Plant Physiol. (1983) 73, 1002-10070032-0889/83/73/1 002/06/$00.50/0

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase inRice LeavesCHANGES IN PHOTOSYNTHESIS AND ENZYMES INVOLVED IN CARBON ASSIMILATION FROMLEAF DEVELOPMENT THROUGH SENESCENCE

Received for publication July 26, 1983

AMANE MAKINO, TADAHIKO MAE, AND KOJI OHIRADepartment ofAgricultural Chemistry, Faculty ofAgriculture, Tohoku University, 1-1 Amamiyamachi-Tsutsumidori, Sendai Japan 980

ABSTRACT

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP)carboxylase level were examined in the 12th leaf blades of rice (Oryzasativa L.) grown under different N levels. Photosynthesis was determinedusing an open infrared gas analysis system. The level of RuBP carbox-ylase was measured by rocket immunoelectrophoresis. These changeswere followed with respect to changes in the activities of RuBP carbox-ylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphatedehydrogenase, and 3-phosphoglyceric acid kinase.RuBP carboxylase activity was highly correlated with the net rate of

photosynthesis (r = 0.968). Although high correlations between theactivities of other enzymes and photosynthesis were also found, theactivity per leaf of RuBP carboxylase was much lower than those ofother enzymes throughout the leaf life. The specific activity of RuBPcarboxylase on a milligram of the enzyme protein basis remained fairlyconstant (1.16 ± 0.07 micromoles of CO2 per minute per milligram at25°C) throughout the experimental period.

Kinetic parameters related to CO2 fixation were examined using thepurified carboxylase. The K.(CO2) and V. values were 12 micromolarand 1.45 micromoles of CO2 per minute per milligram, respectively (pH8.2 and 25C). The in vitro specific activity calculated at the atmosphericCO2 level from the parameters was comparable to the in situ truephotosynthetic rate per milligram of the carboxylase throughout the leaflife.The results indicated that the level of RuBP carboxylase protein can

be a limiting factor in photosynthesis throughout the life span of the leaf.

Leaf photosynthesis shows large fluctuations with leaf age. Asthe leafgrows and chloroplasts are assembled, the photosyntheticactivity rapidly increases to a maximum rate just after fullexpansion. Thereafter, the leaf steadily loses its photosyntheticcapacity during senescence. Changes in the activities of stromalenzymes or electron transport in relation to photosynthesis havebeen studied during leaf senescence in various higher plants (4-6, 11, 27). These parameters also exhibit a large change similarto that in photosynthesis with leaf age. However, the factorsresponsible for the change in photosynthesis have yet to beelucidated.The initial reaction of photosynthetic carbon assimilation is

catalyzed by RuBP' carboxylase (EC 4.1.1.39). Since CO2 level

' Abbreviations: RuBP, ribulose 1,5-bisphosphate; Ru5P, ribulose 5-phosphate; G3P, glyceraldehyde 3-phosphate; PGA, 3-phosphoglycericacid.

in air limits the photosynthetic rate in mature leaves, the in vivofunction of the carboxylase is considered to regulate photosyn-thesis. Some investigators have reported that the in vitro RuBPcarboxylase activity through leaf senescence is highly correlatedwith the photosynthetic rate (7, 29). Friedrich and Huffaker (7)and Peoples et al. (24) found that loss of carboxylase activityduring senescence is caused by loss of enzyme protein, whereasno correlation between them was reported by Hall et al. (9).However, Camp et al. (6) have recently reported that RuBPcarboxylase activity decreases at a much faster rate than thephotosynthetic rate during senescence. Thus, the role of RuBPcarboxylase responsible for the change in photosynthesis withleaf age remains uncertain.The purpose of this study was to clarify whether RuBP car-

boxylase can be a factor that limits photosynthesis in a leafduring its life span. Using the 12th leaf blades on the main stemsof rice, we studied changes in photosynthesis and RuBP carbox-ylase, and their responses to N supply. These changes were alsofollowed in relation to changes in the activities of several otherstromal enzymes (Ru5P kinase, NADP-G3P dehydrogenase, andPGA kinase). In vitro RuBP carboxylase activity, calculated atatmospheric CO2 level from its kinetic parameters, was compa-rable to the in situ true photosynthetic rate during the life spanof the leaf.

