Presence of Chloride Reduces Malate Production in Epidermis during Stomatal Opening

The effects of cations and abscisic acid on chloroplast activity in guard celis of Viia faba were investigated by analysis of the transient of chlorophyli a fluorescence. When epidermal strips containg guard cells as the only living ceDs were incubated in water and wumiatedith strong light, chlorophyl a fluorescence rose rapidbly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K+ or Na+, and the effect of these catons was greater when added with phosphate than with chloride as the anion. Ca'+ suppressed the enhancement by Na+ and, to a lesser extent, that by KV. Abscisic acid also suppressed the enhancement by K+ and Na+. Since the fluorescence decline reflects the increase of intrathylakod H+ concentration necessary for photophosphorylation, the acceleration of the decline by K+ (or Na+ in the absence of Ca2 ) implicates chloroplast activity in ion accumulation by guard celis in the iht. The differenthl effects of phoste and chbride suggest that chloroplast actiity may be Involved In malate formation in guard cells in the light.Stomatal guard cells of most plant species contain chloroplasts. Action spectra (12,20) and inhibitor data (7) suggest that these chloroplasts may play a role in stomatal opening (6), perhaps as a source of reducing or high energy phosphate equivalents for the import of K+ and synthesis of organic counterions (25). The chloroplasts possess both photosystems (23) of photosynthesis and are capable of linear, light-driven electron transport (30). However, they do not contain certain enzymes of the Calvin-Benson cycle (22) and are therefore unlikely to fix CO2 photosynthetically. The specific involvement of the chloroplasts in ion transport and counterion synthesis remains to be demonstrated, however.The kinetics of Chl a fluorescence has been correlated with specific photosynthetic partial reactions and are sensitive indicators of photosynthetic activity (for reviews see 5,16,26 to a quasi-stationary level, S, rises to a maximum level, M, and then declines to a terminal steady state level, T, within a few minutes of illumination. The fast changes, OIDP, are correlated with early photochemical events in the PSII reaction center and electron transport chain, whereas the later changes, PSMT, are correlated with alterations in energy state and membrane structure associated with photophosphorylation.The present study was undertaken to evaluate the effects of cations and ABA on the fluorescence transients in guard cells. The observed dependence of the fluorescence kinetics on conditions known to affect stomatal aperture in vivo suggests that chloroplast activity in guard cells is involved in ion import and counterion production for stomatal opening. MATERIALS AND METHODSPreparation of Sonicated Epidermal Strips. Seeds of Viciafaba L. cv. Long Pod (Burpee Seed Co., Warminster, PA) were germinated in pots and grown in a growth chamber at 28°C day (14 h)/ 18°C night (10 h) at radiation intensities (daylight fluorescent) of 400 ,uE ...

Section: Discussionmentioning

confidence: 73%

Effects of Cations and Abscisic Acid on Chlorophyll a Fluorescence in Guard Cells of Vicia faba

Ogawa¹,

Grantz²,

Boyer³

et al. 1982

“…This explanation is based on the finding that the amount of malate formed in isolated epidermis of V faba during stomatal opening was large when Cl-was absent in the incubation medium, and low when it was present (14). Malate content and stomatal aperture were linearly correlated with each other in the absence of C1-; the correlation broke down in the presence of Cl-.…”

mentioning

confidence: 99%

“…We used established methods: electron probe microanalysis for the determination of K+ and Clin the guard cells (6), and enzymic oxidation coupled to the reduction of NAD for the determination of malate in the epidermal samples (4,14 hr light intensity 85 w m-2. The air temperature was 27 C during the light period and 23 C during the dark period.…”

mentioning

confidence: 99%

Availability of Chloride Affects the Balance between Potassium Chloride and Potassium Malate in Guard Cells of Vicia faba L.

