FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis

1Plant hormones control plant growth and development. Knowledge of the locations of hormone synthesis and transport, and the resulting hormone gradients and distributions in plants is important for understanding how plants respond to their environment via hormone signaling and cross talk of hormone and other small molecule signaling pathways. Among plant hormones, auxin is best characterized in terms of its distribution and transport, analyzed using reporter constructs for auxin-induced gene expression or protein degradation. Reporter genes developed for ABA-induced gene expression (pRD29A/B, pRAB18 and pAtHB6) are also used. Despite their potential, such promoter-linked reporters respond indirectly and slowly to their respective plant hormone. To unequivocally investigate dynamic models of hormone distribution and dissect the complex functions and interconnection of these signaling molecules, direct plant hormone reporters that act instantaneously and reversibly are essential.

2Optogenetic reporters provide a potential solution. These genetically engineered chromogenic proteins (often fluorescent proteins) respond to a specific environmental change via conformationally linked changes in their spectral properties measurable with optical instruments. Such reporters have been developed for a whole palette of molecules and physiochemical processes. However, no reporters for direct visualization of any plant hormone have yet been developed.

3During land colonization, plants adopted ABA as a hormone to signal stress due to limited water supply. ABA is integrated into a complex signaling network that transcriptionally and post-translationally regulates seed germination, root development and stomatal aperture. ABA biosynthesis is a multi-step reaction involving Zeaxanthin epoxidation, isomerization and cleavage to Xanthoxin in the plastid, followed by conversion to abscisic aldehyde and oxidization to ABA in the cytoplasm. In Arabidopsis, ABA is synthesized primarily in vascular tissues of roots and leaves, in guard cells and in seeds. ABA catabolism includes hydroxylation pathways and glucose conjugation leading to less- or inactive compounds. ABA-glucose ester is stored in the vacuole and was reported to be rapidly hydrolyzed by β-glucosidases. However, direct measurements of rapid ABA release are missing.

4ABA moves throughout the plant and crosses cell borders as a function of pH. This ‘ionic trap model’ explains the movement, but excludes cellular efflux of ABA due to low apoplastic pH. Recently identified ABA transporters contribute to ABA export from the vasculature and import into guard cells. Two non-mutually exclusive current models describe how water limitations in the root induce stomatal closure in the leaf: (1) ABA acts as a chemical signal synthesized in the root and transported to the shoot, and (2) a hydraulic signal from the root induces ABA synthesis in the shoot.

5In response to water limitations ABA concentrations increase and decrease upon stress relief. Despite recent progress on ABA synthesis and transport, direct evidence for conditionally triggered changes in local ABA concentrations and time-resolved data for ABA re-distribution in vivo are lacking.

6ABA is perceived by members of a protein family designated as PYRABACTIN RESISTANCE 1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR), which in the presence of ABA negatively regulate Clade A TYPE 2C PROTEIN PHOSPHATASES (PP2Cs). Inhibition of PP2Cs enables the activation of SNF1-RELATED KINASES 2 (SnRK2s), that target transcription factors, ion channels and NADPH oxidases.

7PYR/PYL/RCARs contain an internal ligand-binding pocket flanked by two conserved loops, the ‘Pro-cap/gate’ and the ‘Leu-lock/latch’. ABA binding triggers these loops to rearrange, closing the lid over ABA. The conformational change and rearranged protein surface favors PP2C binding over receptor dimerization. In the resultant ABA receptor–phosphatase complex, a conserved Trp residue from the PP2C inserts between the ‘Pro-cap/gate’ and the ‘Leu-lock/latch’ to further enclose the ABA.

8Here, we report the design, development and application of optogenetic FRET-based reporters for ABA (ABAleons), in which covalently attached PYR1 and the PP2C ABI1 are modulated upon ABA binding, triggering changes in fluorescence emission from attached fluorescent proteins. ABAleons can affect ABA responses at high concentrations and enable the analysis of time-dependent changes in ABA concentration, distribution and transport in live plants with appropriate resolution to monitor endogenous ABA concentration changes.

9Based on structural analyses of PYR1 and the PYL1-ABA-ABI1 complex and a FRET cassette consisting of the fluorescent proteins mTurquoise and Venus circularly permutated at amino acid 173 (cpVenus173;; ), FRET-based reporters for ABA, named ABAleons, were designed (Figure 1A). Full length PYR1 and ABI1 truncated at amino acid S125 (ΔNABI1) were fused via a flexible ASGGSGGTS(GGGGS)4-linker and inserted into the mTurquoise-cpVenus173 FRET cassette using short two amino acid GP- and PG-linkers resulting in the FRET reporter ABAleon1.1 (Figure 1A). Due to the long flexible linker between PYR1 and ΔNABI1 (> 120 Å;; Figure 1A), ΔNABI1 might be free for substrate access in the ABA unbound conformation. Therefore the wild type ABAleon1.1 was mutated to abolish phosphatase activity of ABI1 by introducing a D413L mutation in the catalytic metal-binding site (ABAleon2.1; http://www.uniprot.org/uniprot/P49597) (Figure 1A).

10In vitro application of ABA had no impact on ABAleon2.1 absorbance (Figure 1—figure supplement 1A). However, analyses of fluorescence emission spectra after application of ABA revealed an increase in mTurquoise emission (peak at 476 nm) and a decrease of cpVenus173 emission (peak at 527 nm), indicating that the distance between both fluorescent proteins is increased, or their orientation to each other is changed by the ABA-dependent interaction of PYR1 and ΔNABI1 (Figure 1B–D). Apparent ABA affinities were calculated by curve fitting of ABA-dependent emission ratio plots (Figure 1E) or from fits of the ABA-dependent ratio change (ΔR) relative to the maximum ratio change (ΔRmax) (Figure 1F). For ABAleon1.1 a dynamic range of ∼ 15 % was recorded with an apparent ABA affinity K′d of ∼ 300 nM (Figure 1C,E,F). Moreover, ABA-induced emission ratio changes of ABAleon1.1 were stable in the range of physiological pH conditions (e.g., pH 6.6–8.2; Figure 1—figure supplement 1B). ABAleon2.1 exhibited a dynamic range of ∼ 9 % and a K′d of ∼ 100 nM (Figure 1D–F). In plate reader-based analyses, application of ABA rapidly and clearly induced emission ratio changes of ∼ 8 % when analyzing ABAleon2.1 at 1 µM ABA (Figure 1G). ABAleon1.1 exhibited phosphatase activity comparable to PYR1 and ΔNABI1 when combined in a 1:1 molar ratio (Figure 1H). In the presence of 5 µM ABA phosphatase activity was inhibited to 50 % of initial activity (Figure 1H;;; ). However, the predicted phosphatase inactive ABI1D413L mutation in ABAleon2.1 enabled the design of an ABA-reporter without detectable phosphatase activity (Figure 1H), which was considered to be preferable for use in plants.

