Chlorophyll, Ribulose-1 , 5-Diphosphate Carboxylase, and Hill Reaction Activity in Developing Leaves of Populus Deltoides

Plant Physiol. (1971) 48, 143-145

Chlorophyll, Ribulose-1 , 5-diphosphate Carboxylase, and Hill Reaction Activity in Developing Leaves of Populus deltoides

Received for publication November 24, 1970

DONALD I. DICKMANN1 Institute ofForest Genetics, North Central Forest Experiment Station, United States Department ofAgriculture Forest Service, Rhinelander, Wisconsin 54501

ABSTRACT CO_ Exchange Measurements. Rates of net photosynthesis The synthesis of chlorophyll and ribulose diphosphate car- of individual leaves in the expanding zone of three plants boxylase as well as the development of Hill reaction activity were determined by monitoring CO, concentrations in an open were followed in expanding Populus deltoides leaves and re- gas circuit with an infrared gas analyzer (5). For each leaf, net lated to photosynthetic patterns. Total chlorophyll, which was CO2 flux in light of saturating intensity (5.2 X 10' ergs/cme not correlated with photosynthetic rate in expanding leaves, de- sec) was recorded for 10 min after a constant rate of CO, ex- creased slightly with age in very young leaves, due to a decrease change had been attained. in chlorophyll b, but then increased linearly. The ratio of Chlorophyll and RuDP Carboxylase Determinations. Indi- chlorophyll a to b, which rose sharply in young leaves, was vidual leaves in the expanding leaf zone (LPA 0-7) of four highly correlated with the onset of net photosynthesis. Hill different plants were excised, immediately weighed, and their reaction activity was very low in young leaves and did not in- lengths measured. The midrib was then removed and one-half crease significantly until leaves were about half expanded. of each leaf placed in a Duall tissue grinder containing 3 ml of Ribulose diphosphate carboxylase activity increased in a 80% (v/v) acetone. The leaves were homogenized for 1 to 2 sigmoid fashion with leaf ontogenesis and closely paralleled de- min and the homogenate was centrifuged at lOOOg for 5 min. velopment of the photosynthetic system. The study demon- The supernatant fraction was decanted, and the sediment was strates the importance of chlorophyll a and Calvin cycle en- washed once by centrifugation with 80% acetone. The super- zyme synthesis to photosynthetic development in expanding natants were combined, brought to volume, and chlorophyll a leaves. and b determined spectrophotometrically (1). Chlorophyll determinations were also made for whole leaves on three additional plants. The other one-half of each leaf used for chlorophyll determinations was immediately placed in a Duall tissue grinder with 3 ml of ice-cold 40 mm tris-HCl, pH 7.8, containing 10 mM MgCl,, 0.25 mi EDTA, and 5.0 mm GSH (3). Each leaf sample was homogenized for 1 to 2 min, and the homogenate A comprehensive investigation of the developing apex of was centrifuged at 30,000g for 20 min. All preparative proce- Eastern cottonwood (Populus deltoides Bartr.) is currently dures were carried out at 0 to 4 C. RuDP carboxylase activity underway at this laboratory. As a basis for experiments in this was determined by adding 0.1 ml of the supernatant fraction expanding zone, the first unfolding leaf to reach 2 cm in length at the apex of a plant has been designated the index leaf and to 0.4 ml of a mixture of 10 Mm tris-HCl, pH 7.8, 2.5 /LM assigned a LPA' of 0. The expanding leaf zone extends down- MgCl1, 0.1 Mm EDTA, 1.25 /LM GSH, 1.15 /LM RuDP, and 2.5 Mm NaH"CO. (0.1 Mc/!umole). The mixture was incubated at ward approximately seven leaves from the index leaf, a leaf of 25 C for 5 min, and the reaction was stopped by adding 0.2 LPA 7 being fully expanded (9). In the present study, selected ml 5 M acetic acid. For each leaf extract two replications plus biochemical factors associated with photosynthesis