Originalarbeiten . Original Papers

Department of Botany, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India

Photochemical Activities of Chloroplasts Isolated from Plants with the C4-Pathway of Photosynthesis and from Plants with the Calvin Cyelet )

A. s. RAGHAVENDRA and V. S. R. DAS

With 4 figures

Received 23 August 1977 . Accepted 3 January 1978

Summary

The photochemical activities of chloroplasts isolated from three C4-plants (Setaria italica, Pennisetum typhoides and Amaranthus paniculatus) and two Cs-plants (Oryza sativa and Rumex vesicarius) were examined in detail. Similar rates of the Hill reaction (with NADP, ferricyanide and dichlorophenol indophenol), responses of cyclic and non-cyclic photophosphorylation to inhibitors and pigment composition of the leaves suggest that the basic chloroplast constitution of C4-plants is similar to that of Cs-plants. The chloroplasts of the C4-plants yielded lower Km values for ADP and Pi. Furthermore, the ratio of cyclic/non-cyclic photophosphorylation was more for the C4-plant chloroplasts increased with illumination, whereas it did not for Cs-plants. Active cyclic photophosphorylation, higher chlorophyll a to b ratios and a higher content of P700 suggest an enrichment of photosystem I activity in the leaves of C4-plants.

Key words: Photochemical activities, chloroplasts, C4- and Cs-plants.

Introduction

The rates of carbon assimilation of the leaves of C4-plants reach a maximum at the very high light intensities of 10,000-12,000 ft-c, whereas low light intensities of 1000-4000 ft-c are sufficient to saturate the photosynthesis of most Cs-plan~ (BJORKMAN, 1971; BLACK, 1971; BLACK et aI., 1969). In addition to their response to

light intensity, C4-plants differ from Cs-plants in many other aspects, such as leaf anatomy, optimal temperature for photosynthesis and photorespiratory CO2-evolution (BLACK, 1971, 1973).

1) The results form a part of the Ph. D. thesis of A. S. R. approved by Sri Venkateswara University, Tirupati. This investigation was supported by an U. G. C. Junior Research Fellowship to A. S. R.

z. Pjlanzenphysiol. Bd. 88.1-11. 1978.

2 A. S. RAGHAVENDRA and V. S. R. DAS

The positive response of C,-plants to even high light intensities suggests their greater photochemical potential. But with respeCt to the vast amount of literature concerning the photochemical activities of chloroplasts from Cs-plants (GOODWIN, 1967), the attempts to study the chloroplasts of C,-plants have been very meagre (CHEN et aI., 1969; HEW and GIBBS, 1970; MIFLIN and HAGEMAN, 1963, 1966; MORELAND and HILL, 1962). Furthermore, most of the detailed studies on C,-plant chloroplasts have encompassed only a comparison of mesophyll and bundle sheath chloroplasts (ANDERSON et aI., 1971; BISHOP et aI., 1971; MAYNE et aI., 1971). Preliminary experiments with the isolated chloroplasts from bermuda grass, a C,-plant, indicated the ability of C,-plant chloroplasts to catalyze higher rates of cyclic photophosphorylation (CHEN et aI., 1969).

During the course of the detailed studies of the present authors on photosynthetic systems (RAGHAVENDRA, 1975), the chloroplasts from C,-as well as from Cs-plants were examined in detail. The photochemical activities of chloroplasts from Setaria italica, Pennisetum typhoides (NADP malic enzyme-type C,-plants), Amaranthus paniculatus (a NAD malic enzyme-type C,-plant), Oryza sativa and Rumex vesicarius (Cs-plants) are herewith described.

Materials and Methods Plant Material Plants of Setaria italica Beauv. Var. H-1, Pennisetum typhoides S & H. Var. AKP-2,

Amaranthus paniculatus L., Oryza sativa L. Var. IR-22 and Rumex vesicarius were cultivated in seed pans on soil supplemented with manure (3 parts soil + 1 part farmyard manure) under an approximately 12 h photoperiod (temperature 38°C by day and 20°C by night). The second to fourth leaves beginning with the first fully developed leaf-were harvested from four weeks old seedlings.

Chloroplast isolation The leaves were chilled to 0 °C and cut into pieces of about 1 cm2 in the isolation medium.

