Kurze Mitteilungen' Short Communications

Botany Department, S. V. University , Tirupati, A. P. 517502, India

Inter- and Intracellular Distribution of Carbonic Anhydrase, PEP Carboxylase and RuDP Carboxylase in Leaves of Eleus'ille coracana, A C-4 Plant

C. K. M. RATHNAMl), and V. S. R. DAS

Received September 23 , 1974

Summary

Carbonic anhydrase was found to be associated with the chloroplast envelope membranes in the mesophyll cells, while it was stromal in the bundle sheath chloroplasts of Eleusine coracana. PEP carboxylase and RuDP carboxylase were localized in the mesophyll chloro­plast envelope membranes and stroma of the bundle sheath chloroplasts respectively. The distribution of carbonic anhydrase, consistent with those of the carboxylating enzymes, was interpreted as providing an enzymatic mechanism of concentrating bicarbonate and molecular carbon dioxide at the sites of PEP carboxylase and RuDP carboxylase respectively.

Key words: C-4 photosynthesis, carboxylases, carbonic anhydrase, chloroplast envelope membranes,

Carbonic anhydrase, the enzyme that catalyzes the reversible hydration of CO2,

has been implicated to function in photosynthesis (GRAHAM and REED, 1971). Re­newed interest in the enzyme occurred when the enzyme was reported to be absent in leaves of plants that possess the C-4 dicarboxylic acid pathway of photosynthesis (HESKETH et a!., 1965) which are known to fix CO2 at higher rates (CHEN et a!., 1970). However, recent studies have revealed the presence of the enzyme in all the species assayed but at a distinctly lower level in C-4 plants compared to the C-3 plants (CHEN et a!., 1970). Furthermore, carbonic anhydrase was shown to be cyto­plasmic in C-4 plants, while it is chloroplastic in spinach (EVERSON and SLACK, 1968) where it is localized in the stroma (POINCELOT, 1972 a). However, POINCELOT (1972 b) has reported carbonic anhydrase activity in maize leaves (a C-4 plant)

1) Present address: Horticulture Department, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

Abbreviations: C-3, reductive pentose phosphate; C-4, C-4 dicarboxylic acid; Chi, chlorophyll; PEP, phosphoenolpyruvate; RuDP, ribulose I,S-diphosphate.

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Inter- and Intracellular Distribution of Carbonic Anhydrase 361

comparable to the activity in spinach (a C-3 plant) and that the enzyme is chloro­plastic in maize leaves too. Of the total leaf carbonic anhydrase, 72.5 Ofo was shown to be associated with the mesophyll cells and 14.2 Ofo in the bundle sheath! cells (POINCELOT, 1972 b).

Carbonic anhydrase is considered to operate at the sites of carboxylation. There are two sites of photosynthetic carboxylation in C-4 plants, one in the mesophyll cells accomplished by PEP carboxylase and the other in the bundle sheath cells by RuDP carboxylase (HATCH and SLACK, 1970). The reported absence of carbonic anhydrase in the bundle sheath cells is puzzling and unconvincing since it has been established that carbonic anhydrase is associated with RuDP carboxylase in the chloroplasts of C-3 plants (GRAHAM and REED, 1971).

The reports on the inter- and intracellular localization of the enzymes involved in the C-4 pathway are contradictory. PEP carboxylase has been reported to be associated with the mesophyll chloroplasts (SLACK and HATCH, 1967). But it has also been reported as a cytoplasmic enzyme of the mesophyll cells (HATCH and KAGAWA, 1973) and bulliform cells (COOMBS and BALDRY, 1972). HATCH and SLACK (1970) were of the opinion that it may be attached to the envelope membranes of the meso­phyll chloroplasts.

Hence, a critical study of the inter- and intracellular distribution and localization of carbonic anhydrase, PEP carboxylase, and RuDP carboxylase was made in the present work using Eleusine coracana GAERTN., a C-4 plant (DAS and RATHNAM, 1974).

