by Green ILeaves in Light' )laced in. a leaf - Plant Physiology

Plant Physiol. (1969) 44, 662-670

Determination of the Rate of CO Evolutionby Green ILeaves in Light'

Choy-Sin Hew2, G. Krotkov3, and D. T. CanvinDepartment of Biology, Queen's University, Kingston, Ontario, Canada

Received November 4, 1968.

Abstract. The rate of CO,, evolution in light by green leaves was determined by 2 methodsin a closed system of gas analysis and by measuring the amount of CO, evolved in-to a CO.,free air stream in an open system. All methods gave similar results under comparable oon-ditions.

A light stimulated CO., evolution from green leaves was found in all the plant speciesstudied except corn where there was no apparent CO., evolution in the light.

The magnitude of CO., evolution in light was markedly dependent on air flow rate andlight intensity. At high light intensity and high flow rate, the rate of CO., evolution fromgreen leaves was 1.4 to 1.7 times greater than that in darkness.

In the pas.t, many attemlpts have been made todetermine the rate of respiration of green leaxvesduring photosyntlhesis. In 1951, WNTeigl et al. (40)first employed the tracer technrique to study the effectof light on CO., evolution 'by greeni leaves. Thistechnique was later used extenisivelv bv variouswovrkers 1(2, 20, 24. 31) while other workers have usedmore indirect miiethods to investigate the effect oflight onl CO., evolution or O., uptake (8, 9, 13, 14,23, 35).

The results obtainied ol the effect of light oil CO..evolu,tion. or O., uptake by green leaves are still veryinconclusive. Thev have varied froml pllotoinhibi-tion (20., 213, 26, 40) to no effect (6) to even phlloto-stimnulation (8. 9. 14, 24, 35). This diversitv of conl-clusion,s may be due, in part, to the variety of plantnmaterials used. but mlore variationi is lprobably dueto the man!- methods and elxperimental conditiolnsthalt w\ere used by differenit workers for determinin.gthe rate of CO., evolution or O.. uptake 'by greenleaves in liglht. The objective of the presen,t inivesti-gationl was to examiniiie and com,pare several differentmethods that are used 'to deterninle the rate of CO..evolution. by greenl leaves in liglht.

Materials and Methods

Sunflow-er (Helianthius aniinuus L. 'Menn.onite')and corn (Zca inays L.) seeds were germinated invermiculite anld the l)'lants grown iln a l)lanlt growth

Supported by the National Research Council ofCanada.

2 Present address: D)epartment of Biology, BrandeisUniversity, Waltham, Massachusetts 02154.

3 Deceased January 29, 1968.

662

chamber at day to night temiperature of 250 to 200with relative humidity 0 %. The 'phhotoperiod was16 hr aiid the light in,tensity was 2000 ft-c. Plantswere watered daily. and ever- second day were giveen250 mil comimiercial fer-tilizer 20-20-20 'with miiicro-elemen,ts (Plalnt Products Ltd. Port Credit. Ontario)at a concentration o,f I gramii l)pe liter. The stiun-flower and corn plants used for the experimen,ts werebetween 30 to 40 days old. For sunflower, the full!exl)anded second. leaf nutniberi ng from the bottomwas used.

The folloNwing miietlhods were uie(d to (letermillethe rate of CO.. exolution from green leaves in theliglht.

I) The rate of appatrent photosynthesis at variotusconcentrations of CO., was determiiined tusilig a closedsystemi, the results were graphed and the graplhextrapolated to zero concen;tration of CO.. to estimatethe ra,te of CO.. evolution in light (9). Iden,ticalvalues could be estimiiated mathematically tusinig theequationl derived by Tregunna (37).

II) ]4CO.. w\ias released in a closed svstem all(lthe rate of CO2 evolution in light estinmated from thed(ecrease in the slecific radioactiv-ity of the 14CO..(2, 40).