MATERIALS AND METHODS

Plant Culture. A cultivar of rice (Oryza sativa L., cv Sasani-shiki) was used, and plants were grown to the ripening stage in agreenhouse by water culture method as reported previously (19).Seeds were soaked in tap water at 30'C for 3 d. After germination,the seedlings were grown on a saran net floating on tap water for18 d, and 12 seedlings each were then transported into forty 4-Lporcelain pots containing the nutrient solution. The basal nutri-ent solution contained 1 mm NH4NO3, 0.6 mm Na2HPO4, 0.3mM K2SO4, 0.4 mm MgCl2, and 0.2 mm CaC12, and other minornutrients. The solution was renewed once a week and its pH wasadjusted to 5.0 with 1 N HCl. The strength ofthe nutrient solutionwas varied depending on the age of the plants (days after germi-nation): I/4 strength, 21 to 35 and 140 to 154; 1/2 strength, 35 to49 and 126 to 140; 3/4 strength, 49 to 63 and 112 to 126; and fullstrength, 63 to 1 12. Heading time, defined as the time when halfof the ears on the main stems became visible, was 105 d aftergermination (August 19, 1982).The 12th leaf blades on the main stems were used as samples

throughout the experiments. The tips ofthe leaves emerged fromthe 11th leaf sheaths at the 74th d after germination. Three dlater, plants were transferred to fresh nutrient solution with

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PHOTOSYNTHESIS AND RuBP CARBOXYLASE

different NH4NO3 levels; N-deficient (without N), control, andN-sufficient (2 times the control level). The leaf samples selectedbetween 40 and 43 cm of full length were collected 14 times,respectively, for about 80 d from leaf emergence until full senes-cence. All collections and photosynthetic measurements weremade between 9 AM and 12 PM.

Photosynthetic Measurement. The photosynthetic rate wasdetermined as CO2 exchange using an open IR gas analysissystem. The 12th leaf blade attached to the plant was insertedinto an acryl chamber (4 mm interior thickness, 23 mm width),and its net CO2 exchange was measured with an IR gas analyzer(Hitachi-Horiba ASSA-2). Light was supplied through a 25-cmflowing water filter by nine 500-w flood lamps and the intensitywas adjusted to 1400 ME/mi2' s (70 klux) at the upper surface ofthe leaf. Leaf temperature was maintained at 25 ± 0.5C using afine copper-constantan thermocouple attached to the lower sur-face of the leaf by the temperature of water flowing throughwater jackets on the chamber. The flow rate of the air passingthrough the chamber was kept at 1.5 I/min. The measurementwas performed until a steady-rate ofCO2 exchange was obtained,and the rate at 300 Ml/l CO2 was corrected using the calibrationequation reported by Kahn and Tsunoda (15).Chl and Protein Determinations. Fresh leaf blades (about 2 g

fresh weight) were immediately homogenized in a chilled pestleand mortar with acid-washed quartz sand in 50 mm phosphatebuffer (pH 7.5) containing 5 mm DTT and 12.5% (v/v) glycerolat a ratio of leaves to buffer of 0.25 g/ml. Chl content wasmeasured from part of the homogenate by the method of Arnon(1). The homogenate was centrifuged at 39,000g for 20 min at 0to 4°C. A 50- to 200-til portion of the supernatant was used forthe determination of soluble protein. The pellet was washed oncewith the homogenization buffer, and washed once with 90% (v/v) acetone. This fraction was resuspended in 50 mm phosphatebuffer (pH 7.5) containing 2% (v/v) SDS and 300 mm 2-mercap-toethanol to the same final volume as the supernatant fraction,and heated at 100°C for 3 min. The suspension was centrifugedat 30,000g for 20 min at 25°C. The supernatant was used for thedetermination of insoluble protein.

