Plant Physiol. 2008 June; 147(2): 922–930.

The Stomata of the Fern Adiantum capillus-veneris Do Not Respond to CO2 in the Dark and Open by Photosynthesis in Guard Cells

Plant Physiol. 2008 June; 147(2): 922–930.

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light.

Plant Physiol. 2008 June; 147(2): 922–930.

Stomata are small adjustable pores located on the surface of leaves. They allow the exchange of CO2 and water between the leaf interior and the atmosphere. Through the regulation of stomatal apertures, plants obtain the CO2 required for photosynthesis and prevent excessive water loss through transpiration. Stomata are found in mosses, ferns, and higher plants, and several works have shown their morphological and/or functional diversity (Willmer and Fricker, 1996; Franks and Farquhar, 2007). Such differences may have arisen as the plants evolved to adapt to different environmental factors. While several environmental stimuli, such as light, CO2, humidity, and temperature, have been shown to regulate stomatal movements in higher plants (Willmer and Fricker, 1996), little is known about how these stimuli affect the stomata of ferns.

Plant Physiol. 2008 June; 147(2): 922–930.

Adiantum capillus-veneris belongs to the Leptosporangiopsida, which are newly diversified fern species that grow in shadow beneath angiosperms (Schneider et al., 2004), and usually makes its habitat beneath the canopy, where the ambient light is weak and rich in far-red. The stomata are found only on the lower surface of leaves and lack subsidiary cells, and their guard cells contain densely arranged chloroplasts. Recently, we reported that the stomata of the fern A. capillus-veneris lacked a blue light-specific opening response but did open in response to red light (Doi et al., 2006). A lack of blue light-specific response has commonly been seen in other species of Leptosporangiopsida. We also showed that phy3 (neochrome 1; Suetsugu et al., 2005), a chimeric protein of phototropin and phytochrome, did not work as a photoreceptor for red light-induced stomatal opening in Adiantum. Therefore, the red light-induced stomatal opening in Adiantum could be explained by the photosynthetic activity and/or by the response via phytochrome. Here, we investigated the stomatal responses of Adiantum to light and CO2 in the dark. We demonstrated that the guard cells are insensitive to CO2 and that the guard cell chloroplasts are responsible for stomatal opening in response to light in this plant species.

Plant Physiol. 2008 June; 147(2): 922–930.

Figure 1. Stomatal responses to light in intact leaves of A. capillus-veneris. A, Stomatal conductance, photosynthetic rate, and Ci in response to red (left) and blue (right) light. Each light at an intensity of 600 μmol m−2 s−1 was applied to the upper surface of a leaf as indicated by the upward-pointing arrowhead, and turned off as indicated by the downward-pointing arrowhead. B, PFD response curves for stomatal opening. For each type of light, opening rates were measured at five to seven PFDs with separate leaves of the same plant. Circles, Blue light; squares, red light; triangles, green light. Black, gray, and white symbols represent the separate experiments that were conducted on the different days, respectively. Solid, broken, and dotted lines represent the regression curves for blue, red, and green lights, respectively. Plant Physiol. 2008 June; 147(2): 922–930.

Plant Physiol. 2008 June; 147(2): 922–930.

Figure 4. Dependencies of PFD for stomatal conductance increase and photosynthetic CO2 fixation. Solid and dotted lines represent the increase in stomatal conductance and photosynthetic CO2 fixation, respectively. Red light was irradiated onto the upper (black circles) or lower side (white circles) of the leaf. Plant Physiol. 2008 June; 147(2): 922–930.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

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

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

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 GCKin 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). Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

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). Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

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. Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Plant Cell. 2008 January; 20(1): 75–87.

Transient Transformation of Guard Cells For transient expression of Arabidopsis genes and constructs in V. faba, coding regions for At ROP2, CA1-rop2 (G15V), DN2-rop2 (D121A), RIC7, and RhoGDI1 (At3g07880) were cloned in a pUC vector fused to the C terminus of the GFP or RFP coding region under the control of the 35S promoter. V. faba guard cells were biolistically transformed following the manufacturer's protocol (Bio-Rad Laboratories). In brief, DNA-coated gold particles (diameter, 1 μm) were fired into the abaxial side of V. faba leaves at a helium pressure of 1350 p.s.i. and under a vacuum of 28 inches of mercury. Bombarded leaves were kept at 22°C in the dark. After 12 h to 3 d, epidermal fragments were peeled from the bombarded sites and fluorescence distribution was observed.

Plant Cell. 2008 January; 20(1): 75–87.

Measurement of Stomatal Apertures Stomatal apertures were measured as described by Hwang et al. (1997). The intact rosette leaves of Arabidopsis were floated on an assay buffer containing 50 mM KCl and 10 mM K+-MES, pH 6.1. For stomatal opening in Arabidopsis, leaves were incubated in the dark overnight before starting illumination with white light at an intensity of 250 μmol·m−2·s−1. Stomatal apertures were measured (pore width/length) from abaxial epidermal strips peeled immediately before observation using an eyepiece micrometer or Interactive Measurement software (AxioVision 3.0.6; Zeiss). Stomatal apertures of V. faba were measured from the abaxial epidermal strips peeled from fully expanded young leaves. The epidermal fragments were floated on an assay buffer containing 50 mM KCl and 10 mM K+-MES, pH 6.1, and subjected to stimuli.

Plant Cell. 2008 January; 20(1): 75–87.

Infrared ThermographyArabidopsis plants were grown under well-watered conditions (22°C, 16-h-light photoperiod) for 4 weeks. The temperature of the leaves of intact Arabidopsis plants was measured at the beginning of the photoperiod and after 1 to 6 h of illumination with white light at an intensity of 250 μmol·m−2·s−1 using a ThermaCAM S60 infrared camera (FLIR Systems). The images were analyzed using ThermaCAM Reporter software (FLIR Systems).

Plant Cell. 2008 January; 20(1): 75–87.

ROP2-RIC7 Binding AssayThe MBP-RIC7 fusion protein, GST, and GST-ROP2 protein were expressed in Escherichia coli and purified (Wu et al., 2001). GST (50 μg) or GST-ROP2 (25 μg) was preincubated with 500 μL of binding buffer (40 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 4% glycerol, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 20 mM glycerophosphate supplemented freshly with 2 mM Na3VO4 and 3 mM GTP or GDP) for 30 min and mixed with 2 μL of MBP-RIC7 and 100 μL of agarose-GSH beads. After 2 h of incubation at 4°C, the pellet was washed four times with binding buffer without BSA. Proteins in the pellet were separated by SDS-PAGE, and protein gel blotting was performed using affinity-purified anti-GST-horseradish peroxidase conjugate antibody (1:5000 dilution; Amersham Biosciences) or anti-MBP antibody (1:10,000 dilution; New England Biolabs). Anti-mouse antibodies conjugated to alkaline phosphatase were used to detect anti-MBP (1:5000 dilution; Promega).

Plant Cell. 2008 January; 20(1): 75–87.

Accession NumbersSequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: ROP2 (At1g20090), RIC7 (At4g28556), and RhoGDI1 (At3g07880).