Plant Physiol. (1983) 71, 680-6870032-0889/83/7 1/0680/08/$00.50/0

Influence of Adenosine Phosphates and Magnesium onPhotosynthesis in Chloroplasts from Peas, Sedum, and Spinach1

Received for publication July 16, 1982 and in revised form October 26, 1982

GEORGE J. PIAZZA2 AND MARTIN GIBBSInstitutefor Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT

14CO2 photoasshnilation in the presence of MgATP, MgADP, andMgAMP was investigated using intact chboroplasts from Sedumpraeakwn,a Crassulacean acid metabolism plant, and two C3 plants: spinach andpeas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mge werepresent in the assays, their influence upon COs assimilation was alsoexamined. Free Mg2 was Inhibitory with all chloroplasts, as were ADPand AMP in chloroplasts from Sedun and peas. With Sedun chloroplastsin the presence of ADP, the time course of assimilation was linear.However, with pea chloroplasts, ADP inhibition became progressively moresevere, resulting in a curved time course. ATP stimulated assimilation onlyin pea chloroplasts. MgATP and MgADP stimulated assimilation in allchloroplasts. ADP inhibition of CO2 assimilation was maximal at optimumorthopbosphate concentrations in Sedun chloroplasts, while MgATP stim-ulation was maximal at optimum or below optimum concentrations oforthophosphate. MgATP stimulation in peas and Sedwm and ADP inhibi-tion in Sedun were not sensitive to the addition of glycerate 3-phosphate(PGA).

PGA-supported 02 evolution by pea chloroplasts was not inhibitedhnmediately by ADP; the rate of 02 evolution slowed as time passed,corresponding to the effect of ADP on CO2 assimilation, and indicatingthat glycerate 3-phosphate kinase was a site of inhibition. Likewise, uponthe addition of AMP, inhdbition of PGA-dependent 02 evolution becamemore severe with time. This did not mirror CO2 assimilation, which wasinhibited immediately by AMP. In Sedwn chloroplasts, PGA-dependent 02evolution was not inhibited by ADP and AMP. In chloroplasts from peasand Sedun, the magnitude of MgADP and MgATP stimulation of PGA-dependent 02 evolution was not much larger than that given by ATP, andit was much smaDler than MgATP stimulation ofCO2 assimilation. Analysisof stromal metabolite levels by anion exchange chromatography indicatedthat ribulose 1,5-bisp te carboxylase was inhibited by ADP andstimulated by MgADP in Sedn chloroplasts.The appearance of label in the medium was measured when IU-`4C1

ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, orAMPand their Mge, complexes. The rate of back exchange was stimulated bythe presence of Mg2'. This suggests that ATP, ADP, and AMP penetratethe chloroplast slower than their Mg2e complexes. A portion of the CO2assimilation and 02 evolution data could be explained by differentialpenetration rates, and other proposals were made to explain the remainderof the observations.

There have been a number of studies on the influence ofadenosine-P (ATP, ADP, AMP) on photosynthesis in intact, iso-

'This research was supported by National Science Foundation GrantPMC-8141742.

2Present address: Lawrence Berkeley Laboratory, Division of ChemicalBiodynamics, Bldg. 3, Berkeley, California 94720.

lated chloroplasts. With spinach chloroplasts effects are small ornil (22), although penetration through the chloroplast membranehas been shown to occur via an adenosine-P translocator (14). Incontrast, pea (25, 31) and wheat (9) chloroplasts from very youngplants are sensitive to adenosine-P. In all of these studies, scantattention was paid to the effect of Mg2+, even though MgATP isrequired for the kinases of the reductive pentose-P cycle (26), andMgADP is used by coupling factor (4). In addition, transportacross the chloroplast membrane of externally added adenosine-P(without Mg2+) could result in chelation of chloroplastic Mg2+,affecting the activity of the Mg2"-requiring enzymes, fructose andsedoheptulose bisphosphatase (6, 24) and RuBP3 carboxylase (2).In this paper, the dependence of CO2 assimilation upon theaddition of adenosine-P with and without Mg2' has been investi-gated in isolated chloroplasts from two C3 plants, peas and spin-ach, and Sedum, a CAM species. An attempt was made to pinpointreaction steps of the reductive pentose-P czcle or other factorswhich are responsive to adenosine-P and Mg +, using chloroplastsfrom peas and Sedum.

In studying the interaction of Mg2+ with adenosine-P, it isessential that the real concentration of the Mg2+ complex (asopposed to free adenosine-P) be taken into consideration. Suchan analysis begins by calculation of the apparent metal-ligandstability constant at the medium pH. For any metal binding ligandL which has two different relevant states of ionization, both ofwhich can bind a metal ion M, the following equilibria apply,where Ka is the acid dissociation constant of the ligand, and K1and K2 are the metal stability constants for the more protonatedand less protonated forms of the ligand, respectively.

-Ha Ka -a-i HLHnw- _" Ln-i H

K1JF J[K2

-fa+2 -aolLHnM a LHn-1M

The charge of the protonated ligand is -a, and the metal ion isassumed to be divalent. The apparent metal binding capacity ofthe ligand at any pH is given by 30

K.p = K1 + K2 (Ka/H+)1 + Ka/H+

In this paper, Kpp values (with the subscript dropped) will be used

3Abbreviations: RuBP, ribulose 1,5-bisphosphate; FBP, fructose 1,6-bisphosphate; FCCP, carbonylcyanide-4-trifluoromethoxy phenylhydra-zone; G3P, glyceraldehyde 3-phosphate; HMP, hexose monophosphates;PGA, glycerate 3-phosphate; Ru5P, ribulose 5-phosphate; SBP, sedohep-tulose 1,7-bisphosphate; TP, triose phosphate.

