Planta
Planta I42, 61-65 (1978)
9 by Springer-Verlag 1978
Synergistic Action of Red and Blue Light and Action Spectra for Malate Formation in Guard Cells of Vicia faba L. Teruo Ogawa, Hirohisa Ishikawa, Keizo Shimada, and Kazuo Shibata Laboratory of Plant Physiology, Institute of Physical and Chemical Research (Rikagaku Kenkyusho), Wako-shi, Saitama 351, Japan
Abstract. Malate formation in guard cells of Vicia faba leaves is enhanced by light. The action spectrum for this effect was determined for epidermal strips of Viciafaba, and two different spectra were obtained under different light conditions and with and without background irradiation with high-irradiance red light ( > 6 0 0 nm, 3.0 mW cm -2) superimposed on monochromatic light of other wavelengths. The spectrum obtained at quantum fluxes of 1.7 2.2 nE cm -2 s -~ of monochromatic light without background red light showed a sharp peak at 433 nm with a shoulder around 475 nm and a lower peak at 670 680 nm; the spectrum obtained at much lower quantum fluxes of 0.05-0.07 nE cm 2 S-1 of monochromatic light with red-light background had two peaks of comparable heights at 380 and 460 nm. The formation of malate with 430-nm blue light was saturated at a quantum flux of 3 nE c m - 2 s- ~ without the background red light but at a much lower quantum flux of 0.2 nE cm -2 s 1 with the background red light. At this low intensity, blue light was practically ineffective without background red light. A synergistic action of red light presumably absorbed by the chlorophylls, and blue light absorbed by a yellow pigment is thus demonstrated by these experiments. The action maxima at 380 and 460 nm for the blue-light effect in the presence of background red light agree with the absorption maxima of flavins. Key words: Action spectrum - Guard cells -
action (stomata) - Vicia.
Malate formation -
Light Stomata
Introduction
In 1973, Allaway found that the malate content in guard cells increased on stomatal opening; he suggested that malate serves as counter ion for the
potassium ion taken up by guard cells during stomatal opening. A number of later studies showed that the main product of CO2 fixation by guard cells was malate which was converted to starch during stomatal closure in the dark, and that light promoted malate production (Dittrich and Raschke, 1977; Raschke and Dittrich, 1977; Willmer etal., 1973). It was inferred that light produced N A D P H to reduce oxaloacetate to malate (Salin et al., 1973). There are however no data showing the dependence of this lightinduced malate formation on wavelength which would permit identification of the photoreceptor pigment(s) involved in this process. It has been shown, by Kuiper (1964) in Senecio odoris and Mansfield and Meidner (1966) in Xanthium pennsylvanicum, that blue light is more effective than red light in causing stomatal opening. More recently, Hsiao et al. (1973) showed that both Rb § uptake by guard cells and stomatal opening in epidermal strips of Vicia faba were equally promoted by both red and blue light at high intensity, but that only blue light was effective at low intensity, with maximum efficiency in both processes at 420 440 nm. In this paper, we describe experiments undertaken to determine the action spectrum for malate formation in guard cells of Vicia faba leaves. We were able to obtain preparations of viable guard cells by means of sonication of epidermal strips. The ordinary epidermal cells and any adhering mesophyll cells were broken by the sonication treatment but the guard cells were not and their chloroplasts also remained intact. The formation of malate in guard cells could be measured in such preparations without interference by contamination with other cells. Action spectra for malate formation for this preparation were obtained under two different light conditions, and with and without background irradiation with red light; the results demonstrated a synergistic action of red and blue light.
0032-0935/78/0142/0061/$01.00
62
Materials and Methods Preparation and Sonication of Epidermal Strips Plants of Vicia faba L. were grown from seeds in a greenhouse on a vermiculite bed at 25~ with irrigation with a solution of 0.1% Hyponex (Hydroponic Chemical Co., Copley, O., USA) for a period of 20 30 d. They were placed in darkness for 12 h before harvesting the leaves. Epidermal strips were peeled from the abaxial (lower) leaf surface and put into a solution of 0.1 mM CaC12. The peeled epidermal strips were sonicated for 20 s with a 20-KC ultrasonic disruptor (200 W; Tomy Seiko Co., Tokyo) and washed with fresh CaCI2 solution. Sonication and washing were repeated for a total of 3 times. Microscopic observations showed that no mesophyll cells adhered to the sonicated strips, and vital staining with a solution of 2% fast green FCF (Tokyo Kasei Co., Tokyo) for 2 h indicated that guard cells were the only viable cells. For experiments, the sonicated strips were cut into small pieces (about 20 50 mm 2) with a razor blade.
