Non-autonomous stomatal control by pavement cell turgor via the K+ channel subunit AtKC1

Abstract

Stomata optimize land plants' photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

Figure 1

Impaired control of stomatal aperture and transpirational water loss in atkc1–2-mutant plants. A, Transpirational water loss from excised leaves. The second leaf was excised from WT (Ws ecotype), atkc1–2, and ProAtKC1:AtKC1-complemented atkc1–2 plants. Excised leaf water loss was deduced from the decrease in leaf weight. B, Leaf water conductance measured on intact leaves with a porometer. C, Transpiration rates in whole-plant assays. D, Stomatal aperture in WT, atkc1–2, and ProAtKC1:AtKC1-complemented atkc1–2 plants. Before stomatal aperture measurements, epidermal strips were kept in the dark for 2 h (dark treatment) or in dark for 2 h, followed by 2 h in the light (light treatment) in a 40 mM K⁺ solution. A–D, Means ± se. In (A)–(C), n = 5, 9, and 11, respectively; in (D), n=6 values, each value corresponding to ∼100 stomata. Letters depict significant group values after ANOVA and Tukey’s post hoc test. In C, for the statistical analysis, the data obtained during the four consecutive days were pooled, taking into account the corresponding day cycle.

Figure 2

Shaker-like K⁺ channel activity in guard cells from WT and atkc1–2-mutant plants (Ws ecotype). Guard cell protoplast current/voltage relationships. Means ± se; n = 8 and 10 for the WT and mutant genotypes, respectively. External K-glutamate concentration was 100 mM.

Figure 3

Weakly inwardly-rectifying K⁺ channel activity in pavement cells from WT and atkc1–2-mutant plants (Ws ecotype). A–C, Typical weakly inwardly rectifying K⁺ currents recorded in pavement cell protoplasts and their blockage by 10 mM external BaCl₂. A, Example of inward and outward current traces (right and left panels, respectively), recorded in the presence of 100 mM K-glutamate (total K⁺ concentration: 105 mM) and successively before BaCl₂ addition (top panels), in the presence of BaCl₂ (middle panel), and after BaCl₂ rinse (lower panels). B, Corresponding current/voltage relationships. C, Current inhibition in the presence of BaCl₂ at negative and positive voltages. Means ± se; n = 7. D and E, Effect of change in external K-glutamate concentration on the weakly inwardly rectifying currents in pavement cell protoplasts. D, Example Figure 3: (continued) of inward and outward current traces (right and left panels, respectively) recorded successively in 100 mM K-glutamate (top panels) and 10 mM K-glutamate (lower panels; total K⁺ concentration: 15 mM). E, Current/voltage relationships in the two external K-glutamate conditions. Currents were normalized in each protoplast by the current value obtained in 100 mM K-glutamate at −160 mV. Means ± se; n = 7. F and G, Representative inward and outward (right and left panels, respectively) Shaker-like K⁺ current traces in WT (F) and atkc1–2 (G) pavement cell protoplasts. H, Pavement cell protoplast Shaker-like current/voltage relationship in WT and atkc1–2-mutant plants. External K-glutamate concentration: 100 mM. Means ± se; n = 8 for both the WT and the mutant genotypes. The concentration of K⁺ (essentially as glutamate salt) in the pipette solution and in the bath solution was 140 and 105 mM, respectively, which results in a K⁺ equilibrium potential of close to −7 mV.

Figure 4

The defect in stomatal aperture displayed by the atkc1–2 mutant does not result from loss of AtKC1 expression in guard cells. A, Relative expression of AtKC1 compared with that of other Shaker channels in guard cells (left panel) and relative expression of AtKC1 in guard cells compared with that in leaves (right panel). Expression levels determined by RT-qPCR experiments. B, Stomatal aperture in WT plants, in atkc1–2-mutant plants, and in atkc1–2-mutant plants transformed with either the complementing ProAtKC1:AtKC1 construct (see Figure 1) or with a construct, ProKAT1:AtKC1, rendering AtKC1 expression dependent on the activity of the promoter of KAT1, a Shaker channel gene whose expression in guard cells is specific of this cell type in leaf epidermis (see also Supplemental Figure S4). “Dark” and “light” treatments: stomatal aperture was measured under dark or light as described in Figure 1D. “Light + ABA” treatment: 10 μM ABA was applied for 2 h to light-treated strips before stomatal aperture measurement. A and B, Means ± se. For (A), n = 3 pools of 5–6 plants, and ** and *** denote P < 0.01 and <0.001 in a two-tailed Student’s t test (comparison AtKC1 expression to that of KAT1, KAT2, or GORK, left panel, and AtKC1 expression in guard cells versus AtKC1 expression in leaves, right panel). For (B), n = 6–10 values, each value corresponding to ∼60 stomata. Letters depict significant group values after ANOVA and Tukey’s post hoc test.