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INFLUENCE OF ADENOSINE-P AND Mg2+ ON PHOTOSYNTHESIS

as necessary with reference given for the true acid dissociation andstability constants. This treatment is an oversimplification, sinceit fails to account for competition between Na+ or K+ and thedivalent metal (21). In practice, this results in only a minor errorwhich is partially compensated because K1 and K2 values aredetermined in the presence of some salt. Of somewhat greaterimportance may be differences in the mode of action of Mg2+-adenosine-P complexes with their state of protonation. Such adifferentiation is beyond the scope of this paper, and only the sumof all forms of free or complexed adenosine-P will be considered.It should be noted, however, that the predominant form of aden-osine-P at the pH values used here has its phosphate ester fullyionized.The standard Mg2+ chloroplast medium contains 5 mm EDTA,

as well as Mn2+. At 25°C and pH 7.6, log KEgTA = 6.3, and logKEnTA = 11.2 (28); EDTA strongly prefers to bind Mn2 . In thepresence of 6 mM Mg2+ and 1 mm Mn2+, the concentration ofMnEDTA is approximately 1 mm, and the concentration ofMgEDTA is approximately 4 mm, with only a trace of free Mn2+and approximately 2 mm free Mg2+. Log KmTp, log KQADP, and logK are 4.0, 3.1, and 1.9, respectively, at 25°C and pH 7.6 (29).The stability constant for the strongest Mg2+ chelator, ATP, isabout 2 orders of magnitude lower than that of EDTA. If Mn2+ isconsidered, this difference is much larger. Hence, the binding ofMg2e or Mn2+ to EDTA is essentially unaffected by the additionof any adenosine-P. When 5 mm ATP, ADP, or AMP is added tothe medium containing 2 mm free Mg2+, the concentration ofMgATP, MgADP, or MgAMP is 1.9, 1.7, or 0.6 mm, respectively.The concentration of free Mg2+ is, therefore, 0.1, 0.3, or 1.4 mmwith added ATP, ADP, or AMP. This difference is important,since free Mg2+ can be a good inhibitor of CO2 assimilation inintact chloroplasts (8). In all cases, there is a considerable amountof uncomplexed adenosine-P present in the medium. The experi-mental conditions used here keep free Mg2+ to a minimum butsuffer the disadvantage of variable component composition.

MATERIALS AND METHODS

Chemicals and Supplies. [14CJSodium bicarbonate was suppliedby Amersham. MgCl2.6H20 was from Fisher Scientific. Thesodium salts of ATP (equine muscle), ADP (Grade III), and AMP(Type II) were supplied by Sigma, as was PGA. Aluminumplanchets were purchased from Coy Laboratory Products, AnnArbor, MI. All other chemicals were reagent grade. Water wasdistilled from an all glass apparatus.

Plants. Sedum praealtum and peas (Pisum sativum var. ProgressNo. 9) were cultivated in a greenhouse. Spinach (Spinacia olera-cea) was cultivated under controlled conditions (12 h light, 22°C/12 h dark, 18°C). Spinach and Sedum were grown in a vermiculite-soil mixture (1:1), and peas were grown in vermiculite, afterovernight soaking in aerated water. Pea plant age was measuredfrom the day of planting. All plants were fertilized weekly withone-quarter strength Hoagland solution.

Chloroplast Isolation. Sedum chloroplasts were isolated fromprotoplasts, as previously described (23). Spinach and pea chlo-roplasts were isolated by mechanical homogenization of leavesand were purified on a Percoll cushion (20). Chloroplasts were 80to 90%o intact based on ferricyanide assay (17)."CO2 Assimilation. For those experiments with Mg2+, assimi-

lation with Sedum and pea chloroplasts was conducted in a 1-mlreaction medium containing 0.33 M sorbitol, 50 mm Hepes-NaOH(pH 7.6), 5 mm NaH34C03, 1000 units of catalase, 0.25 mMKH2PO4, 5 mm EDTA, 6 mm MgCl2, 1 mm MnC12, 5 mm adeno-sine-P, and 20 to 50 ,ug Chl. The spinach reaction medium was thesame except that 0.05 M Tricine-NaOH (pH 8.1) was used. Theexperimental protocol was as follows. A stock solution was pre-pared in dim light containing chloroplasts and all of the compo-nents listed above except that the adenosine-P was deleted and

the MgCl2 concentration was only 1 mm. A solution of adenosine-P plus MgCl2 was added into 55 x 17 mm culture tubes held in awater bath. This solution was previously titrated to the appropriatepH with NaOH. After mixing the stock solution, aliquots wereplaced in each culture tube. CO2 assimilation was initiated bybanks of 150-w flood lamps providing 620 w/m2. The temperaturewas 30°C with Sedum and 25°C for peas and spinach. For thoseexperiments lacking Mg2+, CO2 assimilation was conducted asdescribed above, except the EDTA concentration was 2 mm.

Assimilation rates were determined by spotting 100-Pd aliquotsat 3- to 4-min intervals on aluminum planchets containing lenspaper and 0.1 ml 0.5 N HCI. Upon drying, the sample radioactivitywas determined using a Nuclear Chicago gas-flow counter withan efficiency of about 14%. In most cases after an initial lag periodof 3 to 5 min, the rates of assimilation were linear for the next 20min. Unless otherwise specified, assimilation rates were calculatedfrom data taken between 6 and 20 min.PGA-Dependent 02 Evolution. 02 evolution was measured po-

larographically in a Clark-type 02 electrode at 25°C with peasand 30°C with Sedum. The reaction chamber was illuminatedwith a 500-w projector lamp. The incident beam passed through5 cm water and provided 430 w/m2. The assay volume was 1 ml,and the reaction medium was that used for CO2 assimilation,except that 5 mm PGA was substituted for bicarbonate. Solutionsof adenosine-P were prepared in concentrated form such thataddition of 20 p1 gave a final concentration of 5 mm. The influenceof adenosine-P upon 02 evolution was determined as follows.Chloroplasts were assayed in the light with PGA until a lineartrace was obtained (about 4 min). The adenosine-P was added,and the light was switched off for 2 to 3 min. The light was thenswitched on, and 02 evolution was assayed until a linear trace wasobtained (about 4 min). The dark incubation period was requiredfor maximum adenosine-P effect. Control experiments showedthat the intervening dark period had no effect on the basilary rateOf 02 evolution.Transport of Adenosine-P across the Chloroplasts Membrane.

