Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

Abstract

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St.Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both A wide variety of methods were used to prepare specimens of S. secundatum for fluorescence measurements. Both epidermal peels and whole leaf tissues were examined. Epidermal strips were peeled from the leaves through use of a razor blade. Upon separation from the leaves, the epidermal peels were placed on a glass plate and rubbed gently with a pencil eraser to remove adherent mesophyll cells. Ice-cold distilled water was added during the rubbing operation. The epidermal peels were then washed twice and held in either distilled water or Tris buffer (0.37 M sucrose, 25 mM EDTA, 10 mM NaCl, 10 mm Tris, pH 7.6) during a 30 min to 1 h period of dark adaptation before fluorescence measurement. The absence of adherent mesophyll chloroplasts was verified by examination under the fluorescence microscope. Viability ofthe guard cells was confirmed by staining with fluorescein diacetate.Whole leaf tissue to be used for fluorescence measurements was dark-adapted either as detached leaves or as whole plants. Detached leaves were held on wet filter paper to prevent dehydration during dark adaptation. Dark adaptation was carried out at 25°C for times ranging from 30 min to 4 h.Fluorescence induction curves were measured on two different instruments. Most of the measurements were performed with a modified fluorescence microscope as described in Figure 1. Although conceptually similar to the microscope systems used by others (9, 10, 25) to measure guard cell fluorescence, this system had several special features which contributed to improved selectivity and sensitivity. In addition to standard mercury arc illumination, the microscope was equipped with two laser light sources. The argon ion laser was operated in the all-lines mode and emitted a light beam that was filtered at the microscope to provide either blue excitation (several wavelengths in the range 457.9-488.0 nm) or green excitation (514.5 nm). Alternatively, the light beam from a He-Ne laser could be selected to provide 429 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Fluorescence and Delayed Light Emission from Mesophyll and Bundle Sheath Cells in Leaves of Normal and Salt-Treated Panicum miliaceum

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Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels

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