Plant Physiol. (1966) 41, 1623-1631

Increased Rate of Net Photosynthetic Carbon DioxideUptake Caused by the Inhibition of Glycolate Oxidase

Israel ZelitchDepartment of Biochemistry, The Connecticut Agricultural Experiment Station,

New Haven, Connecticut 06504

Received July 22, 1966.

Sumnnmtary. There is considerable variation among species in their rate of photo-respiration, and photorespiration increases greatly at higher temperattures. Theaddition of an inhibitor of glycolate oxidase, a-hydroxy-2-pyridinemethanesulfonicacid, to tobacco leaf disks at 350 stimulated photosynthetic 14,CO2 uptake at least3-fold, but 14CO tuptake was not changed by the inhibitor at 250. The inhibitordid not increase photosynthesis in maize leaf disks at either temperature.

The evolution of COO from glycolate was greatly enhanced in tobacco at 35°compared with 250. Labeling of the glycolate of tobacco with glycolate-1-1'4C and-2-'*C showed that the increased CO evolved in the light (photorespiration) arosespecifically from the carboxyl-carbon atom of glycolate. Maize, a species knownto have a negligible photorespiration, produced 14CO, poorly from glycolate-1-14Cin comparison to tobacco.

Acetate-1-14C, a substrate metabolized by dark respiration, produced similaramounts of '-CO2 in the light in both tobacco and maize. This respiration waschanged little relative to photosynthesis by increasing temperature.

Most plants, such as tobacco, have a high photorespiration. The loss of fixedcarbon causes an increase in the internal concentration of CO, especially at highertemperatures, and results in a lower CO2 concentration gradient and therefore alower net photosynthetic CO2 uptake. Some species, like maize, have a negligiblephotorespiration and are thus morc efficient photosynthetically. The use of aninhibitor of the oxidation of glycolate, the substrate for photorespiration, changedtobacco so that it behaved photosynthetically like maize. Thuls high rates of photo-respiration may limit the net CO., uptake in many plant species.

Ample evidence exists that photosynthetic tissueshave a respiration in the light that results in CO2evoluition (photorespiration) which occurs by reac-tions different from those which occur in darkness.Under normal conditions of photosynthesis, thisphotorespiration is often greater than the darkrespiration, whether measured by 02 uptake or CO2evolution. This paper is concerned with the natureof the substrate of this photorespiration and with therole of photorespiration in net CO, assimilation.

Decker (1) observed that when leaves of sev-eral species including tobacco were transferredfrom the light to darkness, there was a burst ofCOO evolution before CO2 output resumed a lowersteady rate. The CO. burst undouibtedly repre-sented an overshoot of the greater CO2 evolutionthat occurred in the light. By measuiring 0 uptakewith the aid of 180, labeling, Hoch, Owens, andKok (6), have shown that several species ofalgae have a photorespiration which increases withincreasing light intensity and that this is super-

imposed upon and presumably different in mech-anism from the dark respiration. Ozbun, Volk,and Jackson (13) also found a greater 02 uptakeby bean leaves in the light using mass spectrometrictechniques.

Forrester, Krotkov, and Nelson (3, 4) recentlyfound that photorespiration in soybean leaves wasgreatly dependent on the 02 concentration in theatmosphere, whereas dark respiration was essen-tially tunaffected at O, concentrations as low as1 %. Thus photorespiration must occur by reac-tions that are different from those concerned withdark respiration. Maize had an insignificant pho-torespiration at all concentrations of 02 tested.

Several lines of evidence indicate that photo-respiration differs in intensity among variousspecies. Moss (11) measured CO2 evolution in thelight in a CO2-free atmosphere and observed that,in a number of species including tobacco, CO2evolution was at least 50 % greater than darkrespiration. In maize, significant CO, evolution

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PLANT PHYSIOLOGY

could not be detected uinlder the same conditions,again emphasizing the difference in photorespirationbetween these species.

\Vhen a plant is placed in turbulent air in achamber with strong light, the CO., concentrationin the atmosphere surrouinding the plant reaches asteady state (the CO, compensation point). Certainplants known to be efficienit in their photosynthesis,incltiding maize and sugar cane, were foulnd( byMoss (9) to have a CO, compensation point closeto zero. Most other species have a CO, compen-sation point in excess of 50 ppm. Therefore thevalue of the CO, compensation point correlates wellwith the previously indicated knowledge about pho-torespiration in variouis species. In suich specieswhere the internal CO., concentration is considerable,it is reasonable to assulme that photorespiration cancauise a diminished net CO., uptake from theatmosphere through the loss of fixed carbon whichdecreases the CO., concentration gradient on thepathway from the air to the chloroplast.

