elk, exp(- e2k2t/vf) - plant physiology · plant physiology the other hand, if photochemically...

PLANT PHYSIOLOGY

v1vf - exp( - e1klt/vf)elk, - Vlvf

if v is known for both p, and p2 we should have 2equations

G1 + G2 = R a +K1 -

g=Ra' R2911 or

where a, A, a', and /3' stand for the respective bracket-ed terms. These equations can be solved for R1 andR9. If p, and P2 vary with time in an unknown waythen a, /3, etc. cannot be evaluated and we cannotsolve the equations.

If the lag due to resistance to interchange betweengaseous and liquid phases is ignored, that is a, etc.are assumed to be unity, and the value for pi calculatedas cl.

= K2(G1 + G2) - k2 (g1 + g2)C, ~~K2/Kl k.,/k,

then cR1(aK2/Kl - ak2/kl) + R2(/3 - /3)thenc1= K2/K1 - k,k

If subsequent to illumination oxygen productionproceeded at a steady rate pi and carbon dioxide wasconsumed at the same steady rate, that is P2 = - pl,then R1 and R2 in the above equation are replaced byp1 and - pi and

a = { 1 - exp(- EIKIt/vF) }

a = { 1 - exp(- e1k,t/vf) }

A = { 1 - exp(- E2K2t/vF) }

A3' = { 1 - exp(- e2k,t/vf }

and c1 would be an underestimate of p, at least duringthe early stages of equilibration lag. If the vesselswere paired for oxygen (1) then a = a' and

cl = pl { a K,/Kl - k2/k1 }

If, however, there was a big enough burst of CO2during this period then c1 could exceed 3,k. That isthere could be an apparent burst of oxygen.

Sufficient has been said to show that estimatesof rates of gas production from manometer readingsover a relatively short period, subsequent to a changeof conditions which result in a change in rate, canbe misleading.

LITERATURE CITED

1. EMERSON, R. and CHALMERS, RUTH. Transientchanges in cellular gas exchange and the problemof maximum efficiency of photosynthesis. PlantPhysiol. 30: 504-529. 1955.

2. VALENTINE, D. H. Induction phases in photosyn-thesis, Ph.D., dissertation, University of Cambridge,Cambridge, England. 1937.

RELATION BETWEEN RESPIRATION AND PHOTOSYNTHESISIN THE GREEN ALGA, ANKISTRODESMUS BRAUNII'

ALLAN H. BROWN AND DALE WEIS2DEPARTMENT OF BOTANY, UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MINNESOTA

In what way does light affect the respiration ofphotosynthetic cells? In more than a century sincethe reaction of photosynthesis was identified in itsoverall aspect this question has been asked repeatedly.The earliest concern was a purely kinetic one in that"apparent" photosynthesis required correction for res-piration if "true" photosynthetic rates were to be cal-culated and measured as a function of environmentalparameters. The possible influence of light on thephotosynthetic rate again became of paramount im-portance when quantum yield determinations weremade on photosynthesis beginning some 30 years ago.Encouraged by what indirect evidence was available,investigators of photosynthetic efficiency generallyhave come to rely on the assumption that, if rates of

'Received revised manuscript November 26, 1958.2 Present address: Research Institutes, University of

Chicago, Chicago 37, Ill. I

dark metabolism were the same before and after alight period, then during illumination this samerespiratory rate could be used for correcting the rateof apparent photosynthesis.

Less than 20 years ago the basic question was posednot in kinetic but in chemical terms. Thimann (18),in a speculative note, asked in effect if photosynthesiscould not be the reverse of respiration in respect to itsintrinsic mechanism as well as in its overall equation.Since then our rapidly expanding biochemical knowl-edge of respiratory pathways has several times calledfor a rephrasing of the question. The existence ofchemical intermediates common to both photosyntheticC02 reduction and to glycolysis and respiration madeit difficult to feel complacent about their kinetic inde-pendence. Also, within the last decade, has come therecognition that at least some coenzymes and electroncarriers, ubiquitous in the catabolic schemes whichmodern biochemistry uses as working models, may be

224

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

225BROWN AND WEIS-RELATION OF RESPIRATION AND PHOTOSYNTHESIS

photochemically reduced by preparations from greencells and furthermore that the pyridine nucleotide poolsof chemical potential energy, so interrelated withanaerobic and aerobic stages of respiration, are sub-ject to similar photochemical regeneration. All thismakes it seem rather unlikely that strict kinetic inde-pendence of respiratory and photosynthetic gas ex-changes should in most cases obtain, and the kineticists'assumption that respiration and photosynthesis proceedalmost without reciprocal influence (except in rela-tively long term experiments) is hardly to be antici-pated from modern biochemical considerations. Inany case, the opposing processes of respiration andphotosynthesis cannot very well employ potentiallyrate controlling reaction steps in common withoutthere being a most intimate dependence of the rate ofthe one on that of the other. Should this dependencenot be observed, we are almost compelled to assume

either that the mechanisms are truly chemically inde-pendent or that they chiefly operate in separate mor-

phological compartments of the cell. Since we are

dealing with systems which are heterogeneous in bothgross and fine aspects, perhaps whatever kinetic in-dependence of photosynthesis and respiration mav beobserved is but the fortuitous consequence of a cellularmorphology which confines the photosynthetic ap-

paratus to organelles which account for only a minorfraction of the cell's respiratory activity.

\Vhether or not biochemical compartmentalizationon morphological grounds needs to be invoked can

only be determined from kinetic observations, and thebasic question-to what extent does light affect therespiratory rate of photosynthetic cells ?-is one to besettled only by quantitative observations. Also, phy-siological variation between species being what it is,an answer to the question for one plant may not forma secure basis of generalization.

