Localization of carbonic anhydrase in crassulacean acid metabolism plants

Plant Science Letters, 24 (1982) 211--218 Elsevier/North-Holland Scientific Publbhers Ltd.

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L O C A L I Z A T I O N OF CARBONIC A N H Y D R A S E IN CRASSULACEAN ACID METABOLISM PLANTS

M. TSUZUKI, S. MIYACHI*, K. WINTER L** and G.E. EDWARDSa'*** Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku Tokyo 113 (Japan) and aDepartmen t of Horticulture, University of Wisconsin-Madison, Madison, WI 53 706

(U.S.A.) (ReceivedJune 25th, 1981) (Accepted September9th, 1981)

SUMMARY Activity and localization of carbonic anhydrase (CA, EC 4.2.1.1), which is a catalyst for the reversible hydration of CO2, was studied in several Crassulacean acid metabolism (CAM) species. In most species the extractable activity of CA was similar to that of C3 and C4 plants. Using protoplasts and the differential centrifugation technique as means for studying the intracellular compartmentation of CA, we found that the enzyme was localized in the extrachloroplastic fraction of Hoya carnosa and Ananas comosus, species which decarboxylate malate through phosphoenolpyruvate (PEP) carboxykinase. In contrast, the enzyme was localized in the chloroplasts of Sedum praealtum and Mesembryanthemum crystallinum, species which decarboxylate malate through malic enzyme. In both S. praealtum (malic enzyme type) and H. carnosa (PEP carboxykinase type), Diamox and NaCI inhibited CA to a similar extent. The possible role of carbonic anhydrase in the t w o subgroups of CAM plants is discussed in relation to CAM.

*To whom reprint requests should be sent. **Present address: Botanik H der Universitiit, Mittlerer Dallenbergweg 64, 8700 Wiirzburg, W. Germany. ***Present address: Botany Department, Washington State University, Pullman, WA 99164, U.S.A. Abbreviations: CA, carbonic anhydrue; CAM, Crassulacean acid metabolism; Diamox, acetazolamide, 2-acetylamino-l,3,4-thiadiazole-5-sulfonamide; DTE, dithioerythritol; NADP-TPDH, NADP-dependent triolephosphate dehydrogenue; PEP, phosphoenolpyrurate; PGA, 3-phasphoglyceric acid; PVP-40, polyvinylpyrrolidone (average MW 40,000); RuBP, ribulose bisphosphate; veronal, 5,5-diethyl-2,4,6 (1H, 3H, 5H)-pyrimidinetrione (diethylbarbituric acid).

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INTRODUCTION There has been considerable interest in the localization and role of CA in plants. The enzyme is associated with the plasma membrane of Chlamydomonas reinhardtii and Scenedesmus obliquus [1,2]. These algal cells appear to have a HCO] pump in the plasma membrane [2,3] which is not observed in Chlorella cells. In Chlorella vulgaris, 11 h cells, Hogetsu and Miyachi [4] found CA located in the chloroplast. Recent studies also indicate that most, if not all, of the CA is localized in the chloroplasts of the C3 plants wheat and spinach [5,6]. Chloroplasts obtained from spinach and the marine alga Bryopsis maxima use CO2, not HCO], as an external carbon source for photosynthesis [7]. Furthermore, it is well known that ribulose bisphosphate (RuBP) carboxylase reacts with CO2 rather than HCO] [ 8]. CA catalyzes the reversible hydration of CO2. Since CA allows a rapid equilibration between CO2 and HCO], both species can effectively provide carbon to the carboxylase. At physiological pH and at equilibrium the concentration of HCO~ will be considerably higher than CO2. Therefore in the presence of CA the total active pool of inorganic carbon which is available for diffusion and utilization by RuBP carboxylase will be increased. From kinetic analysis of ~4CO2 fixation by Chlorella cells, Tsuzuki et al. [9] concluded that CA increases the supply of CO2 to RuBP carboxylase through an indirect route (conversion of CO2 to HCO], followed by diffusion of HCO], and its reconversion to CO2, see Ref. 9 for details). In ChloreUa this process may reduce the resistance in suppling CO2 to the chloroplast and give a higher rate of photosynthesis under limiting CO~ concentrations [4]. In terrestrial C3 plants, CA in the chloroplast may have a similar function, although a limiting step may be in the cytoplasm where CA is absent [61. In C4 plants CA, like PEP carboxylase, is localized in the cytoplasm of the mesophy11 cells whereas there is little CA activity in bundle sheath cells [10,11]. PEP carboxylase uses HCO~ as the substrate [12]. In C4 plants the hydration of CO2 to HCO~ through CA as atmospheric CO2 enters the cells may increase the gradient for diffusion of HCO~ throughout the cytoplasm where it is directly used by PEP carboxylase. Chloroplast containing cells of CAM plants show high activity of both PEP carboxylase and RuBP carboxylase. Thus far, there have been no reports of the levels of CA and its intracellular localization in CAM plants. MATERIALS AND METHODS Plant materials and growth conditions The CAM plants Hoya carnosa, Stapelia gigantea, S. variegata, Sedum praealtum, Kalanchoe pinnata and K. daigremontiana were grown in a greenhouse. Fruits of Ananas comosus having leaves attached were purchased from a local market in Madison, WI. At least 2 days before the experiments,