Illumination of Sonicated Epidermal Strips The epidermal strips (about 3 4 mg dry weight) were put into 2.5 ml of 5 mM potassium-phosphate buffer, pH 7.0, in a test tube and were irradiated under various light conditions of white light, monochromatic red light, monochromatic blue light _+red background light, and monochromatic light of various wavelengths _+red background light, depending on the purpose of experiment. The time course of malate formation was studied under continuous irradiation with white light from a 400-W high-pressure SnC14 lamp (Toshiba Co., Tokyo) which was passed through a water bath of 5 cm. The irradiance at the sample surface was 6 mW cm-2. The dependence of the rate of malate formation on light energy was studied at 430 and 675 nm without background irradiation with red light, and at 430 nm with background red light. In these experiments, an interference filter (transmission maximum at 430 or 675 nm, half-band width= 10 nm; Nihon Shinku Kogaku Co., Tokyo) was placed in front of a Slide Star projector (Canon Co., Tokyo) equipped with a 300-W incandescent lamp and with a fan-cooled heat-absorbing filter, and the light transmitted through the interference filter and a heat-absorbing CuSor solution (0.5%, 5 cm) was used for irradiation. It was, however, difficult to obtain strong blue light with this light source. The light from a JASCO Spectro-irradiator, model CRM-FA (Nihon Bunko Co., Tokyo), equipped with a 2-kW xenon lamp as the light source was, therefore, used for experiments at high intensities. The background red light (>600 nm) was obtained with a solid glass filter, VR-60 (Toshiba Kasei Co., Tokyo) placed in front of the projector behind a heat-absorbing CuSO 4 solution. The irradiance of the monochromatic light was varied with neutral filters (Fuji Photo Co., Tokyo) placed in front of the sample tube. Action spectra were measured under two different light conditions with and without background red irradiation, which was superimposed on the spectrum from the JASCO Spectro-irradiator. The spectrum from this spectro-irradiator was spread over a stage of 55 cm to irradiate 22 sample tubes with monochromatic light. Twenty two different wavelengths were chosen between 356 and 752 nm without the background irradiation and ten wavelengths between 356 and 544 nm with the background red light. The band width of the monochromatic light was about 12 nm. The quantum flux of the monochromatic light was reduced with neutral filters placed in front of the sample tubes to 0.05-0.07 nE cm -2 s -1 for the measurement of action spectra with background red light, and to 1.7-2.2 nE cm -2 s -~ for the measurement without background light. In the measurement with the background light, three
T. Ogawa et al. : Light and Malate Formation in Vicia Guard Cells projectors, each with a heat-absorbing CuSO4 solution and with a solid glass filter (VR-60) as described above, were used to irradiate the sample tubes uniformly. Irradiances or quantum fluxes were measured with a thermopile (Kipp and Zonen, Delft, Netherlands; model E2). Its sensitivity was standardized with an incandescent standard lamp (U.S. National Bureau of Standards, Washington, D.C.).
Malate Analysis A sample of epidermis was killed in boiling 90% ethanol and malate was extracted from the epidermis with this solvent at 60~ C for 30 min. The extraction was repeated 3 times, and the extracts were combined and evaporated to dryness. The content of malate in the residue was determined by the method of M611ering (1974). The residue was solubilized in a reaction mixture containing 1.6 ml of 0.1 M 3-amino-l-propanol buffer (pH 10.0), 0.20 mI of 0.5 M glutamate (pH 10.0), 0.15 ml of 60 mM fl-NAD and 3.6 units of glutamic-oxaloacetic transaminase (Boehringer & S6hne G.m.b.H., Mannheim, Germany). To this reaction mixture was added 36 units of malate dehydrogenase (Boehringer & S6hne), and the reaction mixture was incubated at 25 ~ C for 30 min. The amount of malate was estimated from the increase in absorbance at 340 nm after incubation with malate dehydrogenase. After extraction, the epidermis was dried in an oven at 60 ~ C for 1 h and weighed. The content of malate in the epidermis was calculated on the basis of this dry weight. About 5 cm 2 of fresh epidermis gave 1 mg dry weight.
Spectrophotometry About 100 strips of sonicated epidermis were sandwiched between two glass plates and absorption spectra were measured with a Multipurpose recording spectrophotometer (model MPS-5000; Shimadzu, Kyoto). The same spectrophotometer was used for the malate analysis.
Results Synergistic Action o f Red and Blue Light on Malate Formation in Vicia faba Guard Cells M a l a t e w a s f o r m e d in V. faba g u a r d cells in t h e p r e s e n c e o f p o t a s s i u m i o n in t h e m e d i u m b u t n o t a t all in its a b s e n c e . F i g u r e 1 s h o w s m a l a t e f o r m a t i o n in s o n i c a t e d e p i d e r m i s o f V. faba d u r i n g 4 h o f i n c u b a t i o n in 5 m M p o t a s s i u m - p h o s p h a t e b u f f e r , p H 7.0, in w h i t e l i g h t o f 6 m W c m - 2 a n d in d a r k n e s s . It is e v i d e n t t h a t l i g h t p r o m o t e s m a l a t e f o r m a t i o n , t h i s light-enhanced process proceeding linearly during 2.5 h a n d s l o w i n g d o w n t h e r e a f t e r (see c u r v e C in Fig. 1). B a s e d o n t h i s r e s u l t , t h e p e r i o d o f i r r a d i a t i o n in f u r t h e r e x p e r i m e n t s w a s c h o s e n t o b e 2.5 h. The dependence of the rate of malate formation in s o n i c a t e d e p i d e r m a l s t r i p s o f V. faba o n l i g h t e n ergy was studied under two different light conditions and with and without background irradiation with r e d light. T h e r e s u l t s o b t a i n e d a t 430 n m a n d 675 n m w i t h o u t t h e b a c k g r o u n d l i g h t a r e s h o w n in F i g u r e 2
T. Ogawa et al.: Light and Malate F o r m a t i o n in Vicia G u a r d Cells
63 I
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z
2 Time( h )
t
o
I..k
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4
Fig. 1. Time course for malate formation in guard cells in sonicated epidermal strips of V.faba during 4 h of incubation in white light (curve A) and in darkness (curve B). Curve C is the difference between curves A and B
I L
I
9
1
Fig. 3. Rate o f m a l a t e formation in ~/:faba guard cells as a function of the light energy of 430-nm monochromatic light with (curve A) and without (curve B) background irradiation with red light ( > 600 nm) at 3.0 m W c m - 2
'
~
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Fig. 2. Rate of malate formation in V.faba guard cells in m o n o chromatic light at 430 n m (curve A) and at 675 n m (curve B) as a function of q u a n t u m flux
Fig. 4. Curve A: action spectrum for the malate formation in V.faba guard cells determined without background red light. The rate is expressed in nmol malate formed per m g dry weight of epidermis during 1 h of irradiation at a q u a n t u m flux of 1.9 nE cm -2 s 1. The rate at each wavelength, as shown by a solid circle, was determined from four separate experiments; the bars indicate the standard deviation of the mean of these data. Curve B : absorption spectrum of isolated epidermis (about 100 strips sandwiched between two glass plates)
(curves A and B, respectively); they indicate that the blue light is much more effective than the red light and that the rate reaches saturation at 3 nE cm -2 s - 1. The effect of background red light superimposed on blue light at 430 nm is shown in Figure 3; it can be seen that the blue light was much more effective with the background red light and that the effect reached saturation at ca. 0.2 nE cm -2 s 1. The rate of malate formation without the background light was in fact nearly zero at this quantum flux and required a much higher quantum flux of 3 nE c m - 2 s 1 for saturation, as shown by curve A in Figure 2.
