Planta
Planta 134, 7 7 - 81 (1977)
9 by Springer-Verlag 1977
Malate Metabolism in Isolated Epidermis of Commelina communis L. in Relation to Stomatal Functioning P. Dittrich Botanisches Institut der Universit/it Miinchen, Menzinger Strage 67, D-8000 Mfinchen 19, Federal Republic of Germany
K. Raschke* Institut ffir Botanik und Mikrobiologie der Technischen UniversitS~t Mfinchen, Arcisstral3e 21, D-8000 Mfinchen 2, Federal Republic of Germany
Abstract. Epidermal strips with closed stomata were exposed to malic acid labelled with 14C either uniformly or in 4-C only. During incubation with [U14C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-14C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to ~4CO2 and then initiating stomatal closure. Of the radioactive fixation products of CO2, only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle. * On sabbatical leave from MSU/ERDA Plant Research Laboratory, East Lansing, MI 48824, USA. Reprint requests to K.R. to this address Abbreviations." ABA=abscisic acid, NAD=nicotinamide adenine dinucleotide, NADP=nicotinamide adenine dinucleotide phosphate, PEP = phosphoenolpyruvate
Key words: C o m m e l i n a c o m m u n i s Gluconeogenesis - Malic acid ment.
-
Epidermis Stomatal move-
Introduction Stomatal opening is the osmotic effect of an import o f K + and C1- into the guard cells and the production of organic acids, predominantly malic acid, in these cells (Raschke, 1975). Guard cells are able to make malic acid by carboxylating PEP (Willmer et al., 1973; Willmer and Dittrich, 1974). During stomatal closing, K § and CI- leave the guard cells. These ions probably disperse in the apoplast of the epidermis, and are taken up by other epidermal cells, particularly subsidiary cells if these are present. The fate of malate during closing is not known. Willmer et al. (1973) reported the presence of NADP-dependent malic enzyme in epidermis of C o m m e l i n a c o m m u n i s . This enzyme catalyzes the decarboxylation of malate to pyruvate. For many decades, botanists have known that starch grains may reappear in guard cells during or after stomatal closing. The occurrence of gluconeogenesis in guard cells is therefore likely. On the other hand, doubt arises whether gluconeogenesis could occur fast enough to remove all the malate from the vacuoles of the guard cells during rapid stomatal closing. Stomata may shut within a few minutes after the application of ABA. Besides being able to make carbohydrates from malate, guard cells must possess an additional mechanism for removing malate, a mechanism faster than gluconeogenesis. The question whether guard cells can make sugars from malate was answered by offering to epidermal strips malic acid either labelled with 14C uniformly or in 4-C only. If gluconeogenesis were to occur, the 4-C of malate would be lost as CO2 and sugars
78
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
could become radioactive only in epidermal strips incubated with [U-14C]malate and not if incubated with [4-1~C]malate. The site of conversion of [U14C]malate to starch was then determined by autoradiography. The removal of malate from guard cells during rapid stomatal closing was followed in another set of experiments. Malate and its metabolites were labelled by exposure of epidermes with open stomata to 14CO2 (Willmer and Dittrich, 1974; Raschke and Dittrich, 1977). Then closing was initiated. The labelling patterns of the epidermes and the solutions on which they floated were determined before and after the stomata had closed.
tion, after 5 min on water or ABA, and after 30 min on water. The water on which the epidermal strips had floated was also subjected to chromatography followed by autoradiography. For the localization by microautoradiography of possible starch formation in epidermis from malic acid, epidermal samples were prepared in the following way. Samples (3 mm x 3 mm) from epidermis with closed stomata were floated on 0.4 m M [U-14C]ma late in the light (15 mW cm -2 from incandescent lamps) at 25~ for 2 h. The strips were then rinsed with distilled water and extracted 3 times with 70% ethanol. After drying, the tissues were takenup by transparent adhesive tape and pressed onto a 2 cm x 3.5 cm piece of Ilford Pan F film (Ilford, Essex, UK) in total darkness. Six days later, the adhesive tape with the epidermal sample was peeled off and the film developed.