They were then ground in a chilled mortar with a pestle together with the isolation medium and a pinch of sand. The macerate was filtered through four layers of cheesecloth and centrifuged at 200 g for 5 min. The supernatant was then centrifuged at 1500 g for 15 min to obtain the chloroplast pellet. The chloroplast isolation medium for the study of photosynthetic electron transfer reactions was composed of 50 mM phosphate buffer (pH 7.4), 0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl2 5 mM dithiothreitol and 0.5 Ofo bovine serum albumin. The chloroplasts were suspended in 50 mM phosphate buffer (pH 7.4) containing 0.33 M sorbitol, 1 mM MgCl2 and 0.5 Ofo abovine serum albumin.

The photochemical activities of the chloroplasts were measured at 25 ± 2°C, following a 3 min period of irradiation at 200 W/m2• Dichlorophenol indophenol (DCPIP) and ferri­cyanide reductions were followed at 620 and 420 nm respectively as previously described (RAGHAVENDRA and DAS, 1976 a). The tedmique of measuring NADP reduction at 340 nm has also been described (RAGHAVENDRA and DAS, 1976 b).

Photophosphorylation The following procedure was optimal for the measurement of the photophosphorylation of

chloroplasts isolated from both C4- and Cs-plants. The isolation medium was composed of 50

z. Pjlanzenphysiol. Bd. 88. 1-11. 1978.

Photochemical activities of C4- and Cs-chloroplasts 3

mM tris-HCl buffer (pH 8.0) containing 0.33 M sorbitol, 5 mM dithiothreitol, 1 mM EDTA, 1 mM MgCI2, 1 mM MnCl2 and 0.5 % bovine serum albumin. The chloroplast pellet was suspended in 20 mM tris-HCl buffer (pH 8.0) containing 0.33 M sorbitol, 1 mM MgCl2 and 0.5 % bovine serum albumin.

The assay medium (3 ml) contained 33 mM tris-HCl buffer (pH 8.0) 5 mM MgCI2, 10 mM NaCl, 0.5 % bovine serum albumin, 1 mM ADP, 2 mM phosphate (containing s2Pi with at least 105-108 cpm), 0.5 mM phenazine methosulphate (PMS) (cyclic photophosphorylation) or 5 mM ferricyanide (non-cyclic photophosphorylation) and chloroplasts equivalent to 10,ug/mI. After irradiation at an intensity of 200 W / m2 for 5 min at 25 DC in air the reaction was terminated with 0.3 ml 20 % (w/v) trichloroacetic acid. After centrifugation at 1200 g for 15 min, the supernatant was assayed for esterified 32p (RAGHAVENDRA and DAS, 1976 b).

Chloroplast components Chlorophyll was estimated after extraction with 80 % (v Iv) acetone (ARNON, 1949). The

chlorophyll alb ratio was estimated according to the method of OGAWA and SHIBATA (1965). Leaf protein was precipitated with 20 % (v/v) trichloroacetic acid and was estimated with phenol reagent (LOWRY et aI., 1951). The methods of extraction and estimation of carotenoids, xanthophylls and P 700 are described (RAGHAVENDRA and DAS, 1978).

Results

No remarkable differences in the photochemical activities of the chloroplasts from Cs- and C4-plants were detected. The rates of photoreduction of dichlorophenol indophenol, ferricyanide and NADP were almost identical (Table 1). However the cyclic photophosphorylation rates of the chloroplasts from C4-plants (S. italica, P. typhoides and A. paniculatus) were slightly higher than those from Cs-plants (0. sativa and R. vesicarius). C4-plants also evidenced a lower chlorophyll/P 700 ratio, indicating a higher content of P 700.

Table 1: Photochemical activities of chloroplasts isolated from crop species.

Activity Setaria Pennisetum Amaranthus Oryza Rumex italica typhoides paniculatus sati'l!a vesicarius

DCPIP reduction*) 268 243 325 293 312

Ferricyanide reduction*) 321 283 432 325 328

NADP reduction*) 54 48 65 55 47

NADP reduction with DCPIP and ascorbate*) 68 62 88 87 71

Cyclic photophos-phorylation**) 518 495 775 434 475

Non-cyclic photo-phosphorylation**) 138 128 346 275 262

Ch1lP 700 (molar ratio) 408 416 321 432 448

*) ,umoles reduced/mg ch1lh. "*) .umoles ATP formed/mg chi/h.

z. Pf/anzenphysiol. Bd. 88. 1-11. 1978.

