oxygen inhibition of photosynthesis - · pdf filephotosynthesis versus (co2-r) ... maximum...

Plant Physiol. (1977) 59, 991-999

Oxygen Inhibition of PhotosynthesisII. KINETIC CHARACTERISTICS AS AFFECTED BY TEMPERATURE'

Received for publication October 28, 1976 and in revised form January 25, 1977

SUN-BEN Ku AND GERALD E. EDWARDSDepartment of Horticulture, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACI

The response of whole leaf photosynthesis of wheat (Tridcum acti-vum L.) in relation to soluble CO2 availble to the mesophyU celLs, underlow (1.5%) 02 at 25, 30, and 35 C, followed Michaelis-Menten kineticsup to saturating CO2 but deviated at higb CO2 levels where the experi-mental Vmax is considerably less than the calculated V... The affinity ofthe leaves for CO2 du photosynthesis was similar from 25 to 35 Cwith Km(CO2) values of approximately 3.5 to 5 atM.

In considering the effect of 02 on photosynthesis at 25, 30, and 35 Cwhere 02 and CO2 are expressed on a solubility basis: (a) the effect of 02on carboxylation effidency was similar at the three temperatures; (b)increasing temperature caused only a slght increase in kinetic constantsKi(02) and Kmn(CO2), while the ratio of Ki(02)/Km(CO2) was dmilr atthe three temperatures; and (c) the reciprocal plots of apparent rate ofphotosynthesis versus (CO2- r) at various 02 levels showed 02 to be acompetitive inhibitor of photosynthesis.A model for sepanting 02 inhibition of photosynthesis into two

components, direct competitive inhibition and inhibition due to photo-respiration, was presented from both simulated and experimental data ofphotosynthetic response curves to varying CO2 concentrations at low 02versus 21% 02. The photorespiratory part of 02 inhibition is consideredas a major component at r and increases with increasing temperatureand with increase in OCO2 solhbility ratio. The competitive componentOf 02 inhibition is considered as a major component of 02 inhibitionunder atmospheric CO2 levels and is relatively independent of tempera-ture at a given OCO2 ratio.

Although temperature is important in environmental regula-tion of photosynthesis, the degree of temperature-dependentvariation in factors controlling photosynthesis in C3 plants at thecellular level such as affinity for C02, nature of 02 inhibition ofphotosynthesis, maximum velocity of photosynthesis based onenzyme potential, and solubility of 02 and CO2 is uncertain. Inorder to make a kinetic analysis of whole leaf photosynthesis inresponse to levels of CO2 and 02 at various leaf temperatures, itis necessary to consider the concentrations of the gases on asolubility basis in the leaf. From such an analysis, the Km(CO2),Ki(02), Vmax for photosynthesis, CO2 compensation point (IF),and effect of 02 on carboxylation efficiency were determined inthe present study for wheat at three temperatures.

In the previous study with various C3 species (16), we reportedthat the magnitude of total percentage inhibition of photosyn-thesis by 02 is dependent on and correlated with apparentsolubility ratio of 02/CO2 in the leaf over a range of solubilityratios from 25 to 45 when the solubility ratio is changed either by

I This research was supported by the College of Agricultural and LifeSciences, University of Wisconsin, Madison.

changing CO2 concentration, by changing 02 level, or by chang-ing leaf temperature. A part of the 02 inhibition of photosyn-thesis is thought to be due to photorespiration (the light-stimu-lated oxidation of photosynthetic intermediates to C02) whilepart may be by direct inhibition of enzymes of the Calvin-Bensonpathway (8, 13). It is not clear what proportion of the totalpercentage inhibition of photosynthesis is due to direct competi-tive inhibition by 02 versus photorespiratory metabolism. Fromthe kinetic analysis in this study, we also propose a method forseparating the total percentage inhibition of photosynthesis by02 into competitive and photorespiratory components in relationto the solubility ratio of 02/CO2 in the leaf.

MATERIALS AND METHODS

Growth Conditions. Triticum aestivum L. was grown in agrowth chamber at a day/night temperature of 30/25 C withother conditions as previously described (16). Newly expandedleaves of 2- to 3-week-old plants were used for all experiments.Gas Exchange Measurements. Measurements of CO2, 02 and

water vapor, determination of intercellular CO2 concentration,and calculation of solubility ratio of 02/CO2 in the leaf were fullydescribed in the preceding paper (16).

