PIanta (1989)178:223 230
Pl~.JIlta 9 Springer-Vertag1989
Inhibition of stomatal opening in sunflower leaves by carbon monoxide, and reversal of inhibition by light M. Pollok, U. Heber, and M.S. Naik* Institute of Botany and Pharmaceutical Biology of the University of Wtirzburg, D-8700 Wiirzburg, Federal Republic of Germany
Abstract. When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO2, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm. s-1. The CO effect on stomatal aperture required a CO/O2 ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibition of cytochrome-c oxidase by CO is competitive in respect to 02. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO2 was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus. In contrast to red light, very weak blue light (405 mn) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a3-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis. * P r e s e n t a d d r e s s : Mahatma Phule Agricultural University, Phulenagar, Rahuri, PIN 413 722, Dist. Ahmednagar, Maharashtra, India
Key words: Adenylate - Blue light (stomata1 opening) - Carbon dioxide and stomatal opening - Helianthus (stomatal opening) - Light and stomatal opening - Stomatal opening
Introduction In C3- and C4-plants stomata open in the light when C O 2 is present, and in the dark when it is absent. The apertures of stomata are influenced by factors such as water potential, intercellular CO2 concentration and the plant hormone abscisic acid. The opening of stomata involves the energydependent pumping of K + into guard cells (Raschke 1979). Several lines of evidence indicate that the ion-transport processes accompanying and actually causing stomatal opening are energized by ATP which must become available in the cytosolic compartment of the guard cells. The main sources of ATP in green cells are the photophosphorylation of chloroplasts and the oxidative phosphorylation of mitochondria. Guard cells contain chloroplasts. However, whereas mitochondria can efficiently export ATP into the cytosol, it is still unknown how effective chloroplasts are in exporting ATP either directly via the adenylate transporter of the chloroplast envelope or indirectly via the phosphoglycerate/dihydroxyacetone phosphate shuttle which is catalyzed by the phosphate translocator of the envelope (see Heber and Heldt 1981 for a review). In guard cells, the ratio of mitochondria to chloroplasts is about 4 times higher than in mesophyll cells (Raschke 1979; Mansfield 1985). Schwartz and Zeiger (1984) have concluded that, whereas stomatal opening in the dark is energized by oxidative phosphorylation which is sensitive to KCN, light-dependent stoma-
224
tal opening requires photophosphorylation. They also suggested that stimulation of opening by blue light is based either on oxidative phosphorylation or a specific membrane-bound electron transporter. Carbon monoxide (CO) is a specific inhibitor of mitochondrial cytochrome oxidase. In its presence, a cytochrome aa-CO adduct is produced (Chance 1953 a, b; Dennis and Richaud 1985; Naik and Nicholas 1986) and cytochrome-c oxidation is blocked. However, plant mitochondria possess a cyanide- and CO-insensitive alternative oxidase which accepts electrons from ubiquinone. This pathway does not use the second and third sites of mitochondrial ATP synthesis, and the yield of ATP synthesis is consequently low. Since CO, unlike KCN, can be administered to whole leaves in the gaseous form, we have used it to investigate the role of mitochondrial and chloroplast energy conservation in providing the ATP for stomatal opening in sunflower leaves. Material and methods Sunflower (Helianthus annuus L.) plants were grown in a greenhouse. Illumination was provided by 400-W halogen lamps (HQI-T; Osram, M/inchen, F R G ) for 10 h per day. Light levels at the stem apex were about 100 W . m 2. Leaves were cut from 8- to 10-week old plants under water and fitted into a temperature-controlled sandwich-type cuvette. The petiole of a leaf was placed in water. The composition of the gas passing through the cuvette was adjusted by mass-flow controllers, Unless indicated otherwise the O2 concentration was 1%. When necessary, CO2 was removed from the gas stream by soda lime. The final concentration of CO added to the gas stream was usually 25%. Contaminating CO2 had to be removed from commercially obtained CO. Gases were made up to 100% with N 2. They were passed through a tube with wet filter paper and subsequently through a cooling trap (usually 12~ C) to adjust the relative humidity of the gas stream entering the cuvette to about 60%. The temperature was usually 20 ~ C. A differential gas analyzer monitored differences in H 2 0 and CO2 between a reference gas stream and the stream which had passed through the cuvette over both surfaces of the leaf. A thermocouple controlled the leaf temperature. The leaf was illuminated by red light (half-bandwidth from 627 to 672 nm, filters: R G 630 cutoff, Schott, Mainz, F R G ; K 65 and Calflex C, b o t h Balzers, Liechtenstein, plus water cuvettes). A 405-nm interference filter (half-bandwidth 12 nm, Balzers) provided blue light of very low irradiance. Weak red light equivalent in photon flux density to the blue light was provided by a 671-mn interference filter. Transpiration and CO z exchange were recorded as described previously (Heber et al. 1986), and stomatal conductivity to water vapor was calculated according to Nobel (1983). The levels of ATP and A D P were determined in leaf discs. As in the gas-exchange experiments, leaves were gassed and illuminated for 1 h as indicated. Leaf discs were cut and frozen in liquid nitrogen, ground to powder in the frozen state and mixed with 4.5 ml 4.5% HC104. After the frozen mixture had thawed, it was centrifuged for 2 rain at 2000-g. Triethanola-
M. Pollok et al. : Inhibition of stomatal opening mine was added to the supernatant to reach a final concentration of 0.05 M. The solution was neutralized with 4 M K2CO3 and kept in ice. Crystalline KC104 was removed, and ATP was determined in the extract by the method of Wulff and D6ppen (1985) and A D P by that of Hampp (1985).
