Promega Corporation, WI,
USA) following the manufacturer’s instructions. To ensure a high quality
and quantity RNA material, Experion RNA Analysis Kit (Biorad) was
performed. After quality control, total RNA was tag, hybridized and
cleaning using GeneChip® 3’ IVT Express Kit. Microarray expression
analysis were performed by Affymetrix GeneChip™ Arabidopsis Genome ATH1
Array Agilent 2100 Bioanalyzer.
Differential expression analysis: The function gcrma(package gcrma) was used to adjust background intensities in Affymetrix
array data, which include optical noise and non-specific binding. The R
package ‘limma’ was used to test the expression data and search for
differentially expressed genes (DEGs) when pairs of experimental groups
were compared. A model matrix was defined by specifying the groups that
belonged to each sample. Then, limma’s functions lmFit andcontrasts.fit , together with the model matrix, were used to
adjust the expression data to a linear model in order to extract fold
changes and confidence statistics associated to the comparisons of
interest. Function eBayes was then used to rank genes in order of
evidence for differential expression. The R libraryath1121501.db was used to include names and descriptions of the
genes associated to the microarray’s features probed. The resulting
p-values were adjusted using the Benjamini and Hochberg’s approach
[31] for controlling the false discovery rate (FDR). To perform a
list of DEGs, genes were filtered by adj p-value (adjusted p-value)
<0.05 and LFC >1 and LFC <-1.
Gene ontology, KEGG pathway and functional protein association
networks analysis: Gene Ontology (GO) enrichment analysis of DEGs was
implemented by AgriGO V2 [32] and FLAME [33]. Significant GO
terms (p-values less than 0.05) were classified into 3 categories:
biological function, molecular process, and cellular component. KEGG
pathway and STRING version 11.0 were used to understand high-level
function and gene interaction network from differential expressed genes
[34].