213 H. carnosa, S. gigantea, S. variegata and A. comosus were transferred to a growth chamber having a 12-h light/12-h dark cycle with a 14-h 20°C/10-h 15°C temperature regime (the temperature was gradually raised to 20°C 1.5 h before onset of the light period and lowered to 15°C 0.5 h after the end of the light period). Light intensity during the light period was 500/~E m -2 X s-1 ( 4 0 0 - 7 0 0 urn). The inducible CAM plant Mesembryanthemum crystallinum was cultivated in the C3 or CAM mode as described elsewhere (Winter et al., unpublished*). The C4 plants, Zea mays and Panicum maximum, and the C3 plant Phaseolus vulgaris, were grown in a growth chamber with a day/night temperature of 24°C/18°C and a light regime of 14-h light/10-h dark. The light intensity was 400 p E m -~ • s-1 ( 4 0 0 - 7 0 0 nm) for the first 12 h and 250 ~E m -2 • s-1 for the last 2 h of the light period. Triticum aestivum (C3) was grown in the greenhouse. Preparation o f leaf extracts Leaf samples (0.3--0.6 g fresh wt.) were ground with pestle and mortar in ice-cold medium (2 ml) which contained 0.1 M Tris--H2SO4 (pH 8.3), 1% (w/v) polyvinylpyrrolidone (tool. wt.av" 40 000) (PVP-40), 1 mM EDTA and 100 mM 2-mercaptoethanol. After centrifugation at 10 000 × g for 2 min, the supernatant was removed and used for assay of enzyme activities. For determination of the effect of Diamox {Japan Lederle, Tokyo) and sodium chloride on CA, the enzyme was partially purified with ammonium sulfate. Protein precipitating between 120 g/1 (~ 23%) and 440 g/1 (~ 70%) of ammonium sulfate was suspended in 20 mM Tris-borate (pH 8.3 at 25°C) 2.5 mM EDTA and 100 mM 2-mercaptoethanol and desalted by passage through a column of Sephadex G-25. Isolation o f pro toplasts and preparation o f protoplast ex tracts The method of Spalding et al. [13] was adapted for isolation and purification of protoplasts of S. praealtum, H. carnosa and A. cornosus. Leaves were cut into strips and washed with 0.3 M sorbitol. The tissue segments were then added to an enzyme mixture containing 0.3 M sorbitol, 1% (w/v) PVP-40 and 1.5% (w/v) Cellulysin (Calbiochem-Behring Co., La Jolla, CA) 1 mM CaC12 and, following vacuum infiltration, were incubated at 30°C for 3--4 h. With A. eornosus, 0.2% (w/v) of Extractase PC (Fermco Biochemics, Elk Grove Village, IL) was also added to the enzyme mixture [ 14]. Protoplasts were collected by filtering the digestion mixture through a 210/~m nylon net. Maximum yields of protoplasts were obtained by washing leaf segments several times with 0.3 M sorbitol before digestion. Protoplasts were purified by floatation as previously described [ 13 ]. Protoplasts from M. crystallinum were isolated and purified as described elsewhere (Winter et al., unpublished*). *Since going to press this paper has been accepted for publication in Plant Physiol.