Soluble protein was measured as the N content with Nessler'sreagent after Kjeldahl digestion, using TCA precipitate, whichhad been washed once with ethanol. Insoluble protein was meas-ured by the same method using a 50- to 200-Ml aliquot of thesupernatant obtained at the final step. The amount of eachprotein was calculated by multiplying its N content by 6.25.Enzyme Assays. A 1.0-ml aliquot ofthe supernatant at 39000g

for 20 min was passed through a small column (1.3 x 5.5 cm)of Sephadex G-25 previously equilibrated with 50 mm Bicine-NaOH (pH 7.8) at 4°C, containing 2 mm DTT and 0.1 mMEDTA. The eluate was used for the enzyme assays.RuBP carboxylase (EC 4.1.1.39) and Ru5P kinase (EC

2.7.1.19) activities were measured at 25°C by the radioisotopicmethod in 1.0-ml screw-capped vials equipped with septa ofTeflon rubbers (Wheaton). Reaction buffers were prepared asC02-free as possible by pH adjustment with carbonate-freeNaOH and flushing with N2 for 2 h prior to the addition ofNaH'4C03. RuBP carboxylase assay (250 Ml final volume) wascarried out for 1 min in 100 mm Bicine-NaOH (pH 8.2), 25 mMMgC12, 5 mm DTT, 10 mm NaH'4 CO3 (0.21 mCi/mmol), and0.5 mM RuBP (20). After activation of the enzyme by preincu-bating with Mg2' and HCO3 , the reaction was initiated byinjecting RuBP. Ru5P kinase assay (400 ul, final volume) wascarried out for 1 min in 100 mm Bicine-NaOH, (pH 8.3), 12.5mM MgCl2, 5 mm DTT, 5 mm Na2HPO4, 5 mm NaH'4CO3 (0.21mCi/mmol), 2 mm ATP, 1 mM RuSP, and 0.3 units RuBPcarboxylase. The reaction was initiated by injecting RuSP. Bothreaction buffers containing NaH'4CO3 were stored at -20'C in1.0-ml screw-capped vials flushed with N2 until each assay to

avoid a change in the specific radioactivity of the bicarbonateduring the experimental period.NADP-G3P dehydrogenase (EC 1.2.1.13) and PGA kinase

(EC 2.7.2.3) activities were measured at 25°C spectrophotomet-rically (340 nm) according to the methods described by Kobay-ashi et al. (16) and Pacold and Anderson (23), respectively, withminor modifications. Both enzymes were assayed after activationof the enzyme preparations by incubating with 5 mm DTT. TheNADP-G3P dehydrogenase reaction mixture contained in a finalvolume of 1200 gl: 100 mM Bicine-NaOH (pH 8.5), 20 mMNa2HAsO4, 1 mm EDTA, 5 mM DTT, 20 mM NaF, 0.6 mMNADP*, and 1 mM D-G3P. Assay was started by adding D-G3P.The PGA kinase reaction mixture contained in a fine volume of1200,Ml: 100 mM Bicine-NaOH (pH 8.4), 10 mm MgCl,, 5 mMDTT, 5 mm ATP, 0.15 mm NADH, 2.5 mM PGA, and 5 unitsNAD-G3P dehydrogenase. Assay was started by adding ATP.Under these conditions, NADH and NADPH oxidase activitiescould not be detected.

Quantitative Determination of RuBP Carboxylase by Immu-nochemical Assay. Antisera against RuBP carboxylase wereraised in two white rabbits using tobacco RuBP carboxylase. Therabbits were injected subcutaneously four times at 3-week inter-vals with 5 mg antigen emulsified with Freund's complete adju-vant, and then were bled by cardiac puncture at 10 d after thelast injection. The antisera were prepared in a conventionalmanner and stored at -25°C in small cryotubes (Nunc). Thespecificity of the antisera to rice RuBP carboxylase was verifiedusing Ouchterlony double diffusion plates and SDS-polyacryl-amide slab gel electrophoresis of the immunoprecipitates afterSDS treatment.The level of RuBP carboxylase protein in the supernatant at

39,000g for 20 min was determined by rocket immunoelectro-phoresis ( 17) according to the LKB procedure (10). After electro-phoresis, the amount of RuBP carboxylase in the crude extractswas determined from the regression line obtained from rice RuBPcarboxylase.

