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Chlorophyll and enzymes of photorespiration in Dendrophthoe falcata seeds
Plant Science Letters, 16 (1979) 165--170 © Elsevier/North-Holland Scientific Publishers Ltd.
165
CHLOROPHYLL AND ENZYMES OF PHOTORESPIRATION IN DENDROPHTHOE FALCA TA SEEDS
D.N. KACHRU* and P.S. KRISHNAN** Department of Biochemistry, Lucknow University, Lucknow-226007 (India)
(Received October 11th, 1978) (Revision received and accepted May 24th, 1979)
SUMMARY
The embryo of mature Dendrophthoe falcata seed contained 0.65 mg chlorophyll/g fresh wt; chlorophyll a : b ratio was 1.6. Extracts of the embryo brought about the hydrolysis of phosphoglycolate and 3-phosphoglycerate, apparently through the agency of distinct phosphatases. Expressed per mg chlorophyll, phosphoglycolate phosphatase activity in the embryo was 16.7 ~mol substrate/min and 3-phosphoglycerate phosphatase activity 6.8 umol substrate/min. Glycolate oxidase activity was not demonstrable in embryo or whole seed extract. NADH-linked glyoxylate reductase activity was demonstrable in the embryo (0.09/~mol substrate/min/g fresh wt), but NADPH-linked activity could not be detected.
INTRODUCTION
The occurrence of chlorophyll in seed seems to be a special feature of many mistletoes. Chlorophyll/green colour has been reported in embryo [1--4] and endosperm [5,6] tissues. It is generally held that light is either required for germination of mistletoe seeds or stimulates them to some degree [3]. Kuijt [5] suggested that the endoserpm of Phoradendron californicum, P. bol~anum, P. juniperium and P. villosum, in which chlorophyll was present throughout, might be photosynthetically active, storing or supplying the embryo with elaborated materials. He believed that Arceuthobium radicle was an actively photosynthesising organ, since its deep red colour changed to green on attachment to the host tree, presumably due to the removal of anthocyanin masking chlorophyll. Scharpf [ 6] considered Present addresses: *Horticulture Research Centre, Chaubatia, Ranikhet, U.P., India. **Department of Botany, Calicut University, Kerala-673635, India.
166
the possibility that the chlorophyll in the embryo of A. occidentale enabled the seed to carry on limited photosynthesis which stimulated and enhanced germination to some degree. Mistletoe seeds have not been studied for the enzyme activities in the pathway of photosynthesis or photorespiration. The fruit of Dendrophthoe falcata is a pseudoberry, the 'seed' lacking a true coat. The embryo is coloured a uniform deep green. On removal of the pericarp, the 'radicular' end of the embryo, a fifth of the whole embryo in length, is visible through the translucent mucilaginous coat as a flattened, button-like projection. The following study relates to the determination of chlorophyll a and b in D. falcata embryo and the activities of selected enzymes in the pathway of photorespiration. MATERIALS AND METHODS
Collection o f fruits and isolation o f embryo. Ripe fruits of Dendrophthoe falcata L. Ettings were collected from the parasite growing on Mangifera indica L. The pericarp was peeled off and the 'seed' freed from the mucilaginous coat by gently rubbing with a dry cloth. The embryo was freed with the aid of forceps and transferred to a chilled container. The embryo constituted, on average, 17% of seed fresh wt. Chlorophyll determination. Chlorophyll extracted in 80% acetone was determined according to Arnon [7]. Preparation o f enzyme extracts. Dispersions (4--8%, w/v) of embryo/ endosperm/whole seed were prepared with medium added in small amounts at a time. The medium was made up of 20 mM Tris--HCl (pH 7.2) or, in the case of glycolate oxidase and glyoxylate reductase, 20 mM phosphate buffer (pH 7.0) containing 20 mM 2-mercaptoethanol, 5 mM ethylenediamine tetraacetic acid (EDTA) and 1% (v/v) Triton X-100. The suspensions were spun at 800 g for 10 min at 4°C and the supernatants filtered through Sephadex G-25 gel. The medium for equilibrating the column and subsequent elution was the same as the dispersion medium, with the difference that EDTA and detergent were omitted. The gel filtrates served as the source of the enzymes. For some tests, protein concentrates were prepared by precipitating the extracts with ammonium sulphate to 80% saturation. Enzyme assays. Phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase were assayed according to Anderson and Tolbert [8] and Randall and Tolbert [9] respectively; inorganic phosphate liberated was estimated with Fiske and SubbaRow reagent [ 10]. Glycolate oxidase (pH 7.0/8.0) and glyoxylate reductase (pH 6.5) were assayed spectrophotometrically according to Hess and Tolbert [ 11]. The assays, when activity was found, were at the predetermined respective pH optima and in the propor. tionality range of activity with respect to amount of enzyme and period of incubation. One unit of enzyme activity was the transformation of 1/~mol of
167 the substrate in 1 min under the assay conditions and reported for I g fresh
weight. RESULTS
Chlorophyll content. The embryo contained 0.647 mg chlorophyll/g fresh tissue or 0.919 mg chlorophyll/g oven
TABLE I DISTRIBUTION OF PHOSPHATASE ACTIVITIES The assays were on gel-filtered extracts, employing 20 mM cacodylate buffer (pH 6.0). The incubation period was 30 rain at 30°C. The values within parentheses are unit(s)/g
equivalent of fresh seed, assuming thatembryo constituted 17% of whole seed. The letters (a) and (b) stand for two experiments with separate batches of seeds. Phosphatase activity (unit(s)/g fresh tissue) Tissue
a
Whole seed Embryo Endosperm an.d.: Not determined.