Carboxylation Efficiency. Carboxylation efficiency (CE) aspreviously defined (10) is the initial slope of response of photo-synthesis to increasing C02:

CE= APSCo2 - r

where APS2 is the apparent rate of photosynthesis, CO2 is theconcentration of C02, and r is the CO2 compensation point. Aspreviously defined, CO2 and F were either atmospheric or esti-mated intercellular (in the leaf air spaces) measurements. Wehave modified the determination of CE in the present studywhere CO2 and r are based on calculated concentration of CO2(AM) in the leaf which is dependent on leaf temperature and CO2level in the intercellular air spaces (see under "Materials andMethods" in ref. 16). Expressing CE on a solubility basis elim-inates variability in carboxylation efficiency which would occurdue to change in stomatal resistance in the case where atmos-pheric CO2 is used and change in temperature (since increasedtemperature decreases CO2 solubility) in cases where eitheratmospheric or intercellular CO2 concentration is used. Carbox-ylation efficiency based on soluble CO2 in the leaf more closelydetermines carbon assimilation efficiency per unit of CO2 availa-ble to the photosynthetic cells and allows comparisons of CEwith varying temperature. In determining CE, slopes were takenfrom the initial phase of apparent photosynthesis at low CO2levels.

2 Abbreviations: RuDP: ribulose 1,5-diphosphate; CE: carboxylationefficiency; r: C02 compensation point; APS: apparent photosynthesis;TPS: true photosynthesis.

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Plant Physiol. Vol. 59, 1977

FIG. 1. a: Dependence of apparent photosynthesis on CO2 under 1.5%02 at three temperatures. Measurements were made progressively from25 to 30 to 35 C and from low to high CO2 concentration at a given temperature. b: Double reciprocal plots of apparent rate of photosynthesis under1.5%02 versus C02 concentration at three temperatures. The best fitting line for each temperature was determined by the least square method.

02 r M]

RESULTS

Dependence of Photosynthesis of Wheat on CO2 under Low02 at Three Temperatures. Figure la shows the photosyntheticresponse of wheat under 1.5% 02 at varying concentrations ofsoluble CO2 (calculated from intercellular C02) at three temper-atures. Low 02 (1.5%) minimizes 02 inhibition of photosyn-thesis and reduces the CO2 compensation point (r). Based on

soluble CO2 in the leaf, photosynthesis saturated at around 7 p.M

CO2 (Fig. la). From the data of Figure la, double reciprocalplots of APS versus (CO2 - r) give linear plots up to CO2concentrations near saturation of photosynthesis (Fig. lb). Al-though r is relatively small under 1.5% 02, subtraction of rfrom CO2 concentration removes the respiratory component(15). Extrapolation of the lines through the axes allows determi-nation of Km for CO2 and Vmax (Fig. lb). Km for CO2 increasedslightly with increasing temperature, being 3.4, 3.7, and 4.8 at25, 30, and 35 C, respectively. The calculated Vmax was 178,277, and 315, while the Vmax obtained experimentally (CO2saturated rate) was considerably lower, being 120, 150, and 155ng C02/cm2 sec at 25, 30, and 35 C, respectively.

Effects of 02 on Photosynthesis at Low CO2 Concentrations atThree Temperatures. In a second experiment (Fig. 2), the effectof 02 on the apparent rate of photosynthesis at 25, 30, and 35 Cin wheat was measured at CO2 concentrations just above thecompensation point at various concentrations of 02. From thedata of Figure 2, several subsequent plots (Figs. 3-6) indicatethe efficiency of photosynthesis at the cellular level at varioustemperatures on the basis of soluble CO2. Figure 3 shows thecarboxylation efficiency with the varying 02 concentration atdifferent temperatures. The effect of 02 on CE measures thedirect effect of 02 on photosynthesis separate from the photores-piratory component (14, 17). As the 02 concentration increases,the CE decreases similarly at three temperatures. This suggeststhat there is a similar direct inhibition of photosynthesis by 02 atall three temperatures. Replotting the data of Figure 3 as CEversus temperature (Fig. 4) shows that CE increases with in-creasing temperature at any given 02 level. An increased CEwith increased temperature would be expected if temperatureincreased Vmax of the enzymes of carbon assimilation. The paral-lel response of CE to temperature at different 02 levels suggeststhat the leaf is not changing its affinity for CO2 relative to 02 atvarying temperatures.The type of inhibition of photosynthesis by 02 with respect to

