Introducing Fluorescent Ion Probes into Attached Leave

抄録

　To develop methods to measure intracellular pH in an attached leaf non-destructively and to construct the pH map when the leaf responds to environment factors, ways of loading fluorescent ion probes into attached leaf cells were tested. Tested fluorescent pH probes included 5-Carboxyfluorescein Diacetate, a ratiometric probe SNARF-1/AM, and a probe for the apoplast FITC-Dextran. Fluorescence of an intact leaf was imaged by a CCD camera attached with a stereomicroscope or by a confbcal laser scanning microscope. Chlorophyll fluorescence analysis and leaf temperature measurements were conducted for evaluating the intensity of leaf damage by introducing the probes. As a result of testing several plant species, it was observed that the feasibility of loading the probes into attached leaf cells depended on species. In the case of species whose cells were easily loaded with the probes, submerging the attached leaf in the probe solution at room temperature for a few hours leaded to bright fluorescence in guard cells, epidermal cells and mesophyll cells. As there was no change in leaf temperature and photochemical efficiency by introducing the probes, it was suspected that no leaf damage was occurred. Path ways through which fluorescent probes can enter into attached leaf cells were also discussed from analysis of the changes in fluorescence distribution patterns in the leaf and from a comparison of the loading results under light and dark conditions.