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

Guard cells emit an alkali-induced, blue fluorescence upon excitation by ul traviolet radiation (emission maximum energy at 365 nm). Fluorescence emiss ion of guard cells was brighter than that of the neighbouring epidermal cel ls in a number of wild and cultivated plants including conifers, but the re lative fluorescence intensity and quality was species-dependent. Three repr esentative plants possessing stomatal complexes which differed morphologica lly were studied: Olea europaea, Vicia faba and Triticum aestivum. Immersin g leaves of these plants in chloroform for 30 s (thereby removing epicuticu lar 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-b ound phenolics), or a thicker cuticular layer covering the guard cells. Giv en 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 con venient method for in situ measurements of the number, distribution and siz e of stomatal complexes. The proposed experimental procedure includes a sin gle 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 obse rvation of the leaf surface under the fluorescence microscope. Fluorescence images were suitable for digital image analysis and methodology was develo ped for stomatal counting using Olea europaea as a model species. (C) 2001 Annals of Botany Company.