Table 1.
Temporal changes in the number of differentially expressed genes (DEGs) in poplar roots in response to PEG-induced drought stress.
Fig 1.
PageMan display of significantly represented functional categories across different time points in response to PEG treatment.
Differentially expressed genes (DEGs) for various time points displaying significant up- and down-regulation were used for the analysis (see materials and methods). The data were subjected to a Wilcoxon test, and the results are displayed as blue-colored bins (significantly over represented), red-colored bins (significantly under represented) and white-colored bin (not significant). Arrow at right side of heatmap represents average level of significance for all the time points. In the figure, non-significant categories have been collapsed to display only significant functional categories. The main functional categories are displayed in the left whereas specific sub-categories are shown in the right.
Fig 2.
PtaJAZ3 and PtaRAP2.6 overexpression affects root growth in response to PEG-induced drought stress.
(a) Root growth of PtaJAZ3 and PtaRAP2.6 overexpressing transgenics in comparison with those of WT-717 in both control (darker bars) and PEG (lighter bars) media. Three independent lines (four replicates per line, see S3 Fig) were measured for calculating the values of each transgenic modification that is shown as mean ± standard error of mean (SEM) (n = 3). Different letters represent means that are statistically different (P< 0.05) as determined by a one-way ANOVA followed by Tukey’s multiple range tests. (b) Representative photos of roots from the three genotypes grown in PEG liquid media for 40 days (see materials and methods).
Fig 3.
Level of PtaJAZ3 and PtaRAP2.6 transgenic overexpression is correlated to the increased root growth under PEG.
Spearman rank correlation (Rs) between relative transgene expression and total root growth in PtaJAZ3 and PtaRAP2.6 transgenic lines. For correlation analysis, 14 and 25 independent transgenic lines were used for PtaJAZ3-oe and PtaRAP2.6-oe, respectively. Analysis was performed in plant roots growing in liquid media (see materials and methods for details). Total root growth represents sum of main root length and lateral root length for each plant. Transcript abundance and total root growth was analyzed in 4 replicates per transgenic line.
Fig 4.
PtaJAZ3- and PtaRAP2.6-centered networks.
Both the regulatory networks were generated using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE) analysis (see experimental procedures). The two superhubs (squares) were connected to four hub genes (hexagons) which in turn were connected to terminal genes (circles). Abbreviations for the four hubs connected to PtaJAZ3 are as follows: 1. PtaCYCB2;4 (Cycline B2;4, Potri.005G251400); 2. PtaSAUR37 (Small Auxin Upregulated RNA 37, Potri.006G278100); 3.PtaARFA1F (ADP-Ribosylation Factor A1F, Potri.008G100000); 4. PtaGH16 (Glycosyl Hydrolases family 16, Potri.006G071200). Abbreviations for the four hubs connected to PtaRAP2.6 are as follows: 1. PtaGRF (Gibberellin-Regulated Family protein, Potri.014G020100); 2. PtaEBF1 (EIN3-Binding F-box protein1, Potri.006G068500); 3. PtaCDC2 (Cell Division Control 2, Potri.004G133500); 4. PtaXTH23 (Xyloglucan Endotransglucosylase/Hydrolase 23, Potri.018G095100). Details of the terminal genes connected to individual hubs are provided in S4 Data and S1 Table.
Fig 5.
PtaJAZ3 and PtaRAP2.6 overexpression affects transcript abundance of the respective hub genes in response to PEG treatment.
Transcript abundance was analyzed in roots of WT-717, PtaJAZ3-oe (left) and PtaRAP2.6-oe (right) transgenic lines under both control and PEG conditions. Hub genes associated with PtaJAZ3 and PtaRAP2.6 are shown in the left and right graph panels respectively. Two independent lines (four replicates per line) were used for each transgenic modification. Values show genotypes’ mean ± SEM (n = 2). Different letters represent means that are statistically different (P< 0.05) as determined by a one-way ANOVA followed by Tukey’s multiple range tests.
Fig 6.
Drought stress and methyl jasmonate treatment affect PtaJAZ3 and PtaRAP2.6 expression.
(a) Transcript levels of PtaJAZ3 and PtaRAP2.6 in response to PEG-induced drought stress. Poplar roots grown in control and PEG liquid media (see experimental procedures) were used for qRT-PCR analysis. Values are show as mean ± standard error of mean (SEM) (n = 2). Asterisks represent statistical significant levels of differences between control and PEG treatment (P< 0.05) calculated using Student’s t test. (b) PtaJAZ3 and PtaRAP2.6 are induced in response to methyl jasmonate (MeJA) treatment but the induction of both genes was abolished in presence of proteasome inhibitor, MG-132. Transcript levels were analyzed in poplar root samples treated with 0.2% DMSO (mock), 100 μM MeJA and 100 μM MeJA+ 80 μM of MG-132 (see experimental procedures). Values show as mean ± standard error of mean (SEM) (n = 2). Asterisks represent the treatments that are statistically different from the mock treatment (P< 0.05) calculated using Student’s t test.
Fig 7.
PtaRAP2.6 expression was increased in response to PEG treatment in PtaJAZ3 overexpressing lines.
Expression of PtaJAZ3 (top) and PtaRAP2.6 (bottom) was analyzed in root samples of WT-717 and PtaRAP2.6-overexpression (oe) and PtaJAZ3-oe transgenic lines, respectively, growing in control and PEG liquid media. Two independent lines (4 replicates per line) were measured for calculating the value of each transgenic modification that is shown as mean ± standard error of mean (SEM) (n = 2). Different letters represent those means that are statistically different (P< 0.05) as determined by a one-way ANOVA followed by Tukey’s multiple range tests.