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Protoplasts were suspended in 0.15 M sorbitol, 1% (w/v) PVP-40, 0.2% (w/v) methylcellulose, 5 mM EDTA, 5 mM dithioerythritol (DTE) and 250 mM Tris--H2SO4 (pH 8.4) and ruptured by passage through a 28 #m nylon net [13]. The protoplast extract was centrifuged at 5000 × g for 30 s and the pellet, containing the chloroplast fraction, was suspended with an equal volume of the same medium. In the case of M. crystallinum protoplast extracts were centrifuged at 820 × g for 2 min. Enzyme assays and .chlorophyll determination CA (EC 4.2.1.1) was determined by following the time-dependent decrease in pH of 12.5 mM veronal buffer at 0°C after the addition of CO2-saturated water [15,16]. The time without enzyme is recorded as To; with enzyme as T. A unit of activity = (To - T)/T. NADP-dependent triose phosphate dehydrogenase (EC 1.2.1.13, NADPTPDH) was assayed by following the decrease in absorbance at 340 nm. The reaction mixture consisted of 0.073 M Tris--HC1 (pH 7.8), 10 mM MgC12, 5 mM GSH, 5 mM ATP, 5 mM NADPH, 10 units/ml 3-phosphoglyceric acid (PGA) kinase and 5 mM PGA. PEP carboxylase (EC 4.1.1.31) was also measured spectrophotometrically at 340 nm. The reaction medium contained 0.1 M Tris--HC1 (pH 7.8), 10 mM MgC12, 2 mM DTE, 10 mM NaHCO3, 0.2 mM NADH, 3 units/ml malate dehydrogenase and 5 mM PEP. Chlorophyll was determined by extraction in ethanol [17].

RESULTS Extractable activities of carbonic anhydrase of CAM plants were generally similar to those of C3 and C4 plants (Table I). Addition of Triton X-100 to 0.5% to the plant extracts to solubilize membranes prior to centrifugation at 10 000 × g did not increase CA activities in the supernatant fraction (data not shown). This indicates there is no loss in activity due to centrifugation nor any latency if the enzyme were associated with membranes remaining in the supernatant fraction. Without PVP and 2-mercaptoethanol in the extraction medium the CA activity in some species was very low (data not shown). In Stapelia gigantea, even when both PVP and 2-mercaptoethanol were added in the extraction medium, the CA activity in whole leaf extracts (Table I, 30 units/rag Chl) was low; however, higher activity was obtained from protoplast extracts (176 units mg Chl-~). The distribution of CA activity between the supernatant fraction and chloroplast pellet after differential centrifugation of protoplast extracts of several CAM species is shown in Table II. NADP-TPDH and PEP carboxylase were assayed as markers for chloroplasts and extrachloroplast fractions, respectively [ 13,14]. In M. crystallinum, grown in a highly saline (400 mM NaCl) rooting medium and exhibiting CAM and in S. praealtum most of the CA was found in the chloroplast fraction. Both species decarboxylate malate via malic enzyme. In M. crystallinum, grown under low NaC1 concen-

215 TABLE I ENZYME ACTIVITIES IN WHOLE LEAF EXTRACTS OF SEVERAL PLANT SPECIES Species

Carbonic anhydrase (unita mg Chl-i )

PEP carboxylase a

NADP-TPDHa

178 134 30

263 968 392

115 201 329

294 150 428 418 317

469 338 81 423 308

390 530 207 118 81

Csplants P. vulgaris T. aestivum

416 320

34 36

499 299

C4 plants P. maximum Z. mays

270 137

345 409

134 488

CAM plants

PEP carboxykinase type A. comosus H. carnosa S. gigantea

Malic enzyme type K. daigremontiana K. pinnata M. crystailinum (C3 mode) M. crystailinum (CAM mode) S. praealtum

aExpressed as ~mol mg-~ Chl • h -~.