III) CO., evolution w\-as determined directlv inlight anid in dlarkniess by maeasuring the amllount ofCO.2 evolved from the leaves into a sitream of CO.-free air in an open system ( 11, 30).

The closed sy-stem used -was a modificationi ofthat described by Lister et al. 1(26). A leaf was)laced in. a leaf chlanhiber in a closed system anid thechlange in concen,tration of CO.. durin,g illuminationio,r darkniess was mleasuired. The leaf chamlber(15 cm X 20 cml X 1 cml) was made of 'plexiglas'with a remlovable to) to allo,w in,troduction of theleaf. A small groove was cut in one of the side wallsfor the leaf petiole. The leaf svas held in the center

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HENW ET AL.-RATE OF CO., EVOLUTION BY GREEN LEAVES IN LIGHT

l)lane of the chamber by nylon threads stretchedacross the chamiiber. The total volume of the wholesystemii was 1.1 liter. The leaf temperature, meas-tured by a colpl)er-constantan. thermocotuple placedagainist the undersurface of the leaf, varied less than

du(Itring any experiment. The air flow througlthe systemi was measured by a haill-type flow mleter.Twelve fluorescent lam,ps (General Electric, coolwhite VHO) wNere used as the light source and lightintensity was measured by a \Weston light meter atthe surface of the leaf.

In the study using "4CO., the chan,ges in 14CO2concentration w%ere measured with a Geiger-1Mullercounter connecte(l to a ratenmeter atnd recorder.\When detached sutnflower or cortn leaves were used.the leaf or petiole was ctut tunder water and the cutends placed in water in a small polyethylene bag.Fortx ,uc of 14CO., were released in-to the closedsystem from w-hich the leaf chanmber was temporarilydisconnected. 14CO., was allowed to circulate for1 to 3 nin until a constant reading otn the ratemeterwas obtained. The pump was stopped. the leafchamber containing the experimnental material vasconnected hack to the svstenui and the air circulationwvas restunied.

In the ol)en systemi studies. the enclosed leaf wascontinuously fluslhed with either air containinig0.03 % CO.. or 0 % CO_. CO.-free air was obtainedby passing atmlospheric air throtugh Indicarb (FisherScientific Company, USA), The difference in CO.,conlcentrationl (ACO) between the ingress and

egress air multiplied by the air flow rate gave therate of CO. uptake or evolution.

In both the open and closed systenms the air streamenitering the CO., analyzer was dried usin,g magne-siuImi perchlorate. The concentration of CO. in thegas streanms was mleasured withl a Becknman (Model215) Infrared Carbon Dioxide Analx' zer (IRGA).

Results

The Rate of CO., Evolu(tion, in Light Obtainedby Extrapolation. The changes in CO., concentra-tion around a single attached sunflower leaf in aclosed system were measured in successive light-darkcycles under varying conditions of light intensityand flow rate. Fach light )eriod lasted between 1Sto 2') miin and the dark lperiod 10 miiin. Betweenleachi successive light-dark cycle, the leaf wsas flushedfor 15; tmim vith atnmospheric air in lighlt.

The rates of apparent pllotosynthesis at variousCO., concentrations were calculated and are shownin Fig. 1. Although CO., concentration was limiting,the rate of app)arent photosynthesis could be furtherincreased if the light intensity were increased (Fig.1A). An increased rate of apparenlt 'photosynthesiswas also observed w-ith in,creased fflow rate (F14ig. 1 B).

By extrapolating eaclh gra.ph in Fig. I to zeroCO., concenitratioin one obtains the rate of aplarenitphotosynthesis at zero CO., concentration. Since therate of photosynthesis at this CO., concentration wvillbe extreniclv low. the rate of apparent lphotosynthesis

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FIG. 1. A and B) Effect of flow rate and light intensity on the rate of apparent photosynithesis of a single at-tached sunflower leaf at various CO., concentrations, as determinied in a closed system. Gas phase was 21 % O.,and at 200.