decreasing CE was determined by a double reciprocal plot of

APS versus (CO2 - F) which is equivalent to a plot of 1/V versus1/S in enzyme kinetics where V = velocity and S = substrateconcentration. These plots, shown in Figure 5 for 25, 30, and35 C, indicate competitive inhibition of photosynthesis by 02 atall three temperatures.By plotting the slopes from these reciprocal plots versus the

concentration of 02, the Ki(02) and Km(CO2) can be deter-mined at the three temperatures. This plot as given in Figure 6ais analogues to the plot of S/V (or 1/CE in this study) versus Iwhere S = substrate concentration, V = velocity, and I =concentration of inhibitor. The Ki(02) as determined from theintercept on the x-axis was 152, 190, and 215 ,UM 02 at 25, 30,and 35 C, respectively, while the Km(CO2) as determined fromthe intercept on the y-axis was 3.2, 4.9, and 5.2 ,UM CO2 at 25,30, and 35 C, respectively. The Km(CO2) and Ki(02) are alsodetermined by plotting the intercepts on the x-axis (Km apparentfor C02) of Figure 5 versus inhibitor concentration (Fig. 6b).Similar results were obtained from this type of plot. The Kmvalues determined in this experiment were similar to those froma different analysis (Fig. 1). Thus with wheat, both Ki for 02 andKm for CO2 increase slightly with increasing temperature al-though the Ki/Km ratio undergoes little change with tempera-ture variations.Model for Partitioning 02 Inhibition of Photosynthesis into

Competitive and Photorespiratory Components. Using the ki-netic constants Ki(02), Km(CO2), and Vmax obtained fromFigures 5 and 6a and F from Figure 2 for wheat, we simulatedvarious rates of photosynthesis as follows.True rate of photosynthesis at 0% 02:

TP=,,, V... E[CQa](TPSoq=02 Km + [CO2] (1)

which is equivalent to:

V= Vmax S (2)

for Michaelis-Menten kinetics.True rate of photosynthesis with 2:

Vmax[C32]02 [CO2] + Km(l + 02/Ki) (

which is according to the equation for competitive inhibition:

V += max'S (4)S + Kmn(1 + I/Ki)

992 KU AND EDWARDS

C0 ]

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OXYGEN INHIBITION OF PHOTOSYNTHESIS. II