3-Phosphoglycerate
Phosphoglycolate
1.43 11.9 (2.0) 0.38 (0.32)
b
a
b
1.07 9.8 (1.6) 0.34 (0.28)
1.3 4.4 (0.75) n.d. a
1.1 4.4 (0.75) n.d.a
168
phosphoglycolate phosphatase activity but 3-phosphoglycerate phosphatase activity remained unaffected. When the extract was held at 55°C for 3 rain, the 3-phosphoglycerate phosphatase activity was completely lost, but 70% of phosphoglycolate phosphatase activity was retained.
Glycolate axidase. Glycolate oxidase activity could not be detected in extracts of embryo or whole seed after incubation for 10 min at pH 6.5 at 30°C. A protein concentrate from the embryo was inactive. Glyoxylate reduetase. Glyoxylate reductase activity linked to NADH or NADPH could not be detected in extracts of embryo and seed. However, NADH-linked activity was found in embryo protein concentrate, but was very weak (0.09 unit/g); there was no activity with NADPH. DISCUSSION
The total chlorophyll content in D. falcata seed embryo (0.92 mg/g dry wt) was 75% of that in Phoradendron shoot (1.2 mg/g dry wt), but was over 2-fold more than in Arceuthobium shoots (0.4 mg/g dry wt). Phoradendron and Arceuthobium shoots are photosynthetically.active [ 12 ] and it may be thought that the chlorophyll content per se would enable the embryo of D. falcata seed to photosynthesise. Chlorophyll a : b ratio found in D. falcata embryo (1.6) was substantially smaller than that reported for Phoradendron (2.5) and Arceuthobium ~3.0) shoots. Black [ 13 ] reported that leaf chlorophyll a : b ratio was 2.8 - 0.4 in C3 plants and 3.9 -+ 0.6 in C4 plants. The smaller chlorophyll a : b ratio in D. falcata seed embryo was indicative of a potentially C~ type of photosynthetic activity.
Phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase in D. falcata embryo. As far as the authors are aware, this is the first report on the occurrence of phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase in any mature seed. That distinct phosphatases in D. falcata seed acted on phosphoglycolate and 3-phosphoglycerate followed from the evidence that Mg2÷ was needed for phosphatase action only on phosphoglycolate, that EDTA completely blocked phosphoglycolate hydrolysis without having any effect on 3-phosphoglycerate hydrolysis and that phosphatase action on 3-phosphoglycerate hydrolysis could be selectively inactivated by heat treatment at 55°C. These properties found in extracts of D. falcata seed embryo were similar to those found by Tolbert [14] for the enzymes purified from various leaves. The predominant localisation of phosphoglycolate phosphatase in the embryo region, contrasting with the occurrence of 3-phosphoglycerate phosphatase both in embryo and endosperm, supports the occurrence of distinct phosphatases. Randall et al. [15] pointed out, after consideration of the properties of the two phosphatases, that the hydrolysis of 3-phosphoglycerate by crude leaf extracts could
169
not be attributed to phosphoglycolate phosphatase and that phosphoglycolate hydrolysis by 3-phosphoglycerate phosphatase could be only 10% of the rate with 3-phosphoglycerate. The activities found in D. falcata seed embryo were not appreciably affected by the action of unspecific phosphatase as appeared from the finding that 0-glycerophosphate was only slowly hydrolysed under the conditions optimal for phosphoglycolate and 3-phosphoglycerate hydrolysis. The phosphoglycolate phosphatase activity in D. falcata seed (16.7 units/ mg chlorophyll) was comparable with the activity of the enzyme in actively photo-respiring green leaves [16]. The ratio of 3-phosphoglycerate phosphatase to phosphoglycolate phosphatase found in D. falcata seed embryo, 1 : 2.4 (Table I), suggested a potentially C3 type of photosynthetic activity
[zs]. Absence of glycolate oxidase activity: block in photorespiration. Glycolate oxidase, found in the leaves of all higher plants, has been reported also in endosperm tissue from germinating castor bean [ 17,18 ]. The failure to detect glycolate oxidase in the embryo tissue of D. falcata seed suggested that notwithstanding a significant content of chlorophyll in the embryo, an apparently satisfactory ratio for chlorophyll a to b [ 13 ] and the ability to readily dephosphorylate phosphoglycolate, the embryo tissue could not photo-respire. Any significant oxidation of glycolate to glyoxylate catalysed by glyoxylate reductase appeared unlikely because of the comparatively weak activity of the enzyme and the unfavourable equilibrium of the reaction [ 19,20 ]. Absence of photorespiration. The embryo of D. falcata does not appear to have the full complement of enzymes to carry out photo-respiration. The absence of photo-respiration in the mature seed of D. falcata would not be surprising, since 80% of the green embryo is enveloped by the massive endosperm and the radicular end which projects out of seed is normally cut off from light by the fairly thick pericarp and the mucilaginous layer which envelope the whole seed. The mechanism whereby chlorophyll was elaborated by mistletoe embxyo, the developmental stage at which chlorophyll was first formed and the mechanism whereby etiolation was prevented in the absence of light, are not known. The presence of chlorophyll and presumably chloroplasts in mature D. falcata seed may have considerable metabolic significance during the germination of the seed. ACKNOWLEDGEMENTS This research was financed by a grant made by the U.S. Department of Agriculture under PL-480 Grant No. FG-IN. 451. This department is grateful to the University Grants Commission, New Delhi, for aid under the Pro-
170 g r a m m e o f Special Assistance t o Selected D e p a r t m e n t s a n d t o t h e R o c k e f e l l e r F o u n d a t i o n f o r g e n e r o u s grants. REFERENCES
1 B.M. Johri and S.P Bhatnapr, Loranthaeeae, CSIR Monograph 8, New Delhi, India, 1972. 2 J. MeLuckie, Bot. Gaz., 75 (1923) 333. 3 J.F. Rigby, Proc. Linn. Soe., 84 (1959) 335. 4 B. Singh, J. Linn. Soc. Bot., 53 (1952) 449. 5 J. Kuijt, Univ. Calif. Berkeley, Publ. ]Sot., 30 (1960) 337. 6 R.F. Scharpf, U ~ . For. Serv. Res. Note PSW, 1970, p. 59. 7 D.I. Arnon, Plant Physiol., 24 (1949) 1. 8 D.E. Anderson and N.E. Tolbert, Methods Enzymol., IX (1966) 646. 9 D.D. Randall and N.E. Tolbert, J. Biol. Chem., 246 (1971) 5510. 10 C.H. Fiske and L. SubbaRow, J. Biol. Chem., 66 (1925) 375. 11 J.L. Hess and N.E. Tolbert, Plant Physiol., 42 (1967) 371. 12 R.J. Hull and O.A. Leonard, Plant Physiol., 39 (1964) 1008. 13 C.C. Black, Annu. Rev. Plant Physiol., 24 (1973) 253. 14 N.E. Tolbert, Curt. Top. Cell. Regul., 7 (1973) 21. 15 D.D. Randall, N,E, Tolbert and D. Gremel, Plant Physiol., 48 (1971) 480. 16 D.D. Randall and N.E. Tolbert, Photosynthesis and Photorespiration, Wiley Interscience, New York, 1971, p. 259. 17 W,H. Tanner and H. Beevers, Plant Physiol., 40 (1965) 971. 18 R.R. Theimer and E. Theimer, Plant Physiol., 56 (1975) 100. 19 L.D. Kohn and W.A. Warren, J. Biol. Chem., 245 (1970) 3831. 20 N.E. Tolbert, R.K. Yamazaki and A. Oeser, J. Biol. Chem., 245 (1970) 5129.