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PLANT PI-IYSIOLOGY

at zero CO., concentration rel)resents the minimunrate of CO., evolution.

The rate of CO., evolution (deterIllined as above)in light wvas founid to increase with increasing lightintensity an(l air flow (table I). An especiallypronouinced increase in the rate of CO., evolutionoccurred w-henl light intensity was increased from500 to 1000 ft-c. Ini contrast to CO., evolutioln inlight, the rate of CO., evolu.tion in darkness was notaffected either by previous light intensity or hv airflow. The rate of CO., evolution at high light inten-sity and higlh flow rate was considerably hliglher thanthe rate of CO., evoluttion in (larkness. The COocompensation l)oint was niot affected by liglht inten-sity or flow rate.

7The Rate of CO., Evoliution. in ILight CalcuilatedUsing 1-CO.. The changes in '4CO.. anid CO., con-cenitration1 in a closed systemi containing either (le-tachied sun1flower or corni leaves are showvn in Fig. 2

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Fic,. 2. The conicentrationi of CO., and( CO,, atnd

the specific activity of CO., around(l a detached stln-flower leaf duritng illuminationi and(i in subsequent (lark-ness. l.ight intetnsity was 1800 ft-c, gas phase was 21 %02 and( at 21 0.

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FIG. 3. The concentration of CO, and '4CO., anldthe specific activity of "4CO., around a detached cornleaf during illumination and in subsequent darkness. Lightintensity was 1800 ft-c,gas lhase was 21 % O., and at 210.

and 3. With the sunflower leaf (Fig. 2), there was

a continiuous decrease in the concentration of 14C0.,and CO., with timle during illumtinlation and thle CO.,compensation point was reached after 8 mii;t. T'hespecific radioactivity of 4CO.. around the leaf alsoshowe(d a conltinluouis decline witlh time indicating a

diluitioli of the 14CO2 by CO., evolved from the leaf.In darkness the specific radioactivity of the "4CO.,continued to declilne. Bothl '4CO.. aild CO., outburstswere observed imminlediately after the light wvas turnedoff.

\When a corn leaf was used (Fig. 3) there wvas

a similar decrease in 4CO., and CO.2 concentrationwith tinme during illumination. However, the CO.2compensation point was zero, and, the specific radio-

Table 1. Effect of Light Inifitnsity (antd 4ir Flow Rate on the Rate of CO, E'olution in Light by a1 Sinigle AttachedLeaf of Sufloiwcr (s i)ctermicind by the Extrapolation llcthod

The gas p)llase was 21% O., and at 200.

CO., evolutionFlow rate Light inltensity Light Dark CO, compensation pointlit m ft-c ug CO, hr/t cm-2PP1m

2.3 500 15 ± 1.51 18 t 0.5 452.3 1000 28 ± 0.2 19 ± 0.7 422.3 2000 33± 1.2 21 ± 1.( 42

0.5 1000 16 21 421.0 1000 24 19 ± 0.5 412.0 1000 28 ± 1.8 20 0.5 42

Mean ± S.D. from 2 or 3 plalnts.

664

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HEW ET AL.-EFFECT OF TEMPERATURE ON PHOTOSY'NTHESIS AND CO2 EVOLUTION OF LEAVES 665

Table II. The Rate of CO., Evolution in) Light by a Single Detached SunflowcerEither by the 14CO, or Extrapolation Methods

Light intensity was 1000 ft-c and the gas phase was 21 % 02 anid at 210.

antd Cornt Leaf as Determined

Time in light CO, Evolutionafter 14CO2 True Apparent Light3 Dark

release plhotosynthesis' photosynthesis2 I I I

sec ,ug CO, hr-1 Curl-2 ug CO. hr-- 1 Cul-r2 Mg CO, hr-I CurI-2Sunflower

45 139 107 32 31115 87 70 17 31 26

Corn75 67 67 0 0 15

115 55 55 0 0

As determined from 14CO, uptake.2 As determined from 12CO2 uptake.3 The rate of CO. evolution, obtained by: 1. True photosynthesis - apparent photosynthesis. IL. From extrapolation

method.

activity of 14CO2 around the corn leaves remainiedconstant during illumination, indicating the abseniceof CO2 evolution by corn leaves inl light. In dark-ness immediately after illumination there was no

14CO or CO.2 outburst and evolution of CO2 and14COO was detected only after a lag period of I to2 min. The specific activity of 14CO2 decreased indarkness as with -the sunflower.