It is evident, from examining curve B in Figure 2 and curves A and B in Figure 3, that the rate of malate formation with blue light applied simultaneously with the background red light is much higher than the sum of the rates in blue and in red light alone. F o r example, the rate at 0.2 nE c m - 2 s- 1 with blue plus red light was 3.3 nmol malate m g - 1 dry weight h or was more than 4 times the sum of the rates, 0 and 0.8 nmol m g - 1 dry wt h-1, respectively, in blue and red light alone. This result thus clearly demonstrates a synergistic action of red and blue light on the malate formation.
64
T. Ogawa et al.: Light and Malate Formation in Vicia Guard Cells I
I
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l
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2 zZ
-d.-. o
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350
I
400
450 500 Wavelength( nrn )
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Fig. 5. Action spectrum for malate formation in V.faba guard cells determined with background red irradiation ( > 6 0 0 n m ; 3.0 m W cm-2). The rate is expressed in nmol malate formed per m g of dry epidermis during 1 h of irradiation at a blue-light quant u m flux of 0.07 nE cm -2 s 1. The rate at each wavelength, as shown by a solid circle, was determined from three separate experiments; the bars indicate the standard deviation of the mean of these data
Action Spectra with and without Background Red Light The quantum fluxes used in the measurement of action spectra for malate formation in epidermal strips (guard cells) of V. faba were chosen from the proportionality range on the curves in Figures 2 and 3: 1.7-1.9 nE cm-2 s 1 without the background red irradiation and 0.05 0.07nE cm -2 s -1 with background red light. The action spectrum determined without the background red light is shown by curve A in Figure 4. It shows a high and a low peak at 433 and 375 nm, with a shoulder around 475 nm in the blue region, and another low peak at 670-680 nm in the red region. The absorption spectrum of isolated epidermal strips is shown by curve B of Figure 4 and is essentially the same as the absorption spectrum of chloroplasts prepared from mesophyll cells (not shown), having distinct peaks at 433 and 675 nm for chlorophyll a and a shoulder around 475 nm for chlorophyll b and carotenoids. The maxima at 433 nm and 670~680 nm of the action spectrum agree with these absorption maxima and both action and absorption spectra show a shoulder around 475 nm. However, the 433-nm peak of the action spectrum is more than 5 times higher than red peak at 670-680 nm whereas the absorption peak at the same wavelength of 433 nm is at most 60% higher than that in the red region. The much higher peak in the blue region of the action spectrum strongly indicates that the photoreceptor pigment responsible for the red band of the action spectrum has also absorption bands in the blue region and that blue light drives two light reactions involved in malate synthesis in the epidermal strips, thus accounting for the synergistic action of red and blue
light demonstrated in the preceding section of this paper. This conclusion is supported by the action spectrum in Figure 5 determined with background red light. This spectrum shows two maxima, around 380 and 460 rim, which are different from both the action maxima of the action spectrum obtained without background red light and the maxima of the absorption spectrum. This indicates that the light reaction limiting the rate of malate formation under irradiation with red light is not photosynthesis but rather a blue-light reaction. The maxima of 380 and 460 nm of this action spectrum agree with the absorption maxima of flavin.