Chemicals. RuBP, Ru5P, PGA, and ATP were obtained assodium salts from Sigma Chemical Co. and NADP+, NADPH,and NADH were from Oriental Yeast Co., Ltd. G3P was pre-pared from the monobarium salt of G3P diethylacetal (SigmaChemical Co.). NaH'4CO3 was obtained from The Radiochemi-cal Centre Amersham. Yeast NAD-G3P dehydrogenase was ob-tained as a (NH4)2SO4 suspension from Sigma Chemical Co.,and RuBP carboxylase was purified from the rice leaves asdescribed previously (20) and stored at -20°C in a buffer con-taining 50% (v/v) glycerol. All the other reagents were fromWako Pure Chemicals Industries, Ltd. Sephadex G-25 (Fine)was the product of Pharmacia Fine Chemicals.

RESULTS

The 12th leaf blade on the main stem emerged from the 11thleaf sheath at 74 d after germination and became fully expanded10 d later. After 80 d from leaf emergence, the leaves senescedcompletely. The fresh weight of the leaves reached maximumjust before the stage of full expansion, and remained constantuntil late senescence, but then declined (Fig. 1A). The dry weightwas constant from soon after full expansion until leaf death (Fig.I B).Changes in Photosynthesis, Chl, and Proteins. Changes in

photosynthetic rate, Chl, soluble protein, and insoluble proteincontents of the 12th leaf blades of rice supplied with different Nlevels from leaf development through senescence are shown inFigure 2. Leaf photosynthesis in each N treatment rapidly in-creased during development and reached its maximum rateabout 20 d after leaf emergence. When the rate at this time wasexpressed on a leaf area basis, CO2 exchange rates of N-deficient,control, and N-sufficient leaves were 18.0, 23.2, and 24.9 mg/

1003

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Plant Physiol. Vol. 73, 1983

0.60

0.40

T 020

TV.10

0.05

4 A

- . '-L-

Io

\ ~~~~~~~~C

x

I . I I

e YX=-

MiO

o 20 40 60 80Days After Leaf Emergence

FIG. 1. Changes in fresh weight (A) and dry weight (B) of the 12thleaf blades on the main stems of rice from development through senes-cence. Plants were grown under different N levels (N-deficient [x],control [0], and N-sufficient [0]). The arrow indicates the time whenthe 12th leaf blades expanded completely.

