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. 2002 Nov;130(3):1276-87.
doi: 10.1104/pp.009480.

Localization, ion channel regulation, and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein, ABH1

Véronique Hugouvieux  1 , Yoshiyuki Murata, Jared J Young, June M Kwak, Daniel Z Mackesy, Julian I Schroeder
Affiliations

Localization, ion channel regulation, and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein, ABH1

Véronique Hugouvieux et al. Plant Physiol. 2002 Nov.

Abstract

Abscisic acid (ABA) regulates developmental processes and abiotic stress responses in plants. We recently characterized a new Arabidopsis mutant, abh1, which shows ABA-hypersensitive regulation of seed germination, stomatal closing, and cytosolic calcium increases in guard cells (V. Hugouvieux, J.M. Kwak, J.I. Schroeder [2001] Cell 106: 477-487). ABH1 encodes the large subunit of a dimeric Arabidopsis mRNA cap-binding complex and in expression profiling experiments was shown to affect mRNA levels of a subset of genes. Here, we show that the dimeric ABH1 and AtCBP20 subunits are ubiquitously expressed. Whole-plant growth phenotypes of abh1 are described and properties of ABH1 in guard cells are further analyzed. Complemented abh1 lines expressing a green fluorescent protein-ABH1 fusion protein demonstrate that ABH1 mainly localizes in guard cell nuclei. Stomatal apertures were smaller in abh1 compared with wild type (WT) when plants were grown at 40% humidity, and similar at 95% humidity. Correlated with stomatal apertures from plants grown at 40% humidity, slow anion channel currents were enhanced and inward potassium channel currents were decreased in abh1 guard cells compared with WT. Gas exchange measurements showed similar primary humidity responses in abh1 and WT, which together with results from abh1/abi1-1 double-mutant analyses suggest that abh1 shows enhanced sensitivity to endogenous ABA. Double-mutant analyses of the ABA-hypersensitive signaling mutants, era1-2 and abh1, showed complex genetic interactions, suggesting that ABH1 and ERA1 do not modulate the same negative regulator in ABA signaling. Mutations in the RNA-binding protein sad1 showed hypersensitive ABA-induced stomatal closing, whereas hyl1 did not affect this response. These data provide evidence for the model that the mRNA-processing proteins ABH1 and SAD1 function as negative regulators in guard cell ABA signaling.