Recognizing that in the respiratory process as

well as in photosynthesis gas exchange quotients may

depart from unity, it seems that an unequivocal answer,if it can be given at all, must be provided with respectto both metabolic gases simultaneously. Also, if theanswer is to be direct, it appears that the only methodfor simultaneous nmeasurement of production and con-

sumption of a gas within a single experimental systemis by the employnment of isotopic tracers.

The essential contribution made possible by theuse of tracers is just this separation of concurrentprocesses of consumption and production of the same

gas. Throughout the present paper, when we speakof gas contsumiption, we are referring to that process

alone ithout regard to whether the same gas alsois being produced. Similarly produiction refers to theevolution of gas whether or not gas uptake occurs as

well. These terms thus do not have here the overallcharacteristic whiclh is conventional in considerationsof niet gas change.

Sonme tracer work havinig a direct bearing on thismatter already has been published. It was shown inthis laboratory, for Clilorella pyrenzoidosa Chick. andfor some other photosyntlhetic species, that illumina-

tion at low to moderate intensities had only minoreffects on the endogenous respiratory rate insofar as02 was concerned (2). These results, obtained undera necessarily limited range of experimental conditions,strongly suggested that respiratory and photosyntheticprocesses functioned quite independently. Neverthe-less evidence from tracer studies was by no means uni-lateral. In certain species, cases of extreme photo-inhibition or of moderate photostimulation of tracer02 uptake were observed (4, 13). Results from radio-carbon labelling experiments by Calvin and associates(1, 5) were interpreted as evidence that the Krebscycle does not operate in bright light and, from kineticmeasurements with tracer C02 (21), it was concludedthat C02 evolution was reduced by intense light toabout one half its value in darkness. This latter find-ing was of special interest when contrasted with meas-urements using tagged 02 (19) on the same kind ofplant (barley) and under rather similar conditions;there a marked photoinhibition of 02 was not observed.

While the various pieces of direct experimental evi-dence are somewhat fragmentary, it is tempting never-theless to assume that C02 production is mediated inphotosynthetic cells by oxidative decarboxylationssuch as occur in the Krebs cycle or the pentose phos-phate cycle, to accept at face value the purportedphotoinhibition of C02 production and to reconcile thereduced C02 evolution with the relatively unchanged02 consumption by proposing a substitution. duringthe light, of photochemically generated reductant forthe "substrate hydrogen" which serves alone to reduce02 in dark metabolism. Speculating that photosyn-thesis and respiration are related in this manner, wemay examine the salient features of a general schemeillustrating the postulated interrelation and determinewhat consequences of its operation ought to be observ-able. Such a model is shown in figure 1. Much moredetailed schemes could be employed suggesting possi-ble interplay between breakdowvn and synthesisthrough, let us say specifically, control of the DPN+/DPNH ratio (leading to enhanced respiratory activityin the light), or through a downward adjustment ofthe ADP/ATP ratio by photophosphorylation (toproduce respiratory inhibition), or perhaps even moredirectly via regulation of the concentration of a ratelimiting common intermediate such as phosphoglycericacid. However, our limited kinetic as well as bio-chemical information about the systems with whichwe are dealing hardly justifies a model more detailedthan is necessary for illustrative purposes. The op-eration of the model (fig 1) is as follows:

If no interrelations between respiratory and photo-synthetic metabolism are postulated except those aris-ing from the existence of a common pool of compoundswhich collectively represent the ultimate respiratorysubstrate andl at the same time the ultimate sink forthe organic prodlucts of photosynthesis, then onlv thesolid lines in figure 1 apply. Respiration and photo-synthesis are seen as independent processes. Photo-chemically produced reductant is then accounted forquantitatively by C02 assimilation (reaction 1). On


PLANT PHYSIOLOGY

the other hand, if photochemically generatedl reductantis capable of reducing directly (or, more likely, afterits reducing potential has been degraded to more orless the appropriate level) any of the respiratory com-ponents as indicated by the dashed lines, then photo-synthesis must, at least under some condlitions, affectrespiration. If the reductant, generated photochemi-cally, is capable of reducing 02 directly (reaction 2),this would tend to increase 02 consunmption in thelight but would not alter the rate of respiratory C02production. If the reductant can react directly withone of the cytochrome chain components (reaction 3),it would tend to stimulate 02 consumption via therespiratory oxidase system. If the carrier-oxidasechain were running at capacity, substitution of photo-chemically produced reductant for the "substrate hy-drogen" normally serving as respiratory reductantwould necessitate a decrease in the rate of oxidativedecarboxylation. In consequence C02 productionwould be reduced. If, by photochemical reduction(reaction 4), the pyridine nucleotide redox potentialshould be forced so low that coenzyme molecules can-not easily be loaded with "respiratory" electrons,similar inhibition of C02 evolution (accompanied byunaltered or even stimulated 02 consumption) wouldbe expected. If the glycolytic intermediate, phos-phoglyceric acid, were to be reduced directly (re-action 5), conceivably this could limit the supply ofpyruvate to the Krebs cycle so that, in strong light,slowing down of the cycle might lead to reduced 02uptake as well as a proportionate inhibition of C02production.

Figure 1 is of course not the only model whichmight be suggested to illustrate possible light effectson respiratory processes. However other models leadto the same or similar expectations. They have incommon the feature that, if photosynthesis and respira-tion are related through the photochemically pridcuced

5PHOSPHATE \v TRIOSE

GLUCOSE-6-PHOSPHATE/

RESERVECARBOHYDRATES

ETC.