Figure 5

Disruption of AtKC1 leads to reduced K⁺ contents in leaf epidermis. K⁺ contents in whole leaf, leaf margin, and leaf epidermis in WT and atkc1–2-mutant plants. Means ± se; n = 3 pools, each one obtained from nine leaves (*P < 0.05, using two-tailed Student’s t test).

Figure 6

Disruption of AtKC1 leads to reduced turgor pressure in pavement cells but not in guard cells. A, Boxplots depicting turgor pressure values obtained with AFM in WT and atkc1–2 pavement cells (left panel) and guard cells (right panel). Upper and lower whiskers: 1.5 times the IQR (first to third interquartile range); border of the boxes: first and third quartile; central line: median. Letters depict different group values after Student’s t test (P < 0.05). For guard cells, n = 46 for the WT genotype and 32 for the atkc1–2-mutant genotype. For pavement cells, n=86 for the WT and 51 for the mutant genotype. B, Disruption of AtKC1 results in decreased osmotic pressures in leaf epidermis as deduced from plasmolysis curves obtained by measuring the percentage of epidermal strips displaying plasmolyzed cells when bathed for 5 min in the presence of mannitol. Ten–twelve strips were examined for each genotype and mannitol concentration. C, Effect on stomatal aperture of adding mannitol to the solution bathing epidermal strips from WT or atkc1–2-mutant plants. n = 92–120 from six leaves for each mannitol concentration and genotype.

Figure 7

The atkc1–2 mutation results in membrane depolarization in pavement cells and in an increased sensitivity of the MP to the external concentration of K⁺. A, MPs recorded in WT and atkc1–2 pavement cells bathed in 0.1 mM or 10 mM K⁺. B, Membrane depolarizations induced by the increase in external K⁺ concentration from 0.1 to 10 mM. Each value corresponded to the difference in the MP that was observed when the external K⁺ concentration was increased from 0.1 mM to 10 mM K⁺ within the same cell. C, Representative trace of a WT pavement cell showing membrane depolarization and repolarization due to changes in external K⁺ concentration. D, Representative trace of an atkc1–2 pavement cell subjected to the same protocol as in (C). White and gray bars depict the periods where the external K⁺ concentration was 0.1 and 10 mM, respectively. In (A) and (B), means ± se are shown. n = 14 cells from five different plants for WT and n = 14 cells from three different plants for atkc1–2. Letters depict significant group values after ANOVA and Tukey’s post hoc test. *** denotes P < 0.001 in a two-tailed Student’s t test.

Figure 8

Restoration of WT stomatal features in the atkc1–2 mutant requires AtKC1 expression in pavement cells and trichomes. A, Stomatal aperture under light in WT Arabidopsis plants (Ws ecotype, black bar), in atkc1–2-mutant plants (white bar), and in atkc1–2-mutant plants transformed with a construct allowing expression of AtKC1 under control of one of the following promoters: ProCER5, ProCYP96A4, ProKCS19, ProOCT3, ProGL2, ProAt1G66460, and ProFMO1 (expression patterns of these promoters: see Table 1 and Figure 8: (continued) Supplemental Figures S1 and S5). Gray bars and dark green bars: transformed plants with rescued or non-rescued stomatal phenotype, respectively. Stomatal aperture was measured following the same procedure as in Figure 1D. B, Stomatal aperture in epidermal strips bathed in mannitol solutions. Transformed lines identified in (A) as displaying stomatal aperture values similar to that of WT plants (transforming constructs: ProAtKC1:AtKC1, ProCER5:AtKC1, and ProCYP96A4:AtKC1) also behaved like WT plants in response to added mannitol (showing a non-monotonous sensitivity to mannitol concentration). In contrast, the transgenic line ProKAT1:AtKC1, shown in (A) to display a stomatal aperture similar to that of atkc1–2-mutant plants, also displayed a monotonous decrease in stomatal aperture in response to increased mannitol concentration, and thus behaved like atkc1–2-mutant plants (see Figure 6C). C, Leaf epidermis K⁺ content in WT plants, in atkc1–2-mutant plants and in atkc1–2-mutant plants transformed with the ProCER5:AtKC1 and ProCYP96A4:AtKC1 complementing constructs. A–C, Means ± se. In (A) and (B), n = 94–131 stomata from six leaves. In (C), n = 3 pools of samples, each one obtained from nine leaves. In (A) and (C), letters depict significant group values after ANOVA and Tukey’s post hoc test.