tration and showing C3 characteristics of photosynthetic carbon metabolism, 86% of the CA was f o u n d in the chloroplast pellet which also contained 8(~o of the NADP-TPDH (data n o t shown). These results on the localization of CA are similar to those for the C3 plants wheat and spinach [5,6]. In contrast, 87--89% of the CA activity in H. carnosa and A. comosus, PEP carboxykinase type CAM plants, occurred in the supernatant fraction; similar to the distribution of PEP carboxylase (Table II). Protoplast extracts from S. gigantea and S. variegata (also PEP carboxykinase type species) also had most of the CA and PEP carboxylase activities in the supernatant fraction (results n o t shown). These data indicate that the localization of CA differs among CAM plants and suggest a distinction in localization of the enzyme between subgroups. As shown in Table III, activity of partially purified CA from S. p r a e a l t u m and H. carnosa was completely inhibited by 0.1 mM Diamox and also strongly inhibited by 50 mM NaCI. In this respect, CA obtained from both species showed similar characteristics. DISCUSSION Previous studies have shown CA to be localized in chloroplasts of Chlorella

216 TABLE II DISTRIBUTION OF CARBONIC ANHYDRASE ACTIVITY FOLLOWING CENTRIFUGATION OF PROTOPLAST EXTRACTS OF CAM PLANTS Species and enzymes

Activity in protoplast extract a

% distribution Pellet

Supernatant

770 425 995 --

80 87 1 98

20 13 99 2

238 106 238 --

99 91 20 90

9 80 i0

257 71 585 --

11 64 19 62

89 36 81 38

202 300 241 --

13 86 14 96

87 14 86 4

Malic enzyme type M. erystaUinum (CAM)

CA NADP-TPDH PEP carboxylase Chlorophyll S. praealtum

CA NADP-TPDH PEP carboxylase Chlorophyll

1

PEP carboxykinase type H. carnoKl

CA NADP-TPDH PEP carboxylase Chlorophyll A. comosu8

CA NADP-TPDH PEP carboxylase Chlorophyll

aCA is reported in units mg Chl- 1; PEP carboxylase and NADP-TPDH are reported in u m o l m g -1 Chl • h -~.

cells and in c h l o r o p l a s t s o f terresterial C3 plants [ 4 - - 6 ] . T h e l o c a l i z a t i o n o f CA in the s t r o m a o f the c h l o r o p l a s t , in association with R u B P c a r b o x y l a s e , has previously been c o n s i d e r e d significant f o r facilitating d i f f u s i o n o f HCO~ t o the sites o f c a r b o x y l a t i o n (see I n t r o d u c t i o n ) . T h e malic e n z y m e t y p e CAM plants e x a m i n e d here also s h o w high activities o f CA in t h e c h l o r o p l a s t . TABLE HI EFFECTS OF DIAMOX AND NaCI ON CA FROM S. P R A E A L T U M A N D H. C A R N O S A Species

S. praealtum H. carnosa

Bovine erythrocytesa

% inhibition of enzyme activity

D~amox (0.I raM)

NaCI (50 raM)

91 100 100

72 84 58

aSigma Chemical Co., St. Louis, MO.