were in- a blank without RuDP were run. The amount of "C fixed was vestigated in the expanding leaf zone of young cottonwood determined by counting 0.2-ml aliquots of the acidified re- plants. Chlorophyll content, RuDP carboxylase activity, and action mixture in a liquid scintillation spectrometer. the Hill reaction were determined in leaves in various onto- Hill Reaction Determinations. Individual leaves in the ex- genetic stages and their correlation with rates of CO2 uptake panding zone from four plants were excised, weighed, and noted. homogenized as above. The extracting medium consisted of 50 MATERIALS AND MEETHODS mm sodium phosphate (pH 7.2), 10 mm KCl, 50 mM sucrose, Cottonwood plants were raised from seed in controlled en- and 2.5 mm MgCl,. The extracts were centrifuged at 250g for vironment rooms in quartz sand cultures (8). When plants 2 min, and the sediment was discarded. The supernatant was reached the 8- to 10-leaf stage, the cotyledons and first four then centrifuged for 10 min at 1OOg, and the pellet was linear washed once by centrifugation with phosphate buffer. All pro- small, leaves above them were excised to maintain uni- cedures were carried out at 0 to 4 C. Two milliliters of acetone formity. Plants used in the present study had 19 to 22 leaves. were added to 0.5 ml of the resuspended chloroplast fragments and shaken. After centrifugation for 5 min at 1000g, total 'Present address: Department of Forestry, Iowa State Univer- chlorophyll concentration of the supernatant was determined sity, Ames, Iowa 50010. (1). The reaction mixture for determination of Hill activity 'Abbreviations: LPA: leaf plastochron age; DPIP: 2,6-dichloro- contained chloroplast fragments (10-15 ,ug chlorophyll) in 2 phenol indophenol; RuDP: ribulose-1, 5-diphosphate. ml of phosphate buffer plus 0.1 Mumole DPIP. Photoreduction 143 Downloaded from on January 16, 2020 - Published by www.plantphysiol.org Copyright © 1971 American Society of Plant Biologists. All rights reserved. 144 DICKMANN Plant Physiol. Vol. 48, 1971 of DPIP by chloroplasts in saturating light after 1 min was measured spectrophotometrically at 590 nm. Because of the small size of leaves LPA 2 or younger (Table I), it was thought that RuDP carboxylase and Hill re- Ls. action activity might be too low to measure when individual 0 leaves were assayed. Therefore, leaves in each of these de- 0 4 velopmental stages from four additional plants were combined - .0 in two separate runs in order to maximize enzyme and Hill 0 activity. However, data from these determinations did not differ significantly from those in which only single leaves were E -a. -i J assayed when expressed on a unit chlorophyll or fresh weight -i 0 basis. I Icr 0 J-I RESULTS AND DISCUSSION 0 -J The average length of leaves of the cottonwood plants used I in this study increased from 2.3 cm at approximately LPA 0 v to 10 cm at full expansion (LPA 7) (Table I). Leaf fresh weight averaged 41 mg at LPA 0 but increased rapidly to 1,178 mg at LPA 7. LEAF PLASTOCHRON AGE Cottonwood leaves of LPA 0 and 1 exhibited little or no FIG. 2. Total chl, chl a, chl b, and chl a/b ratios of 80% ace- capacity for CO2 fixation and evolved CO2 in the light, whereas of cottonwood. net photosynthetic uptake of CO2 was observed by leaves LPA tone extracts from developing leaves 2 or older (Fig. 1). Rates of net uptake increased rapidly between LPA 2 and 4 and then more slowly up to a maximum at LPA 7. The genesis of photosynthesis in expanding leaves of X 600 cottonwood plants of various ages has been discussed in detail by Dickmann (5). ,, 500 The relationship of chlorophyll content to photosynthesis as a- leaves mature and age has long been the subject of investiga- o 400 4,n Table I. Mean Length anid Fresh Weight ofDevelopinig Cottonwood E 300 Leaves Used for Determiniationz of Chlorophyll, RuDP z Carboxylase, antd Hill Reactioni Activity o 200