11ABAleon2.1 was transformed into the Arabidopsis Columbia 0 accession (Col-0) to determine whether it can detect ABA level changes in planta. In a macroscopic view ABAleon2.1 emission ratio maps were recorded from 5 day-old seedlings before (Figure 2A) or 2 h after ABA application (Figure 2B). Emission ratios were also recorded from guard cells of 33-day-old soil-grown plants (Figure 2C). As indicated in the color code of the calibration bar (Figure 2), low ABAleon2.1 ratios (blue) indicate high ABA concentrations and high ratios (red) indicate low ABA concentrations. Comparison of the emission ratio maps before and after ABA application revealed visible ABA uptake into the entire seedling (Figure 2A,B). However the most prominent ratio changes were observed in the root elongation- and early maturation zone, where the yellow-coded regions showed an ABA concentration increase, which is indicated by the yellow-to-blue color shift (Figure 2A,B). Visible ratio changes were also observed in the lower hypocotyl. Here an upward-directed ABA accumulation was detected (Figure 2A,B).

12ABAleon2.1 emission ratios in guard cells were low (Figure 2C, blue), indicating elevated ABA concentrations under the imposed conditions. High ABA concentrations in guard cells were consistent with the constitutive guard cell expression of the ABA-induced reporter pRAB18-GFP when plants were grown at 70 % relative humidity (Figure 2—figure supplement 1A). Expression of pRAB18-GFP was further induced by ABA (Figure 2—figure supplement 1B), however with stronger induction 2 days after transfer to 25 % relative humidity (Figure 2—figure supplement 1C,E).

13ABA-induced ABAleon2.1 responses were then analyzed with higher time- and spatial resolution in guard cells of 45-day-old plants (Figure 3A–C) and in three differentially color-coded regions of the hypocotyl (Figure 3D–F), root differentiation–(Figure 3G–I), root maturation–(Figure 3J–L) and root elongation-zone (Figure 3M–O) of 5-day-old seedlings. Application of ABA induced increases in mTurquoise (Figure 3A,D,G,J,M, mT, solid lines) and decreases in cpVenus173 emission (Figure 3D,G,J,M, cpV, dashed lines), resulting in an up to 12 % decrease in the emission ratios (Figure 3B,E,H,K,N). The ABA-induced ABAleon2.1 emission ratio changes indicate increases in the ABA concentration in all investigated Arabidopsis tissues. These in planta analyses were consistent with in vitro analyses (Figure 1E,G). The ABA-induced ABAleon2.1 ratio changes in guard cells (3–6 %; Figure 3B) and in the root differentiation zone (6 %; Figure 3H) were low compared to changes in the hypocotyl and lower root tissues (9–12 %; Figure 3E,K,N), consistent with data indicating elevated ABA concentrations prior to ABA application (Figure 2A,C). While ABA uptake into guard cells (Figure 3B) and into the root occurred simultaneously (Video 1) in all three analyzed regions (Figure 3H,K,N), a delay in the ABAleon2.1 response was observed in the hypocotyl (Figure 3E; Video 1). These data suggest a directional ‘wave-like’ ABA transport in the hypocotyl, which was also indicated in the ratio images (Figure 3F, Video 1). To describe the ABA transport in the hypocotyl more quantitatively, ABAleon2.1 response curves of all three analyzed regions were fitted by a four parameter logistic curve. From these fits the t1/2 values measure the time point when ABAleon2.1 is half-saturated. These values were used to calculate the delay in ABAleon2.1 responses between the analyzed regions which is a measure for the speed of ABA transport. From three independent experiments the rate of ABA transport in the hypocotyl was calculated as 16.4 ± 0.8 µm/min.

14As ABA was dissolved in EtOH, responses to EtOH as solvent control were analyzed in the hypocotyl (Figure 3—figure supplement 1A–C) and the root maturation zone (Figure 3—figure supplement 1D–F). These treatments did not induce measurable emission ratio changes of ABAleon2.1 (Figure 3—figure supplement 1B,E). In contrast, subsequent application of ABA induced ABAleon2.1 emission ratio changes similar to previous data (Figure 3E,K, Figure 3—figure supplement 1B,E). In control seedlings expressing only the empty FRET-cassette no response to ABA was detected (Figure 3—figure supplement 1G–I). These data support, that PYR1-ΔNABI1D413L incorporated in ABAleon2.1 (Figure 1A) are responsible for the ABA-induced emission ratio changes in Arabidopsis. Taken together, these data clearly demonstrate the direct and instantaneous ABA detection by ABAleon2.1 in various tissues and the visualization of ABA transport in planta.

15ABAleon2.1 was transformed into a PYR/PYL/RCAR ABA receptor quadruple mutant pyr1-1/pyl1-1/pyl2-1/pyl4-1 (pyl4ple;; ). ABA response curves of Col-0 wild type (Figure 4A) and pyl4ple (Figure 4B) were fitted by a four parameter logistic curve using data of four single measurements (Figure 4A,B) or the combined datasets (Figure 4C). Data show that in the pyl4ple mutant ABAleon2.1 exhibited a faster response to 10 µM ABA in the root maturation zone when compared to Col-0 wild type (Figure 4A–C). This finding is also reflected in the t1/2 values (Figure 4D). Half saturation of ABAleon2.1 was reached 16 min after ABA application in Col-0, while this appeared within 7 min in the pyl4ple mutant (Figure 4C,D).

16To investigate whether ABAleon2.1 might affect ABA responses in general, two ABAleon2.1 lines (line 3 and line 10) were compared to Col-0 wild type, YFP-PYR1 and abi1-3/YFP-ABI1 over-expression lines. Analyses of the cpVenus173/YFP fluorescence emissions of the investigated lines indicated, that ABAleon2.1 (line 3) exhibited a ∼ fivefold higher fluorescence emission (expression) when compared to ABAleon2.1 (line 10) (Figure 5A) while emission of YFP-ABI1 was ∼ 7 % compared to YFP-PYR1 (Figure 5A).