Discussion Two different action spectra were obtained with and without background red-light irradiation for the formation of malate in guard cells of V.faba. The action spectrum obtained without background red light resembles the action spectra obtained for stomatal opening and S6Rb§ uptake by guard cells in epidermal strips of Viciafaba by Hsiao et al. (1973). These spectra showed a high maximum around 430 nm and a lower one in the red region. Furthermore, the quantum flux for saturation at 440 nm for stomatal opening was 20 • 1014 quanta cm- 2 s- 1 (3.4 nE cm- 2 s- 1) and thus similar to that which we obtained for malate formation without the background red light. This close agreement indicates that malate formation and stomatal opening are regulated by the same photochemical reactions, this conclusion being in agreement with that drawn by Allaway (1973) and Pearson and Milthorpe (1974). The much greater action in the blue region, compared with that in the red, indicates that malate formation in the guard cells is mediated not only by photosynthesis but also by a blue-light reaction. This view is strongly supported by the synergistic action of blue and red light. In fact, the action spectrum obtained with red background light showed two peaks at 380 and 460 nm which are different from the bands in the Sorer region in photosynthetic action spectra. The maximal wavelengths of 380 and 460 nm for the blue-light effect in the presence of background red light agree with the absorption maxima of flavin. Another system with a similar action spectrum is the enhancement of respiration by light in Chlorella cells (Kamiya and Miyachi, 1974; Kowallik and Gaffron, 1967), in which the maximal effect at 456 nm is attained at quantum fluxes of 400-800 erg c m - 2 s-1 (0.154).30nE cm -2 s - l ) , very close to the energy, 0.2 nE c m - 2 s- 1, for saturation of malate formation in V. faba epidermal strips in the presence of back-
T. Ogawa et al. : Light and Malate Formation in Vicia Guard Cells
ground red light (curve A in Fig. 3). Similar action spectra have also been described for other physiological processes such as phototropism (see, e.g., review by Curry, 1969) and light-induced rearrangement of chloroplasts in cells (Inoue and Shibata, 1973; Zurzycki, 1962). Willmer et al. (1973) demonstrated that phosphoenolpyruvate(PEP)-carboxylase activity of epidermal strips is proportional to the number of stomata in the sample. This indicates that the enzyme is located in the guard cells and mediates the formation of malate. Blue light enhanced PEP-carboxylase activity in Chlorella cells (Kamiya and Miyachi, 1975) and increased 14CO2 incorporation into malate as well as into aspartate, glutamate and fumarate (Kamiya and Miyachi, 1974). These observations indicate the possibility that PEP-carboxylase activity in guard cells is enhanced by blue light. The following mechanism is inferred from these observations to be involved in the synergistic action of red and blue light. The red-light effect could be based on production of ATP or some other energy source for K + influx into guard cells, and the bluelight effect could be based on activation of PEP carboxylase. However, neither of these effects alone enhances malate formation, and only balanced influx of K § by the red-light effect and activation of PEP carboxylase by the blue-light effect accelerate malate formation. The technical assistance of Miss Asayo Suzuki is grately acknowledged. The present paper was supported by grant 211117 from the Ministry of Education of Japan and by a grant for the study of "'Life Sciences" at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho).
References Allaway, W.G. : Accumulation of malate in guard cells of Vicia faba during stomatal opening. Planta 110, 63-70 (1973) Curry, G.M.: Phototropism. In: Physiology of plant growth and development, pp. 245-273, Wilkins, M.B., ed. New York: MacGraw-Hill 1969
65 Dittrich, P., Raschke, K. : Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning. Planta 134, 77 81 (1977) Hsiao, T.C., Allaway, W.G., Evans, L.T. : Action spectra for guard cell Rb + uptake and stomatal opening in Viciafaba.Plant Physiol. 51, 82 88 (1973) Inoue, Y., Shibata, K. : Light-induced chloroplast rearrangements and their action spectra as measured by absorption spectrophotometry. Planta 114, 341-358 (1973) Kamiya, A., Miyachi, S. : Effects of blue light on respiration and carbon dioxide fixation in colorless Chlorella mutant cells. Plant and Cell Physiol. 15, 927-937 (1974) Kamiya, A., Miyachi, S.: Blue light-induced formation of phosphoenolpyruvate carboxylase in colorless Chlorella mutant cells. Plant and Cell Physiol. 16, 729-736 (1975) Kowallik, W., Gaflu H. : Respiration induced by blue light. Planta 69, 92 95 (I966) Kuiper, P.J.C.: Dependence upon wavelength of stomatal movement in epidermal tissue of Senecio odoris. Plant Physiol. 39, 952 955 (1964) Mansfield, T.A., Meidner, H. : Stomatal opening in light of different wavelengths: effects of blue light independent of carbon dioxide concentration. J_ Exp. Bot. 17, 510 521 (I966) M611ering, H. : Determination with malate dehydrogenase and glntamate-oxaloacetate transaminase. In: Methods of enzymatic analysis, vol. 3, pp. 1589 1593, Bergmeyer, H.U., ed. Weinheim : Chemic Verlag 1974 Pearson, C.J., Milthorpe, F.L. : Structure, carbon dioxide fixation and metabolism of stomata. Aust. J. Plant Physiol. 1, 221 236 (1974) Raschke, K., Dittrich, P. : [14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open. Planta 134, 69-75 (1977) Salin, M.L., Campbell, W.H,, Black, C.C., Jr.: Oxaloacetate as the Hill oxidant in mesophyll cells of plants possessing the C4-dicarboxylic acid cycle of leaf photosynthesis. Proc. Natl. Acad. Sci. USA 70, 3730-3734 (1973) Willer, C.M., Kanai, R., Pallas, J.E., Jr., Black, C.C., Jr. : Detection of high levels of phosphoenolpyruvate carboxylase in leaf epidermal tissue and its significance in stomatal movements. Life Sci. 12, 151 155 (1973) Willmer, C.M., Pallas, J.E., Jr., BIack, C.C., Jr.: Carbon dioxide metabolism in leaf epidermal tissue. Plant Physiol. 52, 448~452 (1973) Zurzycki, J. : The action spectrum for the light dependent movements of chloroplasts in Lemna trisulca L. Acta Soc. Bot. Pol. 31, 489-538 (1962)