Y9 40-

~~~~~~30

a))\

20N 35 C|

0 ~~~~~~~~~~~~~~~~~30C

-') S °25C

0~~~~~~~~~~~0).2E1_0 200 300 400 500 600

0

-2 L,"MJ-,O Y FIG. 3. Carboxylation efficiency as a function of 02 concentration at

three temperatures. Carboxylation efficiency was determined from thedata of Figure 2.

4~~ ~,O O s

(3as/au3/203 iJ) SdV 60

U @0M00 0~~~~~~~~~~~~~~~~~~06

SdV\ d̂Ct o0~~~~~~

C4'>a 0- ~~~~~~oU0~~ ~ ~ ~~~

cY cu

n3L0-I 240P°(O r K 1+OJI S

25335 0

-~~~~~~~~2-_6

DCo0 0 0 ,= V) which essentially shifts themreratnre (c)0.~~~~~~~~~~~~~~1

.) bxtionaseargi forsi g e u nctions pea a td

O >o codatant aie3.ue nd6,w btie h

0

Apcrvenforatre ofphotosynthesiatiwt is gven as:

oN \c cthesisat2l% 02 at varying CO2 levels for 25, 30, and 35 C (Fig.

0~~ ~ ~ ~ ~ ~~

E_F\IG.Q7). The partition of total inhibition into competitive and photo-

^ >\ O Xo ~~~~~~respiratory components was then calculated as follows.

@\ X ° E > ~~~~~~~TPS~O% - A.PS2)lO = Total inhibition by 21% 02\ °O TPSo«o, - TPS2jq,0,0 = Competitive inhibition by 21%o 02

\ X 3 aXN TPS21%0, - APS219b2< \4N ; E°~~~a) = Inhibition due to apparent photorespiration at21% 02

The data are replotted as total percentage inhibition, percentage

s;X= competitive inhibition, and percentage inhibition due to appar-

Sent photorespiration at 21% 02 as a function of the 02/C02 ratio'

\ ' ~~~~~~~~~~~(Fig.\ ~~~~~~~reference to TPSo0o2.~~~~~~~~~~The

_<, ~~~~~~~~~~~~~threetemperatures is very similar at solubility ratios of OS/CO2M afrom 15 to 50, but at higher solubility ratios the percentage

.___ .__ .__ .__ .__ .___.___ inhibition increases much more at the higher temperatures.

nN - The percentage competitive inhibition of photosynthesis by 02

(:>YZW3/30 6U) SdV increases with increasing solubility ratio in a similar manner for

0~~~0 o

993Plant Physiol. Vol. 59, 1977



KU AND EDWARDS

( b )

2 3

GO2- r I-M) -


( C )

FIG. 5. Double reciprocal plots of apparent rate of photosynthesis versus CO2 concentration at various 02 concentrations at three temperatures.Apparent rates of photosynthesis were taken from the data of Figure 2.

(a)

-200 -100 0 100 200 300 400 500 600 - 200 -l00 0 l00 200 300 400 50 60

02 [JUMI 02 (PMI

FIG. 6. a: Relationship between the slope of double reciprocal plots from Figure 5 (l/CE) and 02 concentration at three temperatures. For thedetermination of Km (CO2) from the intercept ony-axis (Km(CO2)/Vma) in this plot the Vmax for each temperature was taken from the data of Figure5. b: Relationship between the apparent Km(CO2) from Figure 5 and 02 concentration at three temperatures. In both plots, the best fitting line foreach temperature was determined by the least squares method.

all three temperatures. This similar increase with temperature isexpected if Ki(02)/Km(CO2) is not changing with varying tem-peratures. With competitive inhibitors, the percentage inhibitionis dependent on both the ratio I/S and the substrate concentra-tion S (23) according to the equation:

i + Ki(1 + S/Kin)(100) (6)

where i is the percentage competitive inhibition, I is the concen-tration of inhibitor, and S is the substrate concentration. Thusthe data obtained from this equation are the same as thoseshown in Figure 8b. Because of the dependence of percentagecompetitive inhibition on S as well as I/S, at lower substrateconcentrations it requires a relatively higher ratio of I/S to attain

a certain percentage inhibition. This explains the leveling off ofthe percentage competitive inhibition at higher solubility ratio of02/C02 (Fig. 8b). Although the rate of photosynthesis may varywith temperature, the percentage competitive inhibition is notdependent on velocity (equation 6).The percentage inhibition of photosynthesis due to apparent

photorespiration increases in a linear manner with increasing I/Sand also increases with increasing temperature at any given I/S(Fig. 8c). Percentage competitive inhibition is relatively inde-pendent of temperature while the percentage inhibition due toapparent photorespiration is temperature-dependent.

Photosynthesis of Wheat at Varying CO2 Levels, 1% and 21%02, and Three Temperatures. To test further the above proposalfor separating competitive and photorespiratory components of

994

( )

3 4 5u5-1C0

m

25 C

C.08 /

p .042

K K30 90 4.9| l ~~~~VmGK 35 2 15 5.2