17In seed germination (Figure 5F,G) and cotyledon expansion assays (Figure 5B,C,H) ABAleon2.1 lines were hyposensitive to 0.8 µM ABA when compared to Col-0 wild type and YFP-PYR1. However, the ABAleon2.1 lines exhibited a less pronounced ABA hypersensitivity when compared to abi1-3/YFP-ABI1 (Figure 5G,H). Interestingly, the degree of ABA hyposensitivity of both ABAleon2.1 lines correlated with ABAleon2.1 expression levels (fluorescence emission), as the stronger expressing line 3 exhibited a reduced ABA sensitivity when compared to the lower expressing line 10 (Figure 5).

18In seedling assays 10 µM ABA inhibited growth of all investigated lines (Figure 5D,E,I). Fresh weight of ABAleon2.1 and abi1-3/YFP-ABI1 plants, when grown on media supplemented with 10 µM ABA, was less reduced compared to Col-0 wild type (Figure 5I). Again, the degree of ABA sensitivity of the ABAleon2.1 lines correlated with ABAleon2.1 expression levels (Figure 5A,I). Interestingly, growth and root length of YFP-PYR1 plants was drastically reduced, when grown on 10 µM ABA media (Figure 5D,E,I), suggesting a strong ABA hypersensitivity when over-expressing this receptor.

19In ABA-induced stomatal closure assays ABA sensitivity of ABAleon2.1 lines was comparable to Col-0 wild type responses (Figure 5J), suggesting potential tissue or cell specific effects of ABAleon2.1 on ABA responses, which may be linked to a higher basal ABA concentration in these cells (Figure 2C, Figure 2—figure supplement 1). Taken together, ABAleon2.1 plants exhibit a reduced ABA sensitivity in seed germination and seedling growth which correlated with ABAleon2.1 expression levels.

20To study long-distance ABA transport, modeling clay was placed into the middle of each imaging chamber to generate two isolated chambers (Figure 6A). Seedlings were placed such that the hypocotyl base and root differentiation zone laid over the isolating modeling clay, which isolated the shoot (top chamber) from the root (bottom chamber). Ratio images of the shoot/upper hypocotyl (Figure 6E) and the root maturation zone (Figure 6F) were recorded before and after application of 50 µM ABA. Three regions in the upper hypocotyl and the root maturation zone, indicated by boxes in the t = 0 min ratio images (Figure 6E,F), were used to measure time dependent ratio changes (Figure 6B). Upon ABA application to the upper chamber, ABA concentrations increased in the shoot/upper hypocotyl, as seen by an immediate decrease of the ABAleon2.1 emission ratios (Figure 6B,E). In the root maturation zone a decrease in the emission ratio was observed starting ∼ 90 min after the treatment (Figure 6B,F).

21In additional experiments seedlings were placed such that the hypocotyl and root differentiation zone were located in the top chamber and the root maturation zone in the bottom chamber (Figure 6A, left seedling). In control experiments where 0.05 % EtOH as solvent control was added to the top chamber, no emission ratio changes were detected in the hypocotyl and the root maturation zone, indicating that in these experimental conditions endogenous ABA concentrations were stable (Figure 6—figure supplement 1). However, ABA application to the top chamber induced an ABAleon2.1 emission ratio change in the hypocotyl after 30 min which did not drastically change for the remaining time period (Figure 6C,G). ABA application to the hypocotyl (top chamber) led to a gradual decrease of the ABAleon2.1 emission ratios in the root maturation zone (bottom chamber; Figure 6C,H), providing evidence that ABA is actively transported to the root maturation zone. When ABA was applied to the root maturation zone (bottom chamber), ABAleon2.1 emission ratios rapidly dropped, indicating ABA uptake into the root (Figure 6D,J). However, ABA was not transported upwards to the hypocotyl (top chamber) within 180 min under the imposed conditions (Figure 6D,I). The above data indicate a shoot to root ABA transport (Figure 6B,C). A root to shoot ABA transport could not be detected within 180 min after ABA application (Figure 6D) possibly due to low transpiration when whole seedlings were perfused with buffer solution (‘Materials and methods and Discussion’).

22Based on structural models (Figure 7A, Figure 7—figure supplement 1A), mutations in PYR1 and ΔNABI1D413L of ABAleon2.1 were selected that potentially reduce but not abolish PYR1-ΔNABI1D413L interaction resulting in the new constructs ABAleon2.11–ABAleon2.17 (Figure 7—figure supplement 1A; Table 1). In addition shorter linker versions between PYR1 and ΔNABI1D413L were generated (ABAleon2.2 and ABAleon2.3; Table 1). Recombinant empty FRET control (F3) and all ABAleon versions were purified (Figure 7—figure supplement 2) and biochemical characteristics of these proteins are provided in Table 1.

23Analyses showed that linker deletions slightly changed ABA affinity but also reduced the dynamic range (Table 1). Compared to other investigated ABAleon2.1 mutants, PYR1H60P in ABAleon2.11 strongly impaired ABA-induced emission changes (Figure 7B), which were comparable to ABA-bound ABAleon2.1 (Figure 7C; Table 1). Of the seven analyzed ABAleon2.1 mutants, PYR1V83H in ABAleon2.13 and PYR1H115A in ABAleon2.15 were of particular interest, as these mutants exhibited a reduced ABA affinity (K′d ∼ 3–4 µM of ABAleon2.13 and ∼ 500–600 nM of ABAleon2.15; Figure 7E; Table 1). Also the dynamic range of these ABAleons was not drastically affected (Figure 7D,F; Table 1).

24Unless otherwise stated, all of the above analyses have been conducted using the natural (+)-enantiomer of ABA. In additional experiments, apparent affinities for (+)-ABA (Figure 7E, Figure 7—figure supplement 1B–D) were compared to binding of its unnatural enantiomer (−)−ABA (Figure 7G, Figure 7—figure supplement 1E–G). In these analyses the dynamic ranges and (+)−ABA affinities of ABAleon1.1 (K′d ∼ 300 nM) and ABAleon2.1 (K′d ∼ 100 nM) (Figure 7—figure supplement 1B–D) were comparable to previous analyses (Figure 1E,F; Table 1). However, the binding affinity for (−)−ABA was reduced by about twofold in ABAleon1.1 (K′d ∼ 600 nM) and ABAleon2.1 (K′d ∼ 180 nM) (Figure 7—figure supplement 1E–G). In case of ABAleon2.13, affinities for (+)- and (−)-ABA were comparably low (K′d ∼ 3–4 µM) and ABAleon2.13 did not reach saturating conditions at 200 µM (+)- or (−)-ABA (Figure 7E,G, Figure 7—figure supplement 1B–G). Remarkably, ABAleon2.15 harboring the PYR1H115A mutation exhibited an apparent affinity for (−)-ABA of ∼ 30 µM (Figure 7G, Figure 7—figure supplement 1F,G), which was 50-fold reduced compared to the (+)-ABA affinity (K′d ∼ 0.6 µ M) (Figure 7E, Figure 7—figure supplement 1C,D) suggesting, that PYR1H115 has an important function in ABA stereospecificity. From these analyses, ABAleon2.13 and ABAleon2.15 could be good candidates for analyzing ABA concentration changes in cell types that have higher basal ABA concentrations.