Other workers (5, 15) have described the effectof temperature on the CO., compensation point ina number of species and fouind the valuie to be atleast twice as great at 350 as at 250. Meidner (7)also showe(d there was an effect of temperatuire onthe CO, compensation point of outdoor grown maizealthouigh the qulantities were small, i.e. 2.7 ppm at25° and 5.5 ppm at 350.

The net CO, uptake by maize is twice as greatat 300 as compared to 200 when water is notlimiting (10). In tobacco, however, CO., uptakeis less at 300 to 350 than at 250 (personal com-mulnication from D. N. Mloss). Egle and(l Schenk(2) have shown that the CO., compensation pointin Pelargonium leaves varied greatly with tem-peratture. At 250 they fouind abouit 80 ppm andat 350 185 ppm. Thuis over this 100 range, photo-respiration mulst have initially increased at a ratefaster than the rate of CO., uiptake by the chloro-plasts. These workers also observed a 20 % in-hibition in net CO. uiptake at 350 compared to 250.

Althouigh direct evidence on the natuire of thespecific sulbstrate for photorespiration was thenlacking, I previouisly suiggested (21) on the basisof a number of correlations foulnd in the literatuirethat the CO. produlced coullcl come from glycolateby the glycolate oxidlase reaction as follows:

CH.,OH-C*OOH 4- 0() CHO-C*OOH +(1)

H,O., -* HCOOH + C*O. + 1H.,O(2)

If this were the souLrce of the CO., prodlucedby leaf tissuies with a high photorespiration., in-hibition at either reaction 1 or 2 wouil(d be expectedto lower the internal concentration of CO.,. Thusplants with a high photorespiration could be an-ticipated to show increasecl rates of CO., uiptakewith inhibitor becauise losses of fixe(d carbon wouildbe (liminishe(l and the internial CO. concentration

woulld be decreased. This effect wN-ouldl be im1ag-nifiedl at warmer temperatuires if photorespiratioincreases greatly. Thuis one might be able to con-vert a plant Which behaves like tobacco into onelike maize ancl to obtain increase(l net C(o). uptakeexperimentally at higher temperatulres. Tregunna(16) recently acconiplished the reverse effect whenhe stupplied maize leaves with riboflavin phosphateto increase the CO., compensationi point aind catiseda decreased CO., uiptake.

By adding an effective inhibitor of glycolateoxidase, a-hydroxy-2-pyridinemethanesuil fonic aci(l(19), to tobacco leaf disks, I have now fouind thatthere is a 3-foldl increase in CO., uiptake at 350while at 250 CO., uiptake is the same Nwith or wsithoutinhibitor. Fuirther experiments confirm that thecarboxyl-carbon of glycolate is the primary souirceof the CO., produiced in photorespiration in a specieswith a high rate of photorespiration. These ex-periments also permit a quantitative estimationi ofthe role of photorespiration in the process of netCO., uiptake.

Materials and Methods

Plants. Tobacco (Nicotianial tabacuin, varietyHavana seed) aInd1 maize (Zea nay's, hy-brid PeInn1602A) were grown in sand in a sulbirrigatedc benchin the greenhouise. barge leaxes were excised and(Iplaced with their bases in water in the (lark for-at least several houlrs before disks were cuit fromsymmetrical positions with a sharp punch. Six(lisks, 1.6 cm in (liameter, were threa(ledl on to alength of niylon threadl to facilitate rapid hand(liingan(l placed in large (75 ml) \Warburg xessels con1-tainiing 1 to 2 ml water. The vessels were shakenat 120 oscillations per minute for abouit 1 houir illthe light (2000 ft-c suipplied by tunligsten laampsabove and a mirror directly below the vessels) ina constant temperatuire bath with the vessels openlto the air.

