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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.


Guard cell viability in epidermal peels. Peels were incubated in CO2-free MES-KCl buffer at 22°C and then stained for 30 min in FDA. The x-axis indicates the time from the start of the initial incubation period until microscopic observation of FDA staining. Data represent counts from three areas of approximately 1 mm2 from each piece of epidermis and three independent biological replicates.
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Figure 2: Guard cell viability in epidermal peels. Peels were incubated in CO2-free MES-KCl buffer at 22°C and then stained for 30 min in FDA. The x-axis indicates the time from the start of the initial incubation period until microscopic observation of FDA staining. Data represent counts from three areas of approximately 1 mm2 from each piece of epidermis and three independent biological replicates.

Mentions: Although grasses are less amenable than current model species used for stomatal research, we developed an approach that could be used routinely to generate intact abaxial epidermis isolated from leaves of wheat, barley and Brachypodium seedlings (see “Materials and Methods”). Peels were free of mesophyll cells and contained viable guard cells, subsidiary cells and pavement cells, as determined by FDA staining. Stomata were significantly larger in barley and wheat (typically around 25–50 μm in length) compared to those in the smaller Brachypodium plants (guard cells typically 6–9 μm in length). In order to be useful for measuring guard cell-mediated stomatal responses via an in vitro assay, it is essential that guard cell viability is maintained in isolated epidermis. We monitored viability over the period when assays are typically performed by performing FDA staining at regular intervals for up to 4 h following isolation. Figure 2 illustrates that guard cell viability was around 80% 30 min after isolation for all species. This level was maintained throughout the test period for both barley and Brachypodium, whilst viability gradually declined for wheat.


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)

Guard cell viability in epidermal peels. Peels were incubated in CO2-free MES-KCl buffer at 22°C and then stained for 30 min in FDA. The x-axis indicates the time from the start of the initial incubation period until microscopic observation of FDA staining. Data represent counts from three areas of approximately 1 mm2 from each piece of epidermis and three independent biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Guard cell viability in epidermal peels. Peels were incubated in CO2-free MES-KCl buffer at 22°C and then stained for 30 min in FDA. The x-axis indicates the time from the start of the initial incubation period until microscopic observation of FDA staining. Data represent counts from three areas of approximately 1 mm2 from each piece of epidermis and three independent biological replicates.
Mentions: Although grasses are less amenable than current model species used for stomatal research, we developed an approach that could be used routinely to generate intact abaxial epidermis isolated from leaves of wheat, barley and Brachypodium seedlings (see “Materials and Methods”). Peels were free of mesophyll cells and contained viable guard cells, subsidiary cells and pavement cells, as determined by FDA staining. Stomata were significantly larger in barley and wheat (typically around 25–50 μm in length) compared to those in the smaller Brachypodium plants (guard cells typically 6–9 μm in length). In order to be useful for measuring guard cell-mediated stomatal responses via an in vitro assay, it is essential that guard cell viability is maintained in isolated epidermis. We monitored viability over the period when assays are typically performed by performing FDA staining at regular intervals for up to 4 h following isolation. Figure 2 illustrates that guard cell viability was around 80% 30 min after isolation for all species. This level was maintained throughout the test period for both barley and Brachypodium, whilst viability gradually declined for wheat.

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.