The rate of CO2 evolution in light w,as calculatedfronm the measturenment of the rates of 14CO, and CO.,uptake in light (2). The rate of CO2 evolution inlight is represented by the difference in the rates of14CO., and CO.2 uptake. This is based oln the asstumip-tion that at the tinle '4CO_ is supplied to the leaf, littleor no "4CO, is evolved. Thle rate of '4CO.. uIptakewould theni represent the 'true' photosynthetic rateand the rate of CO2 u'ptake the apparenit photosyn-thetic rate. No attempt was miade to determine therate of CO2 evolutionl at time zero i.e. when the leafwas first exposed to 14CO. because of the uncertaintyinvolved in the extrapolation of the curves for 14CO.,and CO, uptake back to zero time. Instead, the rateof CO.2 evolution was determlined at various times

Light on Light off

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CO Free Air

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FIG. 4. The rate of CO, evolution into CO,-freeair in light and in darkness by a single attached sunflowerleaf. Light intensity was 1800 ft-c, gas phase was 21 %02 and at 210.

after the leaf was exposed to 14CO0,. The resultsobtained are compared to those obtained by theextrapolation method in table II.

The rate of CO2 evolution by a sunflower leaf inlight, calculated using 14CO0 was found to be aboutthe same as that obtained by the extrapolation methodwhen the measurements were made within 45 secafter the exposure of the illuminated leaf to 14CO2.A lower value of CO, evolution was observed whenthe measurements were made 115 sec after 14CO2was supplied to the leaf. In corn, no CO., evolutionin light was detected by either the dilution of 14CO.or by the extrapolation method.

The Rate of CO., Evoluttion in Liglht AleasturedDirectly. An attached sunflower leaf was illumi-nated at 1800 ft-c and flushed with a stream ofnormal air for 3 hr. A stream of CO2-free air wastheln passed over the leaf and the amount of CO.,evollved by the leaf was measured. The rate of CO.,evolution into CO.-free air in light anid in darknessis shown in Fig. 4.

XVhen a leaf was transferred from air containing0.03 % CO, to CO2-free air the initial rate of CO.,evolution was high, but declined within a min. Aconstant rate of CO2 evolution was reached in about20 min when the illumination leaf was darkened, aCO2 outburst vas observed within a few sec. Thiswas followed by a mnuch smaller second outburst anldeventuallv a con,stant rate of CO2 evolution wasreached in albout 15 -to 20 min. WVhen the light wasagain turned on, a CO2 gulip was observed and ittook abotut 15 min to reach a constant rate of CO2,evolution.

The rate of transpiration remained fairly con-stant wlheni the leaf was transferred from atmnosphericair to zero CO2 concenitration indicatin,g that thechan,ges observed were not likely due to stomatalmovemenit (J. L. Ludwig, personal communication).

To ascertain whetlher exposure of leaves to CO-free air had any effects on subsequent rates ofapparent photosynthesis, leaves were placed in CO.,-free air in light or darkness for periods of 2 hr and

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PLANT PHYSIOLOGY

theni the rate of their apparent phiotosynthesis wasdeterminied. No effect of such a pre-treatmeut wasobserved.