FIG. 1. Schematic representation of possible influenceson respiratory processes by means of a reductant producedby a partial reaction of photosynthesis.

reductant, this relation may become apparent either asa light induced deficiency in respiratory C02 produc-tion or as enhancement of 02 consumption or both.Assuming for convenience that the quotients of bothrespiration and photosynthesis normally are unity,then the rate of photochemical generation of reductantcorresponds stoichiometrically to the rate of 02 evolu-tion. The operation of any of the reactions 2, 3, 4, or5 in figure 1 will account for the fate of some of thereductant by a path other than C02 reduction (re-action 1). The rate of photosynthetic reduction ofC02 would diminish and the respiratory portion ofthe gas exchange would be altered-effects whichwould be detectable as enhanced 02 consumption orreduced C02 production or both. These effects shouldbear a stoichiometric relation to the inhibition ofphotosynthetic C02 uptake rate.

If, instead of a reductant, it is the photochemicallyproduced oxidant (vide Gaffron, (10) ) which is post-ulated as influencing the respiratory rate, any coml-ponent with which it reacts-directly or indirectly-must become oxidized, and in such a model, analogouswith figure 1, it is readily shown that the influenceof light would be to depress both production and con-sumption of 02 and to accelerate C02 evolution-changes in respiratory gas exchange which are op-posite from those predicted above. Only wlhen 07uptake and C02 evoluttion remain unchainged in thelight can we conclude that photosynthesis and respira-tion are truly kinetically independent.

What has been said in qualitative terms about theinterrelations suggested by figure 1 may be madequantitative as follows: Let Pco2 andl Uc2 (lesignaterespectively rates of production andl uptake of C02.Similarly P02 and U02 are rates of pro(luction andconsumption of 02. In the light an increase in 02uptake rate as compared with the dark value is referredto as A U02 and a decrease in CO., evolution asA Pco.,. For every 4 units (electron equivalents) ofphoto-reductant formedl in the light there must begenerated 4 units of oxidant corresponding to theproduction of 1 02 unit. The 4 units of reductantmay be accounted for by the uptake of 1 unit of C02,or by the enhancement of U02 to the extent of 1 unit,or by the depression of PC02 to the extent of 1 unit, orby any combination of TUmC2 A PC02, and A U02 whichadds up to 1 unit. Thus in the light according toour model,

P02 = UC02 + A PCO2 + A U02 (1)Another way of looking at the matter is to say thatonly as long as equation 1 is obeyed will the chemicalreduction level of the cell remain unaltered.

The purpose of the present study wvas to test thevalidity of equation 1 by evaluating its 4 componentterms simultaneously for each of a series of experi-ments. These experiments were carried out over arange of light intensities both above and below photo-synthetic compensation. Agreement of the data withequation 1 implies consistency with the general modelshown in figure 1.

226


227BROWN AND WEIS-RELATION OF RESPIRATION AND PHOTOSYNTHESIS

MATERIALS AND METHODSIn a series of contributions from this laboratory,

respiration rates of photosynthetic cells have beenmeasured in the light by a method which employedtracer 02 in the gas phase of the experimental vessel.By thus tagging the environmental 02 consumed bythe cells, the rate of 02 uptake was measured independ-ently of the photosynthetic production of unlabelled02 from water. In principle, analogous experimentscan be performed with tracer C02. However, inpreliminary studies carried out several years ago, C02isotope exchange in the dark was observed to be ofsuch a magnitude that large corrections for this ex-change were required (18). The uncertainty of thesecorrections rendered the interpretation of results ob-tained with tracer C02 much more difficult than in thecase of tracer 02 experiments where isotope exchangein the dark was not detectable under the conditionsprevailing in biological experiments. Numerousstudies carried out subsequent to our earlier publica-tions and on divers photosynthetic species have demon-strated that C02 isotope exchange is not as seriousa drawback to the use of the tracer method as weat first thought, for it has been found that the earlierresults showing excessive C02 exchange were atypical;high exchange rates have since been the exceptionrather than the rule. In short, we usually are able toeffect a kinetic separation of simultaneous C02 uptakeand production without prohibitive corrections forisotope exchange. Fortuitously, certain second ordercorrections which apply to such isotope data (to bedescribed below) are even of much smaller magnitudewith tracer C02 than with tracer 02 measurements.

The organism chosen for the present work wasAnkistrodesmnus brauinii (Naegeli) Collins. This isa green alga similar morphologically and physiologi-cally to the genus, Scenedesmus. The cells were cul-tured autotrophically in medium V of Norris et al(15) at 26 to 290 C. Culture flasks, illuminated frombelow by a bank of white fluorescent lights with ared-orange filter (Corning, no. 348) were agitated bya stream of air enriched with 5 % carbon dioxide andby occasional shaking. Cultures were harvested after3 to 5 days while the growth rate was still high. Cellscollected by slow centrifugation (< 1000 G) werewashedI once and were resuspended in media to be usedin the experiment. Ordinarily this was 0.05 M potas-sium phosphate buffer (pH 5.8) containing 2 %glucose.

A Nier-Consolidated mass spectrometer (Model21-201) continuously monitored the gas phase of thereaction vessel wlhich was a rectangular Warburg-type vessel of 15.8 ml total volunme when attached tothe joint wlhich was in turn connectedI to the massspectrometer inlet leak. The design of this joint andthe method of shaking the vessel in a constant tem-perature water bath did not differ in any essentialmanner from what was illustrated previously (13).The use of the mass spectrometer as adapted for therequired measurements also has been described (3).Labelled 02 andl labelled C02 were intro(luced initially

into the gas phase of the experimental vessel and con-tinuous monitoring of the gas phase with the massspectrometer was carried on thereafter.

When cells were suspended in inorganic bufferonly, the respiratory rate was at first high but de-clined over several hours to a small fraction of theinitial rate. Addition of 2 % glucose to the experi-mental medium prevented this decline. Since thesmaller the respiratory rate the greater was the per-cent error in its measurement, data on glucose stabil-ized respiration was more reliable than were measure-ments on endogenous gas exchange. Also it was con-venient to work with cells whose dark metabolism wasnot changing greatly with time. Therefore, criticallight-dark comparisons were made only with cell sus-pensions containing glucose.