Results Materials and Methods
Uptake and Metabolism of Malic Acid
Plants of Commelina communis L. were grown in a greenhouse at 2 3 ~ 4 ~ during the day and 18~ during the night. Daylength was extended to 14 h by supplementary illumination with mercury vapor lamps. Epidermal samples were taken from leaves of branches that did not flower. [U-14C]malic acid (58.3 Ci mol 1) was purchased from Amersham-Buchler, Braunschweig. [4-14C]malic acid (60 Ci tool-1) was prepared according to Dittrich et al. (1973), coupling x4COz fixation by partially purified PEP carboxylase to reduction catalyzed by malate dehydrogenase. Degradation of the malate so obtained, using NADP-malic enzyme (Dittrich, 1976), showed that 95% of the radioactivity was located in the 4-C position. Epidermal strips were peeled from leaves and then placed in batches of 2 4 cm 2 total area each on drops of solutions of malic acid at the bottom of Petri dishes, with the mesophyll side on the solution. After various incubation periods in light (ca. 15 mW c m - z photosynthetically active light from two 300-W incandescent lamps) or darkness, the epidermal samples were rinsed with distilled water, transferred to boiling 70% ethanol and extracted with three changes of solvent. The extracts were dried and subjected to twodimensional paper chromatography, and radioactive compounds were located by autoradiography according to Willmer and Dittrich (1974). Extracted tissues were hydrolyzed in 2 N HC1 at 100~ for 2 h to determine the radioactivity of the starch formed. The dried hydrolysates were chromatographed on paper in one dimension to show that glucose was the main product. Radioactivity was determined by liquid scintillation counting. For the study of the possibility of leakage of malate from the guard cells of closing stomata, epidermal samples were needed the stomata of which were open and the guard cells of which contained labelled malate. The latter goal was achieved by exposure of epidermis to 14COz. Detached leaves were brought into light while floating on COa-free water at 25~ After 2 h, epidermal strips were peeled; stomatal aperture at this time was between 8 and 10 p.m. The epidermal samples were then placed, cuticle down, on drops of water in Petri dishes, and 14CO2 was injected through a hole in the lid of the dishes, to give a [COz] of about 1.5 x 104 p~l 1-1. The specific activity was 58 Ci tool -1. After an exposure for 2 rain in darkness, the strips were transferred into another Petri dish, where they floated, mesophyll side down, on drops of water in a natural atmosphere of 12CO2, again in darkness. Under this condition, stomata closed within about 30 min. For rapid stomatal closure, occurring within 5 min, epidermal strips were placed on 10-4 M ( _+)-ABA after the fixation of 14CO2. Samples were taken and extracted immediately after 14CO2 fixa-
Malic acid was taken up by epidermal strips at relatively low rates. The accumulation of radioactive malic acid by the tissue did not follow a clear relationship with time, and light did not enhance malate uptake (Tables 1, 2). Metabolism of applied malic acid proceeded only slowly in the epidermal samples. Even 2 h after the beginning of exposure to labelled malate, a large part of the radioactivity (between 61 and 86% of the total activity) was still in the malate fraction. But radioactivity appeared also in two groups of metabolites of malic acid: (i) in the transamination product of oxaloacetate, aspartate, and in compounds associated with the tricarboxylic-acid cycle, such as citrate and
Table 1. Metabolism of [U-14C]malate by epidermal tissue of Commelina communis Incubation with a 0.43 m M solution at pH 3.3 in darkness and in the light. Stomata were closed Incubation (rain)
Dark 10
Light 30
120
10
30
120
1610 5
3470 75
1130 0
1170 45
3150 100
87 0 7 0 0 0 0 0
92 1 4 0 0 0 0 0
78 2 4 2 3 10 0 0
Radioactivity (cpm/cm z) soluble starch
1240 15
Percent of radioactivity of soluble fraction Malate Aspartate Citrate Fumarate Sugar phosph. Glucose Fructose Sucrose
91 2 7 0 0 0 0 0
84 2 6 0 0 0 0 0
61 4 4 2 0 6 5 14
Fig. l a and b. Formation of starch from malate in stomata of Commelina communis. Isolated epidermes were incubated with 0.4 m M [U-l~C]malic acid for 2 h at 25 ~ C in the light, then extracted with 70% ethanol, dried, and brought into contact with photographic film for 6 days. The autoradiograms on the left show the distribution of radioactivity, the photographs on the right that of the stomata in the epidermal sample. Images taken at low magnification a show that the concentration of radioactivity in stomata is not restricted to small patches in the epidermis. High magnification b shows correlation between the distribution of silver grains and that of stomatal complexes. Hydrolysis of the epidermis resulted in glucose as the only radioactive product
80
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
fumarate; and (ii) in carbohydrates, like glucose and starch. Labelled carbon appeared in the tricarboxylicacid cycle within the first ten min of exposure; in sugars after 2 h. There was virtually no difference in the degree to which [U-14C]- and [4-a4C]malic acid were metabolized in the citric-acid cycle, but a striking difference appeared in the degree of labelling of the carbohydrates. After an incubation of epidermal samples with [U-14C]malate for 2 h in the dark, 25% of the total radioactivity was found in glucose, fructose and sucrose (Table 1). In the light, this percentage was smaller (13%). The corresponding incubation with [4-1*C]malate in the dark did not yield radioactive sugars at all; in the light, the radioactivity found in sugars amounted to less than 1% of the total activity (Table 2). Similarly, only 0.6% of the total radioactivity was in the hydrolysate if epidermal samples had been incubated with [4-14C]malic acid for 2 h in the dark, and 0.2% if they had been incubated in the light (Table 2). These values are lower than the relative radioactivities of 2 or 3% in the hydrolysate of epidermes that were exposed to [U-a4C]malic acid (Table 1). Glucose was the only radioactive product of hydrolysis. Radioactivity appearing in the hydrolysate therefore indicated incorporation of 14C into starch. Autoradiograms were prepared of epidermal strips which had been incubated with [U-14C]malate before and then extracted with aqueous ethanol. These films were blackened only above the stomata (Fig. 1). The ability of epidermal tissue to form starch from malic acid is therefore concentrated in the cells of the stomatal complexes.