4 A. S. RAGHAVENDRA and V. S. R. DAS

The characteristics of the photophorylation carried out by the chloroplasts from S. italica, P. typhoides and A. paniculatus differed from that of the o. sativa and R. vesicarius chloroplasts (Table 2). Only small amounts of chloroplasts from the C4-plants in the reaction mixture were necessary for optimal activity (Fig. 1 A, B). But the linearity of activity was maintained only up to 8-15 sec by Crplant chloroplasts, as compared to 16-20 sec for Ca-plants (Fig. 1 C, D). The chloroplasts from Ca-plants were more stable (Fig. 2 A). No great variation in the optimal pH for either cydic or non-cyclic photophosphorylation was observed (Fig. 2 B). C4-plant chloroplasts responded best to slightly higher concentrations of magnesium (Fig. 2 C, D). The Km values for chloroplasts isolated from C4-plants, with respect to both ADP and Pi in the course of cyclic photophosphorylation (40-100 11M), were less than those (0.6-1.2 mM) for Ca-plant chloroplasts (Table 3).

Photophosphorylation activity was sensitive to inhibitors in both Ca- and C4-plants. Cyclic photophosphorylation was inhibited by 2,4-dinitrophenol (2,4-DNP) and salicylaldoxime (Fig. 3 A, B) while 3-(3,4-dichlorophenyl)-1,1-

Table 2: Characteristics of photophosphorylation by isolated chloroplasts.

Source of isolation Observation Setaria Pennisetum Amaranthus Oryza Rumex

italica typhoides paniculatus sativa vesica nus

pH optimum Cyclic 7.8 7.8 7.8 7.5 7.5 Non-cyclic 8.3 8.3 8.2 8.0 8.0

Magnesium concentration (mM) 3.5 3 2 2 1.5

PMS (Cyclic) concentration CuM) 30 40 50 3'0 35

Ferricyanide (non-cyclic) concentration (mM) 0.6 0.8 1.0 3.5 5

Chlorophyll concentration in assay mixture (,ug/ml) 10 15 20 50 30

Maximum period of linearity (min) 8 12 15 16 20

Stability: Period upto which activity retained (min) 100 % activity 15 20 15 JoO 30

75 Ofo activity 90 90 90 120 120

Z. P/lanzenphysiol. Bd. 88. 1-11. 1978.

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Photochemical activities of C,- and Ca-chloroplasts 5

S.ITALICA 16~~~==~----~

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4 8 12 16 20 DURATION OF ILLUMINATION (min)

Fig. 1: Cyclic photophosphorylation activity in relation to the chlorophyll concentration in the reaction mixture (A, B) and the duration of illumination (C, D) by chloroplasts isolat­ed from leaves of S. italica (A, C) and O. sativa (B, D).

dimethyl urea (DCMU) and o-phenanthroline blocked non-cycle photophosphoryla­tion (Fig. 3 C, D).

The response of the cyclic and non-cyclic photophosphorylation of the chloro­plasts to the light intensity was dependent on the source of isolation. Irrespective of the plant from which the chloroplasts were isolated, the non-cyclic photophosphorylation was saturated at 2000-3000 ft-c, whereas the cyclic photophosphorylation was not saturated even at 5000 ft-·c (Figs. 4 A-E). However, the cyclic photophosphorylation by chloroplasts of O. sativa and R. vesicarius (but not of S. italica, P. typhoides and A. paniculatus) exhibited a sigmoid curve in

Z. Pjlanzenphysiol. Bd. 88.1-11.1978.

6 A. S. RAGHAVENDRA and V. S. R. DAS

>- \00 ~~A A soo B I-

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0 I 2 3 0 7-0 7'S 8-0 a-s 9-0 HOURS OF STORAGE AT oOe pH

Fig. 2: Characteristics of photophosphorylation by isolated chloroplasts from the leaves of S. italica and O. sativa. A: Stability of chloroplast preparations as indicated by their cyclic photophosphorylation activity after storage at 0 dc. B: Effect of the pH of the reaction mixture on cyclic and non-cyclic photophosphorylation activities.