Sedum chloroplasts were incubated in 0.1 mM [U-14C]ADP (49.6,uCi/tLmol) on ice for 1.5 h in a 1 ml medium containing 0.33 Msorbitol, 50 mM Hepes-NaOH (pH 7.6), 2 mm EDTA, 2 mMMgC12, 0.5 mm MnC12, and 0.4% (w/v) BSA. After separation ofthe intact and broken chloroplasts on a Percoll gradient (23), thechloroplasts were washed once with Mg2+-free reaction mediumand challenged with exogenously added adenosine-P (5 mM) in amedium (1.15 ml) containing 0.33 M sorbitol, 50mM Hepes-NaOH(pH 7.6), and 6 mM MgCI2, 5 mm EDTA, or 2 mm EDTA whenno Mg2+ was added. After addition of the adenosine-P at 4°C, thechloroplasts were separated from the medium (0.15-ml aliquots)using silicone oil centrifugation at 15- to 20-s intervals for the firstmin, and at 1-min intervals after that. Exchange was about 50%complete in 4 min with MgATP.

RESULTS

CO2 Assimilation. Table I shows the rate of CO2 assimilationby chloroplasts from Sedum, spinach, and peas in the presence ofadenosine-P, with and without Mg2+. The data were taken froma single batch of chloroplasts, except that with Sedum the datawere compiled from a number of experiments. Without exception,MgATP stimulated the assimilation rate, although the magnitudewas variable. In particular, Sedum and young pea chloroplastsshowed the largest stimulation, but the effect was small in spinachchloroplasts. As the pea plants aged, the rate of CO2 assimilationfrom isolated chloroplasts decreased. MgATP could not restorethe rates in chloroplasts from 21-d-old peas to those of chloroplastsfrom younger plants (Table I). The rate of dark CO2 assimilationwith Sedum chloroplasts in the presence ofMgATP was about 3%the light rate versus 0.4% for a control. Hence, the contribution ofdark assimilation was negligible. ATP alone stimulated assimila-

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Plant Physiol. Vol. 71, 1983

Table I. Effect ofAdenosine-P on the Rate of CO2 Photoassimilation byIsolated Chloroplasts

"4CO2 incorporation was measured at 4-min intervals. The rates ofassimilation were calculated from data taken after the lag period exceptfor pea chloroplasts in ADP where rates were calculated from data takenbetween 11 and 23 min. All data were obtained from a single batch ofchloroplasts, except that with Sedum the data were taken from a numberof experiments. The control rates of assimilation in pmol C02/mg Chl hwere 3 to 7 for Sedum, 15 for spinach, 14 for 12-d peas, and 2.5 for 21-dpeas. Normal assimilation mediums were used.

PeaCondition Sedum Spinach

12-d 21-d

Control 100 100 100 100ATP 100 100 266 159MgATP 383 138 894 353

ADP 43 100 21 38MgADP 430 134 132 108

AMP 23 95 2 9MgAMP 41 41 16 19

Table II. Effect ofMgCl2 on CO2 Assimilation in Sedum, Spinach, andPeas

The data for spinach were taken from the work of Demmig andGimmler (8). Assimilation was conducted as usual except that EDTA andMn24 were deleted from the reaction mixture. The control rates of assim-ilation were 2, 82, 5, and 15 ,umol C02/mg Chl.h for Sedum, spinach, pea1, and pea 2, respectively. The pea chloroplasts were prepared from plantswhich were I d old.

MgCl2 Sedum Spinach Pea 1 MgC12 Pea 2

mM % mM %

0 100 100 100 0 1000.05 114 100 0.01 860.5 88 42 96 0.05 481 72 38 101 0.1 435 69 18 73

25 35 55

tion only in pea chloroplasts; no effect was observed with Sedumor spinach. MgADP stimulated assimilation in Sedum, spinach,and pea chloroplasts, but the magnitude was large only in Sedum,suggesting that photophosphorylation was rapid in this case. ADPand AMP were good inhibitors of assimilation in Sedum and peachloroplasts, but no effect was seen with spinach. In the chloro-plasts from older peas, the inhibitory effect of ADP was greatlyreduced. With all chloroplasts except spinach, the addition ofMg2e to AMP resulted in a slight reduction ofAMP inhibition.

Table II shows the effect of free Mg2+ on CO2 assimilation inSedum, spinach, and pea chloroplasts. In all three cases, MgC12inhibited assimilation, except for a small stimulation in Sedumwith a very low concentration ofMgCl2. In addition, the sensitivityto Mg2+ varied in different chloroplast preparations, as shown bythe two experiments with peas. These data show that free Mg2+ isnot a factor in stimulation ofCO2 assimilation by Mg2+-adenosine-P complexes. With spinach chloroplasts, the inhibition by MgAMPof assimilation (Table I) probably resulted from free Mg + (1.4mM) present in the assay. With Sedum chloroplasts, decreasing theAMP concentration from 5 to 1 mm resulted in a very smallincrease in the rate of assimilation (31% of the control). Thisdemonstrates that the diminished inhibition observed withMgAMP cannot only be a consequence of a decreased free AMPconcentration (4.4 mM). Thus, in Sedum, and possibly in peas,

MgAMP is stimulatory, but its influence is masked by free AMPand Mg24. It is also likely that free Mg2+ (0.3 mM) diminished theobserved MgADP stimulation in pea chiloroplasts.

