Figure legends
Figure 1. Genetic and phylogenetic characteristics of sucrose
synthase (SUS) genes of Nicotiana tabacum L. transgenic lines.
(A) Simplified scheme of the construction used in this study, in whichSolanum tuberosum L. StSUS3 isoform was inserted into
tobacco plants in the antisense direction between the KST1 promoter and
OCS terminator. (B) Phylogenetic tree of seven tobacco SUS isoforms
(NtSUS1-7 ) (NtSUS2, grey arrow) and StSUS3 (black
arrow). The numbers refer to the branch length. Similar values between
two isoforms indicate that they are genetically closed to each other.
(C) Expression of NtSUS2 gene in leaves and guard cells of wild
type (WT) and transgenic lines (L3 and L13) plants. NtSUS2 gene
expression was analyzed by qRT-PCR normalized by protein phosphatase 2A
(PP2A ) gene expression as internal control. Data are shown as
relative expression normalized to WT (n = 3 ± SE). Asterisks (*)
indicate significant difference from WT by Student’s t test at
5% of probability (P < 0.05).
Figure 2. Physiological characterization of Nicotiana
tabacum L. wild type (WT) and transgenic lines (L3 and L13) antisense
to NtSUS2 grown under well-watered conditions. (A-B)
Photosynthetic response curves to light (A -PAR) and substomatal
CO2 concentration (A -C i).
The regression line was determined using the equation A =a (1 − e-bx ) + c . (C-F) Steady
state values of transpiration rate (E ), stomatal conductance
(g s), dark respiration (Rd) and
water use efficiency (iWUE)
(A /g s) from gas exchange analysis.
Measurements were taken in plants grown under greenhouse and
well-watered conditions. Asterisks (*) indicate significant difference
from WT by Student’s t test at 5% of probability (P< 0.05). (n = 4 ± SE).
Figure 3. Kinetic curves of physiological parameters and
speediness of stomatal opening during dark-to-light transition ofNicotiana tabacum L. wild type (WT) and transgenic lines (L3 and
L13) antisense to NtSUS2 grown under growth chamber and after
water deficit stress. (A-D) Kinetic of photosynthetic rate (A ),
stomatal conductance (g s), intrinsic water use
efficiency (iWUE) and the ratio of substomatal and
ambient CO2 concentration
(C i/C a). (E) Maximum slope
(Sl max) parameters obtained through the linear
plateau model using the time when g s reach
maximum rate of change in light-induced stomatal opening. (F) Half-time
(t50%) needed to g s reach steady
state in light-induced stomatal opening. Measurements were taken in
plants grown under growth chamber and after water deficit (n = 3 ± SE).
Asterisks (*) indicate significant difference from WT by Student’st test at 5% of probability (P < 0.05).
Figure 4. Whole-plant transpiration (WPT) of Nicotiana
tabacum L. wild type (WT) and transgenic lines (L3 and L13) antisense
to NtSUS2 grown under greenhouse and well-watered conditions. (A)
Daily average of light, temperature and humidity throughout eleven days
of the whole-plant transpiration experiment carried out under greenhouse
conditions. (B and E) Daily WPT per plant (g H2O
day-1 plant-1) (B) or per leaf area
(mg H2O day-1 cm-2)
(E). (C and F). % of daily WPT per plant (C) or per leaf area (F)
relative to WT. (D and G) Accumulated water loss per plant (D) or leaf
area (G) throughout the experiment. Black arrows indicate days of no
watering. One (*) and two asterisks (**) indicate that one or two
transgenic lines are significant different from WT by Student’s ttest at 5% of probability (P < 0.05), respectively. (n
= 5 ± SE).
Figure 5. Whole-plant transpiration (WPT) of Nicotiana
tabacum L. wild type (WT) and transgenic lines (L3 and L13) antisense
to NtSUS2 grown under water deficit stress. (A) Daily WPT per
plant (g H2O day-1plant-1). (B) % of daily WPT per plant relative to
WT. (C) Accumulated water loss per plant throughout the experiment.
Inset, % of accumulated water loss relative to WT on the first three
days of well-watered (WW) and on the next five days of non-watered
condition (WD). White and grey bars on the x-axis indicate WW and WD
days, respectively. Asterisks (*) indicate significant difference from
WT by Student’s t test at 5% of probability (P< 0.05). (n = 4 ± SE).
Figure 6. Orthogonal partial least squares-discriminant
analysis (orthoPLS-DA) of guard cell metabolomics data. (A-B)
OrthoPLS-DA and (C-D) scatter plots (S-plots) of metabolites identified
in WT vs L3 and L13, separately. Highlighted metabolites in
S-plots are the significant ones which have more contribution to the
clustering with high variable confidence (y, correlation) in module
(-0.4 > y > 0.3 for WT vs L3 and -0.4
> y > 0.4 for WT vs L13) and variable
importance in projection (VIP) scores of PLS-DA model above 1 (listed in
figures S5 C and D). These analyses were carried out using the relative
guard cell metabolic changes observed in each genotype in the dark and
after the transition to the light. These analyses were performed using
MetaboAnalyst platform. (n = 5).
Figure 7. Correlation-based metabolic networks of guard cell
metabolomics data from Nicotiana tabacum L. wild type (WT) and
transgenic lines (L3 and L13) antisense to NtSUS2 grown under
greenhouse and well-watered conditions. The networks were created using
data from guard cell metabolite contents observed in each genotype in
the dark (A, C, E) and after the transition to the light (B, D, F).
Thicker links indicate higher r , in module. Bigger nodes indicate
higher degree of connection. (n = 5).
Figure 8. Integration of guard cell metabolic data with
stomatal speediness parameters from Nicotiana tabacum L. wild
type (WT) and transgenic lines (L3 and L13) antisense to NtSUS2grown under greenhouse and well-watered conditions. (A)
Correlation-based network was created using relative guard cell
metabolic changes after dark-to-light transition and maximum slope
(Sl max) and half-time (t50%)
needed to g s reach steady state during
light-induced stomatal opening. Yellow nodes highlight metabolites found
in the S-plots of the orthoPLS-DA. Blue and red links indicate negative
and positive correlation, respectively. (B) Heat map representation of
the relative guard cell metabolic changes during dark-to-light
transition, normalized according to the WT values and (C) relative guard
cell metabolic contents in light, normalized by the dark values, of the
yellow nodes of the figure A. Asterisks (*) indicate significant
difference from WT by Student’s t test at 5% of probability
(P < 0.05). (n = 5).