Phlotosynthetic CO., Upt(ake. At the endl of thepreliminary light period, the water in control vesselswas sometimes removed an(d replace(d with freshwater. In vessels with inhibitor, the water w\,asremoved and quickly replace(d with a soluitionof a-hydroxy-2-p . rid(inemethainesul foinic acid and(1NaH14CO3 was added to the sidlearm. The maniom-eter taps w!ere closed after 30 secoIlIs aind after aperiod of 2 or 3 minu1tes in light, '4CO. of knownconcentration anid( radioactivity was liberated intothe gas phase by the addition of 10 x suilfuiric aci(dto the soluitioin of NaH '4C(>., in the (louble sidlearmof the \Varbuirg Xvessel. At the end(I of the experimeint,the leaf disks on their threads xw-ere rapidly takenfrom the vessels anid pltunged into 20 ml of boiling20 % (v/vr) ethyl alcohol, an(l the mixtulre w%-asboiled for 3 minuites. The killedl disks were homog-enizedl, the suispension made to 25 ml, annd theradioactivitv was oletermiieol by scilntillation coulnt-ing on ta 0.05 m1l sample in 10( ml of a solultion

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ZELITCH-INCREASED CO., UPTAKE BY GLYCOLATE OXIDASF, INHIBITION1

containing 6 g of 2, 5-diphenyloxazole and 100 mgof p-bis [2-(5-phenyloxazolyl)] benzene in 1 liter oftoluene and 500 ml of 95 % ethyl alcohol. Asmall quenching correction was made.

Assimilated-' 4C and 14C09 Produiction. XVhena radioactive substrate was supplied to the leafdisks, this was always done after the disks hadbeen kept for a preliminary period in the lightin water. Then the water was replaced with 1.0ml of radioactive solution. When glycolate-14Cwas added, the '-CO, produced was collected bypassing air saturated with water vapor at the sametemperature throuigh the Warbulrg vessels contin-uially at a rate of 200 ml per minute and trappingthe 14C*09 in the air stream with aeration tubes in25 ml of 1 M ethylenediamine. The radioactivityin the ethylenediamine solution was determined byscintillation cotunting. Control experiments demon-strated that released 14CO9 was trapped quantita-tively under the conditions used.

At the end of the experiment, the disks wereremoved and were briefly immersed in a largevoltume of water to remove adhering 14C-substratebefore being killed in boiling 20 % ethyl alcohol.After grinding the disks with a glass homogenizer,radioactivity in the homogenate was determined,and the stuspension was centrifLuged. The combinedstupernatant flulid and washings were placed on acolumn of Dowex-1 acetate, and the glycolic acidfraction was collected by elution with 4 N aceticacid (19). In some experiments the glycolic acidconcentration was determined colorimetrically onsamples taken from the glycolic acid fraction (19).The amount of assimilated-14C was calctulated fromthe total 14C in the homogenate less the '4C inglycolic acid.

When acetate-1-'4C was the substrate, the ex-periments were carried out exactly as with gly-colate-14C. The acetate-14C remaining in the leafdisks at the end of the experiment was also deter-mined by placing the leaf extract on a column ofDowex-1 acetate. The acetic acid-14C was eluitedwith 4 N acetic acid and appears in the eluate juistprior to the glycolic acid fraction.

Radioactive Suibstrates. These were obtainedfrom Volk Radiochemical Company. Before use,the glycolic acid-l-'4C and -2-'4C were each placedon Dowex-1 acetate coluimns, and the glycolic acidfraction was collected as previouisly described. Thefraction was dried in a stream of air at 450.Carrier glycolic acid was added, and the acid wasnetutralized to about pH 5 with solid KHCO, andmade to a known volume. Potassium acetate-1-14Cadjulsted to pH 5.2 was uised.

Results

Effect of Temiperature ot the CO., Com)pensationPoint for Tobacco. In those species that have asignificant photorespiration, the CO. compensation

Ea

aFz00-QL

0U

N

0

u

130

90

50

1010 20 30 40LEAF TEMP., C

FIG. 1. Effect of temperature on the CO, compen-sation point of excised tobacco leaves. The leaves wereplaced in light (6000 ft-c from an incandescent source)in a chamber in which the air was circulated and theCO9 concentration in the atmosphere was determinedwith an infrared CO9 analyzer (10). The relative hu-midity was about 90 %. Leaf temperatures were deter-mined by thermocouples attached to the leaf. The steadystate CO. concentrations u-ere established in about 30min.

point is considerably greater at 350 than at 250.Figure 1 demonstrates that this generalization alsoholds true for tobacco. The CO2 compensationpoint at 250 was 48 ppm and at 350 was 80 ppm.As others have found, there was a sharp rise above300. This increase in the compensation point couldbe caused by an increase in the internal CO. con-centration from dark as well as photorespiration, orby a decrease in the rate of C00 uptake by thechloroplasts as the temperatture is raised. The re-stilts with maize (10) as well as the increase innet photosynthesis in tobacco that is shown laterin this paper indicate that the latter explanationis most unlikely. Thus one can conclude that in-creased CO, compensation points at higher tem-peratures are caused by a relatively faster prodtuc-tion of CO, by respiration, mostly photorespiration,rather than by decreased rates of photosynthesis.