Release of Endogenous Malic Acid During Stomatal Closing Malic acid in the guard cells was labelled by exposing epidermal samples with open stomata to 14CO2 for 2 min (as described under Materials and Methods). Then these epidermal strips were subjected to two kinds of closing treatments. The first consisted of an exposure of strips floating on water to a normal atmospheric concentration of 12CO2 in darkness. In the second treatment, closure was accelerated by placing the strips on a 10-4M solution of (+)-ABA. The first treatment resulted in stomatal closure within 30 rain. ABA shortened the stomatal response to 5 rain. During slow stomatal closing on water, 43% of the radioactivity in the epidermal sample was released into the water. Although the tissue contained nearly 30% of its radioactivity in aspartate, glutamate, and citrate, the compound released was exclusively malate (Table 3). The amount of radioactivity lost from the epidermal tissue was even larger when stomatal closure was accelerated by ABA. Within 5 min 63% of the radioactivity leaked from the tissue into the water. Again malate was the only labelled product lost from the epidermis. The experiment was repeated with similar results. We conclude that guard cells can release malate faster than it can be metabolized. ABA accelerates the specific leakage of malate.
Table 3. Efflux of [l~C]malate during stomatal closure from epidermes of Commelina communis floating on water or on 10 4 M (+_)-ABA Sequence of Stomata dark treatments
14C (% of total) in: External solution
Epidermis
MaP
Mal
Asp
Glu
Cit
Table 2. Metabolism of [4-14C]malate by epidermal tissue of Com-
melina communis Incubation with a 5 mM solution at pH 5.7 in darkness and in the light. Stomata were closed Incubation (min)
Dark 10
30
120
10
30
120
25,100 13,500 17,420 0 0 100
17,400 17,500 29,400 0 120 64
96 0.3 2 0.5 0 0
Open
0
63
12
8
17
followed by 5 min 12CO2
Open
13
44
14
17
12
followed by 30 min lzCOz
Closed
43
36
0
5
11
0
48
35
4
13
95 0.5 3 0.8 0 0
86 0.9 6 2 0 0
63
22
2
5
6
Rapid closure 2 min 14CO2
Percent of radioactivity of soluble fraction Malate Aspartate Citrate Fumarate Glucose Sucrose
2 min 14CO2 Light
Radioactivity (cpm/cm2) soluble starch
Slow closure
96 0.3 2 0.6 0 0
86 0.5 8 1.1 0 0
84 1.1 7 2 0.3 0.4
Open
followed by 5 rain 12CO2 Closed +10 4 M A B A
a NO labelled compounds other than malate were detected in the external solution
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
Discussion
Our experiments show that gluconeogenesis can proceed in epidermal tissue (Table 1), and that starch formation from malic acid occurs in the stomatal complexes (Fig. 1). Gluconeogenesis involves the loss of 4-C of malate by decarboxylation. This is the reason why carbohydrates became much less radioactive during an incubation of epidermal strips with [4-14C]malic acid than during a sufficiently long presentation (2 h) of uniformly labelled malic acid (Tables 1, 2). One could have expected complete absence of radioactivity from carbohydrates in strips exposed to [4-t4C]malic acid, except for the presence of fumarase. This enzyme catalyzes the exchange of carbon 1 with carbon 4 in malic acid. Once 1-C becomes radioactive through exchange with 4-~4C, radioactive carbon would find its way into carbohydrates. Botanists have known at least since the formulation of the starch ~ osmoticum hypothesis by Lloyd (1908) that starch grains in guard cells may disappear during stomatal opening, and reappear after closure. We reinterpret this observation: Disappearance may be linked to the formation of malate, reappearance to gluconeogenesis from malate. Since guard cells probably possess large pools of soluble carbohydrates, it is not surprising that the rhythm of starch metabolism has occasionally been observed to be considerably out of phase with the rhythm of stomatal movement (Imamura, 1943; Yamashita, 1952). In addition to being able to convert malate into sugars, epidermal tissue can channel endogenous as well as exogenous malate into the tricarboxylic acid cycle (Tables 1, 2, 3). This capability became apparent also in other studies involving the supply of H~4CO3 or 14COz to epidermal strips of Commelina diffusa, Commelina communis, and Tulipa gesneriana (Willmer and Dittrich, 1974; Raschke and Dittrich, 1977). A third and very effective way guard cells possess to dispose of malate is to release a large portion of it into their surroundings. This process occurs during stomatal closing. In our experiments, on the average, about one-half of the labelled malate was lost when stomata closed: somewhat less than one-half when they closed slowly, within 30 min; more than one-half, when closing was accelerated by ABA (Table 3). The loss of labelled malate was specific; other labelled compounds remained in the epidermal tissue. Combining the results of our studies on malate metabolism in epidermes with those of earlier experi-