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Figures

Figure 1
Figure 1
Expression level of the CBC subunits, ABH1 and AtCBP20, is ubiquitous. Northern-blot analyses were performed on approximately 2 μg of poly(A+) RNA (extracted from flowers [F], stems [S], leaves [L], and roots [R]) in WT Arabidopsis. ABH1 or AtCBP20 cDNAs were used as probes. Actin1 probe was used as a loading control.
Figure 2
Figure 2
Analysis of GUS activity in WT plants transformed with the GUS reporter gene under the control of the ABH1 promoter. A through E, GUS activity in the hypocotyl and root of an 8-d old seedling (A), a cotyledon of an 8-d old seedling (B), a mature leaf (C), guard cells (D), and the hypocotyl of 2-d-old seedling (E).
Figure 3
Figure 3
ABH1 expression level is not regulated by ABA. WT leaves from five individual plants were sprayed with 100 μm ABA or water in parallel control experiments. Four hours later, poly(A+) RNA was extracted and 2 μg was used in northern-blot analyses using ABH1 cDNA and the COR47 genomic fragment as a probe. Actin1 probe was used as a loading control. Similar results were obtained in two replicates, and in the WT ecotype Wassilewskija (data not shown).
Figure 4
Figure 4
abh1 plants show a serrated leaf phenotype that is complemented by the ABH1 gene. Two rosette leaves of each plant are shown after 7 weeks of growth. abh1 lines complemented with the ABH1 promoter and gene (abh1:ABH1) were generated as described (Hugouvieux et al., 2001). abh1 lines transformed with the vector control pRD400 only (abh1:pDR400) were used as controls.
Figure 5
Figure 5
abh1 growth is slower compared with WT. A, abh1 and WT rosettes are shown after 3 weeks of growth. For the same age, abh1 rosettes are smaller and show a smaller number of leaves. B, WT and abh1 plants are shown after 6 weeks of growth. The delay in flowering is about 5 to 10 d in abh1. C, When abh1 and WT growth was synchronized, WT and abh1 plants showed similar whole-plant phenotypes (see also Table I). D, Development of the first two leaves in abh1 and WT seedlings grown in petri dishes. Data represent the mean of five experiments ± se (n = 30 seedlings per line for each experiment). E, Root elongation after 2, 6, 9, and 11 d in abh1 and WT. The figure shows a representative experiment ± sd (n = 60). D and E, Error bars are smaller than symbols when not visible. The slower growth in abh1 was complemented by the ABH1 gene (data not shown). A, B, D, and E, WT and abh1 plants were grown in parallel in a growth chamber after stratification for 4 d at 4°C from seeds between 3 to 6 months old.
Figure 6
Figure 6
GFP-ABH1 fusion protein is localized mainly in nuclei in WT and abh1. A through D, Bright-field images of guard cells used to study GFP fluorescence in E through H. Blue and green colors show chloroplast (emission 488 nm) and GFP (emission 522 nm) fluorescence, respectively. F, WT control transformed with GFP fused to the GUS protein (pCAMBIA1303; GenBank accession no. AF23299) shows that GFP-GUS is not localized in nuclei (nu), but rather in the cytosol (cyt). G, abh1-complemented lines with 35S:GFP-ABH1 construct show GFP fluorescence mainly in nuclei. H and I, Epidermal strips from WT transformed with the 35S:GFP-ABH1 construct show GFP fluorescence mainly in the nuclei in guard cells (GC) and epidermal cells (EpC). The same pattern of expression was observed in three independent lines.
Figure 7
Figure 7
Stomatal aperture and guard cell anion and K+ in channel activity are modified in abh1. A, Stomatal apertures in abh1 are smaller than in WT in plants grown at 40% humidity. Leaves were directly harvested from plants grown in a 40% humidity growth chamber and stomatal apertures were measured without any pre-incubation in opening solution. Three independent abh1 lines complemented with the ABH1 locus (abh1:ABH1 nos. 1, 2, and 3) show stomatal apertures similar to WT. abh1 control plants transformed with vector only (abh1:pRD400) show abh1 stomatal apertures. The data represent the mean ± sd of three independent experiments. B, Relative changes in leaf gas exchange in response to different humidity levels are similar in abh1 and WT. Stomatal conductance of WT and abh1 at several humidities was measured in intact leaves using a Li-6400 infrared gas analyzer (LI-COR, Inc., Lincoln, NE). Stomata were acclimated first to a high humidity (≥80% RH), then the humidity was sequentially dropped and new steady-state conductances determined at each humidity level. New steady states were achieved within 5 to 15 min after the humidity step. Data represent the mean ± se of three independent experiments. C and D, Whole-cell current voltage relationships recorded in WT and abh1 guard cells isolated from 40% humidity-grown plants showed a constitutive activation of slow anion channel currents and a decreased inward-rectifying K+ (K+in) channel activity in abh1 compared with WT (WT, n = 26 [C]; n = 13 [D]) and (abh1: n = 35 [C]; n = 14 [D]). Inserts show examples of ion current recordings. E, Whole-cell current voltage relationships recorded in abh1 guard cells isolated from either 40% humidity-grown plants (n = 14 cells) or after 72 h of high humidity (95%) treatment (n = 17 cells). Anion and K+ currents were recorded from 4- to 6-week-old plants as described in “Materials and Methods.”
Figure 8
Figure 8
Stomatal aperture phenotypes in the presence and absence of ABA in the abh1/era1-2 double mutant. A, ABA-induced stomatal closure in the abh1/era1-2 double mutant is similar to abh1. Plants were kept overnight in high (95%) humidity and then leaves were pre-incubated for 2 h in opening solution, under light. ABA was then added and stomatal apertures measured after 2 more hours. Stomatal aperture is expressed relative to the mean of stomatal aperture measured with no ABA for each line. Stomatal aperture ratio (width/length) with no ABA were 0.17 ± 0.016, 0.175 ± 0.015, 0.23 ± 0.029, and 0.21 ± 0.016 for WT, abh1, era1-2, and abh1/era1-2, respectively. B, Stomatal apertures in abh1/era1-2 plants grown at 40% humidity show similar opening to era1-2 and WT. Leaves were directly harvested from plants grown in a 40% humidity growth chamber about 6 h after onset of the 16-h day/night period and stomatal apertures were measured without any pre-incubation in stomatal opening solution. Data in A and B represent the mean ± se of three independent experiments with a minimum of 20 stomatal apertures measured per experiment and condition.
Figure 9
Figure 9
Stomatal aperture phenotypes in the presence or absence of ABA in the abh1/abi1-1 double mutant. A, ABA-induced stomatal closure in abh1/abi1-1 double mutant shows an intermediate response relative to abi1-1 and WT. Leaves were pre-incubated for 2 h in stomatal opening solution, under light, and then ABA was added and stomatal apertures measured after 2 more h. Stomatal aperture is expressed relative to the mean of stomatal apertures measured with no ABA for each line. Stomatal aperture ratio (width/length) with no ABA were 0.187 ± 0.016, 0.194 ± 0.011, and 0.157 ± 0.015 for WT, abh1/abi1-1, and abi1-1, respectively. B, Stomatal apertures in abh1/abi1-1 plants grown at 40% humidity are as wide as abi1-1. Leaves were directly harvested from plants grown in a 40% humidity growth chamber and stomatal apertures were measured without any pre-incubation in stomatal opening solution. A and B, Data represent the mean ± sd of three independent experiments.
Figure 10
Figure 10
ABA-hypersensitive stomatal closing in the sad1 mutant. Plants were kept overnight in high (95%) humidity and then leaves were pre-incubated for 2 h in opening solution, under light. ABA was then added and stomatal apertures measured after 2 more h in sad1 and WT C24 ecotype. Stomatal apertures are expressed relative to the mean stomatal apertures measured without ABA addition for each line. Stomatal aperture ratios (width/length) without ABA addition were 0.158 ± 0.01 and 0.145 ± 0.006 for WT and sad1, respectively. Data represent the mean ± se of three independent experiments. Error bars are smaller than symbols when not visible.
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