Received 7 March; accepted 8 May 1978
Planta I42, 61-65 (1978)
9 by Springer-Verlag 1978
Synergistic Action of Red and Blue Light and Action Spectra for Malate Formation in Guard Cells of Vicia faba L. Teruo Ogawa, Hirohisa Ishikawa, Keizo Shimada, and Kazuo Shibata Laboratory of Plant Physiology, Institute of Physical and Chemical Research (Rikagaku Kenkyusho), Wako-shi, Saitama 351, Japan
Abstract. Malate formation in guard cells of Vicia faba leaves is enhanced by light. The action spectrum for this effect was determined for epidermal strips of Viciafaba, and two different spectra were obtained under different light conditions and with and without background irradiation with high-irradiance red light ( > 6 0 0 nm, 3.0 mW cm -2) superimposed on monochromatic light of other wavelengths. The spectrum obtained at quantum fluxes of 1.7 2.2 nE cm -2 s -~ of monochromatic light without background red light showed a sharp peak at 433 nm with a shoulder around 475 nm and a lower peak at 670 680 nm; the spectrum obtained at much lower quantum fluxes of 0.05-0.07 nE cm 2 S-1 of monochromatic light with red-light background had two peaks of comparable heights at 380 and 460 nm. The formation of malate with 430-nm blue light was saturated at a quantum flux of 3 nE c m - 2 s- ~ without the background red light but at a much lower quantum flux of 0.2 nE cm -2 s 1 with the background red light. At this low intensity, blue light was practically ineffective without background red light. A synergistic action of red light presumably absorbed by the chlorophylls, and blue light absorbed by a yellow pigment is thus demonstrated by these experiments. The action maxima at 380 and 460 nm for the blue-light effect in the presence of background red light agree with the absorption maxima of flavins. Key words: Action spectrum - Guard cells -
action (stomata) - Vicia.
Malate formation -
Light Stomata
Introduction
In 1973, Allaway found that the malate content in guard cells increased on stomatal opening; he suggested that malate serves as counter ion for the
potassium ion taken up by guard cells during stomatal opening. A number of later studies showed that the main product of CO2 fixation by guard cells was malate which was converted to starch during stomatal closure in the dark, and that light promoted malate production (Dittrich and Raschke, 1977; Raschke and Dittrich, 1977; Willmer etal., 1973). It was inferred that light produced N A D P H to reduce oxaloacetate to malate (Salin et al., 1973). There are however no data showing the dependence of this lightinduced malate formation on wavelength which would permit identification of the photoreceptor pigment(s) involved in this process. It has been shown, by Kuiper (1964) in Senecio odoris and Mansfield and Meidner (1966) in Xanthium pennsylvanicum, that blue light is more effective than red light in causing stomatal opening. More recently, Hsiao et al. (1973) showed that both Rb § uptake by guard cells and stomatal opening in epidermal strips of Vicia faba were equally promoted by both red and blue light at high intensity, but that only blue light was effective at low intensity, with maximum efficiency in both processes at 420 440 nm. In this paper, we describe experiments undertaken to determine the action spectrum for malate formation in guard cells of Vicia faba leaves. We were able to obtain preparations of viable guard cells by means of sonication of epidermal strips. The ordinary epidermal cells and any adhering mesophyll cells were broken by the sonication treatment but the guard cells were not and their chloroplasts also remained intact. The formation of malate in guard cells could be measured in such preparations without interference by contamination with other cells. Action spectra for malate formation for this preparation were obtained under two different light conditions, and with and without background irradiation with red light; the results demonstrated a synergistic action of red and blue light.
0032-0935/78/0142/0061/$01.00
62
Materials and Methods Preparation and Sonication of Epidermal Strips Plants of Vicia faba L. were grown from seeds in a greenhouse on a vermiculite bed at 25~ with irrigation with a solution of 0.1% Hyponex (Hydroponic Chemical Co., Copley, O., USA) for a period of 20 30 d. They were placed in darkness for 12 h before harvesting the leaves. Epidermal strips were peeled from the abaxial (lower) leaf surface and put into a solution of 0.1 mM CaC12. The peeled epidermal strips were sonicated for 20 s with a 20-KC ultrasonic disruptor (200 W; Tomy Seiko Co., Tokyo) and washed with fresh CaCI2 solution. Sonication and washing were repeated for a total of 3 times. Microscopic observations showed that no mesophyll cells adhered to the sonicated strips, and vital staining with a solution of 2% fast green FCF (Tokyo Kasei Co., Tokyo) for 2 h indicated that guard cells were the only viable cells. For experiments, the sonicated strips were cut into small pieces (about 20 50 mm 2) with a razor blade.