Table 3: Km-values of isolated chloroplasts for ADP and Pi during cyclic photophosphory­lation.

Plant

Setaria italica Pennisetum typhoides Amaranthus paniculatus Oryza sativa Rumex vesicarius

Km (mM)

ADP

0.04 0.08 0.06 0.60 0.80

Table 4: Chlorophyll and protein content of leaves.

Plant

Setaria italica Pennisetum typhoides Amaranthus paniculatus Oryza sativa Rumex vesicarius

'f) Content in mg/g fresh weight.

Chlorophyll Chloro-alb ratio phyll*)

4.2 3.8 4.5 2.7 2.6

2,438 2.282 1.928 3.643 1.821

Pi

0.06 0.07 0.10 0.81 1.20

Protein")

27.31 24.69 34.95 37.89 28.97

Protein/ chlorophyll ratio

11.2 10.8 18.1 10,4 15.9

relation to the light intensity. Furthermore, the molar ratio of cyclic/non-cyclic photophosphorylation increased more with illumination in C4-plants than that in C3-plants (Fig. 4 F).

Z. Pjlanzenphysiol. Bd. 88. 1-11. 1978.

Photochemical activities of C4- and Ca-chloroplasts 7

200r--------------.

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160

140

120

100

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A

10- 6 10-5 10- 4

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100

80

60

40

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o

c A 0 CYCLIC •• NONCYCLIC

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2,4-DINITROPHENOL (M)

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160

140

120

100

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DCMU(M)

200 ;---------------,

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160

80

60

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SALI CYLALDOXIMECM)

Fig. 3: The response of cyclic and non-cyclic photophosphorylation by isolated chloroplasts to inhibitors.

The contents of chlorophyll and protein per unit fresh weight did not vary greatly among the plants (Table 4). However the chlorophyll alb ratio was higher in C4-plants. f3-carotene also was present at higher levels in leaves of S. italica, P.

z. Pflanzenphysiol. Bd. 88. 1-11. 1978.

8 A. S. RAGHAVENDRA and V. S. R. DAS

typhoides and A. paniculatus than in those of O. sativa and R. vesicarius (Table 5). Lutein, neoxanthin and violaxanthin were present at lower levels in C,-spec'ies than in C3-plants.

N

'0

)C

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R.VESICARIUS C

2

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LIGHT INTENSITY ft. c. x 10-3

4

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Fig. 4: Cylic and non-cyclic photophosphorylation by chloroplasts as a function of light intensity (A-E). Fig. F shows the molar ratio of cyclic to non-cyclic photophosphorylation with increasing light intensity.

Z. P/lanzenphysiol. Bd. 88. 1-11. 1978.

Photochemical activities of C4- and Cs-chloroplasts 9

Table 5: Pigment composition of the leaves.

,umoles/100 ,umoles chlorophyll Pigment Setaria Pennisetum Amaranthus Oryza Rumex

italica typhoides paniculatus sativa vesicarius

Chlorophyll a 81 79 82 73 72 Chlorophyll b 19 21 18 27 28 Chlorophyll alb 4.2 3.8 4.5 2.7 2.6 p-carotene 9.2 8.8 7.8 6.2 6.4 Lutein 5.8 5.7 7.5 9.4 9.3 Neoxanthin 1.2 1.4 2.2 2.7 2.3 Violaxanthin 1.8 1.9 2.4 2.6 2.8

Discussion

The rates of photochemical reduction of NADP, DCPIP and ferricyanide by chloroplasts were similar for C4- and Cs-plants (Table 1). CHEN et aI. (1969) noted that the P/2e- ratios during 'photophosphorylation with different cofactors by bermuda grass (a C4-plant) chloroplasts were near unity, a phenomenon having been previously demonstrated with Cs-plant chloroplasts. Although the optimal conditions for photophosphorylation by chloroplasts were different for C4- and Cs-plants, the responses of cyclic and non-cyclic photophosphorylation were similar in both Oryza and Setaria (Fig. 3). The sensitivity of cyclic photophosphorylation to salicylaldoxime and DNP is in agreement with the known effects of the agents (ARNON, 1969) as was the inhibitory action of DCMU and of o-phenanthroline on non-cyclic photophosphorylation. The qualitative pigment composition of the leaves of C4- and Cs-plants was similar (Table 5). All these observations confirm that the general constitution of the chloroplast system of C4-plants is similar to that of the Cs-type.