02 inhibition, we measured the 02 inhibition of photosynthesis(21% versus 1% 02) in a third set of experiments over a widerange of CO2 concentrations from r to saturating levels at 25,30, and 35 C (Fig. 9). The reason for measuring photosynthesisat 1% 02 instead of 0% 02 iS that stomata of wheat wereunstable in an 02-free atmosphere (1) and 02 is necessary forboth opening and closing of wheat stomata (2). From the differ-ence between photosynthesis at 1% and 21% 02 we determinedthe total percentage inhibition as shown in Figure 1Oa. Competi-tive inhibition was separated from inhibition due to apparentphotorespiration by shifting the curve for APS21qC,o from r to theintercept on x-axis of APSl,/O,,. The percentage inhibition due tothese two components is plotted in Figure 10, b and c. Thetrends of total percentage inhibition, percentage competitiveinhibition, and percentage inhibition due to apparent photores-piration were the same for data from simulated plots based onkinetic constants (Fig. 8) in comparison to the data from experi-mental plots (Fig. 10). However, quantitatively, the total per-centage inhibition was lower from experimental data (Fig. 10)than from the simulated data (Fig. 8). In the experimentaldeterminations, APS1q%02 was taken to indicate TPSO0O2. Thiswould tend to underestimate the total percentage inhibitionsince APS1q0O2 would be lower than the actual TPSA10o2.

(a)

As expected, the C02-saturated rate of photosynthesis under1% 02 (experimental Vmax) in this experiment, being 140, 165,170 ng CO2/cm2-sec at 25, 30, and 35 C, respectively (Fig. 9),was also considerably lower than the calculated Vmax in Figureslb and 7. This is not expected to affect the percentage competi-tive inhibition since velocity is not a component of the equationfor competitive inhibition (equation 6).At high I/S (low CO2 concentrations), apparent photorespira-

tion is the main component of 02 inhibition, whereas at low I/S(high CO2 concentrations), competitive inhibition is the maincomponent of 02 inhibition (Figs. 8 and 10). This is furtherillustrated in Figure 11 (replotting data of Fig. 10) where theproportion of the inhibition due to competitive inhibition andinhibition due to apparent photorespiration is given at varioussolubility ratios of 02/CO2. At CO2 around atmospheric condi-tions as indicated by the arrows in the figures, the main compo-nent of 02 inhibition is suggested to be due to competitiveinhibition and the proportion of inhibition due to apparentphotorespiration increased with increasing leaf temperature.

DISCUSSIONAffinity of Wheat Leaf for CO2 during Photosynthesis. Under

IOW 02 the CO2 required for saturation of photosynthesis of

( c )

to

0tC-

U,0.

4 8 12

8

0l

(n

0 8c02 (yM) C02 ~ M) c02 (yM)

FIG. 7. Simulated CO2 response curves for photosynthetic rates at three temperatures. Calculations of various photosynthetic rates wereaccording to equations 1, 3, and 5 as described in the text. Kinetic constants Km(CO2), Ki(02), and Vmax were taken from the data of Figures 5 and6a, and r from Figure 2.

(a)350 3 250

35 c

c

C:

E0

al2

200 40 80 120 160 200 240 280

Solubility ratio of 02: C02

(b)

40 80 120 160 200 240

Solubility ratio of 0iC02

(C)

O 60

o40

S20

0a

0 40

,

.0C; 20c

280 1 40 80 120 160 200 240 280

Solubility ratio of 0..:C00FIG. 8. Total percentage inhibition of photosynthesis by 02 (a), percentage competitive inhibition of photosynthesis by 02 (b), and percentage

inhibition due to apparent photorespiration (c) as a function of solubility ratio of 02/CO2 at three temperatures. Percentage inhibition ofphotosynthesis by °2 was calculated from the simulated data of Figure 7 as described in the text. Calculation of 02/CO2 solubility ratio from Figure 7was as described in ref. 16.

995

100

80

60

60F

25C30 C

*35 C

35 C

30C25 C


12 16

.-I40




wheat ranged from 7 to 10 AM C02 at three temperatures (Figs.la and 9), which is within the range reported by Jones andSlatyer (15) for cotton (about 5-14.5 AM C02 at 25-28 C). Thephotosynthetic response of wheat leaves is similar to that ofspinach chloroplasts (18) in that the response to CO2 followsMichaelis-Menten kinetics at lower CO2 concentrations but de-viates at higher CO2 concentrations near saturation of photosyn-thesis. Lilley and Walker (18) suggested that this deviation from

0;o Michaelis-Menten kinetics is due to a much higher capacity forA 14** RuDP carboxylase than for in vivo photosynthesis with a major

12Q , limitation on photochemical capacity at saturating CO2. We alsoO C r found that leaf extracts of wheat have substrate-saturated activ-

ity of RuDP carboxylase exceeding the C02-saturated rate ofphotosynthesis (Ku and Edwards, unpublished). Goldstein et al.

E c @ (11) and Edwards et al. (9) also recently reported levels ofRuDP carboxylase in some C., species well above the expected

