The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis

Abstract

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.

Figure 1.

Localization of CHL1 Expression and Protein in Guard Cells. (A) Portion of a leaf from a plant carrying the HaeII CHL1-GUS fusion construct (Guo et al., 2001). Strong GUS staining indicative of CHL1 expression is found in guard cells. (B) Section of a hypocotyl from a 7-day-old transgenic plant carrying the HaeII CHL1-GFP construct (Guo et al., 2001). Strong GFP signals are found only in guard cells using confocal laser scanning microscopy. (C) Immunofluorescence from a portion of a leaf stained with anti-CHL1 antibody. Whole-mount assays were performed with affinity-purified anti-CHL1 antibody and Alexa Flour 488–conjugated secondary antibody (Molecular Probes) using confocal laser scanning microscopy (Guo et al., 2001). (D) Higher magnification of a section of the image in (C) showing immunofluorescence from individual stomata. (E) Control experiment using preimmune serum showing little immunofluorescence from a leaf. (F) Outline of the leaf shown in (E) by propidium iodide staining.

Figure 2.

CHL1 Functions in Light-Induced Stomatal Opening. (A) Stomatal apertures were determined for wild-type and chl1 mutant plants (chl1-4 and chl1-5) undergoing light-induced stomatal opening. Data were averaged from three separate experiments; n = 40 aperture measurements per experiment. Error bars represent standard deviations. (B) Nitrate accumulation was measured in guard cell protoplasts isolated from detached leaves of wild-type and chl1 mutant plants during light-induced stomatal opening in the presence of nitrate. Error bars represent standard deviations (n = 4).

Figure 3.

Stomatal Apertures in Intact Plants. Stomatal apertures were determined for leaves from intact plants during the middle of the light period (16 h of light and 8 h of dark) as described in Methods. Data were averaged from three separate experiments; n = 40 aperture measurements per experiment.

Figure 4.

Stomatal Conductance and Apertures in Plants Depleted of CO₂ in the Dark. (A) Stomatal conductance was measured in wild-type and chl1 mutant plants in the dark when CO₂ levels (400 mL/L) were decreased to 0 mL/L (CO₂-free air). Squares, wild type (n = 6); circles, chl1-4 (n = 5); triangles, chl1-5 (n = 5). Error bars represent standard deviations. (B) Stomatal apertures in leaves of the wild type and chl1 mutants after 40 min of treatment with CO₂-free air. Error bars represent standard deviations (n = 5).

Figure 5.

Abscisic Acid Effects on Stomatal Opening and Closing of Wild-Type and chl1 Mutant Plants (A) Stomatal apertures were determined for wild-type and chl1 mutant plants (chl1-4 and chl1-5) undergoing abscisic acid (ABA)–induced stomatal closing as described in Methods. Data were averaged from three separate experiments; n = 40 aperture measurements per experiment. Error bars represent standard deviations. (B) Light-induced stomatal opening was measured in the wild type and chl1 mutants with or without abscisic acid treatment. Dark control data represent stomatal apertures from leaves taken from seedlings kept in the dark for 5 h. Data were averaged from three separate experiments; n = 40 aperture measurements per experiment. Error bars represent standard deviations.

Figure 6.

Nitrate Induces Depolarization of Membrane Potential in Guard Cells of Wild-Type but Not chl1 Plants. Depolarization of membrane potentials is represented as an increase in fluorescence. (A) Fluorescence from a representative wild-type guard cell at 0, 15, 30, and 60 s after adding CsNO₃ to a final concentration of 100 mM. (B) Fluorescence from a representative mutant (chl1-5) guard cell at 0, 15, 30, and 60 s after adding CsNO₃ to a final concentration of 100 mM. (C) Fluorescence from a representative wild-type guard cell at 0, 15, 30, and 60 s after adding KCl to a final concentration of 100 mM. (D) Fluorescence from a representative mutant (chl1-5) guard cell at 0, 15, 30, and 60 s after adding KCl to a final concentration of 100 mM. (E) Quantified fluorescence signals from the stomates for each genotype or treatment. Error bars represent standard deviations (n = 5).

Figure 7.

Chl1 Mutants Are More Drought Tolerant than Wild-Type Plants under Drought Stress. (A) Water loss from the leaves of wild-type and chl1 plants grown with the indicated nitrogen sources. Data show percentage of initial fresh weight (F.W.) lost from leaves from three individual plants per genotype. Results from one of three experiments are shown. Squares, wild type; circles, chl1-4; triangles, chl1-5. Error bars represent standard deviations. (B) Wild-type and chl1 mutant plants after 5 days of drought stress. Plants were irrigated for 22 days with nitrate-containing medium and then drought stressed by terminating irrigation. Ten pots per genotype were examined in one of three separate experiments. (C) Soil water contents were determined by weighing pots and plotted as a percentage of initial fresh weight in chl1 and wild-type pots during desiccation. Pots were covered to minimize soil evaporation. Squares, wild type; circles, chl1-4; triangles, chl1-5. Error bars represent standard deviations.