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FIG. 5. Effect of the aapparent photosynthesis andin darkness by a single attasured in the open system. Igas phase was 21 % 0, astandard (leviatioii of 3 lea

The rate of apparent photosynthesis, as mieasuredin aln open system, inicreased withi ani ilicrease inflow rate and reached a constant rate at an air-flowat 2.5 to 3 liters per min (Fig. 5). Thle rate ofCO., evolution in light was less than tilat in darknessat 0.5 liters per nill, eqjual to tle (lark rate at 1.0liter per min and greater thlan the (lark rate at lligherflow rate. At a flow rate of abouit 2.5 liters permin, the rate of CO., evolution in lighit became con-stant. In this experiment, the average size of theleaf was 75 cm2.

The rate of CO.. evolution in darkniess (Fig. 5)did not change with flowv rate atid \ as identicalwhen measured in CO.-free air o-r at atmiosphericCO., concentration.

The effects of light inttensitv oil the rates ofW53 o apparent photosynthesis and CO, evolution in light

and iin darkness were examtined under 2 air flowght rates-0.6 and 2.3 literts per niii (Fig. 6A and B).

-i---'Dok ' Under all light intensities at 0.6 liters per min____-__'___ air flow the rate of CO., evolution in light was less

than in darkness (Fig. 6A). It (lecreased withincreasing light intensity fromil zero to about 150 ft-c,

r 2 5 3.0'-thienl slowlv in!creased an,d reached a constan,t valueiir flow oil' th rate of

at 100() ft-c or greater. A.t 2.3 liters per miln air-flowir flo rateonuthe rate of the rate of CO.. evolution. also decreasedl at lighticOhevolutifonw i lightandilea- intensities betweenl zer-o and 150 ft-c, btut then itlched sunflowver leaf, as nica-

Light intenisity was 1800 ft-c, ilclreased to a level above that for CO.. evolution innd at 220. Vertical bars- darknless. Higher rates of CO., evolution at 1800yes. ft-c,, as comipared to those ill darkiness. were also

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FIG. 6. A anid B) Effects of light intcensity on the rate of apl)arent photosynitlhesis anid CO., evolution in lightand in darkness by a single, attached sunflower leaf at 2 air-flows. Gas phase wN-as 21 % 02 and at 220. Photo-synthesis at 300 ppm CO.

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HEW}'ET AL.-RATE OS CO-. EVOLUTION BY GREEN LEAVES IN LIGHT

observed in leaves of egg plant, sovbean, watermelon,and jackbean (18).

At both air-flows the rate of apparent photosyn-,thesis always increased with increasing light inten-sity. Light intensity during the pre-treatment ofexperimental material had little effect on the subse-quent steady rate of CO2 evolution in darkness.

To compare the rates of apparent photosynthesisand CO.2 evolution obtained either in a closed or inan open system, such rates for the same experimentalmaterials wvere first obtained in a closed system andthen in an open one. At the salmle air-flowv, the ratesof apparent photosynthesis. CO.. evoltution in lightanid in darkness measured by either an open systemor a closed svstem were similar (table III).

Discussion

Using a closed system of gas analVsis, the ra.te ofCO., evolution in the light can be estimated by theextrapolation method l(9) or by measuring the dilu-tion in the specific radioactivitv of 14CO., (2, 24, 40).These methods were compared and the estimatedrates of CO. evolution in the light from leaves were

found to be similar. With the method using 14CO.,.however, -the rate of CO.2 evolution that is measuredis depen(lent upon the time of measurement afterexposure of the illuminated leaf to 14CO.,. After2 mim the rate of CO.2 evolution was only abouit 50 %of the rate that was measure(d at 45 sec. This ap-

parent decline in the rate of CO., evolution isattributed to the initiation of '4CO., evolutioin fromlthe leaf andl a subsequent (lecrease in the rate ofdilution of the specific radioactivity of the '4CO., inthe environment. Hence, this 14CO., metlhod canlonly be applied if accurate measurements can beobtained before 14CO., is evolv ed from the leaf.