Approximate LPA Leaf Length Leaf Fresh Weight w 100- Cmn mlg o0 0 2.3 41 oF 1 2 3 4 5 6 7 1 3.1 81 LEAF PLASTOCHRON AGE 2 4.6 176 FIG. 3. Hill reaction activity in isolated chloroplasts from de- 3 6.0 336 veloping leaves of cottonwood. Chloroplast photoreduction of 4 7.5 560 DPIP after 1 min was determined spectrophotometrically at 590 5 8.9 862 nm. 6 9.6 1047 _ ,0 7 10.0 1178 ') I0 x 14 0 Per g Leaf Fresh W x i2- i: 12 10 \ -- a.) 8 a.2 0 IL 6 - 8 w Cy 41 4 C,) 2- _j 6 iPer mg Chlorophyll 0 L I~ 0 2 _ _ _ _ 4 C z m 4- _ -i i6- t) 2 0 a. // 8- c0 /7,

10o, 0 2 3 4 5 6 7 O 2 3 4 5 6 7 LEAF PLASTOCHRON AGE LEAF PLASTOCHRON AGE FIG. 4. RuDP carboxylase activity in developing leaves of cot- FIG. 1. Net CO2 exchange by developing cottonwood leaves in tonwood. Soluble protein extracts were incubated in the presence saturating light. CO2 concentrations in an open gas circuit were of RuDP and NaH'4CO3 for 5 min, and the amount of "C fixed was monitored for 10 min with an infrared gas analyzer. determined by liquid scintillation spectrometry. Downloaded from on January 16, 2020 - Published by www.plantphysiol.org Copyright © 1971 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 48, 1971 PHOTOSYNTHESIS IN DEVELOPING LEAVES 145 tion by plant physiologists (7, 10, 12, 13, 16). Although some rapidly. Togasaki and Levine (18) found that a strain of the of these studies have shown a direct relationship between green alga Chlamydomonas reinhardi, which possessed muta- chlorophyll content and photosynthesis, very young, nonphoto- tions that blocked photosynthetic electron transport, neverthe- synthesizing leaves have not been included. In the present less had normal RuDP carboxylase levels. These results and study cottonwood leaves of LPA 0 had little, if any, capacity data of the present study indicate that the absence of an intact for fixation of CO,, yet they possessed considerable total chlo- photosynthetic electron transport chain has no effect on the rophyll (Fig. 2). As leaves matured, the proportional rise in the level of RuDP carboxylase. rate of photosynthesis was significantly greater than the rise The present study demonstrates the importance of protein in total chlorophyll. Furthermore, when photosynthetic rates metabolism to the onset of photosynthesis during leaf onto- stabilized in mature leaves (LPA 6 and 7), chlorophyll concen- genesis in cottonwood. Apparently, the photosynthetic mecha- tration continued to increase at a rapid rate (Fig. 2). Thus, a nism of newly formed leaves is inoperative until critical levels close relationship between photosynthesis and total chloro- of Calvin cycle enzymes are synthesized, even though consid- phyll content is lacking in expanding cottonwood leaves. erable total chlorophyll may be present. The increase in en- When chlorophyll a to b ratios are compared with the onto- zyme activity, paralleled by a rapid synthesis of chlorophyll a, genetic increase in photosynthetic rate a closer relationship is is followed, after several plastochrons, by a rise in Hill activity noted, however. The ratio of chlorophyll a to b increased as pigment molecules are incorporated into the chloroplast strikingly during early stages of leaf development, first reach- membranes. ing a maximum at LPA 2 (Fig. 2), the same stage that net photosynthesis was first observed. The increase in a/b ratio, a Acknowledgmneets-The author wishes to express his thanks to Drs. Philip phenomenon also noted by Sestak (11) in spinach and radish Larson and John Gordon for their advice and suggestions during the course of these experinments. leaves, was due primarily to an increase in the chlorophyll a component in the present study, probably reflecting synthesis LITERATURE CITED of an active form of this pigment. Hill reaction in isolated from cotton- 1. ARNON-, D. I. 1949. Copper enzymes in isolated chloroplasts. Polysphenoloxi- activity chloroplasts dase in Beta vulgaris. Plant Physiol. 24: 1-15. wood leaves of LPA 0 to 3 was very low but increased rapidly 2. BEN-EDICT, C. R. ANDn R. J. KOHEL. 1969. The synthesis of ribulose-1,5-di- in leaves LPA 4 or older (Fig. 3). Thus, the rapid rise in chlo- plsosphate carboxylase and chlorophyll in virescent cotton leaves. Plant rophyll a/b ratio preceded by several plastochrons the develop- Physiol. 44: 621-622. 3. BJMRKMAN, 0. 