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).