25To investigate the utility of low affinity ABAleons, ABAleon2.13, ABAleon2.14 and ABAleon2.15 were transformed into Arabidopsis Col-0 wild type plants. ABAleon2.14 was included in these analyses, as it exhibited an apparent K′d of ∼ 1.2 µM for ABA, however with a reduced dynamic range (Table 1). Initial analyses were performed in the root maturation zone of T2 lines and compared with ABAleon2.1 (line 10) at comparable expression levels (Figure 8, Figure 8—figure supplement 1). Examples of single representative measurements are presented in Figure 8—figure supplement 1. All investigated ABAleons responded to externally applied 10 µM ABA with a negative emission ratio change (Figure 8, Figure 8—figure supplement 1). While ABAleon2.13 and ABAleon2.14 exhibited a relatively low dynamic range in planta (Figure 8B,C,E, Figure 8—figure supplement 1E,H), ABAleon2.15 responded comparable to ABAleon2.1 (line 10) (Figure 8A,D,E,F, Figure 8—figure supplement 1B,K). Thus, ABAleon2.15 is the best candidate for a low affinity (K′d ∼ 600 nM) ABA-reporter.

26It is well established that plants synthesize ABA in response to water stress. Recent studies reported ABA increases in shoots and roots after 3 h of water stress. In addition, older studies reported ABA concentration increases within 15 min in guard cells of Vicia faba.

2715 min after a drop in humidity ABAleon2.1 emission ratios decreased ∼ 3 % in guard cells and did not change when analyzed at the 30 min time point (Figure 9A,B), indicating fast ABA concentration adjustments in response to humidity changes. In 4 h stress treatments of detached leaves 100 mM NaCl and 10 µM ABA induced a 5–6 % ABAleon2.1 emission ratio change in guard cells (Figure 9C,D). In contrast, 300 mM sorbitol did not induce any detectable changes (Figure 9C,D).

28In root tissues two separate regions labeled by cyan and yellow boxes (Figure 9E,G, left) were measured after 6 h stress treatments. In these analyses 100 mM NaCl or 300 mM sorbitol induced ABAleon2.1 emission ratio changes of 3–6 % in the root maturation zone compared to 7 % changes in response to 10 µM ABA (Figure 9F). In the root elongation zone, responses to 100 mM NaCl and 300 mM sorbitol were 7–8 % while 10 µM ABA induced ABAleon2.1 emission ratio changes of 5–6 % (Figure 9H). These analyses demonstrate the utility of ABAleon2.1 to report endogenous ABA concentration changes in response to low humidity, salt- and osmotic stress (Figure 9).

29Genetically encoded fluorescent protein-based reporters are powerful tools in cell biology. Here we report the design, engineering and application of ABAleons, FRET-based reporters that enable the direct analysis of instantaneous ABA concentration changes in vitro and in planta.

30ABAleons were built on the strictly ABA-dependent interaction of PP2Cs with PYR1 and its phylogenetically close relatives, rather than the other members of the PYR/PYL/RCAR family, which have a higher probability of residing in a ‘monomeric’ state, and which can interact with PP2Cs even in the absence of ABA. PYR1 and close homologs exhibit lower ABA affinities (Kd ∼ 50–100 µM) than those of the ‘monomeric’ homologs (Kd ∼ 1 µM). In comparison, the ABA affinity of PYR/PYL/RCARs when bound to PP2Cs ranges between Kd ∼ 20–125 nM. ABAleons exhibit similar ABA affinities to endogenous PYR/PYL/RCARs or PYR/PYL/RCARs in complex with PP2Cs (Figure 1E,F, Figure 7E, Figure 7—figure supplement 1; Table 1), consistent with our findings, that ABAleon2.1 is able to detect changes in endogenous ABA concentrations (Figure 9).

31ABAleons exhibit higher affinity to the naturally occurring (+)-ABA than to (−)-ABA (Figure 7E,G, Figure 7—figure supplement 1B–G), and could potentially also bind to the synthetic ABA mimics pyrabactin and quinabactin. One of the ABAleon derivatives, ABAleon2.15, carrying the PYR1H115A mutation, exhibited strongly reduced affinity for (−)-ABA (Figure 7G, Figure 7—figure supplement 1E–G), suggesting an important role of this amino acid in ABA stereospecificity. These results are consistent with recent findings that the homologous H139 in PYL3 was important for ABA stereospecificity. ABAleon2.11, carrying the PYR1H60P mutation, exhibited spectral characteristics comparable to ABA-bound ABAleon2.1 (Figure 7C). This is consistent with the notion, that the PYR1H60P protein may form an alternative interaction interface with PP2Cs, even in the absence of ABA.

32In Arabidopsis, over-expression of the ABA receptors PYR1, PYL2 and PYL5 induces ABA hypersensitivity (Figure 5;; ), while ectopic expression of the PP2Cs ABI1 and HAB1 decreases ABA sensitivity (Figure 5;; ). Transgenic Arabidopsis plants expressing ABAleon2.1 exhibited a reduced sensitivity to exogenously applied ABA in seed germination, cotyledon expansion and growth assays, but not in stomatal closure assays (Figure 5). The degree of reduced ABA sensitivity correlated with increasing ABAleon2.1 expression levels in two independent transgenic lines (Figure 5). Because ABAleon2.1 did not exhibit any phosphatase activity in vitro (Figure 1H), it can be speculated, that ABAleon2.1 might sequester a certain amount of physiologically relevant ABA in certain tissues and cell types due to its high affinity (Figure 1E,F, Figure 7—figure supplement 1C,D; Table 1), thus causing ABA hyposensitivity (Figure 5). Future generations of ABAleons and improved imaging sensitivity at lower ABAleon concentrations (Figure 8) could enable the reduction of ABAleon side effects.