Youngest fully expanded leaves of Eleusine coracana GAERTN. were sampled from vigorously growing, 4 to 6 weeks old, plants. The sequential grinding technique of ANDREWS et al. (1971) was used for the step-wise release of the enzymes from the colorless cells (fraction I), mesophyll cells (fraction II) and bundle sheath tissue (fraction III) of the leaves. Structurally intact mesophyll and bundle sheath chloroplasts were isolated as already described (RATHNAM and DAS, 1974). The method of MACKENDER and LEECH (1970) was employed for the isolation of chloroplast envelope membranes. The medium employed for the extraction of carbonic anhydrase consisted of 25 mM veronal buffer at pH 8.2, 1 mM EDTA, and 10 mM 2-mecaptoethanol; and 50 mM Tris-HCI buffer at pH 8.3, 5 mM dithiothreitol, 1 mM EDTA, and 1 mM MgCl2 for PEP carboxylase and RuDP carboxylase.

PEP carboxylase and RuDP carboxylase were assayed according to BJORKMAN and GAUHL (1969). Carbonic anhydrase was assayed basing on the rate of change in pH resulting from the hydration of CO2 as observed with bromothymol blue at 0" by the method of EVERSON (1970). Chlorophyll concentration was determined by the method of ARNON (1949).

Information about the inter- and intracellular distribution and localization of PEP carboxylase, RuDP carboxylase, and carbonic anhydrase was obtained by ana­lyzing the levels of these enzymes in the mesophyll and bundle sheath extracts and their association with the mesophyll and bundle sheath chloroplasts (Table 1). Though carbonic anhydrase was found in equal proportions in both the mesophyll and bundle sheath fractions, the carboxylases were associated primarily with either one of the fractions. The mesophyll cell extract (fraction II) was enriched in PEP carboxylase while RuDP carboxylase was concentrated in the bundle sheath (fraction III) . It is

Z. Pflanzenphysiol. Bd. 75. S. 360-364. 1975.

362 C. K. M. RATHNAM and V. S. R. DAS

also evident that the enzymes were associated exclusively with the chloroplasts of their respective fractions. Thus, PEP carboxylase was found to be localized in the mesophyll chloroplasts, while RuDP carboxylase was concentrated in the bundle sheath chloroplasts. In contrast, carbonic anhydrase was observed to be in equal amounts in both the mesophyll and bundle sheath chloroplasts.

Table 1: Inter- and intracellular distribution of carbonic anhydrase, PEP carboxylase, and RuDP carboxylase in leaves of E. coracana.

Enzyme Activity (p moles/mg Chl/hr)

Chloroplasts Leaf Fractions

Enzyme Whole Mesophyll Bundle Leaf Sheath II III

Carbonic anhydrase"-) 4570 2026 1863 60 2064 1986 PEP carboxylase 378 348 24 36 294 30 RuDP carboxylase 325 38 248 5 38 267

"-) Carbonic anhydrase activity expressed as enzyme units/mg Chi according to POINCELOT (1972 b).

It has often been speculated that PEP carboxylase (HATCH and SLACK, 1970) and carbonic anhydrase (EVERSON, 1970) might be associated with the chloroplast enve­lope membrane. To test this, the mesophyll and bundle sheath chloroplasts were fractionated into their constituent envelope membranes, stroma and lamellae, and the association of the enzymes with these chloroplast fractions was studied (Table 2)_

Table 2: Localization of carbonic anhydrase, PEP carboxylase, and RuDP carboxylase III

the chloroplasts of E. coracana.

Enzyme Activity (fl moles/mg Chljhr)

Enzyme Mesophyll Chloroplasts Bundle Sheath Chlorophats

Envelope Envelope Membranes Stroma Lamellae Membranes Stroma Lamellae

Carbonic anhydrase"-) 1643 280 12 752 1094 8 PEP carboxylase 298 31 0 6 14 0 RuDP carboxylase 0 16 0 22 198 0

Isolation procedures and experimental conditions were as in Materials and Methods. Total chlorophyll content of the intact mesophyll or bundle sheath chloroplasts was taken as the basis for calculating the enzyme activities of the respective chloroplast fractions.

,:-) Carbonic anhydrase activity expressed as enzyme units/mg ChI according to POINCELOT (1972 b) .

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Inter- and Intrace1lular Distribution of Carbonic Anhydrase 363

PEP carboxylase activity was found associated with the envelope membranes of the mesophyll chloroplasts, while RuDP carboxylase was recovered in the stromal frac­tion of the bundle sheath chloroplasts. Carbonic anhydrase was noticed to occur on the envelope membranes of the mesophyll chloroplasts, while it was present in the stroma of the bundle sheath chloroplasts.

