Effects Irradiance thein Vivo Specificity Factor Tobacco

Plant Physiol. (1990) 94, 892-8980032-0889/90/94/0892/07/$01 .00/0

Received for publication May 11, 1990Accepted July 5, 1990

Effects of Irradiance on the in Vivo C02:02 SpecificityFactor in Tobacco Using Simultaneous Gas Exchange and

Fluorescence Techniques

Richard B. PetersonDepartment of Biochemistry and Genetics, the Connecticut Agricultural Experiment Station,

New Haven, Connecticut 06504

ABSTRACT

The effects of gas phase 02 concentration (1%, 20.5%, and42.0%, v/v) on the quantum yield of net CO2 fixation and fluores-cence yield of chlorophyll a are examined in leaf tissue fromNicotiana tabacum at normal levels of CO2 and 25 to 30°C.Detectable decreases in nonphotochemical quenching of ab-sorbed excitation occurred at the higher 02 levels relative to 1%02 when irradiance was nearly or fully saturating for photosyn-thesis. Photochemical quenching was increased by high 02 levelsonly at saturating irradiance. Simultaneous measurements of CO2and H20 exchange and fluorescence yield permit estimation ofpartitioning of linear photosynthetic electron transport betweennet CO2 fixation and 02-dependent, dissipative processes suchas photorespiration as a function of leaf intemal CO2 concentra-tion. Changes in the in vivo C02:02 'specificity factor' (K.p) withincreasing irradiance are examined. The magnitude K.p was foundto decline from a value of 85 at moderate irradiance to 68 at verylow light, and to 72 at saturating photon flux rates. The resultsare discussed in terms of the applicability of the ribulose bis-phosphate carboxylase/oxygenase enzyme model to photosyn-thesis in vivo.

Many studies over the years have confirmed that atmos-pheric (i.e. 21% v/v) levels of 02 are inhibitory to photosyn-thesis in plants possessing the C3 pathway of C02 fixationwhen C02 is present at rate-limiting concentrations. Theprimary biochemical basis for 02 inhibition is the process ofphotorespiration during which glycolate-P is metabolized withconsequent evolution of C02 inside the leaf (14, 22, 28).Glycolate-P arises by oxygenation of RuBP' as catalyzed by

Abbreviations: RuBP, ribulose bisphosphate; Rubisco, RuBPcarboxylase/oxygenase; Fod, Fmax, initial and maximum fluorescenceyields, respectively, for a fully dark-adapted leaf; F.', Fs, Fm', mini-mal, steady state, and maximum fluorescence yields, respectively,during continuous actinic illumination; F,', variable fluorescence(i.e. Fm' - F.'); qp, photochemical quenching coefficient; qN, non-photochemical quenching coefficient; A, CO2 assimilation rate (,umolm-2s-'); C,, Cc, intercellular and chloroplast partial pressures of C02,respectively (gbar); O0, chloroplast partial pressure of 02 (mbar); I,incident photon flux rate (,gmol photons m2s'); 1c, V., maximalvelocities of carboxylation and oxygenation, respectively; Kc, Ko,Michaelis constants for carboxylation and oxygenation, respectively;-t = A/I; qH20, gas phase (boundary plus stomatal) conductance toH20 vapor (jAmols m-2s-'); P, probability level; R, correlation coef-ficient (P < 0.01 ); Ksp, specificity factor.

the enzyme Rubisco (17). The individual enzyme activitiesare related to photorespiratory C02 release (PR) and grossC02 fixation (GPS) by t, the ratio of moles C02 produces permole of glycolate-P metabolized (14). Thus,

PR/GPS = t([02]/[C02])/Ksp. (1)The constant Ksp (specificity factor) clearly determines therelative enzyme-catalyzed velocities of carboxylation (va) andoxygenation (vo) as [02] and [C02] are varied. The Kp equalsVCKO/VXK, where V, and V0 are maximal velocities and Kcand Ko are Michaelis constants for the respective activities ofcarboxylation and oxygenation. The magnitude of Kp isrelatively constant for enzymes isolated from C3 species andis temperature dependent (1, 14).