However, the characteristics of photophosphorylation by isolated chloroplasts from C4- and Cs-plants differed considerably (Table 2). Earlier experiments with C4-plants such as maize and sugar cane indicated the presence of inhibitors interfering with cellular activities (BALDRY et aI., 1970; MIFLIN and HAGEMAN, 1963). The comparative instability of chloroplasts from C4-plants supports this conclusion. The low Km-values of chloroplasts from S. italica, P. typhoides and A. paniculatus for ADP and Pi indicate that the organelles exhibit a great affinity for these compounds, which results in the ability to catalyze more rapid rates of photophosphorylation. The Km-values of 0.6-1.2 mM for ADP and Pi for chloroplasts from Oryza and Rumex and comparable to those observed with spinach and swisschard (AVRON, 1961; AVRON and JAGENDORF, 1959; AVRON and SHAVIT, 1965; AVRON et aI., 1958; MUDD, 1959). The lower Km-values for ADP and Pi observed with Setaria, Pennisetum and Amaranthus are similar to those described for bermuda grass (CHEN et aI., 1969).

Z. P/lanzenphysiol. Bd. 88.1-11.1978.

10 A. S. RAGHAVENDRA and V. S. R. DAS

The higher rates of cyclic photophosphorylation in C4-plants suggest higher levels of PS I in the chloroplasts of these plants. The higher chlorophyll alb ratio in the leaves of C4-plants (Table 4) also indicate a relative enrichment of PS I, as a higher ra1)io of chlorophyll a to b is known to be a feature of PS I (BOARDMAN and ANDERSON, 1964). The lower ratio of chlorophyll to P 700 (Table 5) supports this assumption and indicates that the photosynthetic unit in C4-plants tends to be smaller. MAYNE et aI. (1971) described the possibility of such a smaller photosynthetic unit in C4-plants.

It has been proposed that cyclic photophosphorylation supplies the additional ATP needed during C 4-photosynthesis in C4-plants (HATCH, 1970; CHEN et aI., 1969). Unlike in Ca-plants, the ratio of cyclic to non-cyclic photophosphorylation increased with light intensity in C4-plants (Fig. 4 F). Yet the general response of cyclic and non-cyclic photophosphorylation remained typical in all cases. Cyclic photophosphoryla1)ion with PMS increases with illumination (AVRON, 1961). On the other hand, NADP reduction (TURNER et aI., 1962), DCPIP reduction (AVRON and jAGENDORF, 1959) and non-cyclic photophosphorylation (with NADP and ferredoxin) (TURNER et aI., 1962) were saturated at comparatively lower light intensities. Another significant feature is the sigmoid curve of the response of cyclic photophosphorylation by chloroplasts of Oryza and Rumex to light intensity (Fig. 4 C and D). Such a curve has been obtained with spinach (TURNER et aI., 1962), but not with a C4-plant (CHEN et aI., 1969). The present authors believe that these factors assist in the maintenance of active cyclic photophosphorylation at higher light intensities to effect a continuous supply of the additional A TP required for C4-photosynthesis.

References

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Photochemical activities of C4- and C3-chloroplasts 11

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Agricultural Publishing and Documentation, Wageningen, pp. 215, 1970. HEW, C. S. and M. GIBBS: Can. J. Bot. 48, 1265 (1970). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: J. Bio!. Chern. 193,

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MIFLIN, B. J. and R. H. HAGEMAN: Plant Physio!. 38,66 (1963). - - Crop. Sci. 6, 185 (1966). MORELAND, D. E. and C. L. HILL: Weeds 10, 229 (1962). MUDD, J. B.: Nature 183, 900 (1959). OGAWA, T. and K. SHIBATA: Photochem. Photobio!' 4,193 (1965). RAGHAVENDRA, A. S.: A Comparative Study of C4 and C 3 Photosynthetic Systems in Some

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Dr. A. S. RAGHAVENDRA and Professor Dr. V. S. R. DAS, Department of Botany, Sri Ven­kateswara University, Tirupati 517 502, Andhra Pradesh, India.

Z. PJlanzenphysiol. Bd. 88. 1-11. 1978.