Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

Abstract

At least four genes encoding plasma membrane inward K+ channels (K(in) channels) are expressed in Arabidopsis guard cells. A double mutant plant was engineered by disruption of a major K(in) channel gene and expression of a dominant negative channel construct. Using the patch-clamp technique revealed that this mutant was totally deprived of guard cell K(in) channel (GCK(in)) activity, providing a model to investigate the roles of this activity in the plant. GCK(in) activity was found to be an essential effector of stomatal opening triggered by membrane hyperpolarization and thereby of blue light-induced stomatal opening at dawn. It improved stomatal reactivity to external or internal signals (light, CO2 availability, and evaporative demand). It protected stomatal function against detrimental effects of Na+ when plants were grown in the presence of physiological concentrations of this cation, probably by enabling guard cells to selectively and rapidly take up K+ instead of Na+ during stomatal opening, thereby preventing deleterious effects of Na+ on stomatal closure. It was also shown to be a key component of the mechanisms that underlie the circadian rhythm of stomatal opening, which is known to gate stomatal responses to extracellular and intracellular signals. Finally, in a meteorological scenario with higher light intensity during the first hours of the photophase, GCK(in) activity was found to allow a strong increase (35%) in plant biomass production. Thus, a large diversity of approaches indicates that GCK(in) activity plays pleiotropic roles that crucially contribute to plant adaptation to fluctuating and stressing natural environments.

Fig. 1.

Absence of inward K⁺ channel activity in guard cells of the kincless mutant. (A) Representative macroscopic current traces recorded with the patch-clamp technique in guard cell protoplasts from WT (Upper Left), kat2-1 (knockout mutant disrupted in the KAT2 gene) (Upper Right), domneg-1 (expressing a dominant negative kat2 construct in WT background) (Lower Right), or kincless (expressing the same domneg construct in the kat2-1 background) (Lower Left) plants. In all recordings, the holding potential was −100 mV; voltage steps were applied to potentials ranging between −100 and +120 mV in 20-mV increments (top traces for each genotype) or between −100 and −260 mV in increments of −20 mV (bottom traces for each genotype). (B) Comparison of current–voltage relationships from the different genotypes. Total current (I tot, sampled at time marked by symbols over the recordings in A) is plotted against membrane potential. Data are means ± SE (with the number of repeats in brackets).

Fig. 2.

Disruption of inward K⁺ channel activity affects stomatal opening. Epidermal strips were peeled from WT (filled symbols) or kincless (open symbols) plants at the end of the dark period. Stomatal opening was induced (t = 0) by fusicoccin (10 μM) in the presence or absence of Cs⁺ (30 mM) (A) or by switching on white light (B) or blue or red light (C). K⁺ concentration in the bath solution: 10 mM (A) or 30 mM (B and C). Data are means ± SE. n > 100 in A, n >150 in B, and n > 200 in C.

Fig. 3.

Inward K⁺ channel activity underlies stomatal responsiveness to changes in light, VPD, and CO₂ conditions. (A) Rate of light-induced stomatal opening. The consequences of the absence of GCK_in activity on leaf transpiration were then investigated by using a setup allowing to continuously monitor transpirational water loss in an intact plant (10). (Left) Typical recordings of transpiration in a WT or kincless plant during a whole nycthemeral period (8-h/16-h day/night). (Right) Time constants describing the increase in transpiration rate induced by light (derived by fitting the kinetics with monoexponential functions). Data are means ± SE; n = 4. For each of the four plants tested per genotype, the individual time constant integrated values derived from the recording of transpiration rate during at least five successive photoperiods. (B) Responsiveness to a decrease in VPD. An automated growth chamber [Phenopsis robot (28)] was used to impose rapid changes in VPD. (Left) WT or kincless plant transpiration (bottom) during a climatic scenario comprising (top) a dark-light transition (open arrow) 2 h before a sudden decrease in VPD. Data are means ± SE; n = 60. (Right) Kinetics of the increase in transpiration rate due to stomatal aperture readjustment during the first half hour (boxed region in Left) after the change in VPD. (C) Responsiveness to CO₂ availability. (Left) Epidermal peel bioassays. Epidermal strips were peeled at the end of the dark period and incubated in the dark in 30 mM K⁺. Stomatal opening was induced by bubbling CO₂-free air for 3 h. Stomatal aperture was measured just before (control values, two bars on the left) and after (two bars on the right) this 3-h treatment. (Right) In planta stomatal conductance measurement in WT and kincless leaves. Time courses of stomatal conductance in response to changes in CO₂ concentration under dark (Upper) or light (Lower) conditions. Dark and light periods are indicated by black and white boxes under the graphs. The changes in CO₂ concentration in the air flow are indicated (expressed in ppm) in the gray boxes. The decrease in CO₂ concentration in the air flow under dark conditions mimics depletion of internal CO₂ driven by photosynthetic activity (independent of light signal and photosynthesis).

Fig. 4.

Disruption of GCK_in activity results in impaired stomatal closure upon salt stress. (A and B) Epidermal peel bioassays. Epidermal strips peeled from WT and kincless plants were bathed in either 30 mM KCl (A) or 10 mM KCl and 20 mM NaCl (B). Stomatal opening was induced (t = 0) by switching light on, and stomatal closure was triggered by adding the plant stress hormone ABA (10 μM). (C and D) Experiments on intact plants. White and black boxes under the curves indicate light and dark periods, respectively. The photoperiod was of 8 h in total: 3 h (9 a.m. to 12 p.m.) and five additional hours (3 p.m. to 8 p.m.). Plant transpiration was recorded between 8 a.m. and 3 p.m. Data are means ± SE; n = 15 plants per genotype. (C) Control treatment: 5-week-old plants continuously grown in the absence of Na⁺. (D) NaCl treatment: same plants as in C, but 12 h after introduction of 70 mM Na⁺ in the nutrient solution. (Inset) Transpiration recordings for 3 successive days after Na⁺ introduction in the nutrient solution.

Fig. 5.

Disruption of inward K⁺ channel activity affects stomatal circadian rhythm and renders stomatal opening sensitive to the duration of the preceding photoperiods. (A and B) Effect on stomatal circadian rhythm. (A) Continuous recording of the transpiration rate in a single WT or kincless plant during 7 days. Black and white boxes under the curves indicate dark and light periods, respectively. Two photoperiods were suppressed, on days 4 and 5. (B) Enlargement from A (dotted boxed regions) highlighting stomatal preopening in darkness in the WT plant. (C and D) Sensitivity to the duration of the preceding photoperiods. (C) Transpiration rates recorded in 5-week-old WT or kincless plants exposed to an 8-h photoperiod since sowing. (D) Transpiration rates recorded in the same plants after exposure to a 3-h photoperiod during 2 days. Data are means ± SE; n = 15 plants per genotype.