Effect of Glycolate Oxidaise Inhibitor on 14CoUptake. In previous experiments with tobacco leafdisks in light (22), I have shown that the additionof glycolate oxidase inhibitor at 300 had no sig-nificant effect on the 14CO., uiptake. From the CO.,compensation points, one wotuld anticipate thatphotorespiration in this tissue would greatly increaseabove 300. Accordingly, the rate of 1'CO, uptakeby tobacco leaf disks was measured in the presenceand absence of an effective concentration of inhib-itor at 250 and at 35°. Although this inhibitor isknown to inhibit opcning of leaf stomata (20),

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PLANT PHYSIOLOGY

control experiments showed that there was noclosing effect on stomatal widths as measured bysilicone rubber impressions for at least 20 minutesat 250 and for as long as 40 minutes at 350 underthe conditions of the photosynthesis experiments.

Table I shows that at 350 the rate of 14-COuptake in the presence of inhibitor was stimulatedin the 6 experiments from 2.1- to 6.0-fold, with amean increase of 3.8-fold. In the 3 experimentscarried out at 250, the inhibitor changed the 14CO0uptake from 0.5- to 1.2-fold, with a mean of 0.8-fold, results similar to those previously observedat 300 (22). The 14CO2 uptake took place in aclosed system and varying amounts of the available4CO, were assimilated in the different experiments.The photosynthetic rates given in table I thereforerepresent minimal values. Thus under these experi-mental conditions there was always greater netphotosynthesis at 350 but not at 250. As shown inExperiments No. 4, 5, and 6, disks in water didnot have a greater photosynthetic rate at 350 thanat 250. Since the rate of synthesis of glycolicacid did not increase sufficiently at the highertemperature in the presence of inhibitor, the effect

of the inhibitor in bringing about higher rates of14CO9 uptake at warmer temperattures must berelated to a slowing of the normal production ofCO2 in the glycolate oxidase reaction.

Similar experiments on the effect of temperatureon the rate of 14CO uptake by maize leaves werealso carried out for comparative purposes (table II).In maize, the a-hydroxysulfonate did not cause anincrease in '"CO, uptake, and was in fact somewhatinhibitory and more variable at both 250 and 350in contrast to the results with tobacco. The rateof glycolate synthesis in maize was considerablylower than in tobacco. Nevertheless, significantquantities of glycolate were produced in maize, sothat the differences in the photorespiration of these2 tissues, if glycolate is involved, is more likelydetermined by the metabolic fate of this substraterather than its synthesis.

To account for the results in table I a balancesheet (table III) has been prepared which providesa basis for further experimental tests of the roleof glycolate metabolism in photorespiration. Thetable was- constructed as follows: lines 4 and 5give values of "4CO., uptake that are typical of

Table I. Effect of a-Hydroxysulfonate on 14CO., Uptake by Tobacco Leaf Disks in Light at 250 and 350After a preliminary period in the light at the temperature indicated, the fluiid in the Warburg vessels was replaced

with 001 M a-hydroxy-2-pyridinemethanesulfonic acid (the inhibitor), and after 2 min 3 /moles of 14CO9 (total of4 jumoles in the gas phase to make the initial concentrationi about 0.12 %) containing 502,000 cpm were liberated for thetime shown. In Expt. No. 4, 72 % of the available 14,CO, was taken up by the disks with inhibitor at 350, the highestpercentage of any experiment.

The glycolic acid formed in the presence of inhibitor, in inmoles hr-1 g fr wt-1, at 250 and 350 respectively was:Expt. No. 4, 15.7 and 29.9; Expt. No. 5, 26.9 and 25.8; Expt. No. 6, 5.3 and 9.0.

jumoles 14CO2 uptake per hr per g fr u-t25° 350

Expt Min in Disks in Disks in Disks in Disks inNo 14CO.. water inhibitor water inhibitor

1 5 31 662 2.5 45 2683 3 47 2034 5* 56 67 61 1815 2.5 136 62 58 1286** 2.5 39 34 37 188

* The exposure to 14C,O., was 5 min at 250 and 4 min at 350.** The concentration of inhibitor in this experiment was 0.005 M.

