The Guard Cell Chloroplast: Properties and Function

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

Section: S Rsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

Nothnagel²

1988

“…42 for review) provides an alternative explanation for these observations, pointing to photosynthetically produced sugars as a source of osmoticum. From data on the red light-induced alkalinization and 02 evolution by guard cell protoplasts irradiated with red light, maximal rates of photosynthetic CO2 fixation were estimated at 300 Amol CO2 mg Chl-' h-' (38).…”

Section: Discussionmentioning

confidence: 99%

Light Quality and Osmoregulation in Vicia Guard Cells

Tallman¹,

Zeiger²

1988

111

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K+ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of -0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K+ content or starch degradation in guard cell chloroplasts. At Recent characterization of a blue light-dependent proton pump at the guard cell plasmalemma has linked light perception with the activation of ion transport mechanisms providing guard cells with the capacity for a quantitative transduction of prevailing light fluences into adequate ionic uptake for osmoregulation (2, 37). In addition to the blue light photosystem, guard cells also possess Chl in their chloroplasts, which acts as a separate photoreceptor to induce stomatal opening (9,12,33). Blue lightinduced stomatal opening is the result of combined responses of the two photoreceptor systems. Blue light-dependent opening has been correlated with proton pumping (2, 37), K' uptake (12), starch degradation (22), and malate biosynthesis (23). In Vicia faba, blue light-dependent opening in epidermal peels saturates at 25 umol m-2 s-', is inhibited by KCN but not by DCMU at intensities 0Iusmol m-2 s-', and is partially sensitive to DCMU at intensities 225 umol m-2 s-' (33). Red light-induced stomatal opening relies on sensory transduction mediated by Chl. In Vicia, red light-dependent stomatal opening in peels saturates at 50 ,umol m-2 s-, is insensitive to KCN, and is completely inhibited by DCMU (33). Red light-induced stomatal opening has been correlated with oxygen evolution and photophosphorylation (36,38) and with synthesis of intermediates of the PCRP3 (11).The interconversion of photosynthetically produced sugars and starch was a fundamental hypothesis of stomatal movements that dominated plant physiology p6rior to its abandonment in favor of the K+ hypothesis (16,21

“…The Rubisco activity of a mesophyll-cell protoplast was 32.4 pmol cell-' h-'; it remained unaffected by additions ofguard-cell protoplasts to the assay mixture (34.5 pmol cell-' h-'). There was no evidence for the suggested existence in guard cells of an inhibitor of Rubisco (30). Although guard cells of P. sativum contained Rubisco of a specific activity 3Preliminary experiments had resulted in an activity of0.014 pmol cell-' h-' (19) which, in the light of all determinations that followed, was too low by a factor of 0.1.…”

Section: Contents and In Vitro Activities Of Rubisco In Protoplasts Fmentioning

confidence: 94%

“…The activity of Rubisco in guard-cell protoplasts was determined in experiments in which deliberate contaminations of guard-cell preparations were used to extrapolate to the activity of a pure guard-cell suspension and to examine the possible presence of an inhibitor of Rubisco in guard cells (30). Increasing numbers of mesophyll protoplasts were added to aliquots of a guard-cell suspension which, as determined by microscopy, contained no mesophyll cells (see "Materials and Methods").…”

Section: Contents and In Vitro Activities Of Rubisco In Protoplasts Fmentioning

confidence: 99%

Rubisco Activity in Guard Cells Compared with the Solute Requirement for Stomatal Opening

Reckmann¹,

Scheibe²,

Raschke³

1990

110

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/ oxygenase (Rubisco) were determined by the [14C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to 14C02 for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.