RT-qPCR validation of mRNA sequencing and time course analysis
To validate mRNA-sequencing results and to investigate the temporal
dynamics of PA-IR, twelve genes were chosen for RT-qPCR analysis in a
time-course experiment. These genes were selected based on differential
expression after PA treatment in the mRNA-sequencing dataset, and
because they covered most pathways or groups of functionally related
genes identified in the mRNA-sequencing data (see Supplementary
Table S1 ).
Gene expression was measured at three time points (6, 24 and 72 hours
after PA treatment) in root and shoot to study the spatiotemporal
evolution of the plant response to PA (Figure 4) . There was
significant agreement between mRNA-seq and RT-qPCR data at 24 hours
(Spearman’s ρ = 0.57, P = 0.003; Supplementary Figure S5 ),
which supports the validity of our mRNA-sequencing results.
For most genes assayed via RT-qPCR, PA-treated shoot samples showed the
highest differential expression after 6 and 24 hours. Root samples
showed a smaller fold change at 6 hours, followed by greater differences
after 24 and 72 hours. This hints at a temporal difference in gene
expression changes after PA treatment, with (directly treated) shoots
showing a more rapid response than (systemic) roots.