Raschke¹,

Schnabl²

1978

Self Cite

Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0. Republic of Germany.We tested the effect of the availability of Cl-on the ionic composition of the guard cells of Viciafaba by incubating epidermal samples on solutions free of Cl-or containing Cl-and then determining the amounts of K+ and CF-absorbed as well as the amounts of malic acid synthesized. We used established methods: electron probe microanalysis for the determination of K+ and Clin the guard cells (6), and enzymic oxidation coupled to the reduction of NAD for the determination of malate in the epidermal samples (4,14).MATERIALS AND METHODS Plants. V.faba L. plants (cv. Long Pod; seeds from Lagomarsino Seeds Inc., Sacramento, Calif.) were grown for 3 to 4 weeks in a growth chamber in soil consisting of 2 parts Bacto potting soil (Michigan Peat, Houston, Texas) and I part Perlite (W. R. Grace and Co.) in pots with a soil depth of about 12 cm. The plants were watered once a week with Hoagland solution (full strength, pH 6.5), and twice a week with deionized H20. The day length was 16 hr light intensity 85 w m-2. The air temperature was 27 C during the light period and 23 C during the dark period. Relative humidity was about 85%, day and night.Preparation and Incubation of Epidermal Samples. The second, third and fourth fully expanded leaves were removed from the plants in the morning, immediately before the beginning of the light period. The leaves were washed in deionized H20. Sections measuring about 5 x 10 mm2 were cut from the intercostal areas of the leaves and floated, upper side down, on deionized H20 in the dark. Then the lower epidermis was peeled in room light (from fluorescent tubes), a process during which most of the epidermal cells are ruptured (3). The epidermal strips were rinsed in deionized H20, rubbed with a dissecting needle (bent at an angle of 1200) to remove adhering mesophyll cells, and collected on deionized H20. From there, the strips were transferred to the incubation media in small plastic beakers. The beakers were placed under an inverted crystallizing dish (150-mm diameter, 75-mm height). Humidified, C02-free air was passed through the inverted dish for 4 hr at 50 1 hr-1 and 21 C. The samples were illuminated by mercury vapor lamps (General Electric H 400 RDX 33-1); the irradiance below the water filter (5 cm deep) was 85 w m-2. The

“…In most cases, stomatal closing was induced by addition of ABA to the water. described elsewhere (7). The second, third, and fourth fully expanded leaves were removed, rinsed with distilled H20, and cut into sections of I to 3 x 2 to 4 cm2.…”

mentioning

confidence: 99%

Release of Malate from Epidermal Strips during Stomatal Closure

Kirk

Raschke²

1978

Self Cite

(4,6). The cell walls of the epidermis may also receive and store inorganic ions, although there is doubt whether their capacity suffices (5). The fate of the organic anions during stomatal closure has been studied after 14C labeling of guard cell contents (2). Epidermal samples were exposed to 4C02 for 2 min and then subjected to closing treatments. The analysis of the epidermes as well as of the liquid on which they floated indicated that guard cells may dispose of malate in three ways: (a) catabolism in the tricarboxylic acid cycle; (b) decarboxylation followed by gluconeogenesis and starch formation; and (c) release from the guard cells. However, it was estimated that only a small fraction of the total malate pool was labeled in these experiments. To determine the extent to which these three methods are employed by guard cells during stomatal closure, the fate of the whole malate pool must be examined. In particular, the importance of a specific release of malate was to be assessed because this mechanism would enable guard cells to reduce their turgor faster than metabolism of malic acid would allow (2). Changes in malate content were followed during stomatal closure in epidermal samples and in the water on which they floated. In most cases, stomatal closing was induced by addition of ABA to the water. described elsewhere (7). The second, third, and fourth fully expanded leaves were removed, rinsed with distilled H20, and cut into sections of I to 3 x 2 to 4 cm2. These sections were floated lower side up on distilled H20 in the light (85 w m-2 from mercury vapor lamps, General Electric H400 RDX 33-1) and C02-free air for 4 to 5 hr to allow stomata to open. Then the lower epidermis was removed in strips of about 5 x 10 to 15 mm2. Sixty to 80%o of the ordinary epidermal cells ruptured during peeling. The area of each strip was measured with a ruler. In four of five strips/experiment, stomatal apertures were measured under the microscope; then these strips were plunged into boiling 80%o ethanol to extract malate. The other strips were transferred to distilled H20 or solutions containing ABA. After incubation for various times, the strips were put on a microscope slide for the measurement of 20 to 25 stomatal apertures in each strip and then immediately extracted with boiling ethanol. The extracts were evaporated to dryness and analyzed for malate by enzymical oxidation, coupled to the reduction of NAD; the NADH formed was measured fluorimetrically (3; for details see 7). The solutions on which the strips had floated were also evaporated and assayed for malate.RESULTS AND DISCUSSION Epidermal strips of V. faba with open stomata were exposed in the light to a 0.1 mm solution of (±)-ABA for periods of 15 min to 2 hr (Fig. 1)