Since Sedum and pea chloroplasts showed the greatest sensitiv-ity to adenosine-P, their response to these compounds was inves-tigated in some detail. Figure 1 shows the time course of C02assimilation in the presence of adenosine-P by pea chloroplastsfrom 1 1-d-old plants. Two controls were run, one with 2 mm

15

MgATP12

9

AT_6

qV,control

w4 0

O 3 7 11 15 19 23

~2.5

2.00 A

21.5

1.0

.5

o ~ ~ ~ ~ MAMP

0 3 7 11 15 19 23TIME (min)

FIG. 1. Time course of CO2 assimation in chloroplasts from I 14-oldpea plants in the presence of adenosine-P with and without Mg2+.

I * Lkner Rate

?2I

04

05 0

a a

x1

0 5 10 15 20 25ATP CONCENTRATION (mM)

FIG. 2. Influence ofATP (without Mg2+) on the lag and linear rates ofCO2 assimilation in Sedun chloroplasts. The lag rate was calculated fromthe amount of "4CO2 incorporated during the fuist 8 min of assimilation.

a

682 PIAZZA AND GIBBS

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INFLUENCE OF ADENOSINE-P AND Mg2+ ON PHOTOSYNTHESIS

zo2401.

0

-JZ

ATPCO 140

UwU(~

< ~~~~~ATP ~ ATPI

0 0.5 1.0

FCCP CONCENTRATION (xle M)

FIG. 3. Influence of MgATP and ATP on FCCP inhibition of CO2assimilation in Sedum chloroplasts. FCCP was dissolved in ethanoL and10 i1 was added to each assay containing a total volume of 1.0 ml. Ten

id ethanol was added to the controls. The assays containing MgATP or

ATP are indicated, while the empty bars contain no adenosine-P.

Table III. Back Exchangefrom Sedum Chloroplasts Loaded with [U-'4C1ADP

The indicated adenosine-P was added to chloroplasts, and the rate ofappearance of label in the medium was measured at 4°C in dim light.

Addition Exchange Rate

relativeMgATP 100MgADP 72MgAMP 42ATP 84ADP 18AMP 17

EDTA and 1 mM Mn2+, another with 5 mim EDTA, 1 MM Mn2+,and I mm Mg2+. The rates of assimilation were identical in eithercase, showing that only free Mg2e influences assimilation, and thatEDTA does not affect the chloroplasts directly. Except with ADP,the rate of assimilation was linear to about 23 min. Reproducibly,the initial rate of steady state assimilation at 3 to 7 min with ADPwas the same or somewhat greater than the control rate, but afterII to 15 min, assimilation nearly ceased. In contrast, with Sedumchloroplasts, ADP inhibition was fully evident after the lag period(data not shown).ATP without Mg2e did not enhance the rate of assimilation in

Sedum (Table 1), but the lag period was visibly shortened (Fig. 2).Determination of the exact length of a lag period is difficultexperimentally. However, if the amount of 1 CO2 incorporated ina short time period after the start of illumination is determined,the apparent rate of incorporation reflects the length of the lagperiod, ie. a low rate is indicative of a long lag. The apparent rateof assimilation 0 to 8 min was measured and designated the 'lagrate.' With no ATP added, the lag rate was about one-sixth of thelinear rate (Fig. 2). At concentrations of ATP above 10 mm, thelag rate increased to equal the linear rate, indicating the completeabsence of a lag period. Hence, these data show that ATP canpenetrate the Sedum chloroplast membrane, and ATP is a limitingfactor during the lag period, but not during steady state CO2assimilation.The contention that ATP is not a limiting factor during CO2

assimilation by Sedum chloroplasts is supported by the data shownin Figure 3. Here the effects of MgATP and ATP were observedin the presence of FCCP, an inhibitor of photophosphorylation.

At 0.1 sum FCCP, ATP does provide stimulation, although neitherATP nor MgATP could restore assimilation rates to control values,possibly due to inhibition of the adenosine-P translocator byFCCP. Chlorocarbonylcyanide phenylhydrazone has been re-ported to inhibit the translocator of spinach chloroplasts (14).

Transport of Adenosine-P across the Chloroplast Membrane.Back exchange from Sedum chloroplasts loaded with [U-14CJADPwas measured (Table III). Even when no adenosine-P was addedto the loaded chloroplasts, some label appeared in the medium;all rates were corrected for this. The data show that the rate ofexchange decreased in the order ATP > ADP > AMP. Theaddition of Mg2' had a pronounced stimulatory effect on the rate.The Mg2e-complexed nucleotides may be preferentially ex-changed, but it is also possible that the activity of the translocatoris Mg + dependent.

Because of this finding, it seemed possible that ATP did notstimulate assimilation in pea chloroplasts as well as MgATP, onlybecause ATP had a lower penetration rate, resulting in lowerchloroplastic levels of adenosine-P. To check this idea, pea chlo-roplasts were incubated for various lengths of time in dim light

Table IV. Influence of the Time of Incubation in the Dark upon theStimulation of CO2 Assimilation in Pea Chloroplasts by MgA TP and A TPThe reaction media were prepared as for CO2 assimilation and con-

tained ATP or MgATP. The medium was placed into a 2.4 x 11 cm glasscentrifuge tube covered with aluminum foil and kept at room temperature.At zero time, the chloroplasts (13-d-old peas) were added. At the indicatedtime, a 1.0-ml aliquot was removed to an illuminated reaction tube at25°C. '4C02 incorporation was measured at 4-min intervals.

