SUPPLEMENTARY MATERIAL
Supplementary Figure S1. Fld expression in leaves of transgenic potato plants. (a) Schematic diagram of the T-DNA region of the vector used for transformation. The fld gene was translationally fused to the transit peptide (TP) of pea FNR for plastid targeting, and placed between the constitutive CaMV 35S promoter and the transcriptional terminator of the nopaline synthase gene (NOS terminator) in the binary vector pCAMBIA 2200 (Tognetti et al., 2006). (b) Fld expression in leaf tissue from independent Stpfld lines as determined by SDS-PAGE and immunoblot using Fld antisera (see Materials and Methods). The fourth fully expanded leaves of 30-days old plants were used to prepare the extracts. Soluble fractions corresponding to 10 mg FW were loaded in each lane except for the control, which corresponds to 0.8 pmol of purified Fld (arrow). The intensities of the bands co-migrating with mature-sized Fld were quantified with the Image J software, and expressed in arbitrary units (AU). Data shown are means ± SE of three independent experiments.
Supplementary Figure S2. Long-term drought protocol.Plants were grown in 15-L soil pots and irrigation was stopped at 30 days post germination to initiate the drought regime. One hundred per cent FC was defined as the amount of water necessary to completely moisten a 15-L dry soil pot, and we determined FC throughout the experiment by subtracting weights at each time point of the treatment from the initial weight of each pot.
Supplementary Figure S3. Relative water content during short-term drought. Thirty-days old plants were subjected to water stress and RWC was measured at 0, 3 and 14 days post drought, as described in Materials and Methods. Data are means ± SE of 3 biological replicates. Statistically significant differences with the wild type are shown by asterisks and were determined using ANOVA and Tukey’s Multiple Comparison Test (P < 0.1).
Supplementary Figure S4. Low-populated clusters not shown in the main text Figures. Each gray line of the charts corresponds to a particular gene, and the dark line represents the average behavior of all cluster genes. Labelings of the abscissa and ordinates, total number of genes and the list of pathways over-represented in each cluster are indicated as in the legend to Figure 5. Each cluster is displayedvis-à-vis to other that exhibits the contrasting expression behavior (e.g ., induction vs. repression), with the exceptions of clusters 44, 45 and 46, whose opposite clusters corresponds to 11, 12 and 13 (Fig. 6b), respectively.
Supplementary Figure S5. Clusters formed by genes induced or repressed by drought independently of genotype. Each gray line corresponds to a particular gene, and the dark lines represent the average behavior of all the genes contained in each cluster. Labelings of the abscissa and ordinates, total number of genes and pathways over-represented in each cluster are indicated as in the legend to Figure 5. The list of genes belonging to these clusters and their corresponding descriptions, assignments and FC values in the multiple comparison tests are described in Supplementary Table S4.
Supplementary Figure S6. Clusters formed by Fld-modulated genes not responsive to the drought treatment. Each gray line of the charts corresponds to a particular gene, and the dark line represents the average behavior of all cluster genes. Labelings of the abscissa and ordinates, total number of genes and list of pathways over-represented in each cluster are indicated as in Figure 5. The corresponding data are given in Supplementary Table S5.
Supplementary Figure S7. Amino acid profiling in leaves from WT and Fld-expressing potato plants. Amino acid contents were determined in leaves of 30-days old plants as described in Materials and Methods. Levels are reported as means ± SE of 5-8 plants. Statistically significant differences between lines are shown by asterisks and were determined using ANOVA and Tukey’s Multiple Comparison Test (P< 0.05).
Supplementary Figure S8. Comparison of DE genes associated to the proteasome in leaves from Fld-expressing potato and tobacco plants grown under normal conditions. Mapman representations of the plant proteolytic pathway associated to the proteasome are shown for potato (a) and tobacco (b). Each colored square represents a gene that is induced (red; FC > 2 and FDR < 0.05) or repressed (green; FC < 0.5 and FDR < 0.05) by Fld expression under control conditions. Only DE genes are displayed. The color code is shown in a rectangle using a log2 scale. Genes coding for components of the 26S proteasome are squared. Tobacco data were taken from Pierella Karlusich et al. (2017).
Supplementary Figure S9. Comparison of drought-responsive DE genes in WT and Fld-expressing potato plants. The major functional categories of central and secondary metabolism are shown in Mapman representations. Each colored square represents a gene that is induced (red; FC > 2 and FDR < 0.05) or repressed (green; FC < 0.5 and FDR < 0.05) by drought in WT (a) and Fld-expressing (b) leaves. Only DE genes are displayed. Color schemes are shown as in Supplementary Figure S8.
Supplementary Figure S10. Drought modified the ratios of amino acids related to nitrogen mobilization in WT but not Stpfldplants. Ratios of Gln to Glu and Asn to Asp were determined in leaves of 30-days old plants after 3 days of water restriction and their watered controls, as described in Materials and Methods. Levels are reported as means ± SE of 5-8 plants. Statistically significant differences between lines are shown by asterisks and were determined using ANOVA and Tukey’s Multiple Comparison Test (P < 0.05).