165
CHLOROPHYLL AND ENZYMES OF PHOTORESPIRATION IN DENDROPHTHOE FALCA TA SEEDS
D.N. KACHRU* and P.S. KRISHNAN** Department of Biochemistry, Lucknow University, Lucknow-226007 (India)
(Received October 11th, 1978) (Revision received and accepted May 24th, 1979)
SUMMARY
The embryo of mature Dendrophthoe falcata seed contained 0.65 mg chlorophyll/g fresh wt; chlorophyll a : b ratio was 1.6. Extracts of the embryo brought about the hydrolysis of phosphoglycolate and 3-phosphoglycerate, apparently through the agency of distinct phosphatases. Expressed per mg chlorophyll, phosphoglycolate phosphatase activity in the embryo was 16.7 ~mol substrate/min and 3-phosphoglycerate phosphatase activity 6.8 umol substrate/min. Glycolate oxidase activity was not demonstrable in embryo or whole seed extract. NADH-linked glyoxylate reductase activity was demonstrable in the embryo (0.09/~mol substrate/min/g fresh wt), but NADPH-linked activity could not be detected.
INTRODUCTION
The occurrence of chlorophyll in seed seems to be a special feature of many mistletoes. Chlorophyll/green colour has been reported in embryo [1--4] and endosperm [5,6] tissues. It is generally held that light is either required for germination of mistletoe seeds or stimulates them to some degree [3]. Kuijt [5] suggested that the endoserpm of Phoradendron californicum, P. bol~anum, P. juniperium and P. villosum, in which chlorophyll was present throughout, might be photosynthetically active, storing or supplying the embryo with elaborated materials. He believed that Arceuthobium radicle was an actively photosynthesising organ, since its deep red colour changed to green on attachment to the host tree, presumably due to the removal of anthocyanin masking chlorophyll. Scharpf [ 6] considered Present addresses: *Horticulture Research Centre, Chaubatia, Ranikhet, U.P., India. **Department of Botany, Calicut University, Kerala-673635, India.
166
the possibility that the chlorophyll in the embryo of A. occidentale enabled the seed to carry on limited photosynthesis which stimulated and enhanced germination to some degree. Mistletoe seeds have not been studied for the enzyme activities in the pathway of photosynthesis or photorespiration. The fruit of Dendrophthoe falcata is a pseudoberry, the 'seed' lacking a true coat. The embryo is coloured a uniform deep green. On removal of the pericarp, the 'radicular' end of the embryo, a fifth of the whole embryo in length, is visible through the translucent mucilaginous coat as a flattened, button-like projection. The following study relates to the determination of chlorophyll a and b in D. falcata embryo and the activities of selected enzymes in the pathway of photorespiration. MATERIALS AND METHODS
Collection o f fruits and isolation o f embryo. Ripe fruits of Dendrophthoe falcata L. Ettings were collected from the parasite growing on Mangifera indica L. The pericarp was peeled off and the 'seed' freed from the mucilaginous coat by gently rubbing with a dry cloth. The embryo was freed with the aid of forceps and transferred to a chilled container. The embryo constituted, on average, 17% of seed fresh wt. Chlorophyll determination. Chlorophyll extracted in 80% acetone was determined according to Arnon [7]. Preparation o f enzyme extracts. Dispersions (4--8%, w/v) of embryo/ endosperm/whole seed were prepared with medium added in small amounts at a time. The medium was made up of 20 mM Tris--HCl (pH 7.2) or, in the case of glycolate oxidase and glyoxylate reductase, 20 mM phosphate buffer (pH 7.0) containing 20 mM 2-mercaptoethanol, 5 mM ethylenediamine tetraacetic acid (EDTA) and 1% (v/v) Triton X-100. The suspensions were spun at 800 g for 10 min at 4°C and the supernatants filtered through Sephadex G-25 gel. The medium for equilibrating the column and subsequent elution was the same as the dispersion medium, with the difference that EDTA and detergent were omitted. The gel filtrates served as the source of the enzymes. For some tests, protein concentrates were prepared by precipitating the extracts with ammonium sulphate to 80% saturation. Enzyme assays. Phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase were assayed according to Anderson and Tolbert [8] and Randall and Tolbert [9] respectively; inorganic phosphate liberated was estimated with Fiske and SubbaRow reagent [ 10]. Glycolate oxidase (pH 7.0/8.0) and glyoxylate reductase (pH 6.5) were assayed spectrophotometrically according to Hess and Tolbert [ 11]. The assays, when activity was found, were at the predetermined respective pH optima and in the propor. tionality range of activity with respect to amount of enzyme and period of incubation. One unit of enzyme activity was the transformation of 1/~mol of
167 the substrate in 1 min under the assay conditions and reported for I g fresh
weight. RESULTS
Chlorophyll content. The embryo contained 0.647 mg chlorophyll/g fresh tissue or 0.919 mg chlorophyll/g oven
TABLE I DISTRIBUTION OF PHOSPHATASE ACTIVITIES The assays were on gel-filtered extracts, employing 20 mM cacodylate buffer (pH 6.0). The incubation period was 30 rain at 30°C. The values within parentheses are unit(s)/g
equivalent of fresh seed, assuming thatembryo constituted 17% of whole seed. The letters (a) and (b) stand for two experiments with separate batches of seeds. Phosphatase activity (unit(s)/g fresh tissue) Tissue
a
Whole seed Embryo Endosperm an.d.: Not determined.