81
ments o n C O 2 fixation by epidermes (Shaw and Maclachlan, 1954; Willmer et al., 1973; Willmer and Dittrich, 1974; Raschke and Dittrich, 1977) we conclude: Guard cells can produce malic acid by the carboxylation of PEP which can be derived from sugars and starch. Guard cells are able to dispose of malate by three processes which can occur simultaneously: (i) oxidation in the tricarboxylic acid cycle, (ii) gluconeogenesis after decarboxylation, and (iii) specific release (probably together with K+). The third process enables guard cells to reduce their turgor faster than the metabolism of malic acid would allow. This work was performed while K.R. held a John Simon Guggenheim Memorial Fellowship. The research was further supported by a grant from the Deutsche Forschungsgemeinschaft to K.R. as well as through Contract EY-76-C-02-1338 between Michigan State University and the U.S. Energy Research and Development Administration. We greatly appreciate the careful technical assistance by Carol Holle and G. Ohst, as well as the photographic work by Cheri L. Schmuck. K.R. also wishes to thank Professor H. Ziegler, Institut ffir Botanik der Technischen Universit/it Mfinchen, for laboratory space and plant growth facilities.
References Dittrich, P. : Equilibration of label in malate during dark fixation of CO2 in KalanchoOfedtschenkoi. Plant Physiol. 58, 288-291 (1976) Dittrich, P., Salin, M., Black, C.C., Jr.: Conversion of carbon 4 of malate into products of the pentose cycle by isolated bundle sheath strands of Digitaria sanguinalis L. Scop. leaves. Biochem. Biophys. Res. Com. 55, 104-110 (1973) Imamura, S.: Untersuchungen fiber den Mechanismus der Turgorschwankung der Spalt6ffnungsschliesszellen. Jap. J. Bot. 12, 251 346 (1943) Lloyd, F.E.: The physiology of stomata. Carnegie Inst. Wash. Publ. No. 82 (1908) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309 340 (1975) Raschke, K., Dittrich, P.': [l~C]Carbon-dioxide fixation by isolated leaf epidermis with stomata closed or open. Planta 134, 69-75 (1977) Shaw, M., Maclachlan, G.A.: The physiology of stomata. I. Carbon dioxide fixation in guard cells. Canad. J. Bot. 32, 784-794 (1954) Willmer, C.M., Dittrich, P. : Carbon dioxide fixation by epidermal and mesophylt tissues of Tulipa and Commelina. Planta (Bet1.) 117, 123-132 (1974) Willmer, C.M., Pallas, J.E., Jr., Black, C.C., Jr. : Carbon dioxide fixation in leaf epidermal tissue. Plant Physiol. 52, 448-452 (1973) Yamashita, T. : Influences of potassium supply upon various properties and movement of the guard cells. Sieboldia 1, 51-70
(1952) Received 20 April; accepted 3 November 1976
Planta 134, 7 7 - 81 (1977)
9 by Springer-Verlag 1977
Malate Metabolism in Isolated Epidermis of Commelina communis L. in Relation to Stomatal Functioning P. Dittrich Botanisches Institut der Universit/it Miinchen, Menzinger Strage 67, D-8000 Mfinchen 19, Federal Republic of Germany
K. Raschke* Institut ffir Botanik und Mikrobiologie der Technischen UniversitS~t Mfinchen, Arcisstral3e 21, D-8000 Mfinchen 2, Federal Republic of Germany
Abstract. Epidermal strips with closed stomata were exposed to malic acid labelled with 14C either uniformly or in 4-C only. During incubation with [U14C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-14C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to ~4CO2 and then initiating stomatal closure. Of the radioactive fixation products of CO2, only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle. * On sabbatical leave from MSU/ERDA Plant Research Laboratory, East Lansing, MI 48824, USA. Reprint requests to K.R. to this address Abbreviations." ABA=abscisic acid, NAD=nicotinamide adenine dinucleotide, NADP=nicotinamide adenine dinucleotide phosphate, PEP = phosphoenolpyruvate
Key words: C o m m e l i n a c o m m u n i s Gluconeogenesis - Malic acid ment.