The chloroplast preparations used in the present study were relatively pure with­out much contamination of the extra-chloroplastic contents of the same cell type or of the chloroplastic and extra-chloroplastic contents of the other cell type (RATHNAM and DAS, 1974, 1975).

It has been suggested that carbonic anhydrase may increase fixation of CO2 by in­creasing the availability of the active species of CO! at the site of carboxylation or by increasing the affinity of the carboxylating enzyme for CO2 (GRAHAM and REED, 1971). The enzyme may also overcome a permeability barrier for CO2 at the surface of the chloroplasts thereby stimulating the rate of transfer of CO2 across the chloro­plast envelope membranes.

The presence of carbonic anhydrase in close association with PEP carboxylase and RuDP carboxylase establishes its functional integrity with the carboxylating enzymes and thus results in an enhanced efficiency in their functions. Since bicarbonate anion rather than CO2 is apparently the substrate for PEP carboxylase (COOPER and WOOD,

1971), the role of carbonic anhydrase present on the mesophyJl chloroplast envelope membranes may be to accelerate the hydration of CO2 entering the mesophyll cell and thus making the bicarbonate ion readily available for PEP carboxylase which is strategically associated with the mesophyll chloroplast envelope membranes. It is also well established that molecular CO2 is the active species fixed by RuDP carboxylase (COOPER et al., 1969) and bicarbonate anion is the more logical storage form of CO2

because of its greater stability (ZELITCH, 1971). Hence, the function of carbonic anhydrase in the bundle sheath chloroplasts may be to convert the bicarbonate pool into the active molecular CO2 • The presence of some carbonic anhydrase on the bundle sheath chloroplast envelope membranes may be to facilitate the transport of CO2

across the envelope membranes. Earlier reports regarding the cytoplasmic localization of carbonic anhydrase (EVER­

SON and SLACK, 1968) and PEP carboxylase (HATCH and KAGAWA, 1973) may be due to the fact that the chloroplasts during isolation might have lost their envelope membranes, at least the outer one, and hence to be recovered with the cytoplasmic fraction. The inability to identify carbonic anhydrase in the bundle sheat chloroplasts may be a result of insufficient disruption of the bundle sheath tissue which are more resistant for breakage.

From the present work, it can be envisaged that an enzymatic mechanism of con­centrating bicarbonate and CO2 at the functional sites of PEP carboxylase and RuDP carboxylase respectively is provided by carbonic anhydrase for the effective and rapid fixation of CO2 thus in part accounting for the greater efficiency of C-4 photo­synthesis.

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364 C. K. M. RATHNAM and V. S. R . DAS

Note added in proof

After submission of this manuscript, TRIOLO et al. (TRIOLO, 1., BAGNARA, D., ANSELMI, 1., and BASSANELLI, c.: Physiol. Plant. 31, 86, 1974) presented histOchemical evidence for the localization of carbonic anhydrase in both mesophyll and bundle sheath chloroplasts of several C-4 plants. USUDA et al. (USUDA, H., MATSUSHIM, H., and MIYACHI, S.: Plant and Cell Physiol. 15, 1517, 1974) have shown PEP induction of light dependent CO2 fixation in maize mesophyll chloroplasts. COOMBS et al. (COOMBS, ]., MAW, S. 1., and BALDRY, C. W.: Plant Sci. Letr. 4, 97, 1975) demonstrated bicarbonate anion as the inorganic carbon substrate for PEP carboxylase of Pennisetum purpureum, a C-4 plant. The above results support the data and conclusions reported in this paper.

References

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Agric. Res. Stn., U.S.A. (1965). MACKENDER, R. 0., and R. M. LEECH: Nature 228, 1347 (1970). POINCELOT, R. P.: Biochim. Biophys. Acta 258, 637 (1972 a). - Plant Physiol. 50, 336 (1972 b). RATHNAM, C. K. M., and V. S. R . DAs: Can. J. Bot. 52, 2599 (1974) . - Z . Pflanzenphysiol. 74, 377 (1975). SLACK, C. R ., and M. D . HATCH: Biochem. ]. 103,660 (1967). ZELITCH, I.: Photosynthesis, PhutOrespiration, and Plant Productivity, Academic Press, New

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C. K. M. RATHNAM, Horticulture Depa rtment, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

Z. P/lanzenphysiol. Bd. 75. S. 360-364. 1975