Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua

High rates of both cyclic and noncyclic photophosphorylation were measured in choroplast lamelke isolated from purified guard cell protoplasts from Viciafa L. Typical rates of light-dependent inorportion of 32P into ATP were 100 and 190 micromoles ATP per milm chlorophyll per hour for noncyclic (water to ferricynnide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyHl chloroplasts. Noncyclic photophosphorylation in guard cell chboroplasts was completely idhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylura support the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Sevenl lines of evidence indicated that conamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.A comparison of the photon fluence dependence of noncyclic photophosphoryation in mesophyll and guard cell chloroplasts showed significant differences between the two prepatns with half saturation at 0.04 and 0.08 millile per square meter per second, respectively.Chloroplasts are a constant feature of guard cells (26). Recent studies have shown that guard cell chloroplasts have light harvesting pigments for PSI and PSII (5,15,21,23,27) and the capacity for photosynthetic electron transport (13,21,27) and 02 evolution (7,21). Indirect evidence for photophosphorylation in guard cell chloroplasts includes the presence of the a-and #-subunits of the coupling factor complex (5), the demonstration of typical Chl a fluorescence transients (10,11,13,21), and the light-induced 518-nm electrochromic shift (6). A role of guard cell chloroplasts in stomatal movements is supported by the wavelength dependence of stomatal opening (12,19,20,29), the inhibition of opening by the photosynthetic electron transport inhibitor, DCMU, particularly under red light (19,20), and observations showing that achlorophyllous stomata ofthe orchidPaphiopedilum fail to open under red irradiation (28).The hypothesis that guard cell chloroplasts supply ATP for active ion transport during stomatal movements is a classic concept in stomatal physiology (8,16). Photophosphorylation has been measured in guard cell chloroplasts from epidermal peel preparations (9, 17), but these results have been difficult to interpret because of the possible contamination from mesophyll chloroplasts (27 (6,12,21).Guard cell protoplasts were isolated from the sonicated epidermal strips by a two-step enzymic digestion at 20 ± 1°C (21). In the first step, strips were digested in 4% cellulase (Onozuka R-10), 0.25 M mannitol, and 1 mM CaCl2 for 1 h, with shaking at 60 strokes min-'. After filtration through a 58-;tm nylon mesh and washing in 0.4 M mannitol and 1 mM CaC12, the strips were subjected to a second digestion step for about 20 h, with the mannitol and CaC12 increased to 0.4 M and 5 mm, respectively, and the shaking reduced to about 40 strokes min-'. Released guard cell protoplasts were separated from the epidermal peels by f...

Section: Discussionsupporting

confidence: 73%

mentioning

confidence: 99%

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L.

Shimazaki¹,

Zeiger²

1985

“…The procedure for obtaining both the fast transients of fluorescence induction and the slower oscillations during fluorescence quenching to steady state levels have been previously described (22). Briefly, the fluorescence signal was measured using a microspectrophotometer equipped with epifluorescence and a temperature-controlled microscope stage.…”

Section: Measurements Of Chi a Fluorescence Transientsmentioning

confidence: 99%

“…Acclimation of the photosynthetic apparatus was probed by monitoring in vitro, uncoupled whole-chain and partial reactions of electron transport from isolated thylakoid membranes, changes in the Chl a fluorescence transients (the Kautsky effect) from intact leaftissue, and changes in the temperature-dependency ofthe State 1-State 2 transition which are reported to be closely associated with PSII and PSI activity (11). Changes in the illumination have been previously described (22). Plants were grown for a minimum of 6 weeks.…”

mentioning

confidence: 99%

Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua

Mawson

Cummins²

1989

Self Cite

Thermal acclimation by Saxffraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 200C and 10°C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10°C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20°C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20°C-and 10°C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10°C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20°C-acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10°C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem 11 to photosystem 11 reaction centers. The relative amounts of chlorophyllprotein complexes and thylakoid polypeptides separated electrophoretically were similar for both 20°C-and 10°C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition.Thermal acclimation of photosynthetic activity, usually measured as changes in the optimum temperature for photosynthesis resulting from a change in growth temperature, has been demonstrated in a number of species from diverse environments (for reviews, see Refs. 4 and 25). From an ecological viewpoint, the potential, or capacity for thermal acclimation is considered to be an important strategy for maximizing carbon fixation and seasonal net productivity, ' Supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to WRC. 2Present address:

“…Chl a fluorescence quenching has been widely used for the analysis of GC photosynthesis (5,10,11,13,23). Quenching rates in GC chloroplasts were shown to be much slower than those from their MC counterparts.…”

mentioning

confidence: 99%

Blue Light-Modulation of Chlorophyll a Fluorescence Transients in Guard Cell Chloroplasts

Mawson¹,

Zeiger²

1991

Self Cite

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO2 concentration on the fluorescence transients was investigated in the presence of HC03-at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO2 concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.Light is a primary environmental signal for the modulation of stomatal conductance. Light perception in GC4 activates a H+-pumping ATPase at the GC plasma membrane, and the ensuing electrochemical gradient drives ion uptake, turgor build up, and stomatal opening (19 Some photobiological properties of the stomatal response to light are well characterized. The blue light response has an action spectrum that matches the absorption characteristics of both flavins and carotenoids (7), it saturates at low fluence rates, and its kinetics suggests the operation of a photocycle (6). The photosynthetic response is localized in the GC chloroplasts, which are highly conserved in stomata. Guard cell chloroplasts conduct linear electron transport and cyclic and noncyclic photophosphorylation (see ref. 34 for review). Red light-dependent stomatal opening is inhibited by DCMU, indicating that photosynthetic electron transport is required for that response (18, 21). Red light-stimulated proton pumping in GC is also DCMU-sensitive, and the role of GC chloroplasts in pump activation appear to include the supply of both ATP to fuel the pump and a second photosynthetic product with a regulatory function (20).Recent studies with GC...