-
Epidermis Stomatal move-
Introduction Stomatal opening is the osmotic effect of an import o f K + and C1- into the guard cells and the production of organic acids, predominantly malic acid, in these cells (Raschke, 1975). Guard cells are able to make malic acid by carboxylating PEP (Willmer et al., 1973; Willmer and Dittrich, 1974). During stomatal closing, K § and CI- leave the guard cells. These ions probably disperse in the apoplast of the epidermis, and are taken up by other epidermal cells, particularly subsidiary cells if these are present. The fate of malate during closing is not known. Willmer et al. (1973) reported the presence of NADP-dependent malic enzyme in epidermis of C o m m e l i n a c o m m u n i s . This enzyme catalyzes the decarboxylation of malate to pyruvate. For many decades, botanists have known that starch grains may reappear in guard cells during or after stomatal closing. The occurrence of gluconeogenesis in guard cells is therefore likely. On the other hand, doubt arises whether gluconeogenesis could occur fast enough to remove all the malate from the vacuoles of the guard cells during rapid stomatal closing. Stomata may shut within a few minutes after the application of ABA. Besides being able to make carbohydrates from malate, guard cells must possess an additional mechanism for removing malate, a mechanism faster than gluconeogenesis. The question whether guard cells can make sugars from malate was answered by offering to epidermal strips malic acid either labelled with 14C uniformly or in 4-C only. If gluconeogenesis were to occur, the 4-C of malate would be lost as CO2 and sugars
78
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
could become radioactive only in epidermal strips incubated with [U-14C]malate and not if incubated with [4-1~C]malate. The site of conversion of [U14C]malate to starch was then determined by autoradiography. The removal of malate from guard cells during rapid stomatal closing was followed in another set of experiments. Malate and its metabolites were labelled by exposure of epidermes with open stomata to 14CO2 (Willmer and Dittrich, 1974; Raschke and Dittrich, 1977). Then closing was initiated. The labelling patterns of the epidermes and the solutions on which they floated were determined before and after the stomata had closed.
tion, after 5 min on water or ABA, and after 30 min on water. The water on which the epidermal strips had floated was also subjected to chromatography followed by autoradiography. For the localization by microautoradiography of possible starch formation in epidermis from malic acid, epidermal samples were prepared in the following way. Samples (3 mm x 3 mm) from epidermis with closed stomata were floated on 0.4 m M [U-14C]ma late in the light (15 mW cm -2 from incandescent lamps) at 25~ for 2 h. The strips were then rinsed with distilled water and extracted 3 times with 70% ethanol. After drying, the tissues were takenup by transparent adhesive tape and pressed onto a 2 cm x 3.5 cm piece of Ilford Pan F film (Ilford, Essex, UK) in total darkness. Six days later, the adhesive tape with the epidermal sample was peeled off and the film developed.
