Limits...
Measuring stress signaling responses of stomata in isolated epidermis of graminaceous species.

Shen L, Sun P, Bonnell VC, Edwards KJ, Hetherington AM, McAinsh MR, Roberts MR - Front Plant Sci (2015)

Bottom Line: Our understanding of guard cell signaling in these important species is therefore much more limited.Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon.We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures.

View Article: PubMed Central - PubMed

Affiliation: Lancaster Environment Centre, Lancaster University , Lancaster, UK.

ABSTRACT
Our current understanding of guard cell signaling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signaling in the stomata of graminaceous species (including many of the world's major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signaling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20 and 40°C.

No MeSH data available.


Temperature-dependence of the responses of barley stomata to ABA and CO2. (A) Interaction between temperature and ABA. Promotion of closure assays were performed at a range of concentrations of ABA in CO2-free air at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no ABA). Data from at least 3 sets of independent experiments were pooled and values are means of at least 120 measurements ± SE. (B) Interaction between temperature and CO2. Promotion of closure assays were performed in CO2-free air or at ambient or elevated CO2 at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no CO2). Data from at least three sets of independent experiments were pooled and values are means of at least 120 measurements ± SE.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499840&req=5

Figure 5: Temperature-dependence of the responses of barley stomata to ABA and CO2. (A) Interaction between temperature and ABA. Promotion of closure assays were performed at a range of concentrations of ABA in CO2-free air at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no ABA). Data from at least 3 sets of independent experiments were pooled and values are means of at least 120 measurements ± SE. (B) Interaction between temperature and CO2. Promotion of closure assays were performed in CO2-free air or at ambient or elevated CO2 at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no CO2). Data from at least three sets of independent experiments were pooled and values are means of at least 120 measurements ± SE.

Mentions: Stomatal responses to ABA and CO2 have been shown to be temperature-dependent (e.g., Raschke, 1970; Rodriguez and Davies, 1982; Spence et al., 1984; Honour et al., 1995). We therefore used the epidermal peel assay to examine the temperature-dependence of ABA- and CO2-induced stomatal closure in isolated epidermis of barley. First, we generated dose-response curves for ABA in isolated epidermis incubated at 20, 30, or 40°C. The results presented in Figure 5A, show that in comparison with the response at 20°C, incubation at 30°C significantly increased the sensitivity of guard cells to ABA. Increasing the temperature to 30°C had no effect on apertures in the absence of ABA, but apertures were reduced for all concentrations of ABA tested. By contrast, incubation at 40°C caused a significant increase in apertures in the absence of ABA and with 10–9 M ABA. At higher ABA concentrations, apertures were similar to, or slightly larger than, those observed at 20°C, suggesting a degree of inhibition of ABA sensitivity at 40°C. We also examined the response of barley guard cells to CO2 at these three temperatures. Guard cell responses were again temperature-dependent. However, unlike the response to ABA, we observed maximum CO2-induced stomatal closure at 20°C, with increasing temperatures causing an increasing degree of inhibition of the CO2 response (Figure 5B).


Measuring stress signaling responses of stomata in isolated epidermis of graminaceous species.

Shen L, Sun P, Bonnell VC, Edwards KJ, Hetherington AM, McAinsh MR, Roberts MR - Front Plant Sci (2015)

Temperature-dependence of the responses of barley stomata to ABA and CO2. (A) Interaction between temperature and ABA. Promotion of closure assays were performed at a range of concentrations of ABA in CO2-free air at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no ABA). Data from at least 3 sets of independent experiments were pooled and values are means of at least 120 measurements ± SE. (B) Interaction between temperature and CO2. Promotion of closure assays were performed in CO2-free air or at ambient or elevated CO2 at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no CO2). Data from at least three sets of independent experiments were pooled and values are means of at least 120 measurements ± SE.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499840&req=5

Figure 5: Temperature-dependence of the responses of barley stomata to ABA and CO2. (A) Interaction between temperature and ABA. Promotion of closure assays were performed at a range of concentrations of ABA in CO2-free air at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no ABA). Data from at least 3 sets of independent experiments were pooled and values are means of at least 120 measurements ± SE. (B) Interaction between temperature and CO2. Promotion of closure assays were performed in CO2-free air or at ambient or elevated CO2 at either 20°C (filled circles), 30°C (open circles), or 40°C (filled triangles). To normalize for variation between different experiments, stomatal apertures are expressed relative to that of the control (stomatal aperture at 20°C, no CO2). Data from at least three sets of independent experiments were pooled and values are means of at least 120 measurements ± SE.
Mentions: Stomatal responses to ABA and CO2 have been shown to be temperature-dependent (e.g., Raschke, 1970; Rodriguez and Davies, 1982; Spence et al., 1984; Honour et al., 1995). We therefore used the epidermal peel assay to examine the temperature-dependence of ABA- and CO2-induced stomatal closure in isolated epidermis of barley. First, we generated dose-response curves for ABA in isolated epidermis incubated at 20, 30, or 40°C. The results presented in Figure 5A, show that in comparison with the response at 20°C, incubation at 30°C significantly increased the sensitivity of guard cells to ABA. Increasing the temperature to 30°C had no effect on apertures in the absence of ABA, but apertures were reduced for all concentrations of ABA tested. By contrast, incubation at 40°C caused a significant increase in apertures in the absence of ABA and with 10–9 M ABA. At higher ABA concentrations, apertures were similar to, or slightly larger than, those observed at 20°C, suggesting a degree of inhibition of ABA sensitivity at 40°C. We also examined the response of barley guard cells to CO2 at these three temperatures. Guard cell responses were again temperature-dependent. However, unlike the response to ABA, we observed maximum CO2-induced stomatal closure at 20°C, with increasing temperatures causing an increasing degree of inhibition of the CO2 response (Figure 5B).

Bottom Line: Our understanding of guard cell signaling in these important species is therefore much more limited.Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon.We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures.

View Article: PubMed Central - PubMed

Affiliation: Lancaster Environment Centre, Lancaster University , Lancaster, UK.

ABSTRACT
Our current understanding of guard cell signaling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signaling in the stomata of graminaceous species (including many of the world's major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signaling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20 and 40°C.

No MeSH data available.