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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.


Promotion of closure of cereal stomata by ABA. Following incubation under opening conditions, epidermal peels were exposed to zero (white bars), 10–7 M (gray bars), or 10–6 M (black bars) ABA at 20°C. Values shown are mean stomatal apertures ± SE from n = 240 (barley), n = 90 (wheat), and n = 120 (Brachypodium) measurements. Letters indicate statistically different means within species, determined using one-way ANOVA and a Tukey post-test.
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Figure 3: Promotion of closure of cereal stomata by ABA. Following incubation under opening conditions, epidermal peels were exposed to zero (white bars), 10–7 M (gray bars), or 10–6 M (black bars) ABA at 20°C. Values shown are mean stomatal apertures ± SE from n = 240 (barley), n = 90 (wheat), and n = 120 (Brachypodium) measurements. Letters indicate statistically different means within species, determined using one-way ANOVA and a Tukey post-test.

Mentions: To test the validity of the stomatal assay in isolated epidermis of graminaceous plants, we examined the well-known response of guard cells to ABA in a promotion of closure assay. As expected, we observed that wheat, barley and Brachypodium all exhibit a characteristic dose-dependent response to ABA in the epidermal peel assay (Figure 3). We also measured responses to external CO2. In comparison with CO2-free air, stomatal closure was promoted by ambient CO2 (360 ppm) in barley, but we observed no further response at elevated CO2 (800 ppm; Figure 4A). Brachypodium stomatal apertures were also reduced by CO2 (Figure 4A). We next examined the interaction between ABA and CO2 signaling in barley guard cells. We observed a clear additive effect of ABA and CO2, with lower stomatal apertures at all concentrations of ABA in the presence of either ambient or elevated CO2 relative to CO2-free controls (Figure 4B).


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)

Promotion of closure of cereal stomata by ABA. Following incubation under opening conditions, epidermal peels were exposed to zero (white bars), 10–7 M (gray bars), or 10–6 M (black bars) ABA at 20°C. Values shown are mean stomatal apertures ± SE from n = 240 (barley), n = 90 (wheat), and n = 120 (Brachypodium) measurements. Letters indicate statistically different means within species, determined using one-way ANOVA and a Tukey post-test.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Promotion of closure of cereal stomata by ABA. Following incubation under opening conditions, epidermal peels were exposed to zero (white bars), 10–7 M (gray bars), or 10–6 M (black bars) ABA at 20°C. Values shown are mean stomatal apertures ± SE from n = 240 (barley), n = 90 (wheat), and n = 120 (Brachypodium) measurements. Letters indicate statistically different means within species, determined using one-way ANOVA and a Tukey post-test.
Mentions: To test the validity of the stomatal assay in isolated epidermis of graminaceous plants, we examined the well-known response of guard cells to ABA in a promotion of closure assay. As expected, we observed that wheat, barley and Brachypodium all exhibit a characteristic dose-dependent response to ABA in the epidermal peel assay (Figure 3). We also measured responses to external CO2. In comparison with CO2-free air, stomatal closure was promoted by ambient CO2 (360 ppm) in barley, but we observed no further response at elevated CO2 (800 ppm; Figure 4A). Brachypodium stomatal apertures were also reduced by CO2 (Figure 4A). We next examined the interaction between ABA and CO2 signaling in barley guard cells. We observed a clear additive effect of ABA and CO2, with lower stomatal apertures at all concentrations of ABA in the presence of either ambient or elevated CO2 relative to CO2-free controls (Figure 4B).

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.