Figure 1.
Effect of a change in water availability on leaf hydraulic conductance (Kleaf) in Populus trichocarpa saplings.
Kleaf and the associated leaf water potential (Ψleaf) were measured in 6 well-watered control plants (blue squares), 6 drought-stressed plants (red circles), and drought-stressed plants 2 and 26 h after rewatering (grey squares and diamonds, respectively). Each data point represents a single measurement of Kleaf. The solid line shows the previously established vulnerability curve for Kleaf. A 3 parameter logistic function was used for the curve fit: Kleaf = 6.154/[1+(Ψleaf/0.469)3.337]. An overview of the complete vulnerability curve is shown in the upper right corner of the figure. Individual measurements are shown as crosses; the mean values for each group (±SE, n = 6) are shown using the same symbols as explained above.
Table 1.
Transcript abundance of 12 aquaporin genes expressed in leaves of well-watered control plants.
Figure 2.
Relative expression of aquaporin genes in leaves of plants exposed to a drying-rewatering cycle.
Gene expression was measured in leaves of well-watered control plants (C), drought-stressed plants (D), and 3 h after drought-stressed plants were rewatered (RW). The geometric mean of the expression levels of four reference genes (ACT2, CYC063, TIP41-like, UBQ7) was used to normalize the results. Asterisks denote significant differences in expression level compared to control levels (one-way ANOVA, followed by Bonferroni’s post test, *P≤0.05; **P≤0.01***P≤0.001). Data are means ± SE of three biological replicates.
Figure 3.
Response of leaf hydraulic conductance (A) and stomatal aperture (B) to different perfusion solutions.
Control conditions refer to the Kleaf that was measured after leaves were xylem perfused with filtered (0.2 µm) 20 mM KCl+1 mM CaCl2 solution (subsequently referred to as ‘artificial xylem sap’, AXS) for 2 h. Kleaf was also measured on leaves that were bench-dried for 1 h (Dehydrated) and on leaves that were bench-dried for 1 h and subsequently perfused for 2 h with AXS (RW AXS), AXS+0.2 mM HgCl2 (RW HgCl2), AXS+50 mM H2O2 (RW H2O2) or AXS+50 µM ABA (RW ABA). Values are means ± SE (n = 6). Different letters denote statistically significant differences by one-way ANOVA with Tukey’s test.
Figure 4.
Typical images of transpiring P. trichocarpa leaves that were allowed to take up safranin solution.
(A) A control leaf was excised from a well-watered plant, and the petiole was immersed for 2 h in safranin solution. Transpiration during dye uptake was promoted by placing the leaf near a fan at ∼1,000 µmol m−2 s−1 photosynthetic active radiation. Most leaf veins were stained indicating minimal xylem embolism. (B) Dye uptake in a bench-dried leaf that was subsequently perfused with safranin solution for 2 h. Minor veins exhibited incomplete staining indicating the presence of embolized xylem conduits in minor veins. (C) Dye uptake of a bench-dried leaf subsequently perfused with safranin + HgCl2 solution for 2 h. Mercury is an aquaporin inhibitor. Staining remained even more incomplete than in (B).
Figure 5.
Relative expression of aquaporin genes in detached leaves during a dehydration-rehydration experiment.
Data are from control leaves (C) after they were perfused with artificial xylem sap (AXS) for 2 h, leaves that were dehydrated on the bench top for 1 h (D), and leaves that were dehydrated on the bench top for 1 h and then perfused for 2 h with AXS (RW). The geometric mean of the expression levels of four reference genes (ACT2, CYC063, TIP41-like, UBQ7) was used to normalize the results. Asterisks denote significant differences in expression level compared to control levels (one-way ANOVA, followed by Bonferroni’s post test, *P≤0.05; **P≤0.01***P≤0.001). Data are means ± SE of three biological replicates.
Figure 6.
Relative expression of 12 aquaporin genes in response to dehydration (y-axis) and dehydration + perfusion with abscisic acid (x-axis).
Detached leaves were either dehydrated on the bench top for 1 h or dehydrated for 1 h and subsequently perfused for 1 h with 50 µM abscisic solution (ABA). Data from fully hydrated detached leaves (perfused for 3 h with 20 mM KCl+1 mM CaCl2 solution) were used as the control group, and their expression refers to a value of 1. Pearson’s r = 0.725; P≤0.01. Data are means ± SE of three biological replicates.
Figure 7.
Immunolocalization of AQP proteins in leaves of P. trichocarpa saplings.
Confocal laser scanning micrographs showing the localization of PIP1, PIP2, TIP2 proteins in minor veins of leaf transverse sections (A, B, C respectively). Controls with no primary antibody indicate minimal background fluorescence (D, E, F respectively). Images were taken at an identical setting and were color-coded with an intensity look-up-table (LUT; displayed in A), in which black was used to encode background, and blue, green, yellow, red and white to encode increasing signal intensities. Ph, phloem; PP, palisade parenchyma; Xyl, xylem. Scale bars = 20 µm.