Epicuticular phenolics over guard cells: Exploitation for in situ stomatal counting by fluorescence microscopy and combined image analysis

Abstract

Guard cells emit an alkali-induced, blue fluorescence upon excitation by ultraviolet radiation (emission maximum energy at 365 nm). Fluorescence emission of guard cells was brighter than that of the neighbouring epidermal cells in a number of wild and cultivated plants including conifers, but the relative fluorescence intensity and quality was species-dependent. Three representative plants possessing stomatal complexes which differed morphologically were studied: Olea europaea, Vicia faba and Triticum aestivum. Immersing leaves of these plants in chloroform for 30 s (thereby removing epicuticular waxes) significantly reduced the intensity of the fluorescence emitted by guard cells. This indicates that guard cell fluorescence could be due to either an increased concentration of fluorescing compounds (probably wax-bound phenolics), or a thicker cuticular layer covering the guard cells. Given that the alkali-induced blue fluorescence of the guard cells is a common characteristic of all plants examined, it could be used as a rapid and convenient method for in situ measurements of the number, distribution and size of stomatal complexes. The proposed experimental procedure includes a single coating of the leaf surface by, or immersion of the whole leaf in, a 10% solution of KOH for 2 min, washing with distilled water, and direct observation of the leaf surface under the fluorescence microscope. Fluorescence images were suitable for digital image analysis and methodology was developed for stomatal counting using Olea europaea as a model species. © 2001 Annals of Botany Company.