Assessing Abscisic Acid-Mediated Changes in Stomatal Aperture Through High-Quality Leaf Impressions.

Plants live in highly dynamic surroundings and need to cope with constant environmental challenges. In order to do so, they developed quick reactions to stress that allow them to gain time while mounting a major response. This first line of defense includes the stomata, leaf epidermal pores in charge of regulating water loss and photosynthesis. Stomatal movements are controlled by the stress phytohormone abscisic acid (ABA), which induces fast closure of the stomata upon perception of stress conditions. By modulating plasma membrane ion channels, ABA leads to loss of water from the guard cells surrounding the stomatal pore and a consequent reduction of its aperture. Here, we provide a microscopy-based method to assess the plant's response to ABA through measurements of the stomatal aperture. This protocol describes a simple, quick, and unexpensive method to prepare high-quality impressions of leaves from Arabidopsis thaliana seedlings from long-lasting silicone-based casts, allowing detailed imaging and accurate determination of the aperture of stomatal pores.

Assessing Postgermination Development in Arabidopsis thaliana Under Abiotic Stress.

The abscisic acid (ABA) phytohormone is well known to regulate responses to abiotic stress, particularly tolerance to osmotic stress. Screening for phenotypes at the early plant development stages is fundamental to identify new regulators of the ABA pathway, which in turn is extremely relevant for agriculture in a global climate change context. Typically, under experimental conditions, seeds are germinated in hormone-containing plates, and postgermination development is then assessed through scoring of the appearance of green or expanded cotyledons. However, postgermination phenotypes may be either masked or exacerbated by prior defects in seed germination. To circumvent this, we propose a transfer assay to screen specifically and quickly for postgermination phenotypes affected by exogenous ABA. The assay can be applied to different forms of abiotic stress, and we provide tips to score for postgermination phenotypes in genotypes exhibiting differential development.