Table II. Effect of a-Hydroxysulfonate on 14CO., UI take by Maize Leaf Disks at 250 and 350 in LightThe experiments were carried out in a manner similar to those in table I, except that 2.5 ,umoles of J4CO (total

of 3.5 umoles in the gas phase to make the initial concentration about 0.10 %) were liberated for the time shown.The concentration of a-hydroxy-2-pyridinemethanesulfonic acid in Experiment No. 2, 3 and 4 was 0.005 M.

The glycolic acid formed in the presence of inhibitor, in amoles hrI g fr w-t-1, was: Expt. No. 1, 3.6; Expt. No.4, 3.5 at 25° and 4.9 at 350.

,moles 14CO, uptake per hr per g fr xvt250 350

Expt Min in Disks in Disks in Disks in Disks inNo 14CO., water inhibitor wMater inhibitor

1 5 51 9.8234

435

7840

7511

78 77

57 28

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ZELITCH-INCREASED CO., UPTAKE BY GLYCOLATE OXIDASE INHIBITION

Table III. Model of CO, Budget for Tobacco Leaf inLight at 25° and 350

,umoles CO, per hrper g fr wt

250 350(1) Gross CO., uptake -100 -200(2) CO, output, dark respiration +10 +20(3) CO, output, photorespiration +15 +115

(4) Net CO., uptake observed: -75 -65(5) When photorespiration inhib-

ited, net CO., uiptake observed: -90 -180

observed rates. The rate of dark respiration at250 (line 2) is similar to a published value (19)on the assumption that dark respiration continuesat the same rate in the light. Moss has shown(11) that photorespiration in tobacco at 250 exceedsdark respiration and this is shown in line 3. It isreasonable to assume that the dark respiration isapproximately doubled by an increase of 100, atypical valuLe for enzymatic reactions. I assumethat the rates of enzymatic reactions concerned withCO. tuptake also double at 350 compared with 250,since such an increase occurs normally in maizebetween 200 and 300 (10). To account for thetypical resuilts in lines 4 and 5, it follows that thephotorespirationi (line 3) must increase about 8-foldover this range of 100.

Table III shows that photorespiration accountsfor about 60 % of the gross CO, uptake at 350.If the CO., ouitpuit resulting from photorespirationwere inhibited, there would be little change in theapparent photosynthesis at 250 (lines 4, 5). If theCO, ouitpuit caused by photorespiration were inhib-ited at 350, however, the model shows the expected3-fold increase in the measured CO2 uptake (tableI).

This model was tested further by supplyingleaf disks with tracer qtuantities of glycolate, thesuspected stubstrate of photorespiration, as well aswith acetate, a substrate known to be metabolizedby dark respiration (through the citric acid cycle),in order to see if the results predicted from tableIII would be realized.

Effect of Temtiperature on Metabolisnm of Gly-colate-1-14C aId Glycolate-2-14C. Table III andfigure 2 predict that if the carboxyl-carbon ofglycolate is the primary source of the enhancedCO2 produced at higher temperatures by photores-piration, the "4CO., released from glycolate-1_14Cshouild be about 8-fold greater at 350 than at 250.However, the rate of gross CO. uptake would beincreased 2-fold at the higher temperature. Ac-cordingly, the observed increase in 14CO, otltptltfrom glycolate-1-1"C might only be expected to beapproximately 4 times as great at 350 as at 250.The results were calculated on the basis of theamounlt of assimilated-'+C, because this permits amore meaningful comparison between experiments

when the amount of 14C-substrate taken up by theleaf disks may vary. The assimilated-IlC is thetotal radioactivity in substances other than gly-colate. The results were therefore expressed as

the ratio of the '4CO., produLced to the assimilated-14C.

INTERNALRESISTANCE

>-OH SULFONATE MAIZE

CH20H COOH O?C HO COOH HCOOH +C0

2C02

GLYCOLATE GLYOXYLATE 0O

Io C02ASSIMILATED -14C STO TAL

RESISrANCE

INSIDE LEAF ATMOSPHI

IER

FIG. 2. Representation of the biochemical reactions ofglycolic acid in relation to the CO2 produced by photo-respiration. Glycolate-1-14C (0) or 2-14C (0) was

added to leaf disks in the light and the 14CO., in theatmosphere was swept out by a rapid stream of air andtrapped in alkali. Reaction 1 is the glycolate oxidasereaction that is inhibited by a-hydroxysulfonate. Reac-tion 2 is non-enzymatic, requiring H2O., and is presum-

ably slow in maize relative to Reaction 3. Reaction 3represents all of the biochemical reactions whereby gly-oxylate is converted into other products. Assimilated-14C was determined from the total 14C in the tissue lessthat of the glycolate-14C. Reaction 4 represents theformate dehydrogenase reaction, known to be present inleaves. Reaction 5 indicates the conversion of formateinto products other than 14CO.,. Stomata must be open

in these experiments to minimize the stomatal diffusiveresistance and thus aid the release of 14CO..iv) r