Illumination of Sonicated Epidermal Strips The epidermal strips (about 3 4 mg dry weight) were put into 2.5 ml of 5 mM potassium-phosphate buffer, pH 7.0, in a test tube and were irradiated under various light conditions of white light, monochromatic red light, monochromatic blue light _+red background light, and monochromatic light of various wavelengths _+red background light, depending on the purpose of experiment. The time course of malate formation was studied under continuous irradiation with white light from a 400-W high-pressure SnC14 lamp (Toshiba Co., Tokyo) which was passed through a water bath of 5 cm. The irradiance at the sample surface was 6 mW cm-2. The dependence of the rate of malate formation on light energy was studied at 430 and 675 nm without background irradiation with red light, and at 430 nm with background red light. In these experiments, an interference filter (transmission maximum at 430 or 675 nm, half-band width= 10 nm; Nihon Shinku Kogaku Co., Tokyo) was placed in front of a Slide Star projector (Canon Co., Tokyo) equipped with a 300-W incandescent lamp and with a fan-cooled heat-absorbing filter, and the light transmitted through the interference filter and a heat-absorbing CuSor solution (0.5%, 5 cm) was used for irradiation. It was, however, difficult to obtain strong blue light with this light source. The light from a JASCO Spectro-irradiator, model CRM-FA (Nihon Bunko Co., Tokyo), equipped with a 2-kW xenon lamp as the light source was, therefore, used for experiments at high intensities. The background red light (>600 nm) was obtained with a solid glass filter, VR-60 (Toshiba Kasei Co., Tokyo) placed in front of the projector behind a heat-absorbing CuSO 4 solution. The irradiance of the monochromatic light was varied with neutral filters (Fuji Photo Co., Tokyo) placed in front of the sample tube. Action spectra were measured under two different light conditions with and without background red irradiation, which was superimposed on the spectrum from the JASCO Spectro-irradiator. The spectrum from this spectro-irradiator was spread over a stage of 55 cm to irradiate 22 sample tubes with monochromatic light. Twenty two different wavelengths were chosen between 356 and 752 nm without the background irradiation and ten wavelengths between 356 and 544 nm with the background red light. The band width of the monochromatic light was about 12 nm. The quantum flux of the monochromatic light was reduced with neutral filters placed in front of the sample tubes to 0.05-0.07 nE cm -2 s -1 for the measurement of action spectra with background red light, and to 1.7-2.2 nE cm -2 s -~ for the measurement without background light. In the measurement with the background light, three
T. Ogawa et al. : Light and Malate Formation in Vicia Guard Cells projectors, each with a heat-absorbing CuSO4 solution and with a solid glass filter (VR-60) as described above, were used to irradiate the sample tubes uniformly. Irradiances or quantum fluxes were measured with a thermopile (Kipp and Zonen, Delft, Netherlands; model E2). Its sensitivity was standardized with an incandescent standard lamp (U.S. National Bureau of Standards, Washington, D.C.).
Malate Analysis A sample of epidermis was killed in boiling 90% ethanol and malate was extracted from the epidermis with this solvent at 60~ C for 30 min. The extraction was repeated 3 times, and the extracts were combined and evaporated to dryness. The content of malate in the residue was determined by the method of M611ering (1974). The residue was solubilized in a reaction mixture containing 1.6 ml of 0.1 M 3-amino-l-propanol buffer (pH 10.0), 0.20 mI of 0.5 M glutamate (pH 10.0), 0.15 ml of 60 mM fl-NAD and 3.6 units of glutamic-oxaloacetic transaminase (Boehringer & S6hne G.m.b.H., Mannheim, Germany). To this reaction mixture was added 36 units of malate dehydrogenase (Boehringer & S6hne), and the reaction mixture was incubated at 25 ~ C for 30 min. The amount of malate was estimated from the increase in absorbance at 340 nm after incubation with malate dehydrogenase. After extraction, the epidermis was dried in an oven at 60 ~ C for 1 h and weighed. The content of malate in the epidermis was calculated on the basis of this dry weight. About 5 cm 2 of fresh epidermis gave 1 mg dry weight.
Spectrophotometry About 100 strips of sonicated epidermis were sandwiched between two glass plates and absorption spectra were measured with a Multipurpose recording spectrophotometer (model MPS-5000; Shimadzu, Kyoto). The same spectrophotometer was used for the malate analysis.
Results Synergistic Action o f Red and Blue Light on Malate Formation in Vicia faba Guard Cells M a l a t e w a s f o r m e d in V. faba g u a r d cells in t h e p r e s e n c e o f p o t a s s i u m i o n in t h e m e d i u m b u t n o t a t all in its a b s e n c e . F i g u r e 1 s h o w s m a l a t e f o r m a t i o n in s o n i c a t e d e p i d e r m i s o f V. faba d u r i n g 4 h o f i n c u b a t i o n in 5 m M p o t a s s i u m - p h o s p h a t e b u f f e r , p H 7.0, in w h i t e l i g h t o f 6 m W c m - 2 a n d in d a r k n e s s . It is e v i d e n t t h a t l i g h t p r o m o t e s m a l a t e f o r m a t i o n , t h i s light-enhanced process proceeding linearly during 2.5 h a n d s l o w i n g d o w n t h e r e a f t e r (see c u r v e C in Fig. 1). B a s e d o n t h i s r e s u l t , t h e p e r i o d o f i r r a d i a t i o n in f u r t h e r e x p e r i m e n t s w a s c h o s e n t o b e 2.5 h. The dependence of the rate of malate formation in s o n i c a t e d e p i d e r m a l s t r i p s o f V. faba o n l i g h t e n ergy was studied under two different light conditions and with and without background irradiation with r e d light. T h e r e s u l t s o b t a i n e d a t 430 n m a n d 675 n m w i t h o u t t h e b a c k g r o u n d l i g h t a r e s h o w n in F i g u r e 2
T. Ogawa et al.: Light and Malate F o r m a t i o n in Vicia G u a r d Cells
63 I
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Fig. 1. Time course for malate formation in guard cells in sonicated epidermal strips of V.faba during 4 h of incubation in white light (curve A) and in darkness (curve B). Curve C is the difference between curves A and B
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Fig. 3. Rate o f m a l a t e formation in ~/:faba guard cells as a function of the light energy of 430-nm monochromatic light with (curve A) and without (curve B) background irradiation with red light ( > 600 nm) at 3.0 m W c m - 2
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Fig. 2. Rate of malate formation in V.faba guard cells in m o n o chromatic light at 430 n m (curve A) and at 675 n m (curve B) as a function of q u a n t u m flux
Fig. 4. Curve A: action spectrum for the malate formation in V.faba guard cells determined without background red light. The rate is expressed in nmol malate formed per m g dry weight of epidermis during 1 h of irradiation at a q u a n t u m flux of 1.9 nE cm -2 s 1. The rate at each wavelength, as shown by a solid circle, was determined from four separate experiments; the bars indicate the standard deviation of the mean of these data. Curve B : absorption spectrum of isolated epidermis (about 100 strips sandwiched between two glass plates)
(curves A and B, respectively); they indicate that the blue light is much more effective than the red light and that the rate reaches saturation at 3 nE cm -2 s - 1. The effect of background red light superimposed on blue light at 430 nm is shown in Figure 3; it can be seen that the blue light was much more effective with the background red light and that the effect reached saturation at ca. 0.2 nE cm -2 s 1. The rate of malate formation without the background light was in fact nearly zero at this quantum flux and required a much higher quantum flux of 3 nE c m - 2 s 1 for saturation, as shown by curve A in Figure 2.