WVith the open system of gas analysis, the rateof CO. evolution in the light can be estimated fromthe amount of CO2 evolved into a CO.--free airstream (11, 30).. The rate of CO., evolution meas-ured by this method was found to be similar to thatestimated in the closed svstem using the extrapola-tion method. El-Sharkawy and Heskleth (11) havepresented data with suniflower, at intense light and400, thlat slhows tllat the rates of CO2 evcolutionestimated by these 2 mlethods are similar. In thelatter case the magni.tude of CO.. evolution is about3 timles the magilituide rep)orted in this paper butthis is tundoubtedly due to the Iliulch hiighler lightinten sitv emiiployed. Thlts, un,der coml!parable experi-mental conditions the 2 mietlhods vill yield similaranlswvers. This agreemlienlt w,ould insdicate that i.t isvalid to extrapolate the graph of a,pparent photosyn-thesis against CO2 conicentration to zero CO., con-

centrationi in order to estinmate the rate of CO., evolu-tion l)! green leav es in the light as suggested byDecker (9).

It shouild be nmade perfectly clear at this pointthat all of the above miietlhods mleasure only the CO.ev,olution fromi the leaf into the gaseous environmen.tantid thuls all miethio(ds uniderestiimiate the absolutemagnitude of CO., prodluction by thle cells in thelight by somle unknown amllouint (25 30, 33, 34).In additionl, N%'hen using 14CO.., anly discrimiiinationagainst '4CO.. will further enhance this underestima-tion. Samiiislh and Koller (33) estimate for suIn-flo,er at highl light intensity alnd 0.03 % CO., thatthe CO). exvolution frol the 'leaf only rel)resents66 % of the CO.. prodluction while Bravdo (5) esti-miiates that apparent photosynthesis is reduice(d 21 to25 % 1y respiration if the leaves have a CO., com-

pensation point of 60 ppm. The amlioutlt of CO2,ev,olutio,n froml the leaf not only depends on themagnituide of CO., produiction by the cells but also

Table III. The Rates of Apparcnt Photosvnthesis and CO, Evoilttion in LiOht and ini Darkniess b)Y a Sinlgle AttachedSonflower Leaf as MVeasitred Either in a Closed or in ant Open Svstc),i

Light intensity xvas 1800 ft-c anid the gas phase was 21 % O., anid at 200.

CO., evolution CO., evolution AApparent in light2 in darkness

Air-flow photosynthesis' (A) (B) B

litre mimiin *, CO., hr' cCll 2 IL CO, hrI' t-i!2 ,u. C1O.,hr'I Cr2Closed system

2.8 133 ± 1.13 29 ± 3.0 20 ± 0.4 1.451.9 121 ± 3.0 27 ± 3.0 21 ± 1.2 1.291.0 106 ± 3.5 19 ± 1.7 22 ± 0.9 0.870.6 67 ± 3.0 14 ± 1.5 21 + 1.0 0.67

Open systemii2.8 133 ± 2.0 31 ± 1.1 20 ± 1.5 1.551.9 119 ± 2.0 29 ± 3.0 21 ± 1.5 1.381.0 99 ± 3.0 20 ± 2.4 21 ± 2.0 0.950.6 68 ± 3.0 14 ± 3.0 21 + 2.0 0.67

1 Determined at an average CO2 concentration of 300 ppm in both closed and open systems.2 Determined by the extrapolation method in the closed system and by the amount of CO., evolved into a CO<-

free air stream in the open system.3 Mean + S.D. of 3 replicates.

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PLANT PHYSIOLOGY

on other factors that alter the diffusion rates of CO.,(5, 25, 30, 33). Mathematical discussion of thesecharacteristics 'based on electrical analogue modelshave been presenlted (.5, 25, 30. 33) but it may heuseful to presen,t a brief general dliscussion of thesefactors as they affect the rates of CO., evolutionestitmiated by the various miletlhods.