For present purposes it is necessary to reconsiderthe principles involved in the tracer method of meas-uring independently both uptake and output of a givengas. Whether 02 or C02 is considered, the presenceof isotopically enriched gas in the environment insuresthat gas produced will have an isotopic compositiondifferent from that of gas consumed. If isotopicenrichment is large, it is possible to ignore the influ-ence of the small natural concentrations of rare iso-topes of 02 and of C02. It is readily seen that theactual rates of production, P, and of consumption, U,are functions of the rates of metabolism of both iso-topes. These "true" rates are given by the equations,

M*P = h (R -R* M

U = h R* (1 ± AIM

(2)

(3)where R and R* are rates of change of the 2 isotopicforms of the gas being metabolized, M and M* are themean concentrations of the respective isotopes whichprevail during the period of measurement at the siteof gas consumption, and h is a dimensional constant.While it is approximately correct to consider U alinear function of R*, as was done in some of theearlier work from this laboratory, when only relativerates of gas exchange are required, it should be keptin mind that this simplification is valid only as longas M/M* remains essentially constant during the ex-periment. This ratio, measured in the gas phase, canbe expected to remain adequately constant only if thetotal tension of the gas is relatively high and the rateof change of either isotopic form of the gas relativelylow.

Furthermore, it is the ratio, WI/AI*, at the site ofreaction which is critical rather than the ratio whichprevails in the gas phase where the mass spectrometercan measure it. Under any single set of steadv stateconditions a difference in this ratio on passing fromthe gas phase to the reaction site is of minor conse-quence for relative measurements. But, if it is neces-sary to compare data taken under different steadystate conditions (viz. light vs. darkness), the M/M*ratio at the reaction site can be appreciably differentunder the separate conditions. WVith photosynthesiz-


I-z

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SECONDS AFTER LIGHT PERIOD BEGINS

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44 &&&Xx i. x'~~~~&

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SECONDS VESSEL EXCURSION CGM.

FIG. 2 (top, left). Apparent induction period for photosynthesis measured in liquid and in the gas phase.FIG. 3 (beloze). A. Time course of equilibration of 02 in a rectangular Warburg type vessel (15.8 ml) containing

3 ml 0.1 M KH2PO4. Gas phase: He, 02, H20 vapor. The parameter of the 4 curves was shaking rate as indicatedin oscillations per minute. B. Semilogarithmic plot of data above. C. Relation between equilibration and shaking rates.D. Relation between equilibration rate and vessel excursion distance for 3 different shaking rates. Ordinate valueswere multiplied by the factors shown so as to superimpose curves.

FIG. 4 (top, right). Example of mass spectrometer data for 2 isotopic forms of CO2 (masses 44 and 45) and 2 of02 (masses 32 and 34). Expt. 2607. 8.4 ,ul cells. 26.00 C. Gas phase, C02 : 02 : He (1 : 4 : 328). Tungsten illu-mination through red filter (Corning no. 2403) at intensity corresponding to 2.6 times compensation.

- 240

220

200

180

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140

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.


BROWN'N AND WEIS-RELATION OF RESPIRATION AND PHOTOSYNTHESIS

ing cells the ratio will be smaller in the gas phase thanat the site of reaction so that the 02 utilization rateas calculated by equation 3 must be considered a mini-mal value; the true rate may be higher. A qualitativeway of looking at this discrepancy between calculatedand true rates is to think of 02, as it is evolved butbefore it can escape into the gas phase, being utilizedby the respiratory enzymes in preference to the 02of the gaseous environment which must cross a sig-nificant diffusion barrier before the enzymes haveaccess to it. The use of the tracer method for experi-ments such as ours has been criticized along these linesby Warburg (20). Decker (6) has also raised ob-jections which are more easily considered in theseterms.

The difference between M/M* ratios in the gasphase and at the site of reaction can be minimized byvarious devices but never eliminated and, when thedifference is large, it leads to what has been termed"preferential reutilization" of the gas produced (2).In previous studies, keeping the metabolic rate lowand the prevailing tension of the gas being measuredrelatively high (insofar as technical considerationswould permit) offered assurance only that the errors inrelative metabolic rate determinations were as small aspossible. It now seems that errors from this causewere sometimes larger than we had thought and, wheneither more precise or absolute measurements areneeded, corrections for "preferential reutilization"usually are mandatory.

In the present work, absolute rather than merelyrelative measurements were required. Also, as canbe seen from inspection of equations 2 and 3, computedchanges in production or consumption of the 2 meta-bolic gases depended upon 4 essentially simultaneousmeasurements of concentrations of different mass com-ponents of the gas phase and upon 4 measurementsof the rates of change of these components as com-pared with the result of similar computations from acomparable set of 8 experimentally determined valuestaken from another condition being tested. Thus theconclusion to be arrived at from a single experiment(e.g., an individual point in fig 7) is sensitive to errorin any of 16 items of data. Under such circumstancesthe necessity to reduce all known errors to a minimumwas unusually compelling. Therefore corrections ofM/M* in equations 2 ancl 3 have been introduced sothat the computed values approach as closelv as possi-ble the ratios prevailing at the site of reaction. Ifthese corrections are ignored, when relativelv highmetabolic rates are determined by the tracer method,the estinmated rates can be in error by over 100 % inthe case of 02 at least. This error arises from the in-terrelated effects of diffusion gradients which existin the experimental system and the corrections arebased on an approximate evaluation of these gradients.