Dark Experiment ia Experiment 2'Incubation

Time MgATP ATP MgATP ATP

min pmol C02/mg Chl.h0.5 76.2 (60) 29.0 (63) 81.3 (64) 59.0 (100)2.2 81.3 (64) 32.2 (70)5.5 92.7 (73) 59.6 (86) 90.2 (71) 58.4 (99)8.5 108 (85) 42.3 (92)11.0 127 (100) 46.0 (100) 127 (100) 58.4 (99)18.0 127 (100) 55.5 (94)26.0 108 (85) 38.9 (66)

a Per cent incorporation is shown in parentheses.

Table V. Effect ofAdenosine-P on the Rate ofPGA-Supported 02Evolution

The data for Sedum were obtained from a single batch of chloroplasts,and the assays were conducted at 30°C with 51 isg Chl/ml. Pea chloroplastswere obtained from 11-d-old plants. PGA-dependent 02 evolution (16.5yg Chl/ml) and CO2 assimilation in the absence of PGA (22.0 ,ug Chl/ml)were conducted with a single batch of chloroplasts at 25°C.

02 Evolution' CO2 AssimilationCondition

Sedum Pea (Pea)aumol/mg Chl h

Control 12.1 (100) 67.8 (100) 10.7 (100)MgATP 36.9 (294) 111.0 (164) 75.0 (701)MgADP 31.4 (260) 71.4 (105) 14.3 (134)MgAMP 24.2 (200) 48.8 (72) 0.34 (3.2)

ATP 19.2 (159) 102.2 (150) 39.0 (364)ADP 20.1 (166) 91.7 (135; 0-2 min) 16.3 (152; 3-7 min)

52.4 (77; 2-6 min) 5.8 (54; 7-11 min)27.3 (40; 6-12 min) 1.8 (16; 11-23 min)

AMP 12.3 (102) 53.9 (79; 0-2 min) 0.31 (2.9)a Per cent incorporation is shown in parentheses.

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Plant Physiol. Vol. 71, 1983

with either MgATP or ATP, and then the rate ofCO2 assimilationin the light was determined. In Table IV are shown the results oftwo such experiments with 13-d-old peas. In the first experiment,stimulation with both ATP and MgATP increased somewhat withincreasing incubation time, but by less than 2-fold; this increase issmall compared to the 9-fold stimulation given by MgATP com-pared to control rates (Table I). In the second experiment, increas-ing incubation time did not influence the rate with ATP, but didslightly with MgATP. Hence, these data demonstrate that the roleof Mg2+ in the stimulation of C02 assimilation is not due to itsability to enhance chloroplastic levels of adenosine-P.PGA-Supported 02 Evolution. In the reductive pentose-P cycle,

MgATP is required for the'conversion ofPGA to G3P and for thesynthesis of RuBP from Ru5P. The rate of MgATP utilization inthe production of G3P can be monitored by measuring the rate ofPGA-supported 02 evolution (5 mm PGA is substituted for 5 mMbicarbonate in the reaction medium). The results of such studieson Sedum and pea chloroplasts are shown in Table V. In addition,for peas, CO2 assimilation rates are shown which were obtainedwith the same batch of chloroplasts and at the same time as the 02

evolution data. In order to mimic the conditions of CO2 assimi-lation, the chloroplasts were subjected to a dark incubation period(2 min for peas, 3 min for Sedum) with adenosine-P, before 02

evolution in the light was recorded. Unlike CO2 assimilation, lagperiod in 02 evolution was less than 0.5 min.The control rates of PGA-dependent 02 evolution were much

higher than rates of CO2 assimilation for peas and somewhathigher for Sedum. However, during steady state C02 assimilationonly- two-thirds of the available MgATP is utilized for G3Pformation. Multiplying the control and MgATP 02 evolution rates(Table V) for pea chloroplasts yields 45.2 and 74 pmol/mg Chl.h. For MgATP, the corrected rate of G3P formation is equal tothe rate of CO2 assimilation. Hence, the rate of G3P formation isa limiting factor for C02 assimilation in the presence of MgATP,possibly because the supply of NADPH is limiting. This conclu-sion assumes, however, that the penetration rate of PGA into thechloroplast during the 02 evolution assay is not a limiting factor;this seems likely, given the high rates ofPGA transport measuredwith spinach chloroplasts (10). For the control, the 'corrected' rateof G3P formation was substantially higher than the rate of CO2assimilation. This suggests that some step(s) of the reductivepentose-P cycle after G3P formation is rate determining. Additionof MgATP may accelerate G3P formation, but its main influenceis expected in other parts of the reductive pentose-P cycle. Thereis a possible pitfall to this argument, however, and this concernsthe levels of reductive pentose-P cycle intermediates, and whetherthese are saturating to the same degree in the presence of 5 mmPGA as with HCO. It is possible that the acceleration in C02assimilation with MgATP results from increased levels of cycleintermediates as well as ATP. To check this possibility, the stim-ulation in CO2 assimilation by MgATP was measured in thepresence ofa range ofPGA concentrations. With pea chloroplasts,MgATP had the ability to accelerate assimilation even in thepresence of PGA, and the per cent stimulation by MgATP was

only slightly lower with PGA pr'esent than without PGA (Fig. 4).Both MgATP and MgADP also provided substantial stimulationin CO2 assimilation in the presence of 2 mM PGA or FBP plusaldolase with Sedum chloroplasts (data not shown). With Sedumchloroplasts, ATP was less effective in the stimulation of PGA-dependent 02 evolution than MgATP (Table V), although anystimulation is unexpected, because ATP had no effect on C02assimilation (Table I). This is explicable if the rate of G3Pformation is fast relative to other reaction steps and not ratedetermining for CO2 assimilation, even before ATP is added. Aswith pea chloroplasts, this suggests that MgATP (or MgADP afterphotophosphorylation to ATP) enhances CO2 assimilation byaccelerating a reaction step(s) of the reductive pentose-P cycle