\Vith th,e extrapolation miietlho(d in a closed systemthe rate of CO., evolution in the light is a functionof the slope of the rate of apl)arent photosynthesisversius CO., concenttration. As Saimishi a;nd Koller(34) have l)oin'ted out,, miuclih higher rates of CO,evolution can be estiniated if the rate of ap,parentphotosynithesis is plotted versuts the internal CO..However, the slope of these graphs were also in-creased 'by light intensity, (Fig. 1; 3, 41). The netresuilt of this effect was thiat at low lighlt intensityCO., evolution in the lighit was less thail dark res-

piration whereas at hiighi light intensity it was greaterthan dlark respiration. Similar resuilts were obtainedin the open system (Fig. 6) and the genieral shapeof cuirve resemibled that obtained for RutimiexHolmlgrein and Jarvis (21) and th'at of O., uptake atvarious light initensities reported by Hochi et al. (20).According to Hoch et al. (20) tilis pattern indicates2 nmechanlisimis of O., uyptake or CO., evolu-tion inlight, I that is sulpl)ressed b1 ligigt and I that isstimulated as liglht intensity increases.

In our system the rate of apparenit photosyn-thesis of a sunflower leaf was 13.3 mg CO., hr-1dm--2at 180I0 ft-c and 300 ppm CO.2 (table IfI). rThisrate is sitimilar to that published for stunflower atequivalent light intensity by Waggoner, Moss, andHesket,h ( 39) anid above that reported by \Vhitemanand( Koller 4(41) . The rate of CO., evolution inlight was 2.9 mig CO., hr1'd1dn-2 which is 1.43 timiesthe rate of dark res;piration or 22 % of the rate ofal)l)arent photosynthesis (table III). In stinflower,the rate of ap,parent 'photosynthesis continues to in-crease with lighlt intensity up to ftill stinlight and atthat high lighlit initensity the rate of apparen,t photo-syniithesis is approximately .50 mig CO., hr-V1dnl (39).At light intensity presumabl)l equivalent to ftull stin-light (in the I)apler the only indicationi of lightintensity given was "intense") the rate of CO., evo-

lutionl in light was abotut 9 img CO., hbr-1d(1'm2 (11).This rate, although greater than oturs, wvas about18 % *of the rate of apparent p)hotosynthesis andabout twice the rate of dark respiration at 30° (11).Thus the mlagnitude of CO., evolution in the lightfromii sunflo%ver leaves is a function of the lightintensity and exceeds the rate o,f dark respirationiexcept at very low%, ligh,t inteInsities.

Light-stimulated CO., evolution fromii leaves hlasbeen previously os)served (9, 11, 13, 14, 21,30, 36).Althlouigh Ozbuin et al. (31, 32) report that the rate

of CO., evolution was reduiced by 300 ft-c of lighttheir data (table 1, 32) also show that the rate ofCO., evolution from beain leaves at 1500 ft-c was

3 timiles highler thain the rate at 300 ft-c and 21 %higher thalnl dark resl)iration. Recently, it wvas re-

por-ted that a ligh,t-stimulated CO.-, evolution wasobserved only in leaves which possess the functionalcapacity for photosynthesis (19). It would be pos-sible, when the CO., supply is adequate, to observeconcomi tan,t increases in both apparent photosyn-tlhesis and CO. evolution with ligh.t intensity anld anaplparent association of the rate of CO., evolutionwith that of apparent photosynthesis. This, how-ev.er, miayle miiisleadiing because increased CO.,evolu.tion with increased liglht initensity nmay occurwithout the accominpanying increase in CO., atssimiila-tioin under severelyN CO., limiiite(d conditions CO.,-freeair). Thus the effect of light on CO., evolution waslikely not due only to increased substrate suppliesas the result of tlle incre.ased CO., assimiiilation butwas p)robably dtue to a direct effect of light Onl CO.,evolution mediated through the plhotosy'nthetic ap-paratus ( 19).