3-Phosphoglycerate
Phosphoglycolate
1.43 11.9 (2.0) 0.38 (0.32)
b
a
b
1.07 9.8 (1.6) 0.34 (0.28)
1.3 4.4 (0.75) n.d. a
1.1 4.4 (0.75) n.d.a
168
phosphoglycolate phosphatase activity but 3-phosphoglycerate phosphatase activity remained unaffected. When the extract was held at 55°C for 3 rain, the 3-phosphoglycerate phosphatase activity was completely lost, but 70% of phosphoglycolate phosphatase activity was retained.
Glycolate axidase. Glycolate oxidase activity could not be detected in extracts of embryo or whole seed after incubation for 10 min at pH 6.5 at 30°C. A protein concentrate from the embryo was inactive. Glyoxylate reduetase. Glyoxylate reductase activity linked to NADH or NADPH could not be detected in extracts of embryo and seed. However, NADH-linked activity was found in embryo protein concentrate, but was very weak (0.09 unit/g); there was no activity with NADPH. DISCUSSION
The total chlorophyll content in D. falcata seed embryo (0.92 mg/g dry wt) was 75% of that in Phoradendron shoot (1.2 mg/g dry wt), but was over 2-fold more than in Arceuthobium shoots (0.4 mg/g dry wt). Phoradendron and Arceuthobium shoots are photosynthetically.active [ 12 ] and it may be thought that the chlorophyll content per se would enable the embryo of D. falcata seed to photosynthesise. Chlorophyll a : b ratio found in D. falcata embryo (1.6) was substantially smaller than that reported for Phoradendron (2.5) and Arceuthobium ~3.0) shoots. Black [ 13 ] reported that leaf chlorophyll a : b ratio was 2.8 - 0.4 in C3 plants and 3.9 -+ 0.6 in C4 plants. The smaller chlorophyll a : b ratio in D. falcata seed embryo was indicative of a potentially C~ type of photosynthetic activity.
Phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase in D. falcata embryo. As far as the authors are aware, this is the first report on the occurrence of phosphoglycolate phosphatase and 3-phosphoglycerate phosphatase in any mature seed. That distinct phosphatases in D. falcata seed acted on phosphoglycolate and 3-phosphoglycerate followed from the evidence that Mg2÷ was needed for phosphatase action only on phosphoglycolate, that EDTA completely blocked phosphoglycolate hydrolysis without having any effect on 3-phosphoglycerate hydrolysis and that phosphatase action on 3-phosphoglycerate hydrolysis could be selectively inactivated by heat treatment at 55°C. These properties found in extracts of D. falcata seed embryo were similar to those found by Tolbert [14] for the enzymes purified from various leaves. The predominant localisation of phosphoglycolate phosphatase in the embryo region, contrasting with the occurrence of 3-phosphoglycerate phosphatase both in embryo and endosperm, supports the occurrence of distinct phosphatases. Randall et al. [15] pointed out, after consideration of the properties of the two phosphatases, that the hydrolysis of 3-phosphoglycerate by crude leaf extracts could
169
not be attributed to phosphoglycolate phosphatase and that phosphoglycolate hydrolysis by 3-phosphoglycerate phosphatase could be only 10% of the rate with 3-phosphoglycerate. The activities found in D. falcata seed embryo were not appreciably affected by the action of unspecific phosphatase as appeared from the finding that 0-glycerophosphate was only slowly hydrolysed under the conditions optimal for phosphoglycolate and 3-phosphoglycerate hydrolysis. The phosphoglycolate phosphatase activity in D. falcata seed (16.7 units/ mg chlorophyll) was comparable with the activity of the enzyme in actively photo-respiring green leaves [16]. The ratio of 3-phosphoglycerate phosphatase to phosphoglycolate phosphatase found in D. falcata seed embryo, 1 : 2.4 (Table I), suggested a potentially C3 type of photosynthetic activity