Mixing within the liquid is so rapid in a wellshaken vessel that appreciable diffusion gradients arenot established within the liquid proper and, for sev-eral reasons, it seems likely that, of the various diffu-sion barriers which must exist between gas phase and

enzyme, that which is by far the most important existsat the liquid-gas interface (shearing layer) in the re-action vessel.

The effect of this barrier alone is illustrated infigure 2 in which time course data from gas phasemass spectrometer measurements are compared withliquid phase determinations obtained by a differentmethod (21). Chlorella cells were utilizing 02 inthe dark until, at zero time, the suspensions were il-luminated. The much greater response lag in the caseof the mass spectrometric data is attributable almostentirely to the diffusion gradient across this barrierfor which we must make correction.

Provisionally, we shall assume that with smallcells the barrier at the cell-liquid interface generallyis unimportant in comparison with the barrier whichexists at the gas-liquid interface.

The effects of the interfacial barrier in manometricexperiments have been considered both theoreticallyand experimentally by Roughton (16) and by Myersand Matsen (14) in terms of the usual stationaryfilm theory. Only a brief explanation is in orderhere. The influence of the barrier can be describedquantitatively by an equilibration constant, K, whichcharacterizes the rate at which a concentration differ-ence spontaneously disappears across the interfacialfilm according to the equation,

(p - p) = e- (4)in which p is the partial pressure of the gas at anygiven time, t, and p. is the final value of p afterequilibration. The constant, K, is a function of thechemical nature of the 2 phases, the surface area, theamplitude and frequency of shaking the vessel, andthe volumes of liquid and gas space.

In order to determine K experimentally, one nmakesa sequence of measurements of the partial pressure ofa gas which is undergoing equilibration across theinterfacial diffusion barrier. Plotted semilogarith-mically, the data on departure from equilibrium arelinear with time. The value of K which applies tothe conditions under which the measurements weremade can be computed readily from the slope of thislinear plot as described by Roughton. If desired,values of K are easily converted to the half-times forgas phase response to liquid phase changes-the fa-miliar way of expressing equilibration lag. Usingthis procedure variables such as vessel shape, liquidvolume and composition, presence of surface activematerials, gas phase composition, vessel shaking rate,and excursion distance were studied systematically todetermine their repective influences on K-i.e., onequilibration lag. Figure 3 illustrates by example thismethod of observing effects of the last two variablesmentioned.

When making measurements of K usinig a milassspectrometer instead of a manometer, one gains thesignificant advantage that, of the several gases whichmay be involved in complete pressure equilibration,data are taken only on the single gas with which oneis concernedl. However, 2 additional points must be

229


PLANT PHYSIOLOGY

considered; instrument response time and gas phasemixing.

The rate of response of a manometer to an instan-taneous pressure change is limited by manometer fluidviscosity and several other minor factors. In prac-tice the Warburg manometer responds so fast to gasphase pressure change that, for nearly all ordinaryuses, the inherent instrumental response lag is negligi-ble. On the other hand, most commercially availablemass spectrometers have gas inlet systems which offermajor resistance to diffusion either on the highpressure side of the leak or in the low pressure regionwhere molecular diffusion occurs. In the presentadaptation of a Nier-Consolidated mass spectrometerthese instrumental response lags have been minimizedby reducing the diffusion barriers as much as was fea-sible by altering the design of the leak and gas inletsystem to remove unnecessary constrictions. Thespeed of the mass spectrometer diffusion pump is highand the pump-out time of the mass spectrometer tube(time for half replacement of the gas in the ion sourceregion) was in our case 2.5 seconds which is smallcompared with the half-time for equilibration acrossthe gas-liquid interface in the reaction vessel.

The response of a mass spectrometer is somewhatslower than that of a manometer also because gasphase mixing is a much more important factor in theformer. The most rapid response was observed atnear vacuum pressures in the reaction vessel. Vari-ous background or carrier gases slowed down the re-sponse. The effect was a function of the carrier gasviscosity. As might be expected, the 02 and CO2equilibration constants depended on the total pressurein the gas phase but not on the partial pressure of thegas being measured nor on the direction of diffusion(into or out of solution). Helium was chosen (inpreference to nitrogen) as the carrier gas to be usedin all experiments reported here. Measurementswere carried out in a gas phase which was 96 to 98 %He and the remainder was a mixture of CO2 and O2,both isotopically enriched. The total pressure wasapproximately 1 atmosphere.

All experiments were run in the same rectangularWarburg-type reaction flask under essentially thesame conditions except as regards light intensity.The volume of the cell suspension was 2 ml with acell concentration of about 0.5 %. The shaking ratewas 172 complete oscillations per minute at a vesselexcursion of 17 mm. The value of the constant, K,in equation 4 was, for O2 in this system, 0.06. Thiscorresponds to a half-equilibration time of about 11seconds.

As was anticipated above, knowledge of the Kvalue characteristic of the system used in our experi-ments makes it possible to compute the ratio, MI/M*,in solution from measurements of the ratio made inthe gas phase. For a particular gas being measuredthe diffusion rate, R, across the interface is a linearfunction of the concentration difference through thebarrier. The proportionality constant, referred to as

the interface transfer constant, is k which is relatedto K by

K = k (a v + V )v V (5)

where v and V are liquid and gas volumes respectivelyand a is the solubility coefficient. Therefore k iseasily computed from K as determined experimentallyand the concentration of dissolved gas, M., is givenby

M = aM - Rs ~~k (6)

in which R is the steady state rate of gas transportacross the interface and M is the concentration of thegas prevailing in the gas phase. The values of k forour experimental conditions were, for 02, 0.13 and,for C02, 0.072 vol. units sec'.