33By definition, a hormone transmits a signal from the site of hormone synthesis to its place of action. Although long-distance ABA transport has been studied for many years (;;; and references therein), no method for the direct detection of ABA concentration changes and ABA transport rates in planta has been available. The detailed characterizations of ABAleons demonstrate the utility of ABAleon2.1 (K′d ∼ 100 nM) and ABAleon2.15 (K′d ∼ 600 nM), which exhibit a sufficient ABA-specificity and dynamic range (9–10 %) upon ABA binding to monitor instantaneous ABA-induced or environmentally-triggered ABA concentration changes.

34ABAleon2.1 and ABAleon2.15 enabled measurements of rapid ABA-induced changes in ABA concentrations in various tissues (Figure 3, Figure 8), ABA uptake into whole seedlings (Figure 2A,B), directional ABA transport from the hypocotyl base towards the shoot (Figure 3E,F; Video 1) and from the hypocotyl or shoot to the root (Figure 6B,C,E-H). Under the imposed conditions the speed of ABA transport within the hypocotyl was ∼ 16 µm/min. Furthermore, ABA transport from the root maturation zone to the shoot could not be detected within three hours (Figure 6D,I,J). Because the experimental setup (Figure 6), in which both shoot and root were perfused with buffer, would compromise the transpirational stream, the present data do not exclude concomitant ABA transport from roots to shoots, as has been found in other plant species.

35In response to water stress, Arabidopsis plants synthesize ABA in the shoot, which has been reported to be transported to the root. ABA accumulation in roots and leaves was detected 2.5–3 h after stress initiation. Older studies, using manually dissected guard cells, measured ABA concentration increases in guard cells 15 min after passive dehydration of leaves. ABAleon2.1 enabled the rapid detection of ABA concentration changes in guard cells in response to a humidity drop (Figure 9A,B) and the visualization of long term ABA accumulation in response to salt in guard cells (Figure 9C,D) and in response to salt- and osmotic stress in roots (Figure 9E–H).

36Surprisingly, ABA-induced ABAleon2.1 ratio changes in the root maturation zone were accelerated in the pyr1-1/pyl1-1/pyl2-1/pyl4-1 mutant (Figure 4). Mutants defective in ABA signaling may compensate by up-regulating ABA levels, as reported previously and could also up-regulate ABA transport activity (Figure 4C,D). Alternatively, knock out of ABA receptors may also affect ABA buffering capacity (Figure 4C,D). ABA reporter analyses of ABA uptake (Figure 4) or long-distance ABA transport (Figure 3D–F, Figure 6) could be utilized for the characterization or identification of ABA transporters and their regulation mechanisms in planta or in heterologous systems.

37ABA induces cytoplasmic alkalinization of guard cells. In guard cells of Vicia faba, cytoplasmic pH was found to be 7.67 and increased 0.27 units upon ABA treatment. Cytoplasmic pH in roots was 7.3 and could increase to 7.7. In these pH ranges ABA-induced ABAleon1.1 emission ratio changes were stable in vitro (Figure 1—figure supplement 1B).

38In vivo ABA concentrations in cellular compartments of specific plant cells and tissues are currently unknown. Overall ABA levels range from 30–50 ng/g dry-weight in non-stressed plants, which can increase up to 30-fold in response to limited water conditions. ABAleon2.1 and ABAleon2.15 exhibit a sufficient dynamic range for in vitro calibrations (Figure 1E,F, Figure 7E, Figure 7—figure supplement 1B–D) that permit approximations of cellular ABA concentrations, for example in the range of ≤ 25 nM in the root elongation zone. In Vicia faba guard cells ABA concentrations were ∼ 0.7 fg/cell pair in unstressed and ∼ 17.7 fg/cell pair in stressed guard cells. ABA concentrations in stressed guard cells were estimated to be in the range of ∼ 15 µM. Extrapolating from these values, unstressed guard cell ABA concentration would be ∼ 500 nM. Such approximations would be consistent with the partial saturation and reduced response of ABAleon2.1 in guard cells (Figure 3A–C) and with strong expression of the ABA-induced reporter pRAB18-GFP (Figure 2—figure supplement 1).

39Our results demonstrate that ABAleons affect ABA signaling to certain extent, but can analyze changes in ABA concentrations in diverse tissues and cell types and measure ABA transport in vivo. ABAleons will thus allow hitherto challenging investigations of ABA synthesis and transport in planta, in response to changes in environmental conditions or treatment with synthetic compounds designed to improve plant survival and crop yields under adverse climate conditions. Further, in combination with other genetically encoded reporters, ABAleons can be used to decipher the cross talk between ABA and other signaling molecules. During the course of our ABAleon research we found, that Jones et al. developed ABACUS-type ABA reporters, however with biochemical properties complementary to ABAleons. Thus, ABAleons and ABACUS could be utilized to study novel aspects of ABA signaling in intact plants.

40Fluorescent protein coding sequences were re-amplified from the pF40 plasmid and ligated into Xba I/Apa I (mTurquoise) or Xma I/Sac I sites (cpVenus173) of a modified pUC19 plasmid resulting in the pUC-F3 and pUC-F3_II empty FRET-cassettes with the latter containing a Nde I-site downstream of the Xba I-site and a StrepII-tag fusion of cpVenus173 at its free end. PYR1-GGSGG and (GGGGS)4-ΔNABI1 and mutants were cloned and inserted between Apa I/Spe I and Spe I/Xma I sites of pUC-F3 and pUC-F3_II to obtain pUC-ABAleon and pUC-ABAleon_II, respectively. Escherichia coli expression vectors were obtained by sub-cloning ABAleon_II versions Nde I/Sac I into pET-24b(+) (Novagen, Darmstadt, Germany). For expression in plants, the pUBQ10 promoter (AT4G05310;; ), inserted between Hind III/Spe I sites of a modified pUC19 plasmid (kindly provided by Jörg Kudla, University of Münster), was mutated to remove a Sac I site within the pUBQ10. In addition, the HSP18.2 terminator (T) (AT5G59720; ) was inserted between the Sac I/Eco RI sites of pBluescript II (Stratagene, La Jolla, CA) and the Hind III site within the HSP18.2T was deleted resulting in pKS-HSP18.2TΔHind III. Both pUBQ10ΔSac I and HSP18.2TΔHind III were sub-cloned into plant compatible vectors pGPTVII.Bar, which confers glufosinate (BASTA) resistance, and pGPTVII.Hyg, which confers hygromycin resistance, resulting in the barII-UT and hygII-UT plasmids. Finally, ABAleon2.1, ABAleon2.1 mutants and the empty FRET-cassette were sub-cloned from pUC-ABAleon2.1, pUC-ABAleon2.1x_II and pUC-F3 plasmids into the barII-UT and hygII-UT plasmids to obtain the barII-UT-ABAleon2.1, barII-UTF3 and hygII-UT-ABAleon2.1 and mutant plasmids for expression in plants. More detailed information about oligo-nucleotides and plasmids used and generated in this work is provided in Supplementary file 1A and Supplementary file 1B.