Results
Gas exchange of leaves after addition of CO. In air, the stomata of sunflower leaves were largely closed in the dark. Figure I shows that they opened after removal of COz from the atmosphere. Stomatal opening increased transpiration, and the leaf temperature decreased accordingly. When transpiration was constant, CO was flushed over the leaf (gas flow rate 1 l.min 1, composition of the atmosphere 25% CO, 1% 02, 74% N2). This decreased respiratory CO2 release after a transient increase (Fig. 1, lower part), and stomata closed slowly (Fig. 1, middle). The decreased transpiration caused an increase in the leaf temperature. Effects of illumination will be considered below. On average, CO doubled the stomatal resistance to watervapor diffusion in the dark when CO2 was absent. Table I shows the sum of the stomatal and boundary-layer resistances to water loss and respiratory COz release before and after addition of CO. Complete stomatal closure was not observed under the influence of CO. The data of Table 1 indicate an average reduction in CO2 output by 16%. It is important to note, that carbohydrate can be oxidized to CO2 in a cyanide- and CO-resistant alternative respiratory pathway which yields only onethird of the ATP synthesized during electron transport mediated by the cytochrome-oxidase pathway. Release of the CO inhibition of stomatal opening by 0 2 . Oxygen counteracted the inhibitory effect of CO on stomatal opening. When the ratio of CO to O2 was decreased below a value of about 25, stomates reopened, and when a low ratio of CO to O2 was increased, stomata closed. Figure 2 shows the relationship between stomatal conductivity to water vapor and the CO/O2 ratio. The data indicate that the reaction of CO with plant cytochrome oxidase, which is involved in the opening of stomata, is reversible. Photodissociation is not required for reversibility as it is in animal mitochondria (Chance and Erecinska 1971). Effects of red light on stomatal opening in the presence of CO. Since CO is an inhibitor of cytochrome oxidase, the closure of stomata under the influence of CO is likely to be caused by ATP deficiency.
M. Pollok et al. : Inhibition of stomatal opening
225
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Pl~.JIlta 9 Springer-Vertag1989
Inhibition of stomatal opening in sunflower leaves by carbon monoxide, and reversal of inhibition by light M. Pollok, U. Heber, and M.S. Naik* Institute of Botany and Pharmaceutical Biology of the University of Wtirzburg, D-8700 Wiirzburg, Federal Republic of Germany
Abstract. When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO2, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm. s-1. The CO effect on stomatal aperture required a CO/O2 ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibition of cytochrome-c oxidase by CO is competitive in respect to 02. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO2 was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus. In contrast to red light, very weak blue light (405 mn) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a3-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis. * P r e s e n t a d d r e s s : Mahatma Phule Agricultural University, Phulenagar, Rahuri, PIN 413 722, Dist. Ahmednagar, Maharashtra, India
Key words: Adenylate - Blue light (stomata1 opening) - Carbon dioxide and stomatal opening - Helianthus (stomatal opening) - Light and stomatal opening - Stomatal opening
Introduction In C3- and C4-plants stomata open in the light when C O 2 is present, and in the dark when it is absent. The apertures of stomata are influenced by factors such as water potential, intercellular CO2 concentration and the plant hormone abscisic acid. The opening of stomata involves the energydependent pumping of K + into guard cells (Raschke 1979). Several lines of evidence indicate that the ion-transport processes accompanying and actually causing stomatal opening are energized by ATP which must become available in the cytosolic compartment of the guard cells. The main sources of ATP in green cells are the photophosphorylation of chloroplasts and the oxidative phosphorylation of mitochondria. Guard cells contain chloroplasts. However, whereas mitochondria can efficiently export ATP into the cytosol, it is still unknown how effective chloroplasts are in exporting ATP either directly via the adenylate transporter of the chloroplast envelope or indirectly via the phosphoglycerate/dihydroxyacetone phosphate shuttle which is catalyzed by the phosphate translocator of the envelope (see Heber and Heldt 1981 for a review). In guard cells, the ratio of mitochondria to chloroplasts is about 4 times higher than in mesophyll cells (Raschke 1979; Mansfield 1985). Schwartz and Zeiger (1984) have concluded that, whereas stomatal opening in the dark is energized by oxidative phosphorylation which is sensitive to KCN, light-dependent stoma-
224