Parenthetically, another method of evaluating kmay be mentioned. If the density of the cell suspen-sion is increased until R reaches a maximal value, itmay be assumed that this condition prevails only whenAMS in equation 6 becomes vanishingly small. Thusk = R/aM approximately. It was noted that the2 methods led to essentially the same value of k inspite of the fact that very different cell densities wereemployed. While this was the case for Ankistrodes-

TABLE ISAMPLE DATA AND COMPUTATION RESULTS*

Ex]DARK

45 MIN

R32 -0.49M39 143R 34 -0.22M34 121R44 1.1M44 115R45 0M45 84.5P02 -0.15**PCO2 1.2U02 0.6Uco2 0

A PC02A UC02A PCO2 + A U02 + UC02

DPERIMENTAL PERIODSLIGHT DARK29 MIN 31 MIN

2.7 -0.68278 318

-0.04 -0.22111 102.5

-1.2125

-0.92725.70.43.12.80.92.15.8

1.31270.0559.50.3**1.41.30.2

* Expt. 2107. 5.3 Pul cells. 26.00 C. Gas phase, CO, :02 He (3 : 4: 493). Constants (cf. equations 7 and8) j02, 0.932; F02, 36 X 10-6 per chart div. ;f02, 6.36 X10-6 ml (N.T.P.) 02 sec-1 per chart div. min-1; kOD0.13 ml sec-1; iC02, 1.121; FCOy, 22.4 X 10-6, per chartdiv.; fC02, 5.17 x 10-6 ml (N.T.P.) CO, sec-1 per chartdiv. min-'; kC2, 0.072 ml sec-1. Units: M, chart div.;R, chart div. min-; all others, /Aml sec'- /Al-'.

** Oxygen production may be assumed not to occur indarkness; departure of tabular values from zero indicatesexperimental error.

230


BROWN AND WEIS-RELATION OF RESPIRATION AND PHOTOSYNTHESIS

mus in phosphate buffer, it may have been fortuitous.Other experimental material or different media mayinfluence interfacial properties in such a way that kbecomes a significant function of cell concentration.We found this to be the case with another organism(Ochromonas). Complications of this kind alsowere reported by Habermann for chloroplast suspen-sions ( 11 ), and were noted by Dr. Robert Emerson forChlorella suspensions (personal communication).

For a given kind of gas we were concerned in theseexperiments with 2 isotopic species; these must beconsidered separately. A pair of Ms values were cal-culated from measurements of M and R for the respec-tive isotopic masses. Thus a pair of isotope concen-trations-consequently a ratio-in the liquid phase wasdetermined. Ratios calculated in this way, beingmuch better approximations of the ratios existing atthe site of gas consumption, were used in preferenceto the gas phase ratios as measured. From theseconsiderations it is possible to improve on equations2 and 3, which were first approximations involvingonly gas phase quantities, by substitution of corres-ponding expressions based on liquid phase quantitiesobtained by taking into account the diffusion barrierat the gas-liquid interface. Corrections of this sortalso were applied to mass spectrometer data by Hor-witz and Allen (12).

Values of MI and M* and of their rates of changewere measured in the gas phase in arbitrary unitsof pen excursion on the recorder chart. For this andfor other reasons several conversion factors must beintroduced in order that results may be expressed inthe desired units. The overall calculations are indi-cated by the following equations wrhich are revisionsof equations 2 and 3:

P = (Rfj) - (R*fj)

a lF - Rf

a \I*F - R*f( k )

*a F - Rf (-)

U = R*fj 1 + (8)

a MI*F - R*f (-)

for which the following symbols are defined (for thease where 02 is the gas considered):

P and U = production and consumption rates for). Units, ml per sec per ,ul cells X 10-6.M and M* = concentrations of the 2 isotopic

forms of 02 measured on the recorder chart as mass

spectrometer peak heights for the respective masses.

Units, arbitrary scale divisions.R and R* = rates of change of M and M* respec-

tively. Units, scale divisions per minute.

= a v + V 273 where v and VVv T ,weea V

are liquid and gas volumes (ml) and T is the absolutetemperature.

F = C/S, where C is the oxygen fraction of thegas used for calibration and S is the mass spectrometerpeak height (in scale div) for mass 32 when measuringthe calibration gas at 1 atmosphere.

f = F V/60.k = interface transfer constant as previously de-

fined. For analogous measurements on C02, masses44 and 45, a set of similar equations with comparableconstants was applied. The units of P02, U02, PCO29and UC02 are ml sec-1. The final values are expressedon a unit cell volume basis.

RESULTS ANI) DISCUSSIONFigure 4 shows an example of mass spectrometer

data for the metabolism of 2 isotopic forms of 02(masses 32 and 34) and 2 of C02 (masses 44 and 45).The essential measurements on a chart record such asfigure 4 are recorded in table I along with results ofcomputations leading to the true production and con-sumption rates of both 02 and C02. The table alsoshows the final result of light-dark comparisons.Note that the 02 production rate in the light period,Po2 is nearly the same as the sum of: deficit in CO.production, enhanced 02 consumption, and C02 uptakeas shown by the last entry in the table. This agree-ment is predicted by equation 1. Data on 13 light in-tervals, such as that on which table I was based, withdark periods before and after for comparison, wereobtained over a range of light intensities. Values ofUC0°, A PCO2, and A U02 calculated for each illumina-tion period, were plotted against corresponding valuesof Po2 in figures 5 to 7.

Figure 5 shows the relation between rate of C02utilization and 02 production. If the photosyntheticquotient were actually unity, the points would all liealong the diagonal. This was not the case as the up-take of C02 fell short of being equivalent to 02 produc-tion especially at higher rates of photosynthesis.

Figure 6 shows the light induced deficit in respira-tory C02 production. Compared with the dark rateC02 production was reduced to about one half by light,an effect which varied little with photosynthetic rate.