It is evident, from examining curve B in Figure 2 and curves A and B in Figure 3, that the rate of malate formation with blue light applied simultaneously with the background red light is much higher than the sum of the rates in blue and in red light alone. F o r example, the rate at 0.2 nE c m - 2 s- 1 with blue plus red light was 3.3 nmol malate m g - 1 dry weight h or was more than 4 times the sum of the rates, 0 and 0.8 nmol m g - 1 dry wt h-1, respectively, in blue and red light alone. This result thus clearly demonstrates a synergistic action of red and blue light on the malate formation.
64
T. Ogawa et al.: Light and Malate Formation in Vicia Guard Cells I
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Fig. 5. Action spectrum for malate formation in V.faba guard cells determined with background red irradiation ( > 6 0 0 n m ; 3.0 m W cm-2). The rate is expressed in nmol malate formed per m g of dry epidermis during 1 h of irradiation at a blue-light quant u m flux of 0.07 nE cm -2 s 1. The rate at each wavelength, as shown by a solid circle, was determined from three separate experiments; the bars indicate the standard deviation of the mean of these data
Action Spectra with and without Background Red Light The quantum fluxes used in the measurement of action spectra for malate formation in epidermal strips (guard cells) of V. faba were chosen from the proportionality range on the curves in Figures 2 and 3: 1.7-1.9 nE cm-2 s 1 without the background red irradiation and 0.05 0.07nE cm -2 s -1 with background red light. The action spectrum determined without the background red light is shown by curve A in Figure 4. It shows a high and a low peak at 433 and 375 nm, with a shoulder around 475 nm in the blue region, and another low peak at 670-680 nm in the red region. The absorption spectrum of isolated epidermal strips is shown by curve B of Figure 4 and is essentially the same as the absorption spectrum of chloroplasts prepared from mesophyll cells (not shown), having distinct peaks at 433 and 675 nm for chlorophyll a and a shoulder around 475 nm for chlorophyll b and carotenoids. The maxima at 433 nm and 670~680 nm of the action spectrum agree with these absorption maxima and both action and absorption spectra show a shoulder around 475 nm. However, the 433-nm peak of the action spectrum is more than 5 times higher than red peak at 670-680 nm whereas the absorption peak at the same wavelength of 433 nm is at most 60% higher than that in the red region. The much higher peak in the blue region of the action spectrum strongly indicates that the photoreceptor pigment responsible for the red band of the action spectrum has also absorption bands in the blue region and that blue light drives two light reactions involved in malate synthesis in the epidermal strips, thus accounting for the synergistic action of red and blue
light demonstrated in the preceding section of this paper. This conclusion is supported by the action spectrum in Figure 5 determined with background red light. This spectrum shows two maxima, around 380 and 460 rim, which are different from both the action maxima of the action spectrum obtained without background red light and the maxima of the absorption spectrum. This indicates that the light reaction limiting the rate of malate formation under irradiation with red light is not photosynthesis but rather a blue-light reaction. The maxima of 380 and 460 nm of this action spectrum agree with the absorption maxima of flavin.