tal opening requires photophosphorylation. They also suggested that stimulation of opening by blue light is based either on oxidative phosphorylation or a specific membrane-bound electron transporter. Carbon monoxide (CO) is a specific inhibitor of mitochondrial cytochrome oxidase. In its presence, a cytochrome aa-CO adduct is produced (Chance 1953 a, b; Dennis and Richaud 1985; Naik and Nicholas 1986) and cytochrome-c oxidation is blocked. However, plant mitochondria possess a cyanide- and CO-insensitive alternative oxidase which accepts electrons from ubiquinone. This pathway does not use the second and third sites of mitochondrial ATP synthesis, and the yield of ATP synthesis is consequently low. Since CO, unlike KCN, can be administered to whole leaves in the gaseous form, we have used it to investigate the role of mitochondrial and chloroplast energy conservation in providing the ATP for stomatal opening in sunflower leaves. Material and methods Sunflower (Helianthus annuus L.) plants were grown in a greenhouse. Illumination was provided by 400-W halogen lamps (HQI-T; Osram, M/inchen, F R G ) for 10 h per day. Light levels at the stem apex were about 100 W . m 2. Leaves were cut from 8- to 10-week old plants under water and fitted into a temperature-controlled sandwich-type cuvette. The petiole of a leaf was placed in water. The composition of the gas passing through the cuvette was adjusted by mass-flow controllers, Unless indicated otherwise the O2 concentration was 1%. When necessary, CO2 was removed from the gas stream by soda lime. The final concentration of CO added to the gas stream was usually 25%. Contaminating CO2 had to be removed from commercially obtained CO. Gases were made up to 100% with N 2. They were passed through a tube with wet filter paper and subsequently through a cooling trap (usually 12~ C) to adjust the relative humidity of the gas stream entering the cuvette to about 60%. The temperature was usually 20 ~ C. A differential gas analyzer monitored differences in H 2 0 and CO2 between a reference gas stream and the stream which had passed through the cuvette over both surfaces of the leaf. A thermocouple controlled the leaf temperature. The leaf was illuminated by red light (half-bandwidth from 627 to 672 nm, filters: R G 630 cutoff, Schott, Mainz, F R G ; K 65 and Calflex C, b o t h Balzers, Liechtenstein, plus water cuvettes). A 405-nm interference filter (half-bandwidth 12 nm, Balzers) provided blue light of very low irradiance. Weak red light equivalent in photon flux density to the blue light was provided by a 671-mn interference filter. Transpiration and CO z exchange were recorded as described previously (Heber et al. 1986), and stomatal conductivity to water vapor was calculated according to Nobel (1983). The levels of ATP and A D P were determined in leaf discs. As in the gas-exchange experiments, leaves were gassed and illuminated for 1 h as indicated. Leaf discs were cut and frozen in liquid nitrogen, ground to powder in the frozen state and mixed with 4.5 ml 4.5% HC104. After the frozen mixture had thawed, it was centrifuged for 2 rain at 2000-g. Triethanola-
M. Pollok et al. : Inhibition of stomatal opening mine was added to the supernatant to reach a final concentration of 0.05 M. The solution was neutralized with 4 M K2CO3 and kept in ice. Crystalline KC104 was removed, and ATP was determined in the extract by the method of Wulff and D6ppen (1985) and A D P by that of Hampp (1985).
Results
Gas exchange of leaves after addition of CO. In air, the stomata of sunflower leaves were largely closed in the dark. Figure I shows that they opened after removal of COz from the atmosphere. Stomatal opening increased transpiration, and the leaf temperature decreased accordingly. When transpiration was constant, CO was flushed over the leaf (gas flow rate 1 l.min 1, composition of the atmosphere 25% CO, 1% 02, 74% N2). This decreased respiratory CO2 release after a transient increase (Fig. 1, lower part), and stomata closed slowly (Fig. 1, middle). The decreased transpiration caused an increase in the leaf temperature. Effects of illumination will be considered below. On average, CO doubled the stomatal resistance to watervapor diffusion in the dark when CO2 was absent. Table I shows the sum of the stomatal and boundary-layer resistances to water loss and respiratory COz release before and after addition of CO. Complete stomatal closure was not observed under the influence of CO. The data of Table 1 indicate an average reduction in CO2 output by 16%. It is important to note, that carbohydrate can be oxidized to CO2 in a cyanide- and CO-resistant alternative respiratory pathway which yields only onethird of the ATP synthesized during electron transport mediated by the cytochrome-oxidase pathway. Release of the CO inhibition of stomatal opening by 0 2 . Oxygen counteracted the inhibitory effect of CO on stomatal opening. When the ratio of CO to O2 was decreased below a value of about 25, stomates reopened, and when a low ratio of CO to O2 was increased, stomata closed. Figure 2 shows the relationship between stomatal conductivity to water vapor and the CO/O2 ratio. The data indicate that the reaction of CO with plant cytochrome oxidase, which is involved in the opening of stomata, is reversible. Photodissociation is not required for reversibility as it is in animal mitochondria (Chance and Erecinska 1971). Effects of red light on stomatal opening in the presence of CO. Since CO is an inhibitor of cytochrome oxidase, the closure of stomata under the influence of CO is likely to be caused by ATP deficiency.
M. Pollok et al. : Inhibition of stomatal opening
225
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