Figure 7 shows the photostimulated fraction of the02 consumption which was negligible at low light in-tensities but which became large at higher rates of02 evolution. It may be noted that, at low light in-tensities, neither stimulation nor inhibition of respira-tory 02 uptake occurred. Under these conditions re-liable measurements of photosynthetic 02 productioncould have been made on such Ankistrodesmus sus-pensions by ordinary methods not involving the useof tracers. However, in the case of C02 metabolism,results were less reassuring. Because of the suppres-sion of C02 production in light of even low intensity,measurements without tracer of net C02 exchangeare not readily interpreted.

131


PLANT PHYSIOLOGY

Each point in figure 8 is the absolute sum of ordi-nate values in figures 5 through 7 plotted against 02production rate. These points lie along the diagonalindicating that, over the range of light conditionsemployed, the rate of production of photosynthetic re-ductant (corresponding to the rate of oxidant produc-tion and measured as 02 evolution) was equivalentto the sum of the C02 consumption rate, the deficit in

8 . . . . . -,

7 ,,

6

d,

U - ~~~~~04 J,8

o 3 410

the rate of C02 production, and the increase in therate of 02 absorption. This is the condition imposedby equation 1. Thus, figure 8 demonstrates that thedata were generally in agreement with the equationover a relatively wide range of light intensities (upto about 6 times compensation). The data are con-sistent with the assumption of an effect of light onrespiratory metabolism mediated by a reductant rather

7

60S0.

.0

.0

6v 2U

r) L

6 l

02 production (PO2)

2 3 4 5

02 production CPoL)

6 7

0.

e

-a

0 1 2 3 ,4 5 6 7

02 production (Po) °2 production (P)

FIG. 5 (top, left). Relation between rates of CO2 uptake and 02 production in the light. If the photosyntheticquotient actually were unity, all points would lie on the diagonal.

FIG. 6 (top, right). Relation between deficit in CO2 production rate caused by light and the rate of 02 production.FIG. 7 (below, left). Relation between the enhancement by light of the rate of 02 consumption and the 02 production

rate.FIG. 8 (below, right). Comparison of light induced deficit of CO2 evolution rate, increment in 02 consumption rate,

and C02 uptake rate with the rate of 02 production. Prediction according to equation 1 requires that the points should

lie along the diagonal.

232

-, 0.0

0,

0

'-

t>

u~~~~~ 0

7 ,"0

6 0',,

4 / 0

2 .CO/w#o

O,0/, , , ,


BROWN AND WEIS-RELATION OF RESPIRATION AND PHOTOSYNTHESIS

than by an oxidant. At low light intensities-lowrates of reductant generation-only CO2 evolution isaffected. At higher light intensities enhancement of02 consumption occurs as well.

From kinetic data of this type it is of course notpossible to decide where in the oxidative process thephotosynthetic reductant interacts with respiratorymetabolites, coenzymes, or electron carriers. It maybe noted also that the enhanced 02 consumption ob-served at the higher light intensities is not readilydismissed as photooxidation not directly related tophotosynthesis and respiration, since the increment in02 consumption was matched by increasing 02 pro-duction. If the usual kind of photooxidation weresuperimposed on photosynthetic and respiratory gasexchange, results would not agree stoichiometricallywith equation 1.

During some of the experiments described abovefurther data were taken while the cell suspensions wereallowed to photosynthesize until the C02 was depletedto the level of C02-compensation. As this conditionwas approached, the CO2 isotope ratio. M44/M45, be-came so large that calculations of production and con-sumption rates were subject to large error as the netrate of CO2 uptake approachel zero. Oxygen com-putations were as reliable as in other experiments.Figure 9 illustrates the changes which were observedin 2 representative experiments. After C02-compen-sation was attained, the rate of 02 consumption usual-ly was higher and 02 production invariably lowerthan was the case for normal photosynthesis with anadequate concentration of C02. Evolution and uptakeof 02 were then steady and essentially in balance. Justbefore the condition of compensation was reached, atransient stimulation of 02 exchange sometimes wasobserved (as in experiment 1707 of fig 9). Whensuch experiments were performed on suspensions

EXPT. '2107 EXPT. 1707

140 0~~~~~1 02 Cvolutiono140 0,03 uptake

120

A eoo ea.~ 0

o 60

40-~ 0

0-cnough low CO2 enough low C02C02 C02 compensation C02 C02 compensation

FIG. 9. Comparison of rates of 02 production and con-sumption under conditions of C02 depletion. Two sepa-rate experiments are shown.

without supplementary glucose, this transient usuallywas especially pronounced.

It is reasonable to suggest that results such asthese, obtained as the cells run out of C02, demon-strate a change in fate of the photochemical reductantwhich, when deprived of its normal oxidant, reduces02 instead. The steady state which finally obtains isone in which 02 consumption is elevated sufficientlyto match-or nearly match-02 evolution; i.e., netgas exchange approaches zero and an 02 exchange inratio 1 :1 prevails. The rates of 02 consumptionobserved under such compensation conditions, whilehigher than normal for the light intensity used, wereabout equivalent to-but did not exceed-the maximalrate of extra 02 uptake observed in any light periodwhere sufficient C02 was present. This maximalrate was observed only at a significantly higher lightintensity in the case of normal photosynthesis. Fromthe fact that 02 production rates were not maintainedunder C02 deficient conditions, as illustrated in figure9, we may deduce that 02 reduction by photochemicalreductant either is less efficient than C02 reductionor, more probably, when C02 compensation prevails,secondary changes may inhibit energy transfer fromchlorophyll, increase the probability of back reactions,and thus lower the 02 production rate as suggestedby Franck and coworkers (7, 8, 9, 17).