The rate of CO,. evolution in the li-lht measturedin either the open or closed svstemis was also affectedby air flow rate through tlle cuvette (Fig. 1 and 5).At less than 1 liter per nmim the rate of CO., evolutionwas always lower than that in darkness. However,at 2 liters per mmin the rate of CO., evolution in lightwas 1.5 times greater than that observed in darkness.El-Sharkawv, Loomis atnd \\Tilliami.s ('12) have pre-viouslv reported that at low air-flow the rate ofCO., evolutio,n in the light w\,tas about 40 % of darkrespiration whereas at high air-flow it was almosttwice the dark rate. The flow, rate of air aroundthe leaf exerts its effect b\y decreasing the lanminarresistance of the leaf anXd ren,ewing the gas in thecuvette and thereby nmaintaining the difftusion gradi-ents for CO., into or out of the leaf ( 1, 16, 17, 21).In the open system xvith CO.2-free air a higlh air-flowwould rapidly remilove the evolved CO., fronm thevicinity of the leaf and it would be mieasuired in theIRGA. At low air-flow the CO., concen-trationaround the leaf would increase, less CO., woulddiffuse fro-m the leaf and miore woulld be refixed byphotosynthesis. In addition evolved CO., maxy bereabso,rbed bv the leaf-both events would decreasethle mieastired CO2 evolution froml the leaf. This issupported by the observation that in darkness wherethere was little or no refixation of CO.,, the flowrate had no effect o,n the rates of CO., evolution. Itis difficult to understand the results of EI-Sharkawvet al. ( 12) that shlow dark respiratioin to be inlhibitedby 50 % with increasing flow rates. rhey claimithat the decreased rates are due to stomiiatal closureand a consequent decrease in. CO., diffusion due tothe increased stoinatal resistance. During the tranisi-tory period when stomilata are closinig their explana-tion is quite v;alid but the stead, rate of dark respira-tionI will remain the samiie sinice the diffusio,n ratewill againl increase because of rapid establishmient ofa. larger CO., gradien,t (30).

The adequacy of air flow rates in a gas exchangesystenm is an importan,t question In 1947 Decker(7) showed that the rate of photosyn!thesis in,creasedhyperbolically with increased air supply and Avery

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(1) and Hesketh and Musgrave (17) have verifiedthis pattern. A hyperbolic response pattern of ap-parent photosynthesis and CO., evolution in the lightto air flowv was also observed in the present studies(Fig. 5). Obviously, flow rates are adequate wlhennlo further increases in the processes are observedwith further increases in flowv rates. In our cuvettesystem.s this I)oint corresponded to ain air flow greaterthan 2 liters per mnil. This does not meani that thelaminar resistance is as low as possible but onlyr thata further decrease in laminiar resistance cannot beachieved by increasing the flowv rate.

Some studies with corn leaves wvere included inthe present study since it has previously heen re-ported (15. 27, 28, 29) that they di(d not exllibit alight-stimiulated CO., evolution. This was corrobo-rated as the CO., compensation point was near zeroas reported by many workers (15, 27.28, 29, 30)anid the specific radioactivity of the 4CO., arounldthe leaf during illumination remained conxstantt (Fig.3). This may be due either to the absence of 12CO.0evolution during illumination or to an efficient photo-synthetic mechanism that is able to reabsorb tlhe CO.,evolved within the leaf before it can escape to theoutside. It has 'been observed previously (4, 14, 36,38) that an increase in the 0., con,tent of the gasstream suppresses photo.synthesis in many plants in-cluding corn. However, in corn, increasing 0., coIn-centration had no effect on the CO., conmpensationpoint (15). It appears unlikely theni that cornexhibits any light-stimiulated CO., evolution. TlliscoInclusioII wvas also reached by Downton and Tre-gunna (10) on the basis of other evidence and moreextensive experimentation.

Acknowledgment

The sunflower seeds were supplied by Dr. E. D. Putt,Department of Agriculture, Mforden, Manitoba, Cana(la.

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by Green ILeaves in Light' )laced in. a leaf - Plant Physiology