1968. Carboxydismutase in and sun- ment of Hill activity in the critical zone where photosynthetic activity shade-adapted adapted species of higher plants. Physiol. Plant. 21: 1-10. CO2 uptake increased rapidly. A similar lag in Hill activity was 4. BRADBEER, J. W. 1969. The activities of the photosynthetic carbon cycle noted by Thorne and Boardman (17) in greening pea leaves. enzymes of greening bean leaves. New Phytol. 68: 233-245. They concluded that during chloroplast development there is 5. DICKMANN. D. I. 1971. Photosynthesis and respiration by developing leaves of cottonwood (Populus deltoides Bartr.). Bo-. Gaz. In present in the plastid some chlorophyll which has not been press. 6. FULLER, R. C. AND M. GIBBS. 1959. Intracellular and phylogenetic distribution incorporated into the pigment assemblies of either photosys- of ribulose-1,5-diphosphate carboxylase and D-glyceraldehyde-3-phosphate tem I or II. The genesis of the energy conversion system of dehydrogenases. Plant Physiol. 34: 324-329. maturing cottonwood leaves in the present study might then 7. IRVING, A. A. 1910. The beginning of photosynthesis and the development of chlorophyll. Ann. Bot. 24: 805-818. be as a two sequence: initial envisaged step rapid synthesis of 8. LARSON, P. R. AND J. C. GORDON. 1969. Leaf development, photosynthesis, and chlorophyll a followed by assembly of the grana membranes C"4 distribution in Populus deltoides seedlings. Amer. J. Bot. 56: 1058-1066. in the chloroplasts, with the concomitant increase in Hill ac- 9. LARSON, P. R. AND J. G. ISEBRANDS. 1971. The plastochron index as applied tivity. to developmental studies of cottonw ood. Can. J. Forest Res. 1: 1-11. RuDP carboxylase, the major enzyme responsible for the 10. SESTAK, Z. 1963. Changes in the chlorophyll content as related to photosynthetic activity and age of leaves. Photochem. Photobiol. 2: 101-110. initial fixation of CO2 in photosynthesis, is localized in the 11. SESTAK, Z. 1969. Ratio of photosystem I and II particles in young and old chloroplasts of higher plants (6, 15) and it may constitute 5 leaves of spinach and radish. Photosynthetica 3: 285-287. to 10% of the total soluble protein of leaves (19). Thus, this 12. SESTAK, Z. AN-D J. CATSKY. 1962. Intensity of photosynthesis and chlorophyll enzyme should be correlated with net content as related to leaf age in Nicotiana sandarae Hort. Biol. Plant. 4: highly photosynthesis 131-140. in developing leaves, a statement borne out by the present 13. SINGH, R. N. AND K. N. LAL. 1935. Investigation of the effect of age on study (Fig. 4). RuDP carboxylase levels were low in young assimilation of leaves. Ann. Bot. 49: 291-307. cottonwood leaves incapable of net photosynthesis, whereas 14. SMILLIE, R. M. 1962. Photosynthetic and respiratory activities of growing the development of enzyme activity closely paralleled the in- pea leaves. Plant Physiol. 37: 716-721. crease in rate in older leaves 15. SMILLIE, R. M. 1963. Formation and function of soluble proteins in chloro- photosynthetic (Fig. 4). These plasts. Can. J. Bot. 41: 123-154. are findings in agreement with those of Smillie (14) for grow- 16. SMILLIE, R. M. AN-D G. KROTKOV. 1961. Changes in the dry weight, protein, ing pea leaves and Bradbeer (4) for greening bean leaves. nucleic acid and chlorophyll contents of growing pea leaves. Can. J. Bot. In contrast, a close relationship of RuDP carboxylase to 39: 891-900. 17. total chlorophyll in expanding leaves has not been established THORNE, S. WV. AND N. K. BOARDMAN. 1971. Formation of chlorophyll b, and the fluorescence properties and photochemical activities of isolated were (2). Similar results shown by the present study, particu- plastids from greening pea seedlings. Plant Physiol. 47: 252-261. larly in later stages of leaf development when total chloro- 18. TOGASAKI, R. K. -ND R. P. LEVINE. 1970. Chloroplast structure and function phyll increased at a faster rate than did RuDP carboxylase. in ac-20, a mutant strain of Chlamydomonas reinhardi. I. C02 fixation and Furthermore, the development of Hill activity in chloroplasts ribulose- 1, 5-diphosphate carboxylase synthesis. J. Cell Biol. 44: 531-539. isolated from cottonwood leaves lagged behind RuDP carbox- 19. WEISSBACH, A., B. L. HORECKER, AND J. HURWITZ. 1956. The enzymatic formation of phosphoglyceric acid from ribulose diphosphate and carbon ylase activity in the zone where net photosynthesis increased dioxide. J. Biol. Chem. 218: 795-810.