Discussion Two different action spectra were obtained with and without background red-light irradiation for the formation of malate in guard cells of V.faba. The action spectrum obtained without background red light resembles the action spectra obtained for stomatal opening and S6Rb§ uptake by guard cells in epidermal strips of Viciafaba by Hsiao et al. (1973). These spectra showed a high maximum around 430 nm and a lower one in the red region. Furthermore, the quantum flux for saturation at 440 nm for stomatal opening was 20 • 1014 quanta cm- 2 s- 1 (3.4 nE cm- 2 s- 1) and thus similar to that which we obtained for malate formation without the background red light. This close agreement indicates that malate formation and stomatal opening are regulated by the same photochemical reactions, this conclusion being in agreement with that drawn by Allaway (1973) and Pearson and Milthorpe (1974). The much greater action in the blue region, compared with that in the red, indicates that malate formation in the guard cells is mediated not only by photosynthesis but also by a blue-light reaction. This view is strongly supported by the synergistic action of blue and red light. In fact, the action spectrum obtained with red background light showed two peaks at 380 and 460 nm which are different from the bands in the Sorer region in photosynthetic action spectra. The maximal wavelengths of 380 and 460 nm for the blue-light effect in the presence of background red light agree with the absorption maxima of flavin. Another system with a similar action spectrum is the enhancement of respiration by light in Chlorella cells (Kamiya and Miyachi, 1974; Kowallik and Gaffron, 1967), in which the maximal effect at 456 nm is attained at quantum fluxes of 400-800 erg c m - 2 s-1 (0.154).30nE cm -2 s - l ) , very close to the energy, 0.2 nE c m - 2 s- 1, for saturation of malate formation in V. faba epidermal strips in the presence of back-
T. Ogawa et al. : Light and Malate Formation in Vicia Guard Cells
ground red light (curve A in Fig. 3). Similar action spectra have also been described for other physiological processes such as phototropism (see, e.g., review by Curry, 1969) and light-induced rearrangement of chloroplasts in cells (Inoue and Shibata, 1973; Zurzycki, 1962). Willmer et al. (1973) demonstrated that phosphoenolpyruvate(PEP)-carboxylase activity of epidermal strips is proportional to the number of stomata in the sample. This indicates that the enzyme is located in the guard cells and mediates the formation of malate. Blue light enhanced PEP-carboxylase activity in Chlorella cells (Kamiya and Miyachi, 1975) and increased 14CO2 incorporation into malate as well as into aspartate, glutamate and fumarate (Kamiya and Miyachi, 1974). These observations indicate the possibility that PEP-carboxylase activity in guard cells is enhanced by blue light. The following mechanism is inferred from these observations to be involved in the synergistic action of red and blue light. The red-light effect could be based on production of ATP or some other energy source for K + influx into guard cells, and the bluelight effect could be based on activation of PEP carboxylase. However, neither of these effects alone enhances malate formation, and only balanced influx of K § by the red-light effect and activation of PEP carboxylase by the blue-light effect accelerate malate formation. The technical assistance of Miss Asayo Suzuki is grately acknowledged. The present paper was supported by grant 211117 from the Ministry of Education of Japan and by a grant for the study of "'Life Sciences" at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho).
References Allaway, W.G. : Accumulation of malate in guard cells of Vicia faba during stomatal opening. Planta 110, 63-70 (1973) Curry, G.M.: Phototropism. In: Physiology of plant growth and development, pp. 245-273, Wilkins, M.B., ed. New York: MacGraw-Hill 1969
65 Dittrich, P., Raschke, K. : Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning. Planta 134, 77 81 (1977) Hsiao, T.C., Allaway, W.G., Evans, L.T. : Action spectra for guard cell Rb + uptake and stomatal opening in Viciafaba.Plant Physiol. 51, 82 88 (1973) Inoue, Y., Shibata, K. : Light-induced chloroplast rearrangements and their action spectra as measured by absorption spectrophotometry. Planta 114, 341-358 (1973) Kamiya, A., Miyachi, S. : Effects of blue light on respiration and carbon dioxide fixation in colorless Chlorella mutant cells. Plant and Cell Physiol. 15, 927-937 (1974) Kamiya, A., Miyachi, S.: Blue light-induced formation of phosphoenolpyruvate carboxylase in colorless Chlorella mutant cells. Plant and Cell Physiol. 16, 729-736 (1975) Kowallik, W., Gaflu H. : Respiration induced by blue light. Planta 69, 92 95 (I966) Kuiper, P.J.C.: Dependence upon wavelength of stomatal movement in epidermal tissue of Senecio odoris. Plant Physiol. 39, 952 955 (1964) Mansfield, T.A., Meidner, H. : Stomatal opening in light of different wavelengths: effects of blue light independent of carbon dioxide concentration. J_ Exp. Bot. 17, 510 521 (I966) M611ering, H. : Determination with malate dehydrogenase and glntamate-oxaloacetate transaminase. In: Methods of enzymatic analysis, vol. 3, pp. 1589 1593, Bergmeyer, H.U., ed. Weinheim : Chemic Verlag 1974 Pearson, C.J., Milthorpe, F.L. : Structure, carbon dioxide fixation and metabolism of stomata. Aust. J. Plant Physiol. 1, 221 236 (1974) Raschke, K., Dittrich, P. : [14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open. Planta 134, 69-75 (1977) Salin, M.L., Campbell, W.H,, Black, C.C., Jr.: Oxaloacetate as the Hill oxidant in mesophyll cells of plants possessing the C4-dicarboxylic acid cycle of leaf photosynthesis. Proc. Natl. Acad. Sci. USA 70, 3730-3734 (1973) Willer, C.M., Kanai, R., Pallas, J.E., Jr., Black, C.C., Jr. : Detection of high levels of phosphoenolpyruvate carboxylase in leaf epidermal tissue and its significance in stomatal movements. Life Sci. 12, 151 155 (1973) Willmer, C.M., Pallas, J.E., Jr., BIack, C.C., Jr.: Carbon dioxide metabolism in leaf epidermal tissue. Plant Physiol. 52, 448~452 (1973) Zurzycki, J. : The action spectrum for the light dependent movements of chloroplasts in Lemna trisulca L. Acta Soc. Bot. Pol. 31, 489-538 (1962)
Received 7 March; accepted 8 May 1978