daCC~~~mh15 * Sol. Protein

~~~, ~ +

1.X 0

10. A

.0~~~0X

1.0-~~~

E J

0 ~~ ~~~~-0

0 20 40 60 80 0 20 40 60 80Days After Leaf Emergence

FIG. 2. Changes in CO2 exchange rate, Chl, soluble protein, andinsoluble protein contents in the 12th leaf blades on the main stems ofrice from development through senescence. Plants were grown underdifferent N levels (N-deficient [x], control [0], and N-sufficient [0]).The steady-state net rate of CO2 exchange was measured by open IR gasanalysis under the conditions of 1400 MuE/in2.* s, 25°C, and 300 Ml/l CO2.ChI content was measured by the method of Amnon (1). Leaves werehomogenized in 50 mM phosphate buffer, (pH 7.5) containing 5 mMDII and 12.5% (v/v) glycerol. The insoluble fraction was solubilized inthe presence of 2% (w/v) SDS at 100§C for 3 min.

din2 *h, respectively. Therefore, photosynthesis steadily declinedduring senescence.

Leaf Chl content increased proportionally with the photosyn-thetic rate during development. However, the loss of Chl duringsenescence was not always correlated with that of photosyn-thesis.This trend was most obvious in the N-sufficient leaf. Forexample, from days 21 to 45, ChI content decreased only 10%

T2.0

_. 1.0c

0u00E

2z0

' 1.0CN0

01

R~jPcxbox44ase or =O.968 9

x xx

Ru5Pkinase 0 0 Sr=0974 O

oDr9

0 xoO.A x~a. 0

yo 0~~~00*0

0A I a

4 8,

T 10 20Units-leaf bae

NADP G3P * ,0

r = 0.947 0xxxw

w x 0hX 0O x

PGA kinase 0 ar= 0.940 0

61

000X

xx x

0 0X . x .X 0 X 00 0 0 0

20 40 10 20 30Units-leaf blade-1

FIG. 3. Relationships between CO2 exchange rate and enzyme activ-ities in the 12th leaf blades on the main stems of rice from developmentthrough senescence. Plants were grown under different N levels (N-deficient [x], control [0], and N-sufficient [0]). The steady-state net rateof CO2 exchange was measured by open IR gas analysis under theconditons of 1400 ME/Mm2-s, 25°C, and 300 ul/l CO2. Each enzyme assayis described in the text. One unit of each individual enzyme was definedas the amount that changed I gmol substrate/min at 25C. Regressionanalysis was performed using first-order kinetics.

A Control40

. NADP G3PA (',dehydogenase

A,

30

(1 0 I \ v.\.A-

Ru5PJ) V\kinase kinase (

10 6RJBP

"'0

1020 40 60

Days After Leaf EmergenceFIG. 4. Changes in the activities of RuBP carboxylase (0), Ru5P

kinase (@), NADP-G3P dehydrogenase (A), and PGA Kinase (V) and inthe steady-state net rate ofCO2 exchange (-) in the 12th leafblades on themain stems of rice from development through senescence. Experimen-tal procedure is given in Figure 3. One unit of each individual enzymewas defined as the amount that changed 1 Amol substrate/min at 25'C.One unit ofCO2 exchange was defined as the rate that exchanged I gmolC02/min under the conditions of 1400 ME/m2.s, 25°C, and 300 ul/l CO2.

while 40% of the maximum photosynthetic activity was lost.Loss of Chl is the most commonly observed symptom of senesc-ing leaves. Our results show, however, that loss of Chl does notnecessarily indicate the decrease in photosynthetic activity.

1004 MAKINO ET AL.

I

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PHOTOSYNTHESIS AND RuBP CARBOXYLASE

The amounts of soluble protein exhibited larger changes thanthose of insoluble protein from leaf emergence through senes-cence, and were apparently correlated with photosynthesis.

Relationships between the Activities of Stromal Enzymes andPhotosynthesis. Activities of RuBP carboxylase, Ru5P kinase,NADP-G3P dehydrogenase, and PGA kinase in the 12th leafblades were measured throughout the leaf life under optimalconditions. The relationships between the activities of theseenzymes and photosynthesis are shown in Figure 3. The in vitroactivities of all these enzymes, notably RuBP carboxylase andRu5P kinase, showed a high positive correlation with leaf pho-tosynthesis (r = over 0.9).

Figure 4 shows the changes in the activities of these enzymesand the rate of photosynthesis per leaf blade in control leavesfrom leaf emergence through senescence. Here, 1 unit of photo-synthesis was defined as the rate that exchanged lAmol C02/min. All enzymic activities measured in vitro were enough toaccount for leaf photosynthesis observed in situ throughout theexperimental period. The activities of RuBP carboxylase weremuch lower than those of other enzymes throughout the leaf life.The activity was only about 3 times the measured photosynthesisalthough the enzyme after full activation was measured underthe condition of CO2 saturation (about 14-fold the atmosphericCO, level). In contrast, the activities of other stromal enzymesin crude extracts were 7 to 9 times greater than the amountneeded to account for the measured apparent photosynthesis.These results were also found for N-deficient and N-sufficientleaves (data not shown).RuBP Carboxylase Protein and Activity. The relationship be-