E

-15 350

S~~> 10>I/0

W5 250

0 20 40 60 80MIN. IN GL_YCOLATE-I-'4C

FIG. 3. Rate of 14CO., production by tobacco leafdisks in light from glycolate-1-14C at 250 and 350. Sixleaf disks were shaken in the light in water for about1 hr, then the fluid was replaced by 1.0 ml of K gly-colate-1-14C (0.04 M containing 1.85 X 106 cpm). Moist-ened air at the same temperature was passed throughthe vessels at a rate of 200 ml per min, and the 14CO,evolved was trapped in 25 ml of 1 M ethylenediamnine.

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Table IV. Effcct of TInipcratureon Metabolism of Glycolate-1-14C and G/v u/u/c-2-14C by Tuluit ( I'fDisks in Light

Experiment 4 was carried out in darkness. In an experiment carried out like those in the tabile, it vas foull(ithat after 60 min in the light in H.,O followed by 60 min in 0.04 M K glycolate at pH 5.0 the meall stcmatal xidthwas 7.3 jg at 250 and 7.2 at 350.

Expt Position ofNo glycolate-' 4C

-1--1--2--2-

-1--1--2--2-

-1--1--2--2-

-1--1--2--2-

2

3

4

Temp,0

25352535

25352535

25352535

25352535

Total"4C supplied,106 Cpm21

1.041.041 001.00

1.041.041.001.00

2562 562.89289

2.512.512.792.79

14CO.0Time, released,min cpm

80808080

60606O60

60606060

60606060

13809250750

3630

100047009001900

325017.30015096130

18.50065 70014506290

Assimilated-1 4C,cpm

18 20029 60012 40028 200

16 20)25 30013 40025 500

32600.5 80923 30066,100

20 50039 60055 700

122 000

Ratio 14COreleased:

1 4C-assimilated

0.0760310.0600.13

0 062( 190)07)0 075( 100310 0640093

0.9017.0 0260 052

Glycolate-2-"4C may servethe ratio with this substrate

as a control, sinceshould not change

significantly (ffig 2) if the methylene-carbon ofglycolate does not serve as the primary source ofthe increased CO., in photorespiration at highertemperatuires. Figuire 2 also indicates that if reac-tion 2 were blocked in maize, in the light little "CO2woould be produced compared with tobacco.

Experiments to test this hypothesis with gly-colate-14C are shown in figture 3 and table IV.The rate of 14CO.. release from glycolate-1-_4Cwas constant after a short lag period and was

greatly dependent on the temperatture. No correc-

tioIn for assimilated-l-C was made in figure 3. InExperiment 1 (table IV), the ratio of the 14CO,prodtuced to the assimilated-14C at 350 compared to250 with glycolate-1-"4C increased 4.1-fold, and inExperiments 2 and 3 the ratio was 3.1 times asgreat. These figures agree reasonably well withthe anticipated increase of about 4-fold expectedfrom the model in table III. The yield of "4CO2from glycolate-2-14C was similar at 250 to thatproduiced from glycolate-1-'4C but there was no

great increase in '4CO. from methylene-labeledglycolate at 350. In the dark (Experiment 4), atleast 6 times as much 14CO9 was evolved from thecarboxyl-carbon as in light, and the ratio increasedless in the dark than in light when the temperaturewas raised although there could not have been

significant "4CO., uptake in darkness. Thus thegreatly enhanced 14CO., output observed from gly-colate-1-14C during photorespiration at higher tem-peratulres probably does not represent all of the

14COQ prodluced within the tissule. The small changein ratio with glycolate-2-14C as substrate in thelight serves as a control for comparison.

If the carboxyl-carbon of glycolate is the primarysource of CO2 in photorespiration, one would ani-ticipate that little 14CO., wouild be liberated fromglycolate-l-l'C by a leaf such as maize whichnormally has only a slight photorespiration (fig 2).The rate of 14CO. slhoulld be small from either carbonatom of glycolate, and shouild be relatively inde-pendent of temperatuire. This prediction wvas con-firmed by the data in table V. Maize leavesassimilated as much '4C from glycolate-'4C as didtobacco, but the qvantity of 14C0, produticed fromglycolate-1-'4C at 350, for example, was only 2 %as much in maize as in tobacco. There vas alsoa relatively small effect of the position of thelabel or of the temperature on the quantity ofi4QCO produced in maize in comparison with to-bacco.