51

I ~ ~ ~G COCET ATIO P(M

0)390w

-i 27

in chorpassfrmlSdolreapans

0 15

0

3

0 0.3 0.5 0.9 3 7 15PGA CONCENTRATION (mM)

FIG. 4. Influence of PGA on MgATP stimulation Of CO2 assimilationin chloroplasts from 15-d-old pea plants.after G3P formation.With pea chloroplasts in the presence of ADP, the trace of

PGA-supported 02 evolution was similar to that obtained withCO2 assimilation (Table V). Initially, the rate of 02 evolution wassomewhat higher than the control rate, but it decreased graduallywith time. The 02 evolution rates are shown only to 12 min. Afterthis time, curvature was apparent in the control as well, probablydue to broken chloroplasts. It seems likely, therefore, that inhibi-tion of PGA kinase by ADP is at least partially responsible forinhibition in CO2 assimilation.When AMP was added, curvature in the 02 evolution rate was

also noted, although Table V contains only the initial rate ofevolution. This is in contrast to CO2 assimilation which wasseverely inhibited by AMP at the beginning of the light period.Because the chloroplast contains adenylate kinase (13), AMP canreact with ATP to yield ADP. It is therefore not possible todetermine whether AMP or ADP is responsible for the progressiveinhibition of 02 evolution. Inasmuch as the influence of AMPupon CO2 assimilation was much different than the influence ofADP, it seems likely that AMP has some specific site of inhibitionother than PGA kinase, and this site is in the portion of thereductive pentose-P cycle after G3P formation.With Sedum chloroplasts, Table V shows that ADP stimulated

02 evolution, and AMP had almost no effect. Hence, PGA-sup-ported 02 evolution did not show the inhibition that was observedin CO2 assimilation. The possibility that PGA is the agent respon-sible for this difference was examined by repeating CO2 assimi-lation in the presence ofPGA. The sensitivity ofCO2 assimilationto ADP was not changed (data not shown).When Mg2+ was added to either AMP or ADP, little or no

inhibition of PGA-supported 02 evolution was observed with peachloroplasts. In addition, the traces were linear or only veryslightly curved in the case ofAMP. With Sedum chloroplasts, theaddition of Mg2+ to either AMP or ADP resulted in stimulationof the rate of 02 evolution (Table V). Reference back to Table Ishows that MgADP also stimulated the rate of CO2 assimilation.At least part of this stimulation can be traced to a stimulation inthe rate of G3P formation. On the other hand, MgAMP inhibitedCO2 assimilation in Sedum, although not as severely as AMP. Thefailure of CO2 assimation to reflect the increase in G3P produc-tion with MgAMP is probably due in large measure to secondaryinhibition by free Mg +.

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INFLUENCE OF ADENOSINE-P AND Mg2+ ON PHOTOSYNTHESIS

Metabolite Levels in Sedwn. As discussed above, PGA-sup-ported 02 evolution was not inhibited by the addition ofADP toSedum chloroplasts. To gain an understanding of the site ofADPinhibition, 14CO2 assimilation was conducted for 20 min and thelabeled products were separated by anion exchange chromatog-raphy (23). For comparison, experiments were also performedwith MgADP and FCCP. In Table VI, the levels of a number ofreductive pentose-P intermediates are shown. These intermediatescontained the bulk of labeled carbon; the remainder was found instarch and neutral saccharide fraction. Most of the label in TPwas found in dihydroxyacetone phosphate, indicating the activityof triose-P isomerase was large enough to establish near thermo-dynamic equilibrium. Fructose 6-P was the main component ofHMP, with the remainder being glucose 6-P.On the right side of Table VI are shown the metabolite levels

Table VI. Metabolite Levelsfrom '4Co2 Photoassimilation with SedumChloroplasts

After "'CO2 assimilation for 20 min, the stroma and medium wereseparated and analyzed by anion exchange chromatography as describedpreviously (23). Experiments with MgADP and ADP contained 59 ygChl/ml; those with FCCP contained 29 ,ug Chl/ml. The values in paren-theses are the percentage change from the control.

Control ADP MgADP Control FCCP

jumol carbon/100 mg ChlMediumHMP 17.4 9.2 (-47) 72.8 (+318) 17.6 10.4 (-41)TP 39.7 27.3 (-31) 77.9 (+96) 39.8 7.1 (-82)PGA 3.8 2.2 (-42) 10.1 (+166) 7.9 7.0 (-11)SBP 1.6 0.91 (-43) 4.8 (+200) 1.2 0.26 (-78)FBP 6.4 3.5 -45) 13.1 (+105) 4.6 0.62 (-87)

StromaHMP 4.2 3.1 (-26) 13.0 (+210) 31.0 22.5 (-27)TP 0.88 0.46 (-48) 0.87 (-1) 2.6 0.84 (-68)PGA 0.90 0.84 (-7) 1.5 (+67) 2.1 1.0 (-52)SBP 0.42 0.18 (-57) 0.75 (+79) 0.76 0.36 (-53)FBP 0.71 0.25 (-65) 1.1 (+55) 1.0 0.23 (-77)RuBP 0.41 1.3 (+217) 0.38 (-7) 1.5 0.43 (-71)

