title: A Rapid and Simple Method for Microscopy-Based Stomata Analyses.
abstract: There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.
authors.affiliation: Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany.
authors.forename: Jochen F
authors.initials: JF
authors.lastname: Eisele
authors.affiliation: Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Fäßler
authors.affiliation: Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany.
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Bürgel
authors.affiliation: Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Chaban
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D029681:Arabidopsis Proteins / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D000071677:Histidine Kinase / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000191:economics / Q000379:methods*; D018515:Plant Leaves / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D054046:Plant Stomata / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000648:ultrastructure; D012235:Rhodamines / Q000032:analysis; D013194:Staining and Labeling / Q000191:economics / Q000379:methods; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D005456:Fluorescent Dyes; D012235:Rhodamines; C026188:rhodamine 6G; D000071677:Histidine Kinase; C000605494:AHK5 protein, Arabidopsis