0 maximum rates of leaf photosynthesis.m o Previous studies (15, 22) have considered intracellular flux of£ Q-. CO2 on the basis of transfer resistance from the cell wall to the

X chloroplasts and carboxylase resistance. The high affinity of0 wheat leaves for CO2 under low 02 (Km 3-5 Am, Figs. 1 and 6)o suggests that the transfer resistance may be a minor component,

> since these Km values are lower than those thus far determinedQ °eN in vitro for RuDP carboxylase (4, 6, 17). The absolute values of

CZ Km for RuDP carboxylase in vitro are dependent on degree ofactivation of the enzyme (19) although the lowest values thus far

c .g= determined are 7 to 20 AM C02 (4, 6, 17). In the present study,ev o we consider the Km(CO2) determination approximating maxi-s$.1 mum values for RuDP carboxylase since the CO2 solubility is

0 based on calculated intercellular levels of CO2 in the air spaces ofthe leaf. Without a CO2 concentrating mechanism in the leaf, the

2 ° ,o O actual soluble CO2 in the chloroplast may be equivalent to oro 8 n :, somewhat lower than the solubility values as determined de-O0n X X pending on the transfer resistance. Lilley and Walker (18), whoCX considered a limitation on the carboxylase activity in vivo at CO2

saturation of photosynthesis, also suggested that the transfer~X<, resistance is a minor component (also see ref. 22). Jones and

E Slatyer (15) initially suggested that transfer resistance of CO2from the cell wall to the chloroplast may be a larger component

o 5 than the carboxylation resistance during photosynthesis. How-cu . ever, their analysis assumed experimental V1max represents sub-o ,, strate saturation of the carboxylase, predicted very low Km*.0 values for the carboxylase of about 0.2 to 1.8 ,UM C02 on a

s >2 soluble basis, and suggested a transport limitation rather than a= N < carboxylase limitation at Vinax.o8 0 Goldsworthy (12) previously determined the apparent Mi-

Cu chaelis constants for CO2 during photosynthesis in several spe-cies by experimentally measuring the Vniax and the atmospheric

0O CO2 concentration required to give '/2 Vinax under nitrogen.X e e However, such Michaelis constants would not be indicative ofCu E Z carboxylation efficiency or affinity of the photosynthetic cells for

. CO2 since both boundary layer and stomatal resistances to CO2T , ( Q = transfer and photochemical limitations at high CO2 were notr considered. Thus, Michaelis constants based on atmospheric

°X 0 levels of CO2 cannot be reasonably compared with those of the+o° present study. Akita and Moss (2) also noted some limitations in

. o X using experimental Vmax for whole leaf photosynthesis to deter->~> X = mine apparent Michaelis constant for CO2.E Oxygen Effect on Carboxylation Efficiency, Km(CO2), and

. f Ki(02) in Wheat. It is necessary, when analyzing the effect of 02on leaf photosynthesis with respect to CE, Km(CO2), and Vinaxat varying temperatures, to make comparisons on a solubilitybasis for 02 and CO2. Previous methods of determining CE haveused either atmospheric or calculated intercellular levels of CO2(3, 10, 17). If CE is to represent the rate of true photosynthesisper unit of available CO2 as an indication of biochemical effi-ciency, then determinations should be made on the basis ofsoluble CO2. CE determinations from external CO2 concentra-

996 KU AND EDWARDS




-6H

c

tn

-VS?0

2

c

c

40L 40

Z 20E

(b)

D 40 80 120 160 200-^.. sr-rn

240 280

Solubility ratio of 02:C02 ZUOUUility raTio of u2 *.u2 Solubility ratio of °2 CO2

FIG. 10. Total percentage inhibition of photosynthesis by 02 (a), percentage competitive inhibition of photosynthesis by 02 (b), and percentageinhibition due to apparent photorespiration (c) as a function of solubility ratio of 02/CO2 in the leaf at three temperatures. Percentage inhibition ofphotosynthesis by 02 was calculated from the data of Figure 9 and 02/CO2 solubility ratio as described in ref. 16.

(a) (b)1.0

0.8

,c 0.6c

2 040

a: Q

0.2

40 80 120 160 200 240 280

Solubility ratio of 02:C02

0 40 80 120 160 200 240

Solubility ratio of 02:C02

1.0

( c )

0.8.c

-0 0.6

w.?OA

tl

280 0 40 80 120 160 200 240

Solubility rotio of 02 C°2

FIG. 11. Relative proportion of the 02 inhibition of photosynthesis due to competitive inhibition and due to apparent photorespiration as afunction of solubility ratio of 02/CO2 in the leaf at three temperatures. Calculations were made from the data of Figure 10. The total percentageinhibition of photosynthesis by 02 at various solubility ratios of 02/CO2 was always taken as 1.0. Arrows indicate the relative proportion of the twocomponents at atmospheric conditions while data at highest solubility ratio indicate proportion of two components at CO2 compensation point.

