3.10 Comparative expression of selected DE transcripts in WT andStpfld252 leaves
The expression of several DE genes was evaluated by qRT-PCR to validate
the results obtained with the RNA microarray experiments. Genes were
selected on the basis of their stress responses. Out of 10 DE genes
evaluated, 4 showed a strict correlation between the microarray and
qRT-PCR data, including the FC, whereas other 5 displayed similar
expression patterns in both procedures, but quantitative differences in
FC were observed between them (Figure 7). In all 5 cases, the observed
differences did not modify cluster assignment.
Chloroplast-targeted Fld prevented the drought-dependent repression of
photosynthetic genes to various extents (Figures 5, 6). The PSII core
complex protein psbY and the photosynthetic Fd isoform 1 were
down-regulated in WT leaves under water limitation, as members of
cluster 7 (Figure 5). While expression patterns of the two genes
determined by microarray analysis and qRT-PCR were similar, the latter
procedure gave significantly lower FC values for drought-dependent
repression in the wild type (Figure 7). Instead, the two methods yielded
a good quantitative correlation for the PSI component psaK belonging to
cluster 12 (Figure 6b). Cluster 7 also contained genes encoding two
stress-responsive enzymes of central metabolism regarded as drought
molecular markers: a granule-bound starch synthase and nitrate reductase
3 (Azedo-Silva, Osório, Fonseca & Correia 2004). Microarray results for
both genes were quantitatively validated by qRT-PCR (Figure 7).
Genes primed by Fld expression included the drought-associated
transcription factor ATHB7 (cluster 15, Figure 6c), which has been
reported to promote stomatal closure among other abiotic stress
responses (Ré, Capella, Bonaventure & Chan 2014). The ATHB7 gene
was induced by Fld in the absence of stress and by drought in both
lines, displaying one of the highest FC values (Figure 7). The opposite
behavior (e.g. , Fld- and drought-dependent repression) was
exhibited by a gene encoding an aminocyclopropane-1-carboxylate (ACC)
oxidase involved in ethylene biosynthesis, which was a member of cluster
17 (Figure 6c). In both cases, microarray analysis and qRT-PCR displayed
quantitative differences that did not modify cluster assignment. Another
gene displaying a large FC in the transcriptional profiling wasGAST1 , encoding a protein involved in plant development and used
as a molecular marker to monitor changes in endogenous active
gibberellin levels (Shi & Olszewski 1998). Stress-dependent repression
in both WT and Stpfld leaves matches the patterns observed in
cluster 2 (Supplementary Figure S5), although lower FC values were
obtained for the drought condition in the qRT-PCR experiment compared to
the microarray (Figure 7).
Further, two genes encoding components of the proteasomal system
(cluster 3, Supplementary Figure S6) were evaluated: subunit PSα2β,
which contributes to the assembly of the 20S proteasome core complex,
and RPN9b, a regulatory subunit of the 26S proteasome that is involved
in the ATP-dependent degradation of ubiquitinated proteins (Geng, Wenzel
& Tansey 2012). Whilst the RPN9b-encoding transcripts showed a good
quantitative correlation between the microarray and qRT-PCR assays,
those corresponding to PSα2β would instead be allocated to cluster 34
instead of cluster 3 on the basis of the qRT-PCR assay (Figure 7).
Noteworthy, the change in cluster assignment was caused by a single
significant difference in the FC obtained for stressed WT plants (Figure
7).