Results Materials and Methods
Uptake and Metabolism of Malic Acid
Plants of Commelina communis L. were grown in a greenhouse at 2 3 ~ 4 ~ during the day and 18~ during the night. Daylength was extended to 14 h by supplementary illumination with mercury vapor lamps. Epidermal samples were taken from leaves of branches that did not flower. [U-14C]malic acid (58.3 Ci mol 1) was purchased from Amersham-Buchler, Braunschweig. [4-14C]malic acid (60 Ci tool-1) was prepared according to Dittrich et al. (1973), coupling x4COz fixation by partially purified PEP carboxylase to reduction catalyzed by malate dehydrogenase. Degradation of the malate so obtained, using NADP-malic enzyme (Dittrich, 1976), showed that 95% of the radioactivity was located in the 4-C position. Epidermal strips were peeled from leaves and then placed in batches of 2 4 cm 2 total area each on drops of solutions of malic acid at the bottom of Petri dishes, with the mesophyll side on the solution. After various incubation periods in light (ca. 15 mW c m - z photosynthetically active light from two 300-W incandescent lamps) or darkness, the epidermal samples were rinsed with distilled water, transferred to boiling 70% ethanol and extracted with three changes of solvent. The extracts were dried and subjected to twodimensional paper chromatography, and radioactive compounds were located by autoradiography according to Willmer and Dittrich (1974). Extracted tissues were hydrolyzed in 2 N HC1 at 100~ for 2 h to determine the radioactivity of the starch formed. The dried hydrolysates were chromatographed on paper in one dimension to show that glucose was the main product. Radioactivity was determined by liquid scintillation counting. For the study of the possibility of leakage of malate from the guard cells of closing stomata, epidermal samples were needed the stomata of which were open and the guard cells of which contained labelled malate. The latter goal was achieved by exposure of epidermis to 14COz. Detached leaves were brought into light while floating on COa-free water at 25~ After 2 h, epidermal strips were peeled; stomatal aperture at this time was between 8 and 10 p.m. The epidermal samples were then placed, cuticle down, on drops of water in Petri dishes, and 14CO2 was injected through a hole in the lid of the dishes, to give a [COz] of about 1.5 x 104 p~l 1-1. The specific activity was 58 Ci tool -1. After an exposure for 2 rain in darkness, the strips were transferred into another Petri dish, where they floated, mesophyll side down, on drops of water in a natural atmosphere of 12CO2, again in darkness. Under this condition, stomata closed within about 30 min. For rapid stomatal closure, occurring within 5 min, epidermal strips were placed on 10-4 M ( _+)-ABA after the fixation of 14CO2. Samples were taken and extracted immediately after 14CO2 fixa-
Malic acid was taken up by epidermal strips at relatively low rates. The accumulation of radioactive malic acid by the tissue did not follow a clear relationship with time, and light did not enhance malate uptake (Tables 1, 2). Metabolism of applied malic acid proceeded only slowly in the epidermal samples. Even 2 h after the beginning of exposure to labelled malate, a large part of the radioactivity (between 61 and 86% of the total activity) was still in the malate fraction. But radioactivity appeared also in two groups of metabolites of malic acid: (i) in the transamination product of oxaloacetate, aspartate, and in compounds associated with the tricarboxylic-acid cycle, such as citrate and
Table 1. Metabolism of [U-14C]malate by epidermal tissue of Commelina communis Incubation with a 0.43 m M solution at pH 3.3 in darkness and in the light. Stomata were closed Incubation (rain)
Dark 10
Light 30
120
10
30
120
1610 5
3470 75
1130 0
1170 45
3150 100
87 0 7 0 0 0 0 0
92 1 4 0 0 0 0 0
78 2 4 2 3 10 0 0
Radioactivity (cpm/cm z) soluble starch
1240 15
Percent of radioactivity of soluble fraction Malate Aspartate Citrate Fumarate Sugar phosph. Glucose Fructose Sucrose
91 2 7 0 0 0 0 0
84 2 6 0 0 0 0 0
61 4 4 2 0 6 5 14
Fig. l a and b. Formation of starch from malate in stomata of Commelina communis. Isolated epidermes were incubated with 0.4 m M [U-l~C]malic acid for 2 h at 25 ~ C in the light, then extracted with 70% ethanol, dried, and brought into contact with photographic film for 6 days. The autoradiograms on the left show the distribution of radioactivity, the photographs on the right that of the stomata in the epidermal sample. Images taken at low magnification a show that the concentration of radioactivity in stomata is not restricted to small patches in the epidermis. High magnification b shows correlation between the distribution of silver grains and that of stomatal complexes. Hydrolysis of the epidermis resulted in glucose as the only radioactive product
80
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
fumarate; and (ii) in carbohydrates, like glucose and starch. Labelled carbon appeared in the tricarboxylicacid cycle within the first ten min of exposure; in sugars after 2 h. There was virtually no difference in the degree to which [U-14C]- and [4-a4C]malic acid were metabolized in the citric-acid cycle, but a striking difference appeared in the degree of labelling of the carbohydrates. After an incubation of epidermal samples with [U-14C]malate for 2 h in the dark, 25% of the total radioactivity was found in glucose, fructose and sucrose (Table 1). In the light, this percentage was smaller (13%). The corresponding incubation with [4-1*C]malate in the dark did not yield radioactive sugars at all; in the light, the radioactivity found in sugars amounted to less than 1% of the total activity (Table 2). Similarly, only 0.6% of the total radioactivity was in the hydrolysate if epidermal samples had been incubated with [4-14C]malic acid for 2 h in the dark, and 0.2% if they had been incubated in the light (Table 2). These values are lower than the relative radioactivities of 2 or 3% in the hydrolysate of epidermes that were exposed to [U-a4C]malic acid (Table 1). Glucose was the only radioactive product of hydrolysis. Radioactivity appearing in the hydrolysate therefore indicated incorporation of 14C into starch. Autoradiograms were prepared of epidermal strips which had been incubated with [U-14C]malate before and then extracted with aqueous ethanol. These films were blackened only above the stomata (Fig. 1). The ability of epidermal tissue to form starch from malic acid is therefore concentrated in the cells of the stomatal complexes.