[zs]. Absence of glycolate oxidase activity: block in photorespiration. Glycolate oxidase, found in the leaves of all higher plants, has been reported also in endosperm tissue from germinating castor bean [ 17,18 ]. The failure to detect glycolate oxidase in the embryo tissue of D. falcata seed suggested that notwithstanding a significant content of chlorophyll in the embryo, an apparently satisfactory ratio for chlorophyll a to b [ 13 ] and the ability to readily dephosphorylate phosphoglycolate, the embryo tissue could not photo-respire. Any significant oxidation of glycolate to glyoxylate catalysed by glyoxylate reductase appeared unlikely because of the comparatively weak activity of the enzyme and the unfavourable equilibrium of the reaction [ 19,20 ]. Absence of photorespiration. The embryo of D. falcata does not appear to have the full complement of enzymes to carry out photo-respiration. The absence of photo-respiration in the mature seed of D. falcata would not be surprising, since 80% of the green embryo is enveloped by the massive endosperm and the radicular end which projects out of seed is normally cut off from light by the fairly thick pericarp and the mucilaginous layer which envelope the whole seed. The mechanism whereby chlorophyll was elaborated by mistletoe embxyo, the developmental stage at which chlorophyll was first formed and the mechanism whereby etiolation was prevented in the absence of light, are not known. The presence of chlorophyll and presumably chloroplasts in mature D. falcata seed may have considerable metabolic significance during the germination of the seed. ACKNOWLEDGEMENTS This research was financed by a grant made by the U.S. Department of Agriculture under PL-480 Grant No. FG-IN. 451. This department is grateful to the University Grants Commission, New Delhi, for aid under the Pro-
170 g r a m m e o f Special Assistance t o Selected D e p a r t m e n t s a n d t o t h e R o c k e f e l l e r F o u n d a t i o n f o r g e n e r o u s grants. REFERENCES
1 B.M. Johri and S.P Bhatnapr, Loranthaeeae, CSIR Monograph 8, New Delhi, India, 1972. 2 J. MeLuckie, Bot. Gaz., 75 (1923) 333. 3 J.F. Rigby, Proc. Linn. Soe., 84 (1959) 335. 4 B. Singh, J. Linn. Soc. Bot., 53 (1952) 449. 5 J. Kuijt, Univ. Calif. Berkeley, Publ. ]Sot., 30 (1960) 337. 6 R.F. Scharpf, U ~ . For. Serv. Res. Note PSW, 1970, p. 59. 7 D.I. Arnon, Plant Physiol., 24 (1949) 1. 8 D.E. Anderson and N.E. Tolbert, Methods Enzymol., IX (1966) 646. 9 D.D. Randall and N.E. Tolbert, J. Biol. Chem., 246 (1971) 5510. 10 C.H. Fiske and L. SubbaRow, J. Biol. Chem., 66 (1925) 375. 11 J.L. Hess and N.E. Tolbert, Plant Physiol., 42 (1967) 371. 12 R.J. Hull and O.A. Leonard, Plant Physiol., 39 (1964) 1008. 13 C.C. Black, Annu. Rev. Plant Physiol., 24 (1973) 253. 14 N.E. Tolbert, Curt. Top. Cell. Regul., 7 (1973) 21. 15 D.D. Randall, N,E, Tolbert and D. Gremel, Plant Physiol., 48 (1971) 480. 16 D.D. Randall and N.E. Tolbert, Photosynthesis and Photorespiration, Wiley Interscience, New York, 1971, p. 259. 17 W,H. Tanner and H. Beevers, Plant Physiol., 40 (1965) 971. 18 R.R. Theimer and E. Theimer, Plant Physiol., 56 (1975) 100. 19 L.D. Kohn and W.A. Warren, J. Biol. Chem., 245 (1970) 3831. 20 N.E. Tolbert, R.K. Yamazaki and A. Oeser, J. Biol. Chem., 245 (1970) 5129.