tween RuBP carboxylase protein and its activity was examinedfrom leaf emergence through senescence. Figure 5A shows thechanges in the levels of RuBP carboxylase protein in the 12thleaf blades of rice supplied with different N levels. RuBP carbox-ylase in each N treatment rapidly increased during development.About 20 d after leaf emergence, RuBP carboxylase contentreached maximum, accounting for 53 to 55% of the soluble

Days After Leaf Emergence

FIG. 5. Changes in the level of RuBP carboxylase protein (A) andspecific activity (B) in the 12th leaf blades on the main stems of rice fromdevelopment through senescence. Plants were grown under different Nlevels (N-deficient [xl, control [0], and N-sufficient [0]). The level ofRuBP carboxylase protein was determined by rocket immunoelectropho-resis (17) according to the LKB procedure (10). RuBP carboxylaseactivity was determined by measuring the initial rate of incorporation of'4C02 into acid-stable products in a 1.0-ml screw-capped vial. Specificactivity was calculated from the level of RuBP carbxylase protein.

ca,. 1.0QL f Km(C02)=12E i E lO Vmx1.45

c~~~~~IC

E 0.5 E 50x4 E 5 l

E0 50 100 150

(PiM)0 II0 50 100 150 200

CO2, PMFIG. 6. K,4CO2) and VmOA of RuBP carboxylase purified from rice

leaves. Assay (300 .u, final volume) was carried out at 25"C in 100 mMBicine-NaOH (pH 8.2), 25 mM MgC2, 5 mM DTT, 0.5 mM RuBP, anddifferent NaH'4CO3 (0.32 mCi/mmol) concentrations in a 1.0-ml vialflushed with N2. Enzyme reaction was initiated by injecting 10 Ml ofenzyme (25.5 Mg), which had been passed through a small column (1.3x 5.5 cm) ofSephadex G-25 previously equilibrated with 100 mm Bicine-NaOH (pH 8.2), 25 mM MgCl2, 10 mm NaHCO3, and 0.2 mM NADPHat room temperature. After 30 s, the reaction was stopped by adding 60Ml HCI. The CO2 concentration in the assay buffer was calculated fromthe NaHCO3 solution added, using the Henderson-Hasselbach equationat pK' of6.37 (25"C). The slope and intercept ofthe plot was determinedby calculating the first-order regression line from the obtained data.

protein or 28 to 32% of the total leaf protein. When its contentat this time was expressed on a leaf area basis, the carboxylasecontents of N-deficient, control and N-sufficient leaves were18.1, 24.9, and 29.1 mg/dmi2, respectively. Thereafter, it de-creased rapidly during senescence. This pattern was similar tothat of photosynthesis (Fig. 2). Figure 5B shows the changes inthe enzymic activity of RuBP carboxylase/mg enzyme protein.The specific activity remained fairly constant at 1.16 ± 0.07units/mg throughout the experimental period. These results in-dicate that the change in the carboxylase activity during devel-opment and senescence was caused by a change in the level ofthe enzyme protein.RuBP Carboxylase Activity Calculated from Its Kinetic Pa-

rameters and True Photosynthetic Rate. Although the kineticproperties of the carboxylase have been studied for a number ofphotosynthetic organisms, those of the rice leaf enzyme havenot. Kinetic studies of CO2 fixation reaction were conductedusing the purified carboxylase. Prior to assay, the enzyme wasfully activated by passage through a small column of SephadexG-25 previously equilibrated with 100 mm Bicine-NaOH (pH8.2), containing 25 mM MgC92, 10 mm NaH'4CO3, and 0.2 mMNADPH at room temperature. The assay was run at 25°C for 30s to minimize deactivation of the enzyme during the reaction,especially under low CO2 concentrations. As shown in Figure 6.the measured Km for C02 was 12 Mm and the V,"a, was 1.45 MmolC02/min- mg. These values are similar to those obtained fromother higher plant carboxylases ( 12).When the carboxylase specific activity at 300 Mi/l ofCO, (10.2

Mm at 25°C) is calculated frm the kinetic parameters, the valueof 0.65 units/mg is obtained. When the photosynthetic rate/mgofRuBP carboxylase is calculated from the data shown in Figures2 and 5, its value becomes 0.38 ± 0.08 units/mg. Assuming thatthe ratio of photorespiration to apparent photosynthesis is 0.3 to

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Plant Physiol. Vol. 73, 1983

0.5 at 25-C (13, 14), the true photosynthetic rate on the carbox-ylase protein basis is between 0.49 and 0.57 units/mg. This valueis comparable to the in vitro carboxylase activity at the atmos-pheric CO2 level.

DISCUSSION

Our experimental results indicate that the RuBP carboxylaselevel can be a limiting factor in photosynthesis throughout thelife span of the leaf based on the following three findings.

First, in vitro RuBP carboxylase activity was highly correlatedwith photosynthesis from leaf development through senescence(Fig. 3). Although high correlations between the activities ofother stromal enzymes and photosynthesis were also found, theactivity ofRuBP carboxylase was much lower than those of otherenzymes (Fig. 4).

