Generation of Active Pools of Abscisic Acid Revealed by In Vivo Imaging of Water-Stressed Arabidopsis

Activation of pRD29B::LUC and pATHB6::LUC by ABA and water stress. Seedlings expressing either the pRD29B::LUC or pAtHB6::LUC reporter gene were exposed to ABA (30 μm), water stressed (Ψ = −0.8 MPa), or not treated in the control for 24 h prior to measurement of reporter activity. The analysis was performed in ABI1 wild-type (wt; A) and abi1-1 background (B; n = 45). Activity is expressed as relative light units captured by the CCD camera within 10 min for pRD29B::LUC (white bars) and pAtHB6::LUC reporter (black bars).

Reporter response and ABA levels in dependence of water potential. LUC expression of 4-d-old pAtHB6::LUC seedlings was recorded 24 h after exposure to various water potentials calibrated by supplementing the solidified medium with mannitol. Light emission (A) and ABA (B) levels of shoots were recorded in wild-type (black circles), ABA-deficient aba2-1 (white squares), and ABA-insensitive abi1-1 (white triangles) background. Values are means of 3 independent measurements, each comprising 15 seedlings.

Differential activation of ABA signaling induced by water stress. Seedlings of the pAtHB6::LUC line were exposed to control conditions (A), ABA (100 μm; B), and water stress (Ψ = −0.8 MPa; C) for 24 h prior to measurement of reporter activity. Luminescence intensity is depicted in false colors ranging from blue to red, indicating low and high intensity, respectively. In A, the scale of light emission is 5× enhanced compared to B and C. The nonenhanced image is shown as the inset. The seedling analyzed in B was deficient in ABA biosynthesis due to the aba2-1 mutation. The signals induced by water stress (C) are primarily confined to the stomata and vasculature of the cotyledons. The signal of the root and hypocotyl was hardly detectable. The pictures were taken by a CCD camera mounted to an inverted microscope with 2.5× magnification of the objective and an exposure time of 10 min with 4 × 4 pixel binning. The scale bars correspond to 1 mm.

ABA signaling in the root tip: response specificity toward ABA. Seedlings containing the pRD29B::GUS expression cassette were either nontreated or exposed to 30 μm ABA (ABA) or their roots were water stressed (stress; Ψ = −1.0 MPa) for 24 h prior to histological staining for reporter activity. The analysis included seedlings with wild-type ABA response (wt), ABA deficiency, and ABA insensitivity provided by the aba2 and abi1 mutant background, respectively. The pictures arranged in a vertical row represent photographs of the same specimen with one specific treatment, while the horizontal rows represent photographs of the same organ or organ parts of seedlings treated differently. The first horizontal row (A–E) shows a close-up view of the cotyledon (scale bars represent 100 μm), the second the shoot (scale bars represent 1 mm), the third a section of the root (scale bars represent 100 μm), and the fourth the root tip (scale bars represent 100 μm). The specimens were stained for identical periods (1 h) to visualize reporter activity, with the exception of Figure 5, K, N, and O, which were stained for 3 h.

Induction of ABA pools and ABA signaling in shoots by water-stressing roots. Seedlings of lines pRD29B::LUC and pRD29B::GUS were exposed to water stress (Ψ = −0.8 MPa) via the roots for 24 h prior to the analysis of shoots and roots. The analysis included determination of ABA levels (A), GUS reporter activity (B), and LUC activity (C), with both reporters assayed enzymatically in tissue extracts, and light emission analyzed ratiometrically by in vivo measurement (D). The values are shown for water-stressed seedlings (black bars) and controls (white bars) of either the pRD29B::LUC (A, C, and D) or pRD29B::GUS (B) line. The values are given relative to the highest value obtained within one type of analysis for better comparison with 100% marking in 148 fmol ABA μg protein⁻¹ (A; this equals 164 nmol ABA kg⁻¹ shoot fresh weight or 435 nmol ABA kg⁻¹ root fresh weight), 8.12 RLU s⁻¹ μg protein⁻¹ (B), 1,890 RLU s⁻¹ μg protein⁻¹ (C), and 31 CCD-RLU s⁻¹ μg protein⁻¹ (D). The analysis was performed on 3 groups of 15 seedlings per data point (± sd).

Reporter response in Arabidopsis rosette plant. The pAtHB6::LUC reporter plants cultivated for 4 weeks under sterile conditions were exposed to water stress (Ψ = −1.0 MPa) via their root system for 24 h prior to in vivo measurement of reporter activity shown in false colors (A) for a single plant and the corresponding brightfield image (B). Comparatively developed and nonstressed plants served as a control and revealed only background LUC activity, while p35S::LUC transgenic plants showed high reporter activity in root and shoot (not shown). Color code is as introduced in Figure 4. The scale bars correspond to 1 cm.