41pET-F3_II (empty FRET) and pET-ABAleon_II versions in BL21-CodonPlus (DE3)-RIL cells (Stratagene) were grown at 37 °C in 2 L Luria Broth (LB) medium containing 25 µg/mL Kanamycin. At an optical density at 600 nm (OD600) of 0.5, cells were induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and shaken for additional 4-6 h at 24 °C. Cells were collected by centrifugation (15 min 5.000×g and 4 °C) and stored at −80 °C. Proteins were extracted by sonification after 60 min incubation in 20 mL extraction buffer (30 mM Tris–HCl [pH 7.4], 250 mM NaCl, 1 mM Ethylenediaminetetraacetic acid [EDTA], 1 mM Phenylmethylsulfonyl fluoride [PMSF], 1x protease inhibitor [Roche, USA] and 1 mg/mL Lysozym). Cell debris was removed by centrifugation (2 × 30 min, 20.000×g and 4 °C) and by filtration through 0.45 µm syringe filters.

42Protein extracts were mixed with 2.5 ml 50 % Strep-Tactin Macroprep resin (IBA, Göttingen, Germany) pre-equilibrated in wash buffer I (30 mM Tris–HCl [pH 7.4], 250 mM NaCl, 1 mM EDTA) and protein/resin mix was incubated for 1 h at 4 °C while shaking in a 50 ml tube. The suspension was run twice through a 20 mL gravity column (BioRad, Hercules, CA) followed by two washes of the remaining protein/resin mix with 10 column volumes (CV) of wash buffer I and one wash with 10 CV of wash buffer II (30 mM Tris–HCl [pH 7.4], 250 mM NaCl, 10 mM MgCl2 and 1 mM MnCl2). Proteins were eluted 3x in 1 CV wash buffer II supplemented with 2.5 mM Desthiobiotin (Sigma, USA) and concentrated to ∼ 1 mL volume by centrifugation at 3.000×g and 4 °C using Amicon Ultra-4 30K or 10K centrifugal filters (Millipore, Billerica, MA). Purified proteins were run through a Superdex 200 HiLoad 16/60 column (GE Healthcare) in wash buffer II using an ÄKTA purifier fast protein liquid chromatography (FPLC) system (GE Healthcare) with 0.8 MPa column pressure limit, 1 mL/min flow rate and 2 mL fraction size volume. Fractions exhibiting 280 nm, 445 nm and 516 nm absorbance were analyzed by SDS-PAGE and Instant Blue (Cole–Parmer, USA) protein staining and selected for further concentration using Amicon Ultra-4 centrifugal filters. Protein aliquots were flash frozen in liquid N2 and stored at −80 °C. Protein purity was analyzed by SDS-PAGE, immuno-blotting using anti-GFP antibody (Life Technologies, Darmstadt, Germany) and PageBlue staining (Thermo Scientific, Rockford, IL; ). Results of F3 empty FRET and ABAleon purifications are provided in Figure 7—figure supplement 2.

43The coding sequence of ΔNABI1 corresponding to amino acid residues 125–429 was inserted via Nde I/Bam HI into pET28a (Novagen; Supplementary file 1A, Supplementary file 1B) and transformed into E. coli BL21 (DE3). E. coli were grown at 37 °C, and protein expression was induced by 1 mM IPTG at OD600 of 0.6–0.8. After overnight incubation at 25 °C, cells were harvested by centrifugation (15 min 5.000×g and 4 °C) and re-suspended in 50 mM Tris–HCl (pH 8.0) and 500 mM NaCl. Cells were sonicated on ice and lysates were obtained after centrifugation at 12,000×g for 1 h 6xHis-ΔNABI1 extracts were applied to a Ni-NTA column (Qiagen, Hilden, Germany) and washed with five bed volumes of 50 mM Tris–HCl (pH 8.0), 500 mM NaCl and 10 mM imidazole. Bound proteins were eluted in 50 mM Tris–HCl (pH 8.0), 500 mM NaCl and 300 mM imidazole. 6xHis-ΔNABI1 was further purified using Sephacryl S-200 (GE Healthcare) in 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl. 6xHis-PYR1 and 6xHis-ΔNABI1 proteins were re-buffered and concentrated into wash buffer II using Amicon Ultra-4 centrifugal filters. Protein purity and concentrations were analyzed by SDS-PAGE and PageBlue staining and quantified according to a 0–2000 ng BSA (NEB, Ipswich, MA) standard calibration.

44ABA titration experiments were conducted in a TECAN Infinite M1000 PRO (TECAN, Männedorf, Switzerland) using 1 µM ABAleon protein in wash buffer II with 0.1 % EtOH or DMSO as solvent for (+)-ABA (TCI, Portland, OR), generally used in assays unless otherwise stated, or (−)-ABA (Sigma). Protein samples were excited with 440 ± 5 nm and emission 450–700 nm was measured in 1 nm steps with 5 nm bandwidth and 10 flashes of 20 µs and 400 Hz. Gain settings to obtain optimal emission spectra were calculated by the TECAN software from unbound ABAleon emission. Emission bands of mTurquoise (470–490 nm) and cpVenus173 (518–538 nm) were used to calculate cpVenus173/mTurquoise emission ratios. Apparent ABA affinities (K′d) were calculated from emission ratio plots by fitting a four parameter logistic curve or from ΔR/ Δ Rmax plots by fitting a three parameter sigmoidal Hill equation using the SigmaPlot 10.0 version (Systat, San Jose, CA). Dynamic ranges were calculated from experimentally determined values as.

