Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

Abstract

The lack of detectable variable fluorescence from guard cell chloro-plasts 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 fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secun-datum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts. Chloroplasts are the primary site of light absorption in most guard cells. Several lines of evidence, particularly observations of variable Chl fluorescence, indicate that guard cell chloroplasts contain PSII and are capable ofphotosynthetic electron transport (9, 11, 13, 18, 25). The involvement of guard cell PSII activity in the regulation of stomatal aperture, however, remains a point of controversy. Inhibition of stomatal opening by DCMU, an inhibitor of PSII, has been reported by some investigators (4, 8), but not by others (1, 27). Results of measurements of the action spectrum for stomatal opening have likewise proven inconclusive. A variety of results have been reported (3, 4, 16, 20, 21, 24) for the ratio of stomatal openings in blue and red light, a ratio which is considered to reflect the relative contributions of the blue light and photosynthetic systems to stomatal control. Other ' reports (2, 6, 22) indicate that photosynthesis is not always necessary for light-control of stomatal aperture. We report here the absence of detectable variable fluorescence from chloroplasts in functioning guard cells of Stenotaphrum secundatum var variegatum A.S. Hitchc., a fast growing, NADP+ malic enzyme type of C4 plant (23). These results indicate that the guard cell chloroplasts of this plant do not contain an active PSII and provide evidence that PSII activity in guard cells is not required for stomatal regulation. MATERIALS AND METHODS The plants of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) used in this study were collected from the Botanic Garden on the campus of the University of California at Riverside. These and the other plants used in this study were grown in a mixed soil in one-gallon pots under natural lighting in a greenhouse. The plants were watered daily and fertilized weekly with half-strength Hoagland solution. Young, fully-expanded leaves were selected for all experiments. 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 selec-tivity 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.plant.org on May 28, 2015-Published by www.plantphysiol.org Downloaded from