I 8 H MgATP

S\.

e.)m -

Ij

2[,

0 .15 .25 .75 1 ' 6 15 25

PHOSPHATE CONCENTRATION (mM)FIG. 5. Influence of Pi on MgATP stimulation and ADP inhibition of

CO2 assimilation in Sedum chloroplasts.

with 0.1IsM FCCP, which inhibited CO2 assimilation about 50%o.The percentage change in PGA was much smaller than that ofTP, this being most evident in the chloroplast medium where thebulk of these two compounds was found. As expected, these dataindicate a block in TP formation which results from inhibition ofPGA kinase, as a consequence of a reduced chloroplastic ATP/ADP ratio.The metabolite levels with 5 mm ADP or MgADP are shown

on the left side of Table VI. ADP inhibited the rate of CO2assimilation by 30%o. Examination of the stromal levels of PGAand TP revealed a relatively large decrease in TP. In this case,

however, the medium levels did not reflect this change, and theTP/PGA ratio of the medium increased by about 18% over thecontrol value. Consideration of the amount of PGA exported tothe medium and the assimilation rate shows that the flux fromPGA to TP in the stroma is greatly reduced in the presence ofADP. Yet the stromal level of PGA is not much lower than thecontrol value. Thus, an inhibition by ADP of enzymic activity isindicated. This results in a paradox, since PGA-supported 02evolution was not inhibited in the presence of ADP. It was

estimated that the concentration of binding sites ofRuBP carbox-ylase lies between 1.5 and 4 mm in spinach chloroplasts (27), butin Sedum in the presence of ADP, the sum of the concentrationsofPGA and RuBP is only 0.2 mm (assuming a stromal volume of25 pi/mg Chl). Thus, a considerable amount of PGA may bebound to carboxylase, and that available for conversion to TP ismuch lower than the value displayed in Table VI. In earlier '4CO2-labeling studies of the reductive pentose-P cycle (3), it was foundthat PGA was initially labeled to a higher degree than expected.We concluded that only one molecule of newly formed PGA was

in equilibrium with the PGA pool, and that the other threecarbons of RuBP were either bound or were directly converted tosome other molecule.The percentage increase in stromal RuBP was over 200%o in the

presence of ADP, indicating that the primary site of inhibition isRuBP carboxylase. Inasmuch as inhibition by ADP was observedin the presence of exogenously added PGA which serves toincrease the levels of reductive pentose-P intermediates, it is likelythat the catalytic capacity ( V,,,) of carboxylase is being affectedby ADP.

In the presence of MgADP, the stromal level of RuBP was

slightly lower than the control value, in spite of an over 2-foldincrease in the rate of CO2 assimilation; the activation of carbox-ylase is thus indicated. The net flux of TP to the medium in thepresence ofMgADP is larger than the control by about a factor of2. Since the stroma concentration was not increased, the activityof the Pi translocator toward TP must be enhanced. The stromallevel ofHMP increased by over 2-fold and the medium level over

3-fold, but increases in stromal levels of FBP and SBP were

considerably smaller. This suggests that a reaction step after HMPformation is rate determining.

Effects of Pi. Figure 5 shows that 6 mm Pi depressed stimulationof CO2 assimilation by MgATP in Sedum chloroplasts. Thissuggests that the rate-determining step of the reductive pentose-Pcycle in the presence of high Pi is not affected by MgATP. Analternative explanation is that Pi inhibits the penetration ofMgATP into the chloroplast through competitive inhibition of theadenosine-P translocator. To be effective at a concentration ofonly 6 mm (the MgATP concentration is 1.9 mM) indicates a tightbinding constant which seems unlike>, given the structural dis-parity involved. Since at pH 7.6 log K Mg is only 1.6 (29) versus 4.0for ATP, it is also unlikely that Pi exerts its effect by sequesteringM2+.MgTDpThe inhibition ofADP was apparent at optimum Pi concentra-

tions. Perhaps at very low Pi, photophosphorylation becomes ratedetermining. At very high Pi concentrations, ADP inhibition wasalso minimal, which supports the contention that the rate-deter-

*

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Plant Physiol. Vol. 71, 1983

mining step of the reductive pentose-P cycle has changed and isnot affected by ADP. The possibility that ADP andPi competefor a common inhibitory binding site on carboxylase can be ruledout, because product analysis with ahigh concentration ofPi hasshown no inhibition of carboxylase (23).

DISCUSSION

Stimulation of PGA-Supported 02 Evolution. A one-to-oneexchange of adenosine-P via a translocator of the chloroplastmembrane will not result in a net increase in chloroplastic aden-osine-P. It has been shown, however, that PPi can exchange foradenosine-P (25), and there may be other compounds in thechloroplast capable of this. It is likely that stimulation of PGA-supported 02 evolution by MgADP, ADP, and MgAMP in Sedum,and MgATP and ATP in Sedum and peas (Table V) results fromincreases in the chloroplastic levels of adenosine-P.