45Absorbance spectra (275–700 nm, slit 0.2 nm) of ABAleons were analyzed with a UV-VIS-Spectrophotometer (UV-2700) (Shimadzu, Columbia, MD). Absorbance at 434 nm (mTurquoise) was used to calculate concentrations of the empty FRET and ABAleon proteins and the ratio of Abs515 (cpVenus173) and Abs434 was used to estimate protein purity.

46pH titrations were performed by addition of 1 µL concentrated ABAleon1.1 protein (final concentration 200 nM) to 100 µL wash buffer II adjusted to a pH range between pH 5.0–8.2 with 1 M HCl and MES powder or with 2 M NaOH. After recordings of ABA-free ABAleon1.1 emission spectra, using the TECAN Infinite M1000 PRO as mentioned above, 1 µL of 1 mM ABA (final concentration 10 µM ABA) was added and emission spectra were recorded using identical settings. Experiments were performed in duplicate and fitted by a four parameter Hill equation in SigmaPlot 10.0 (Systat, San Jose, CA). Ratio change was calculated by subtraction of the ABA-free from the ABA-bound equation values.

47ABA-induced ABAleon2.1 kinetics were analyzed using 2.77 µM ABAleon2.1 in a Berthold Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany) with the following settings: Lamp energy 5000, counting time 0.05 s, excitation 440 ± 10 nm, emission 470 ± 10 nm (mTurquoise) and 530 ± 20 nm (cpVenus173) measured in cycles of 6.12 s. At cycle 25, 50 µL 3 µM ABA in wash buffer II and 0.3 % EtOH was applied with low injector speed to result in the final 1 µM ABA in 0.1 % EtOH.

48Phosphatase assays were performed using the serine/threonine phosphatase assay system (Promega, Madison, WI). In brief, 50 µL reactions containing wash buffer II, 10-50 pmol protein, 5000 pmol Ser/Thr phosphopeptide ± 5 µM ABA with 0.005 % EtOH as solvent were incubated for 10–30 min at room temperature. Reactions were stopped by addition of 50 µL of the supplied molybdate dye/additive mixture and phosphate release was measured according to a standard curve with a Berthold Mithras LB 940 plate reader (Absorbance 600 ± 10 nm, lamp energy 50,000 and counting time 2 s).

49Three-dimensional coordinates of major components of ABAleon were built with known crystal structures of mTurquoise (pdb: 2YE0, ), PYL1-ABA-ABI1 (pdb: 3JRQ, ) and Venus (pdb: 1MYW, ). Each component of ABAleon was manually assembled using COOT. In the assembly, PYL1 was replaced by PYR1 (pdb: 3K3K, ) by tracing the Cα backbone. The unstructured C-terminus of mTurquoise was placed in distance corresponding to the PG linker and the PYR1 N-terminus. Venus was placed between ABI1 and mTurquoise with the N-terminus of Venus facing towards the C-terminus of ABI1. All structural figures were drawn with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.).

50Seeds were sterilized in 70 % EtOH and 0.04 % sodium dodecyl sulfate (SDS), washed three times in 100 % EtOH and sown on 0.5 Murashige and Skoog (MS) media (Sigma) adjusted to pH 5.8 with 1 M KOH and supplemented with 0.8 % phytoagar. After at least 4 days of stratification in the dark at 4 °C plants were cultivated in a growth room (16 h day/8 h night cycle, 25 °C, 50–100 µEm−2s−1 and 26 % relative humidity) or in a CMP4030 plant growth chamber (16 h day/8 h night cycle, 22 °C, 50 µEm−2s−1 and 25 % relative humidity; Conviron, Winnipeg, Manitoba). 6-day-old seedlings were transferred to soil and grown either in the growth room or in a CMP3244 plant growth chamber (16 h day, 22 °C/8 h night, 18 °C cycle, 50–100 µEm−2s−1 and 30–50 % relative humidity; Conviron).

51barII-UTF3 empty FRET, barII-UT-ABAleon2.1, hygII-UT-ABAleon2.13, hygII-UT-ABAleon2.14 and hygII-UT-ABAleon2.15 were transformed into Arabidopsis Columbia 0 accession and hygII-UT-ABAleon2.1 was transformed into pyl4ple [pyr1-1 (Q169 stop)/pyl1-1 (SALK_054640)/pyl2-1 (GT2864)/pyl4-1 (SAIL_517_C08)] by the floral dip method. Transformants were selected on 0.5 MS media supplemented with either 10 µg/mL glufosinate or 25 µg/mL hygromycin and further cultivated in soil in a CMP3244 plant growth chamber. Positive transformants were further selected by fluorescence intensity at a confocal microscope (see below). A list of transgenic Arabidopsis lines generated and used in this work is provided in Supplementary file 1C.

52For ABA seed germination assays seeds were sown on 0.5 MS agar media supplemented with 0.08 % EtOH as solvent control or 0.8 µM (+)-ABA (TCI, Portland, OR). After stratification, plants were grown in the growth room. Germinated seeds and seedlings with expanded green cotyledons were counted for a time period of 7 days with blinded genotypes. Analyses represent mean values ± SEM of four technical replicates normalized to the seed count (48–50 seeds) of each experiment.

53For growth assays, 4-day-old seedlings grown on 0.5 MS agar media were transferred to 0.5 MS agar media supplemented with 0.1 % EtOH as solvent control or 10 µM (+)-ABA and grown vertically in the growth room. Images were acquired 5 days after seedling transfer. Fresh weight was measured from pools of seven seedlings/experiment (means ± SEM, n = 5) normalized to the 0 µM ABA control conditions.

54ABA-induced stomatal closure analyses were performed with 20-23-day-old plants grown vertically on 0.5 MS agar media in the CMP4030 plant growth chamber. Six detached leaves were floated in assay buffer (5 mM KCl, 50 µM CaCl2 and 10 mM MES-Tris pH 5.6) at 22 °C and 100 µE m−2s−1 for 2 h. Subsequently, (+)-ABA or EtOH as solvent control was added to a final concentration of 10 µM (+)-ABA in 0.1 % EtOH followed by additional 2 h incubation. Leaves were blended 4x ∼10 s in ∼50 ml deionized water and leaf epidermal tissue was collected through a 100 µm nylon mesh (Millipore, Billerica, MA) and mounted on a microscope slide for imaging. Images were acquired using an inverted light microscope (Nikon Eclipse TS100) equipped with a 40x/0.65 ∞/0.17 WD. 0.57 objective and connected to the Scion camera and Scion VisiCapture Application Version1.3 (Scion Corporation, Frederick, MD). Experiments were performed with blinded genotypes and treatments and ≥ 24 stomatal apertures were measured per experiment using Fiji. Data represent mean stomatal apertures ± SEM of three experiments normalized to the solvent control.

