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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.


Technique for removing abaxial epidermis. (A) The first true leaf is removed from the plant, bent over the finger adaxial side up, and a cut made across the lamina using a scalpel blade. The tip of the leaf blade is bent back and forth to detach a small section of the mesophyll from the lower epidermis. (B) The upper layer was then peeled back using forceps to leave the lower epidermis attached to the leaf tip. The size of the epidermal peel obtained typically varies from around 1–3 cm. (C) Bright field micrograph illustrating open stomata in a typical abaxial epidermal peel from wheat (20 μm scale bar).
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Figure 1: Technique for removing abaxial epidermis. (A) The first true leaf is removed from the plant, bent over the finger adaxial side up, and a cut made across the lamina using a scalpel blade. The tip of the leaf blade is bent back and forth to detach a small section of the mesophyll from the lower epidermis. (B) The upper layer was then peeled back using forceps to leave the lower epidermis attached to the leaf tip. The size of the epidermal peel obtained typically varies from around 1–3 cm. (C) Bright field micrograph illustrating open stomata in a typical abaxial epidermal peel from wheat (20 μm scale bar).

Mentions: Isolated epidermis was obtained from the abaxial surface of the first true leaf of 8–14 day old wheat and barley plants, when the first true leaf had stopped expanding (5–8 cm long). For Brachypodium distachyon, the youngest fully expanded leaves of 3–4 week old plants were used. Leaves were cut from the plant and bent over the forefinger with the adaxial surface facing upward. A shallow cut was made with a sharp razor blade horizontally across the leaf and a flap of leaf tissue lifted with a razor, leaving the lower epidermis intact (Figure 1). The leaf tissue was removed from the epidermis with forceps. Once a section of epidermis approximately 1 cm long was exposed, it was cut from the leaf and floated cuticle-side-up in 10 mM MES/KOH (pH 6.2), 50 mM KCl.


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)

Technique for removing abaxial epidermis. (A) The first true leaf is removed from the plant, bent over the finger adaxial side up, and a cut made across the lamina using a scalpel blade. The tip of the leaf blade is bent back and forth to detach a small section of the mesophyll from the lower epidermis. (B) The upper layer was then peeled back using forceps to leave the lower epidermis attached to the leaf tip. The size of the epidermal peel obtained typically varies from around 1–3 cm. (C) Bright field micrograph illustrating open stomata in a typical abaxial epidermal peel from wheat (20 μm scale bar).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Technique for removing abaxial epidermis. (A) The first true leaf is removed from the plant, bent over the finger adaxial side up, and a cut made across the lamina using a scalpel blade. The tip of the leaf blade is bent back and forth to detach a small section of the mesophyll from the lower epidermis. (B) The upper layer was then peeled back using forceps to leave the lower epidermis attached to the leaf tip. The size of the epidermal peel obtained typically varies from around 1–3 cm. (C) Bright field micrograph illustrating open stomata in a typical abaxial epidermal peel from wheat (20 μm scale bar).
Mentions: Isolated epidermis was obtained from the abaxial surface of the first true leaf of 8–14 day old wheat and barley plants, when the first true leaf had stopped expanding (5–8 cm long). For Brachypodium distachyon, the youngest fully expanded leaves of 3–4 week old plants were used. Leaves were cut from the plant and bent over the forefinger with the adaxial surface facing upward. A shallow cut was made with a sharp razor blade horizontally across the leaf and a flap of leaf tissue lifted with a razor, leaving the lower epidermis intact (Figure 1). The leaf tissue was removed from the epidermis with forceps. Once a section of epidermis approximately 1 cm long was exposed, it was cut from the leaf and floated cuticle-side-up in 10 mM MES/KOH (pH 6.2), 50 mM KCl.

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.