tions will give lower values than use of intercellular levels. Inparticular, use of atmospheric levels of CO2 would underesti-mate CE when stomatal resistance increases as may occur athigher CO2 levels and higher temperatures. Use of intercellularCO2 concentrations for determining CE will also underestimateCE as temperature increases due to a decreased solubility ofCO2. Since as temperature increases the solubility ratio of 02/CO2 increases (Fig. 1 of ref. 16), use of intercellular levels of 02and CO2 to determine the sensitivity of CE to 02 would overesti-mate the effect of 02 on CE at higher temperatures. Thus, whenCE is expressed on the basis of atmospheric levels of CO2 and02, CE could become increasingly more sensitive to atmospheric02 with increasing temperature either due to increased stomatalresistance which gives lower intercellular C02, and/or due toincreased solubility ratio of 02/CO2. This could in part be thebasis for the previously reported higher sensitivity of CE to 02

with increasing temperature in soybean (17).The kinetic analysis of the effect of 02 on photosynthesis at

varying temperatures when considered on the basis of solubleCO2 and 02 (Figs. 2-6) allowed several conclusions: (a) part ofthe 02 inhibition of photosynthesis is by competitive inhibition(Fig. 5); (b) there is a similar decrease in CE at 25, 30, 35 C as

02 iS increased (Fig. 3); and (c) both the inhibition constant ofphotosynthesis for 02 (Ki) and the kinetic constant for CO2(Km) increase slightly with increasing temperature from 25 to35 C, while the ratio of Ki(02)/Km(CO2) with varying tempera-ture is similar (Fig. 6). The Ki/Km ratio did not change in favorof increased 02 inhibition at higher temperature.

From equation 4 for competitive inhibition, rearranging gives:

S 1 S +Km(l + I/Ki)Vi CE Vmax (7)

From equation 2 for Michaelis-Menten kinetics without inhibi-tor, rearranging gives:

S 1 S+ KmV CEO Vmax

(8)

where CE1, is the carboxylation efficiency without inhibitor.Combining equations 7 and 8 gives:

1 1 +Kmn ICE CEO KijVmax (9)

Therefore, the plot of 1/CE versus I is expected to give a linearrelation as shown in Figure 6a, where 1/CE,, is the intercept ony-axis. Rearranging and combining equations 2 and 4 gives:

CEO(S + Km)S + Km(l + I/Ki)

(10)

Since 02 is a competitive inhibitor of photosynthesis and sincereciprocal plots of APS versus (CO2 - F) give linear relations(Fig. 5), as expected from equation 4 for a competitive inhibitor,equation 10 appears to be a reasonable expression of the effectOf 02 on CE. Laing et al. (17) used an empirical equation todescribe CE which they adapted from a derivation of Forrester et

al. (10) in analyzing the apparent rate of photosynthesis versus

250C30 C

35 C

1.0

0.8

c- 0.6

0

a) 0.4

Zlr0.

)~~~~~~ X * *Total

tW~~~~~~~~2 C

rIeltv

I

Total

Photorespirotion

Coompetitive

30 C

A. "A... & ITotol

Photorespiration

Competitive 35C

280

997Plant Physiol. Vol. 59, 1977




02 concentration. This equationCE = CE0e-Olk

or

In CE = In CEo - O/k

suggests a linear relationship between In CE versus 0 where 0 =oxygen and k = an arbitrary constant. However, the log CEversus I does not give a linear plot either using the velocityequation for competitive inhibition

CE= VmaxS + Km(i + I/Ki)

with theoretical values of Km, Ki, and Vmnax or using the experi-mental data in this paper (for example, from Fig. 2). In bothcases, with increasing 02 there is a decrease in log CE in acurvelinear manner. Thus, any determination of k as an indica-tion of relative effect of 02 on CE at various temperatures by thismethod is rather arbitrary, and also fails to provide a quantita-tive evaluation of Ki. Since 1/CE versus 02 provides a linear plot(equation 9) as expected for a competitive inhibitor (Fig. 6a),the empirical equation derived by Forrester et al. (10) is notnecessary for defining CE in relation to 02 effect. Peisker andApels (22) also reported a linear increase in intracellular resist-ance, ri (equivalent to 1/CE in the present study), with increas-ing 02, which supports a model of photosynthesis proposed byPeisker (21) based on competitive interactions of CO2 and 02with RuDP carboxylase-oxygenase.