SUMMARY

The gaseous metabolism of the alga, Anikistrodes-7mls braunii, was measured in darkness and in light ofvarious intensities. Cells were suspended in phosphatebuffer containing glucose and the gas phase, enrichedwith 02 and C02 isotopes, was monitored with a re-cording mass spectrometer. In contrast to earlierresults with other experimental material, isotope ex-change involving C02 was not prohibitively rapid sothat with this gas as well as with 02 it was possibleto calculate simultaneous production and consumptionduring the light. These calculations dependedI onknowledge of the isotope ratio prevailing at the siteof gas consumption and, since the ratio (especially inthe case of 02) generally was not the same in liquidand gas phases, corrections were introduced whichtook into account the effect of the diffusion barrier atthe gas-liquid interface; calculations therefore werebased on isotope ratios prevailing in the liquid phasewhich were considered close approximations to thoseat the intracellular reaction site. It was found thatlight induced an inhibition of C02 evolution which wasnearly independlent of light intensity whereas 02 con-sumption in the light was unaffected at low intensitybut enhanced at high intensities. The results werequantitatively in agreement with interaction betweena photosynthetic reductant and the respiratorymechanism.

This work was aided by a contract between theOffice of Naval Research, Department of the Navy,and the University of Minnesota (NR104-030) and

233


PLANT PHYSIOLOGY

also was supported by a grant from the GraduateSchool.

The authors are grateful for the isotopically en-

riched 02 which was supplied by Dr. A. 0. C. Nier.It was prepared under a grant from the AmericanCancer Society through the Committee on Growth ofthe National Research Council.

A pure culture of the experimental organism was

kindly supplied and identified by Dr. R. E. Norris.Support in the form of a post doctoral fellowship

for one of us (D. W.) from the Charles F. KetteringFoundation is gratefully acknowledged.

LITERATURE CITED

1. BENSON, A. A. and CALVIN M. The path of carbonin photosynthesis. VII. Respiration and photo-synthesis. Jour. Expt. Bot. 1: 63-68. 1950.

2. BROWN, A. H. The effects of light on respirationusing isotopically enriched oxygen. Amer. Jour.Bot. 40: 719-729. 1953.

3. BROWN, A. H., NIER, A. 0. C. and VAN NORMAN,R. W. Measurement of metabolic gas exchangewith a recording mass spectrometer. Plant Physiol.27: 320-334. 1952.

4. BROWN, A. H. and WEBSTER, G. C. The influenceof light on the rate of respiration of the blue-greenalga, Anabaena. Amer. Jour. Bot. 40: 753-758.1953.

5. CALVIN, M. and MASSINI, P. The path of carbonin photosynthesis. XX. The steady state. Ex-perientia 8: 445-484. 1952.

6. DECKER, J. The effects of light on respiration usingisotopically enriched oxygen: an objection and alter-nate interpretation. Plant Science Bull. 4 (No. 4):3-4. 1958.

7. FRANCK, J. The relation of the fluorescence ofchlorophyll to photosynthesis. In: Photosynthesisin Plants, J. Franck and W. E. Loomis ed. Chap.16. Iowa State College Press, Ames. 1949.

8. FRANCK, J. and FRENCH, C. S. Photoxidation pro-

cesses in plants. Jour. Gen. Physiol. 25: 309-324.1941.

9. FRANCK, J., FRENCH, C. S. and PUCK, T. T. Thefluorescence of chlorophyll and photosynthesis.Jour. Chem. Phys. 45: 1268-1300. 1941.

10. GAFFRON, H. Studies on the induction period ofphotosynthesis and light respiration in green algae.Amer. Jour. Bot. 27: 204-216. 1940.

11. HABERMANN, H. M. Investigation of the Mehler re-action and related light dependent oxygen metabo-lism of chloroplast preparations. Thesis, Univer-sity of Minnesota, Minneapolis 1956.

12. HORWITZ, L. and ALLEN, F. L. Oxygen evolution andphotoreduction in adapted Scenedesmus. Arch.Biochem. Biophys. 66: 45 63. 1957.

13. JOHNSTON, J. A. and BROWN, A. H. The effect oflight on the oxygen metabolism of the photosyntheticbacterium, Rhodospirillztn rubrumn. Plant Physiol.29: 177-182. 1954.

14. MYERS, J. and MATSEN, F. A. Kinetic characteris-tics of Warburg manometry. Arch. Biochem.Biophys. 55: 373-388. 1955.

15. NORRIs, L., NORRIs, R. E. and CALVIN, M. A surveyof the rates and products of short-term photosyn-thesis in plants of nine phyla. Jour. Expt. Bot.6: 67-74. 1955.

16. ROUGHTON, F. J. W. A method of allowing for theinfluence of diffusion in manometric measurementsof certain rapid biochemical reactions. Jour. Biol.Chem. 141: 129-145. 1941.

17. SHIAU, Y. G. and FRANCK, J. Chlorophyll fluores-cence and photosynthesis in algae, leaves, andchloroplasts. Arch. Biochem. 14: 253-295. 1947.

18. THIMANN, K. V. The absorption of carbon dioxidein photosynthesis. Science 88: 506-507. 1938.

19. VAN NORMAN, R. W. and BROWN, A. H. The rela-tive rates of photosynthetic assimilation of isotopicforms of carbon dioxide. Plant Physiol. 27: 691-709. 1952.

20. WARBURG, 0. Energetik der Photosynthese.Naturwiss. 15: 337-341. 1952.

21. WEIGL, J., WARRINGTON, P. M. and CALVIN, M. Therelation of photosynthesis to respiration. Jour.Amer. Chem. Soc. 73: 5058-5063. 1951.

22. WHITTINGHAM, C. P. Photosynthesis in Chlorelladuring intermittent illumination of long periodicity.Plant Physiol. 29: 473-477. 1954.

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