Release of Endogenous Malic Acid During Stomatal Closing Malic acid in the guard cells was labelled by exposing epidermal samples with open stomata to 14CO2 for 2 min (as described under Materials and Methods). Then these epidermal strips were subjected to two kinds of closing treatments. The first consisted of an exposure of strips floating on water to a normal atmospheric concentration of 12CO2 in darkness. In the second treatment, closure was accelerated by placing the strips on a 10-4M solution of (+)-ABA. The first treatment resulted in stomatal closure within 30 rain. ABA shortened the stomatal response to 5 rain. During slow stomatal closing on water, 43% of the radioactivity in the epidermal sample was released into the water. Although the tissue contained nearly 30% of its radioactivity in aspartate, glutamate, and citrate, the compound released was exclusively malate (Table 3). The amount of radioactivity lost from the epidermal tissue was even larger when stomatal closure was accelerated by ABA. Within 5 min 63% of the radioactivity leaked from the tissue into the water. Again malate was the only labelled product lost from the epidermis. The experiment was repeated with similar results. We conclude that guard cells can release malate faster than it can be metabolized. ABA accelerates the specific leakage of malate.
Table 3. Efflux of [l~C]malate during stomatal closure from epidermes of Commelina communis floating on water or on 10 4 M (+_)-ABA Sequence of Stomata dark treatments
14C (% of total) in: External solution
Epidermis
MaP
Mal
Asp
Glu
Cit
Table 2. Metabolism of [4-14C]malate by epidermal tissue of Com-
melina communis Incubation with a 5 mM solution at pH 5.7 in darkness and in the light. Stomata were closed Incubation (min)
Dark 10
30
120
10
30
120
25,100 13,500 17,420 0 0 100
17,400 17,500 29,400 0 120 64
96 0.3 2 0.5 0 0
Open
0
63
12
8
17
followed by 5 min 12CO2
Open
13
44
14
17
12
followed by 30 min lzCOz
Closed
43
36
0
5
11
0
48
35
4
13
95 0.5 3 0.8 0 0
86 0.9 6 2 0 0
63
22
2
5
6
Rapid closure 2 min 14CO2
Percent of radioactivity of soluble fraction Malate Aspartate Citrate Fumarate Glucose Sucrose
2 min 14CO2 Light
Radioactivity (cpm/cm2) soluble starch
Slow closure
96 0.3 2 0.6 0 0
86 0.5 8 1.1 0 0
84 1.1 7 2 0.3 0.4
Open
followed by 5 rain 12CO2 Closed +10 4 M A B A
a NO labelled compounds other than malate were detected in the external solution
P. Dittrich and K. Raschke: Malate Metabolism and Stomatal Functioning
Discussion
Our experiments show that gluconeogenesis can proceed in epidermal tissue (Table 1), and that starch formation from malic acid occurs in the stomatal complexes (Fig. 1). Gluconeogenesis involves the loss of 4-C of malate by decarboxylation. This is the reason why carbohydrates became much less radioactive during an incubation of epidermal strips with [4-14C]malic acid than during a sufficiently long presentation (2 h) of uniformly labelled malic acid (Tables 1, 2). One could have expected complete absence of radioactivity from carbohydrates in strips exposed to [4-t4C]malic acid, except for the presence of fumarase. This enzyme catalyzes the exchange of carbon 1 with carbon 4 in malic acid. Once 1-C becomes radioactive through exchange with 4-~4C, radioactive carbon would find its way into carbohydrates. Botanists have known at least since the formulation of the starch ~ osmoticum hypothesis by Lloyd (1908) that starch grains in guard cells may disappear during stomatal opening, and reappear after closure. We reinterpret this observation: Disappearance may be linked to the formation of malate, reappearance to gluconeogenesis from malate. Since guard cells probably possess large pools of soluble carbohydrates, it is not surprising that the rhythm of starch metabolism has