55For guard cell imaging, 4-week-old detached leaves without mid vein were glued with the abaxial side on a cover glass using medical adhesive (Hollister, Libertyville, IL) and upper cell layers were dissected away with an industrial razor blade. Epidermal strips were incubated in assay buffer (5 mM KCl, 50 µM CaCl2, 10 mM MES-Tris, pH 5.6) and 0.01 % EtOH, as solvent control for ABA, for 1 h. Glass cover slips were mounted on a microscope slide with a central hole (Ø = 13 mm) using vacuum grease silicone (Beckman, Pasadena, CA) and analyzed in 200 µL of buffer mentioned above. For ABA application, epidermal strips were perfused by washing (pipetting) four to five times with assay buffer supplemented with 10 µM ABA in 0.01 % EtOH. Low humidity drop experiments were conducted on 19-27-day-old seedlings grown vertically on 0.5 MS agar media in the CMP4030 plant growth chamber. Low humidity was induced by opening the lid of the 0.5 MS agar plates. At time points 0, 15 and 30 min after plate opening two seedlings were blended 4x ∼10 s in ∼50 mL deionized water and leaf epidermal tissue was collected through a 100 µm nylon mesh (Millipore, Billerica, MA) and mounted on a microscope slide for imaging. Experiments were performed in triplicates and ≥ 27 guard cell pairs were analyzed per experiment. Long term stress treatments were conducted on detached leaves of 20-24-day-old plants grown vertically on 0.5 MS agar media in the CMP4030 plant growth chamber. Four leaves were pre-incubated for 1–4 h in assay buffer at 22 °C and 100 µEm−2s−1 and treatments were performed by addition of assay buffer supplemented with 10-fold concentrated EtOH (as solvent control for ABA), ABA, NaCl or sorbitol to obtain final concentrations of 0.01 % EtOH, 10 µM ABA, 100 mM NaCl or 300 mM sorbitol. 4 h after the treatments leaves were blended (see above) and leaf epidermal tissue was collected for imaging. Experiments were performed in triplicates with blinded treatments and epidermal fractions used for ABAleon2.1 emission ratio imaging were selected using the bright field channel. 24–40 guard cell pairs were analyzed per experiment.

56For seedling imaging, 4-day-old seedlings were transferred to glass bottom dishes (MatTek, Ashland, MA) supplemented with 200 µL 0.25 MS, 10 mM MES-Tris (pH 5.6) and 0.7 % low melting point agarose (Promega) and grown vertically for an additional day in the CMP4030 plant growth chamber. Before microscopic analyses, 90 µL assay buffer was added. Treatments were conducted by pipetting 10 µL ABA solution or the respective amount of EtOH as solvent control diluted in assay buffer to reach a final concentration of 10–50 µM ABA and 0.01–0.05 % EtOH. To apply ABA to defined tissues, transparent modeling clay was used to divide each glass bottom dish into two isolated chambers before application of the growth media. Seedlings were placed on top of the growth media and modeling clay, such that either the hypocotyl base and root differentiation zone (shoot to root transport) or the root maturation zone (hypocotyl to root and root to hypocotyl transport) laid on the dry modeling clay. Long term treatments were performed on 5-day-old seedlings, which were transferred to glass bottom dishes (MatTek, Ashland, MA) supplemented with 200 µL 0.25 MS, 10 mM MES-Tris (pH 5.6) and 0.7 % low melting point agarose with addition of 0.01 % EtOH (as solvent control for ABA), 10 µM ABA, 100 mM NaCl or 300 mM sorbitol. 6 h after transfer 100 µL assay buffer supplemented with 0.01 % EtOH, 10 µM ABA, 100 mM NaCl or 300 mM sorbitol was added before the root maturation- and elongation zone were imaged. Regions of the root maturation zone with similar distance from the root tip were selected in the bright field channel before ratio images were acquired. Treatments were performed blinded and 8–10 seedlings were analyzed per treatment.

57pRAB18-GFP plants were grown in soil in the CMP3244 plant growth chamber. To ensure high relative humidity (RH) conditions (70%) plants were kept under a plastic cover and sprayed with water twice a day. 2 days before the analyses, plants were removed from the growth chamber, placed in 25 % RH conditions and withheld from water supply. For the ABA treatment, detached leaves were floated for 4 h in 50 µM ABA prior to microscopic analyses.

58Expression analyses based on cpVenus173, YFP or GFP emission were performed using an Eclipse TE2000-U microscope equipped with Plan 20x/0.40 ∞/0.17 WD 1.3 and Plan Apo 60x/1.20 WI ∞/0.15–0.18 WD 0.22 objectives (Nikon), a CascadeII 512 camera (Photometrics), a MFC2000 z-motor (Applied Scientific Instruments, Eugene, OR), a QLC-100 spinning disc (VisiTech international, Sunderland, UK), a CL-2000 Diode pumped crystal laser (LaserPhysics Inc., West Jordan, UT), a LS 300 Kr/Ar laser (Dynamic Laser, Boston, MA) and guided by Metamorph software version 7.7.7.0 (Molecular Devices). Images were analyzed, processed and calibrated in Fiji.

59ABAleon ratio-imaging was conducted according to using an Eclipse TE300 microscope equipped with a Plan Fluor 10x/0.30 DIC L ∞/0.17 WD 16.0 for seedlings or a Plan Fluor 40x/1.30 Oil objective DIC H ∞/0.17 WD 0.2 for guard cells (Nikon, Tokyo, Japan), a Cool SNAP HQ camera (Photometrics, Tucson, AZ), a Mac 2002 System automatic controler, a CAMELEON filter set 71007A (D440/20, D485/40, D535/30; Chroma, Bellows Falls, VT) and guided by the MetaFluor software version 7.0r3 (Molecular Devices, Sunnyvale, CA). Images were acquired in intervals of 6 s using 200-250 ms exposure, Binning 4, Gain 2x (4x) and 20 MHz transfer speed. Images were analyzed and processed using Fiji. Analyses of seedlings were standardized as treatments were performed 4 min after the experiments started and regions used for emission ratio analyses had identical areas and distances from each other. ABA response curves in the root maturation zone and the hypocotyl were analyzed by fitting, using SigmaPlot 10.0 (Systat, San Jose, CA), to either data of single measurements or combined datasets.