217 No difference was found in the level or localization of CA in M. crystallinum exhibiting photosynthetic characteristics of either a C3 or a CAM plant. In PEP carboxykinase type CAM plants like A. comosus, CA was found in the 5000 X g supernatant fraction indicating the enzyme is extrachloroplastic and probably occurs in the cytoplasm along with PEP carboxylase, similar to the compartmentation of both enzymes in mesophyll cells of C4 plants; [10,11]. CAM plants having CA in the cytoplasm may utilize the enzyme during dark fixation of CO2 through PEP carboxylase. As atmospheric CO2 diffuses into the cell it would be readily converted to HCO~ in the cytoplasm through CA. HCO~ could then diffuse throughout the cytoplasm where it would serve as a substrate for PEP carboxylase. Since both malic enzyme and PEP carboxykinase type CAM plants fix CO2 via a cytoplasmic PEP carboxylase it is uncertain whether the absence or presence of CA in the cytoplasm is significant for dark CO2 fixation. In both subgroups of CAM plants the decarboxylases NADP-malic enzyme and PEP carboxykinase are localized in the cYtoplasm [13,14; Winter et al., unpublished]. The CO2 concentration in the leaf during decarboxylation is considered several fold higher than that of C3 plants such that it would likely be unlimiting for photosynthesis [ 18]. Thus, for either CAM subgroup, it is difficult to conceive of a need for CA in the cytoplasm for facilitating diffusion of CO2--HCO~ during the decarboxylation phase. In certain cases when the CO2 concentration is high (Chlorella cells grown on high [CO2] or in bundle sheath cells of C4 plants) the CA activity is very low. By analogy one might expect there to be little need for CA in the cytoplasm or chloroplasts of CAM plants during deacidification when the internal [CO2] is high. By comparing the localization of CA in CAM species with those of C3 and C4 plants the most obvious speculations are: (a) the malic enzyme type CAM plants examined may utilize their chloroplastic enzyme during direct fixation of atmospheric CO2 (to the degree they function in the C3 mode). In this respect, localization of CA in the chloroplast of certain CAM plants could be most important when the relative capacity for photosynthesis in the C3 mode is high. (b) the PEP carboxykinase type CAM plants examined may utilize their cytoplasmic CA for converting CO2 to HCO~ in the cytoplasm during carbon dioxide fixation in the dark through PEP carboxylase. This latter role of CA in CAM plants could be most important when the stomatal conductance in the dark is relatively low and when the internal capacity for CO2 fixation is reasonably high. Further studies are needed to see if these differences in compartmentation of CA in CAM plants are reflected in different physiological characteristics of carbon assimilation (e.g. in response curves to [CO2] for nocturnal CO2 fixation and daytime FL~ation of atmospheric CO2). ACKNOWLEDGEMENT The authors appreciate the advice of Dr. Joyce Foster and help of Mrs. Shizuko Miyachi. The research was supported by Japan-U.S. Cooperative

218

Research Program (The Japan Society for the Promotion of Science, NSF Grant INT 78-17245), by NSF Grant PCM 77-09384 to G.E.E. and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison and by the Japanese Ministry of Agriculture, Forestry and Fisheries (GEP 55-II-1-31). REFERENCES 1 G.R. Findenegg, Z. Pflanzenphysiol., 79 (1976) 131. 2 M. Imamura, M. Tsuzuki, D. Hogetsu and S. Miyachi, Proc. 5th, Int. Congr. on Photosynthesis, 1980, Greece, in press. 3 M.R. Badger, A. Kaplan and J.A. Berry, Plant Physiol., 66 (1980) 407. 4 D. Hogetsu and S. Miyachi, Plant Cell Physiol., 20 (1979) 747. 5 B.S. Jacobson, F. Fong and R.L. Heath, Plant Physiol., 55 (1975) 468. 6 M. Tsuzuki, S. Muto and S. Miyachi, Proc. 5th Int. Congr. on Photosynthesis, 1980, Greece, in preu. 7 Y. Shiraiwa and S. Miyachi, FEBS Lett., 95 (1979) 207. 8 D.L. Filmer and T.G. Cooper, J. Theor. Biol., 29 (1970) 131. 9 M. Tsuzuki, Y. S~iraiwa and S. Miyachi, Plant Cell Physiol., 21 (1980) 677. 10 M. Gutierrez, S.C. Huber, S.B. Ku, R. Kanai and G.E. Edwards, in: M. Avron (Ed.), HI Int. Congr. on Photosynthetic Res., Elsevier Science Publishing Co., Elsevier, Amsterdam, 1974, p. 1219. 11 S.B. Ku and G.E. Edwards, Z. Pflanzenphysiol., 77 (1975) 16. 12 T. Whelan, W.M. Sackett and C.R. Benedict. Plant Physiol., 51 (1973) 1051. 13 M.H. Spalding, M.R. Schmitt, S.B. Ku and G.E. Edwards, Plant Physiol., 63 (1979) 738. 14 M.S.B. Ku, M.H. Spalding and G.E. Edwards, Plant Sci. Lett., 19 (1980) 1. 15 M. Tsuzuki, S. Miyachi, F. Sato and Y. Yamada, Plant Cell Physiol., 22 (1981) 51. 16 Worthington Enzyme Manual, Worthington Biochemical Corporation, Freehold, New Jersey, 1972, p. 159. 17 J.F.G.M. Wintermans and A. De Mots, Biochim. Biophys. Acta, 109 (1965) 448. 18 M.H. Spalding, D.K. Stumpf, M.S.B. Ku, R.H. Burris and G.E. Edwards, Aust. J. Plant Physiol., 6 (1979) 557.