occasionally been observed to be considerably out of phase with the rhythm of stomatal movement (Imamura, 1943; Yamashita, 1952). In addition to being able to convert malate into sugars, epidermal tissue can channel endogenous as well as exogenous malate into the tricarboxylic acid cycle (Tables 1, 2, 3). This capability became apparent also in other studies involving the supply of H~4CO3 or 14COz to epidermal strips of Commelina diffusa, Commelina communis, and Tulipa gesneriana (Willmer and Dittrich, 1974; Raschke and Dittrich, 1977). A third and very effective way guard cells possess to dispose of malate is to release a large portion of it into their surroundings. This process occurs during stomatal closing. In our experiments, on the average, about one-half of the labelled malate was lost when stomata closed: somewhat less than one-half when they closed slowly, within 30 min; more than one-half, when closing was accelerated by ABA (Table 3). The loss of labelled malate was specific; other labelled compounds remained in the epidermal tissue. Combining the results of our studies on malate metabolism in epidermes with those of earlier experi-
81
ments o n C O 2 fixation by epidermes (Shaw and Maclachlan, 1954; Willmer et al., 1973; Willmer and Dittrich, 1974; Raschke and Dittrich, 1977) we conclude: Guard cells can produce malic acid by the carboxylation of PEP which can be derived from sugars and starch. Guard cells are able to dispose of malate by three processes which can occur simultaneously: (i) oxidation in the tricarboxylic acid cycle, (ii) gluconeogenesis after decarboxylation, and (iii) specific release (probably together with K+). The third process enables guard cells to reduce their turgor faster than the metabolism of malic acid would allow. This work was performed while K.R. held a John Simon Guggenheim Memorial Fellowship. The research was further supported by a grant from the Deutsche Forschungsgemeinschaft to K.R. as well as through Contract EY-76-C-02-1338 between Michigan State University and the U.S. Energy Research and Development Administration. We greatly appreciate the careful technical assistance by Carol Holle and G. Ohst, as well as the photographic work by Cheri L. Schmuck. K.R. also wishes to thank Professor H. Ziegler, Institut ffir Botanik der Technischen Universit/it Mfinchen, for laboratory space and plant growth facilities.
References Dittrich, P. : Equilibration of label in malate during dark fixation of CO2 in KalanchoOfedtschenkoi. Plant Physiol. 58, 288-291 (1976) Dittrich, P., Salin, M., Black, C.C., Jr.: Conversion of carbon 4 of malate into products of the pentose cycle by isolated bundle sheath strands of Digitaria sanguinalis L. Scop. leaves. Biochem. Biophys. Res. Com. 55, 104-110 (1973) Imamura, S.: Untersuchungen fiber den Mechanismus der Turgorschwankung der Spalt6ffnungsschliesszellen. Jap. J. Bot. 12, 251 346 (1943) Lloyd, F.E.: The physiology of stomata. Carnegie Inst. Wash. Publ. No. 82 (1908) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309 340 (1975) Raschke, K., Dittrich, P.': [l~C]Carbon-dioxide fixation by isolated leaf epidermis with stomata closed or open. Planta 134, 69-75 (1977) Shaw, M., Maclachlan, G.A.: The physiology of stomata. I. Carbon dioxide fixation in guard cells. Canad. J. Bot. 32, 784-794 (1954) Willmer, C.M., Dittrich, P. : Carbon dioxide fixation by epidermal and mesophylt tissues of Tulipa and Commelina. Planta (Bet1.) 117, 123-132 (1974) Willmer, C.M., Pallas, J.E., Jr., Black, C.C., Jr. : Carbon dioxide fixation in leaf epidermal tissue. Plant Physiol. 52, 448-452 (1973) Yamashita, T. : Influences of potassium supply upon various properties and movement of the guard cells. Sieboldia 1, 51-70
(1952) Received 20 April; accepted 3 November 1976