Acetate-1-14C Aletabolism in Light. The modelin table III shows that if a substrate known to beoxidized to CO, by clark respiration mechanismswere labeled with '4C, the rate of 14CO., releasedlin the light would Inot be greatly affected bv achange in temperatulre from 250 to 350. Thus at350, one would expect an increase in the rate of'+CO2 production of 2-fold compared with 250, butthe gross CO., uiptake would also be twice as rapid.Therefore table III predicts that the ratio of the"4CO2 produced woould be the same at 350 as at 250.Acetate-l- 4C was therefore added to leaf disks oftobacco andl maize in the same manner as in the

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ZELITCH-INCREASED CO., UPTAKE BY GLYCOLATE OXIDASE INHIBITION 1629

Tab'e V. Effect of Temiperature on Metabolism of Glycolate-1- 4C and GlIcolate-2-4C by Maize Leaf Disks in Liqhi

TotalExpt Position of Temp, 14C supplied,No glIcolate-14C 0 106 cpm

-1--1--2--2-

-1--1--2--2-

2

25352535

25352535

1.041.041.001.00

9789.78

10.410.4

14CO0)Time, released,min

60606960

60606060

cpm

400200200100

250012501000709

Assimilated-' 4C,cpm

27,40054,10018,70046 300

194,000475,000228 000348,000

Ratio 14CO.,released:

I 4C-assimilated

0.020.0040.010.002

0.010.0030.0040.002

Table VI. Effect of Temtterature on Mletabolism of Acetate-1-14C by Tobacco and Mlai.-e Leaf Disks in Light

l 4CO., Raitio 14CO.,Expt Temp, Total 14C, Time, released, Assimilated-'4C released:No Species ° 10e cpm min cpm cpm 14C-assimilated

1 Tobacco 25Tobacco 35

Tobacco2? Tobacco

MaizeMa.-ize

TobaccoTobaccoMaizeMaize

25352535

25352535

3.953.95

0.7900 7900.7900.790

13.013.013.0130

80 428080 7020

60606060

60606060

5001000200400

690014,00056002900

226,000284,000

16,80027,50081009400

143,000308,000

61 000

0.0190.025

0.030040.020.04

0.0480.045

0.048

previous experiments with glycolate-1"C, and therestults were expressed in the same manner. TableVI (Experiments 1, 2, 3) shows that the ratio of'4CO., produced to the 14C-assimilated was about thesame in tobacco at 250 and 35°. Experiments 2and 3 demonstrate that the relative proportion of14C09 produced in tobacco and maize was also thesame at both temperatures. This is in sharp con-trast to the restults obtained when the glycolic acidin the leaf was labeled with glycolate-1-14C (tablesIN' and V).

Discussion

Glycolate is an important early product of pho-tosynthesis produced from CO, only in the light(22). There are a number of suiggestions thatglycolic acid serves as the primary substrate forphotorespiration, and is hence the main soturce ofCO, inside the leaf in most species includingtobacco. Species known to be efficient in photo-synthetic CO. uptake appear to lack the reactionswhereby CO., is produced from glycolate. Moss(12) has recently found that addition of a glycolateoxidase inhibitor, a-hydroxy-2-pyridinemethanesul-fonic acid, to tobacco leaves slowed the photores-piration but did not affect the dark respiration.The quiantity of glycolate formed in the light agreed

well with the amouint that would normally havebeen needed to produce CO, in the leaf withouttinhibitor. Photorespiration increases relative tophotosynthesis especially at temperatures above 300.When the oxidation of glycolate was inhibited intobacco leaf disks at 350, there was at least a3-fold increase in the rate of photosynthetic CO9uptake. The inhibitor did not stimulate photosyn-thesis in tobacco at 250 or in maize at eithertemperatulre. This increase in photosynthesis intobacco was undoubtedly brought about by inhibitingthe CO9 outputt from glycolate, so that fixed carbonwas conserved and the CO9 concentration gradientbetween the atmosphere and the chloroplast wasthereby increased. Since maize has only a negli-gible photorespiration, large increases in CO9 up-take with higher temperatures occur normally (10).Increases in gross photosynthesis also occutr athigher temperatutres in tobacco, but this is maskedby the high photorespiration.