Stimulation of CO2 Assimilation. It was shown thatMg2+ en-hances the penetration rate of adenosine-P (Table III). An in-creased penetration rate can explain why MgATP and MgADPenhanced CO2 assimilation in spinach, while ATP and ADP hadno effect. If penetration rate is the only factor involved, it isdifficult to understand why free ADP is a potent inhibitorof CO2assimilation in both Sedum and pea chloroplasts (TableI, Fig. 1).The experiments in which pea chloroplasts were incubated in

MgATP or ATP before assimilation (Table IV) negate the possi-bility that differences in chloroplastic levels of adenosine-P areresponsible for the increased stimulation by MgATP relative toATP alone. It is possible, however, that MgATP or ATP areproviding substrate amounts of ATP during assimilation, and theincreased penetration rate of MgATP, compared to ATP, explainsthe superiority ofMgATP in CO2 assimilation by pea chloroplasts(Table I). With Sedum chloroplasts, this explanation is not viable,since ATP was shown to penetrate the chloroplast by the backexchange experiment (TableIII), and by its enhancement ofPGA-supported 02 evolution (Table V). ATP was also shown to havean effect upon the lag periodofCO2 assimilation (Fig. 2); yet ATPprovided no stimulation in Sedum chloroplasts, while MgATP did(Table I). Furthermore, an enhancement ofassimilation in Sedumby ATP could only be observed when photophosphorylation wasdeliberately crippled by FCCP (Fig. 3).That MgATP and MgADP have special effects upon assimila-

tion other than just the contribution of adenosine-P is supportedby the metabolite analysis with Sedum chloroplasts (Table VI)which demonstrated that RuBP carboxylase is stimulated byMgADP. This observation is completely consistent with the CO2assimilation (Table I) and PGA-supported 02 evolution data(Table V) from peas and Sedum which showed that some reactionstep of the reductive pentose-P cycle after TP formation is stimu-lated by MgADP or MgATP. One possible explanation for thedata is that the free Mg2e concentration in Sedum and peachloroplasts is suboptimal, and that Mg2+ is carried into thechloroplast along with either ATP or ADP.

Various measurements of stromal Mg2+ have revealed that itsconcentration is rather high and can increase by several millimolarupon illumination (15). This increase is partially responsible forlight activation ofCO2 assimilation. All of these studies have beenperformed with spinach chloroplasts, the plastids least sensitive toMgATP. Studies using the ionophore A23187 and EDTA suggestthat about one-half of the Mgz+ and all Ca2+ are bound in thespinach chloroplast stroma (19). It is possible that Sedum or youngpea chloroplasts are deficient in free Mg2+, but the total concen-tration of Mg2+ is still high.

31P NMR in intact Ehrlich ascites tumor cells indicated thatadenosine-P are not saturated with Mg2+ (12). Measurement ofadenosine-P levels has indicated an energy charge which isstrongly inhibitory toward nitrogenase in vivo (7). To explain thehigh activity of this enzyme, it has been suggested that low Mg2+

limits the concentration of MgADP (the inhibitory form of ADP).In liver cells, less than 10% of total Mg2+ is free (33). Thus, thereis some evidence in other biological systems that Mg2+ concentra-tion is not always excessive.

It cannot be ascertained whether the proposed deficiency ofMg2e isan artifact of chloroplast preparation. However, the peachloroplasts were prepared quite rapidly by mechanicalginding,and the whole procedure required only about 15 min. The lack ofany significant stimulation by free Mg2+ suggests thatMe2+ byitself does not penetrate the chloroplast membrane and, therefore,would not leak out during preparation. The membrane of spinachchloroplasts has been shown to be nearly impermeable to freeMge (11).

Recently, it has been reported that MgPPi can stop the deacti-vation of RuBP carboxylase in older (about 20 min) spinachchloroplasts (32). Perhaps as the chloroplasts age, Mg2+ leaks outor is bound to the thylakoids, and MgPPi penetrates the chloro-plast to replenish freeMg2+. Thus, Mg2+ may be transported intothe chloroplist by a variety of carriers.Inhitin by ADP and AMP. ADPinhibited CO2 assimilation

in Sedum and pea chloroplasts (Table I). In pea chloroplasts,inhibition by ADP of PGA kinase (Table V) was partially orperhaps completely responsible for inhibitionofCO2assimilation.The kinetics are strange, however, and initially ADP often stim-ulated PGA-supported 02 evolution and CO2 assimilation. It ispossible that ADP initially increased chloroplastic levels of aden-osine-P but as time proceeded depleted MgATP from the chloro-plast through counterexchange. If this were occurring, it is difficultto understand why ATP does not have a similar effect, unless it ishypothesized that the adenosine-P translocator is selective in itsrelease of Mg2+.

It is possible that ADP serves two roles. One is to penetrate thechloroplast membrane, increasing internal levels of adenosine-P.A second role is to bind to the external (sorbitol-impermeable)membrane and thereby affect internal processes. A pertinentexample is the binding of ADP (without Mg2+) to a surfacereceptor of human blood platelets which in turn inhibits adenylatecyclase (18). If this scheme is operative, Mg2+ must regulate thebinding or action of adenosine-P on the outer membrane. In thiscontext, the role of Mg2+ is indirect. A differentiation betweendirect penetration of Mg2+ and an indirect effect must awaitexperiments with radiolabeled Mg2e.AMP is a more effective inhibitor than ADP in both Sedum

and pea chloroplasts (Table I). In pea chloroplasts, AMP inhibi-tion was immediate upon illumination (Fig. 1), whereas ADPinhibition was not. One site of AMP inhibition must be after TPformation in chloroplasts from either plant. In a reconstitutedspinach chloroplast system, AMP was shown to inhibit the reduc-tive pentose-P cycle after FBP formation (16). However, chloro-plastic fructose bisphosphatase of spinach (5) as well as Ru5Pkinase of peas (1) have been reported to be insensitive to AMP.The relevance of these studies is questionable, inasmuch as theywere performed with activated enzyme. In the studies discussedhere, AMP was added to the chloroplasts in the dark when theenzymes were in the inactive, nonreduced state, and the state ofactivation may influence the sensitivity to inhibitors. Alternatively,AMP may be inhibiting at some other as yet undetected site.

Acknowledgpent-We thank Dr. G. E. Edwards for providing the stimulus for thework on pea chloropLasts.

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INFLUENCE OF ADENOSINE-P AND Mg2+ ON PHOTOSYNTHESIS

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