Perchorowicz et al. (25), using wheat seedlings, observed thatRuBP concentration in the stroma was 8 to 10 mm under thesaturating light condition. Although RuBP concentration wasnot measured in this study, it (8-10 mM) is much higher thanthe K,,, (RuBP) of 25 gM of the purified rice leaf enzymes (datanot shown). Because the production of RuBP depends on variousstromal enzymes, those enzymes probably do not limit photo-synthesis.During senescence, RuBP carboxylase activity decreased at a

much faster rate than Chl content, and the loss of Chl was notalways correlated with that of photosynthesis (Fig. 2). Theseresults are similar to those obtained with barley (7). Recently,Camp et al. (6), using wheat leaves, reported that the change inChl content most closely paralleled the change in photosynthesisand RuBP carboxylase activity was not highly correlated withphotosynthesis. One possible reason for this discrepancy is thedifference between the photosynthetic measurement methods.Camp et al. (6) used leaf segments under CO2 saturation (10 mmHCO3), whereas we used intact leaves and an open IR gasanalyzer. Air levels of CO2 are known to limit the rate ofphotosynthesis.

Second, the change in the in vitro carboxylase activity wascaused by a change in the level of the enzyme protein; the specificactivity on a mg of the carboxylase basis remained constantthroughout the experimental period (Fig. 5). Much has beenreported on the change in the enzymic activity of RuBP carbox-ylase per mg of the enzyme protein during senescence (7, 9, 24,26, 29) but much disagreement exists among the findings. Inaddition, the activity per mg of the carboxylase in crude extractsrange widely from 0.03 to 1.7 units/mg at 25°C (7, 9, 24-26, 29),whereas the specific activity of the purified carboxylase fromhigher plants is generally between 1.0 and 1.5 units/mg at 25°C(2, 8, 20-22, 29). Presumably, this is due to the lability of thecarboxylase activity in crude extracts (20). Since glycerol additionwas very effective for preserving enzyme activity (20), we used a

homogenization buffer containing 12.5% (v/v) glycerol and con-

ducted the assay within 2 h after homogenization. Thus, thedecrease in the enzyme activity was negligible. As shown inFigure 4B, the specific activity remained fairly constant at 1.16± 0.07 units/mg throughout the experimental period, and was

comparable to the Vnac (1.45 units/mg) obtained from the puri-fied enzyme (Fig. 6).

Third, the in vitro specific activity calculated at atmosphericCO2 level from the kinetic parameters was comparable to the insitiu true photosynthetic rate per mg of the enzyme protein duringthe life span of the leaf. Kinetic studies of the carboxylationreaction were conducted using the fully activated enzyme (Fig.6). However, a number of studies on the enzymic propertiessuggest that the amount of the carboxylase as well as the degreeto which the enzyme is activated under given field conditionsshould be considered as regulating photosynthesis (12). The ini'ivo activation state of the carboxylase responds to limiting light

(18, 25), and the enzyme is maintained in a substantially acti-vated state under saturating light conditions (3, 28). Since, inthis study, leaf photosynthesis was measured under a saturatinglight condition, most of the RuBP carboxylase was in the acti-vated state. Thus, the true photosynthetic rate per mg of thecarboxylase determined immunologically in this study may havebeen close to the specific activity of the fully activated enzyme.In addition, a high correlation between RuBP carboxylase activ-ity in vitro and photosynthesis (Fig. 3) indicates that activationof the enzyme cannot be a major factor responsible for thechange in photosynthesis with leaf age.A decrease in leaf conductance to CO2 diffusion was observed

during senescence but this change was not closely related to thedecline in photosynthesis (data not shown). However, since theintercellular CO2 level depends on both leaf conductance andthe photosynthetic rate (30) the Km(CO2) value of the carboxylaseis close to the atmospheric CO2 level, interaction between thechanges in the levels of RuBP carboxylase and intercellular CO2is considered to be an important factor. Further work aimed atresolving this problem is in progress.

Acknowledgmenis-We wish to thank Professor S. Tsunoda and Mr. M. Fuku-shima of the Department of Agronomy of this university for the use of the IR gasanalyzer, and gratefully acknowledge the assistance of Dr. T. Inamoto in prepara-tion of the RuBP carboxylase antisera. Additionally, we wish to thank Dr. K.Shimamoto for reading the manuscript and for his valuable discussions.

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10. Immunoelectrophoretic techniques with the LKB 2117 multiphor. 1978 LKBApplication Note 249

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