The CO, budget that was constrtucted to accountfor the results with tobacco (table III) indicatesthat at 350 the rate of CO., produced by photores-piration woutld represent 60 % as much as the rateof the gross CO9 tuptake by the chloroplasts. Thusin most plants, especially at higher temperatutre, theloss of assimilated carbon by photorespiration isprobably the limiting factor in net photosyntheticCO., utptake. The anticipated increase in 14CO0,

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10PLANT PHYSIOLOGY

formation at 350 compared to 250 wxas foulntd withglycolate-1-'4C as substrate buit not with glycolate-2-14C. In the dark, L4CO., ratios increased equiallyat the higher temperatuire from 1)oth carbon atomsof glycolate.

The experimenits in wlich glycolate-14C was uisedan(d the '4CO., released was meastured were carrie(douLt uindler contditionis that were optimal for stomatalopeniing. Control experiments (lemonstrate(l thatthe addlitioin of glycolate soluition had a tendlenicy tocauise even somewhat larger stomatal wi(dths com-pared with leaf disks floated on water alone. Theseconditions were essential to permit the demonstra-tion that the carboxyl-carbon of glycolate was thespecific souirce of the increase(d CO., ouitplut inphotorespiratioil at higher temperattures in tobaccobut not maize (tables I V, V) . Acetate oxi(latioIndid not ftulfill these requiiremeilts (table VTI). AnI-other attempt was made to test the role of glycolatein photorespiratioln by) uise of ain inhibitor thatm:ght block CO., uiptake in the light but not photo-respiration. For example 3-(4-chlorophenyl)-1, 1-dlimethyluirea (CMIU), at 5 X 10) N', completelyinhibited '4CO. uiptake by tobacco leaf disks. Thisconcentration also close(d stomata significailtly, hencethis inhibitor was not suitable to permit a validtest of the role of glycolate in photorespiratioI,since CO. produiced would likely be refixed whenstomatal diffulsive resistance is high (fig 2).

It is known that if leaf tissues are adequatelyhydrated, stomata open wider in the light at highertemperattures (14, 18). Also in tobacc) leaf, thestomata open wi(ler at stuch higher temperatuiresalthouigh it is clear that in a closedI system theinternal concentration of CO., muist increase greatlyas the leaf is warnmed. Nccordingly, the hypothesis(8) that closing of stomata is the resuilt of increasedconceintratioins of CO., ithin the tissules lea(ds tocertain dlifficuilties which are presently not fullyresolved.

MWaize assimilatecl glycolate effectiv,ely but di(dnot produtce CO., from this substrate in the light(fig 2). IInmaize, the fuirther oxidatioin of gly-oxylate is presuimably slow relative to the rate atwhich it is converted into other produLcts. Tre-gulnIna (16) suiggested that the prosthetic grouip ofglycolate oxidase (riboflavin phosphate) is (lisso-cclated in maize so that glycolate oxidase activity islimiting, hence only small amoLlnts of Ci(O., areproduticed. Data in table V, howeever, in(licate thatglycolate is oxidize(d effectively in \viv\o. Hencethe failuire to produlce CO. b)y this reactioni mustrestult from a block as shownl in figuire 2, sincereactioins for the direct ultilization of glycolateare ulnknown.

The high rate of CO., evolution by photorespira-tion, especially at elevated temperatures, serves toemphasize that the internial tuirnover of carbon mtustbe conlsiderable in many species. These dlifferencesin the photorespiratioin among species may, wellaccouinlt for the high ph 3tosynithetic efficienlcy of

maize, as compare(l wvith plaints like tobacco, thathas often been observed (1/7). The glycolate oxi-(lase reaction is presuimably- essential for plantsgrowing at lower temperattires but appears to beharmfuli at warmer temperatulres when the reactionprodtucts are altere(l. The (lemoinstration in thelaboratory that the efficiency in absorbing C().,from the air by- a planit wvith a high photorespiratioi(tobacco) can be made similar to oIne wvith a lowphotorespiration (nmaize), provides another approachto the possibility of obtaining large increases inphotos)ynthetic yields.

Acknowledgments

I thanik mny colleague Dale N. NMoss for carrying outthe experiment oIn the CO., coII)enisatioIl point, BoInnieGamiibs for skillful technlical assistanice, and 111v associatesfor their helpful (lisciiSsionI.

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ZELITCH-INCREASED CO., UPTAKE BY GLYCOLATE OXIDASE INHIBITION

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