In vitro assays of RuDP carboxylase-oxygenase activities ofsoybean suggested that Ki(02) decreased, Km(CO2) increased,and RuDP oxygenase-carboxylase activities increased with in-crease in temperature (17). These results are not compatiblewith calculated changes in Ki(02) and Km(CO2) with wheat inthe present study (Figs. 1 and 6) where both Ki(02) andKm(CO2) increased with increase in temperature. A change inKi and Km in the same direction with variable temperature isgenerally expected for reactions involving competitive inhibition(26). For example, Singer et al. (24) found that Ki for malonateincreased as a competitive inhibitor of succinate dehydrogenasefrom 20 to 38 C as did the Km for succinate. Recently, Cholletand Anderson (7) also found that there is no differential regula-tion of the RuDP carboxylase-oxygenase by physiological levelsof some plastid metabolites.

Competitive and Photorespiratory Components of 02 Inhibi-tion of Photosynthesis. The results of the present study supportprevious suggestions that 02 is a direct inhibitor of photosyn-thesis and 02 inhibits photosynthesis through photorespiration(14, 17). Around atmospheric conditions, most of the 02 inhibi-tion appears to be due to competitive inhibition (Fig. 11).Ludwig and Canvin (20) suggested that under atmospheric con-ditions about 2/3 of the 02 inhibition was due to direct inhibitionand 1/3 due to photorespiration in sunflower. With wheat theresults suggest that if a constant 02/CO2 ratio is maintained atvarying temperatures the percentage competitive inhibition issimilar (Figs. 8 and 10). Also at a given solubility ratio aroundatmospheric conditions (20 to 45) the total percentage inhibitionof photosynthesis by 02 iS the same at varying temperatures(Figs. 8 and 10, and ref. 16), since competitive inhibition is themain component under these conditions. Lack of temperatureeffect on percentage competitive inhibition at a given solubilityratio of 02/CO2 is expected with similar Ki/Km and similar effectOf 02 on CE at varying temperatures. The percentage competi-tive inhibition of photosynthesis by 02 is similar in both thetheoretical plots generated from kinetic constants (Fig. 8b) andthe experimental plots (Fig. 10b). It seems that the rate of truephotosynthesis under 02 is equivalent to the response curve ofAPS shifted from F to the origin.

The percentage inhibition due to apparent photorespirationshows a linear increase with increasing 02/CO2 solubility ratioand also increases with temperature as well. These responsesmay be characteristics of photorespiration but they have notbeen interpretated on a biochemical basis. Laing et al. (17) havesuggested that F may be a function of V,nax oxygenase/V0naxcarboxylase and Km(CO2)/Km(02). If temperature does nothave differential effects on these parameters, then an additionaltemperature-dependent component may be involved in photo-respiration.The temperature-dependent increase in percentage inhibition

of photosynthesis by 02 due to apparent photorespiration as wellas rate of photorespiration becomes particularly significant atlow CO2. The total percentage inhibition reaches 100% (effec-tively CO2 compensation point) at increasing solubility ratios of02/CO2 as the temperature decreases (Fig. 10a). Recalculatingthe data of Bjorkman et al. (5) and Laing et al. (17) for F atvarying temperatures also shows that the 02/CO2 solubility ratioat F increases as temperature decreases. Ludwig and Canvin(20) suggested that the rate of photorespiration is constant withincreasing CO2 up to atmospheric lovels in sunflower, althoughresults of the present study would suggest a progressive decreasein the rate of apparent photorespiration with increasing CO2.Ludwig and Canvin (20) determined the level of photorespira-tion as the difference between true photosynthesis, measured asdirect fixation of '4CO2, and apparent photosynthesis by IR gasanalysis. However, the true rate of photosynthesis may be un-derestimated at low external CO2 concentrations due to in-creased dilution of 14CO2 by high photorespiration and recyclingof '2CO2. This could lead to an underestimation of photorespira-tion at low CO2 concentrations. Tamas and Bidwell (25) suggestthat the rate of photorespiration decreases with increasing CO2in bean leaves.

In the present study, the carboxylation efficiency (Fig. 3),Vmax (Fig. 5), and percentage inhibition of photosynthesis due toapparent photorespiration (Figs. 10 and 11) all increased withincreasing temperature. However, the kinetics of these increasesneeds to be evaluated over a broader temperature range.

Acknowledgments -The authors assume responsibility for interpretations of the data al-though useful discussions with S. C. Huber, W. W. Cleland, and J. C. Servaites are acknowl-edged.

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