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).