Simultaneous determination ofC02 exchange and PSII Chla fluorescence yield at room temperature offers a new oppor-tunity for assessment of the proportion of total linear electrontransport expended for collective 02-dependent processes suchas photorespiration and the Mehler reaction (21, 22). Diffi-culties inherent in estimation of photorespiration have beendiscussed previously (22). A recently developed modulationtechnique permits separation and quantitation of major flu-orescence quenching processes during steady state C02 fixa-tion (25). Specifically, 'photochemical' quenching (expressedas the coefficient qp) is related to the redox state of QA, thefirst stable quinone electron acceptor in PSII (2, 24). 'Non-photochemical' quenching is dominated by reversible proc-esses which regulate the extent of thermal dissipation ofabsorbed excitation in PSII (9, 13). Alternative means ofexpression ofthis quantity are as the coefficient, qN, or variablefluorescence yield, Fv'/Fm'. Recent evidence (5) indicates thatthe quantum yield of linear electron transport is directlyrelated to the proportion of PSII centers which are open (i.e.qp) and to the efficiency of energy capture by these centers(i.e. Fv'/Fm').

This study examines interactive effects of [02] and irradi-ance on quantum yield of net C02 uptake, qp, qN, and Fv'/Fm' in leaf tissue from Nicotiana tabacum at normal levels ofC02. At elevated 02 levels the product qp X Fv'/Fm' isemployed as an empirical means of predicting the quantumyield of total linear electron transport (5). Thus, allocation ofphotosynthetic reducing equivalents to O2-dependent, dissi-pative processes may be assessed as the difference betweenthe predicted quantum yield of PSII electron transport basedon fluorescence yield and that observed based on net C02

892

Dow

nloaded from https://academ

ic.oup.com/plphys/article/94/3/892/6088565 by guest on 26 N

ovember 2021

EFFECTS OF 02 ON FLUORESCENCE AND QUANTUM YIELDS

exchange. This approach constitutes a more accurate meansof studying effects of 02 on carbon metabolism since it takesinto account changes in the partitioning of excitation in PSIIwhich may result from associated changes in acceptor avail-ability and Pi-recycling as the [02] is varied. This is in contrastto conventional measurements of02 inhibition which rely ongas exchange alone. Assessment of allocation of reducingequivalents to 02-dependent processes combined with meas-urements of leaf internal [02]/[CO2] permits calculation ofthe in vivo specificity factor, K,s. An earlier study (21) indi-cated that discrepancies between observed electron allocationand that predicted by the Rubisco model occurred at saturat-ing irradiance. This could indicate enhanced Mehler reactionactivity (19) or a change in the proportion of glycolate-Pcarbon converted to CO2 at very high light intensity. Thedependence ofKp on irradiance is examined in greater detailin this study.

MATERIALS AND METHODS

Nicotiana tabacum var Havana seed was grown in a green-house as described previously (21). For each experiment a leafwas removed at 0730 (EST), washed with hand soap, andrinsed with distilled H20. Leaf discs were prepared and CO2and H20 exchange measurements were conducted using anopen, flow-through system using a gas flow rate of 2.0 L min-'(23). The light responses of steady state net CO2 assimilation,transpiration, and fluorescence yield ofChl a were determinedfollowing stepwise increases in irradiance of continuous whitelight over the range of '80 to 1900 ,mol photons m-2s-'.The 02 concentrations in the gas stream were 1.6, 20.5, and42.0% (v/v) corresponding to 16, 210, and 430 mbar, respec-tively. The mean steady state partial pressure for CO2 in thechamber gas phase was 355 (SD = 8) jsbar. The water vaporpressure deficit was 10 to 11 mbar. Three replicate experi-ments were performed at each [02] for each of two leaftemperatures of 25 and 30°C (18 experiments total).Gas phase (i.e. combined boundary layer and stomatal)

conductances to H20 vapor (qH2o) and CO2 (qco2) werecalculated according to Long and Hallgren (16). The value ofqco2 differs from that of qH20 over the same diffusion pathwaydue to differences in diffusivity between the two gases. Asdescribed previously (16), the H20:CO2 conductance ratioacross the stomatal barrier is 1.61 and that across the bound-ary layer (where turbulent transfer contributes) is 1.37. Inter-cellular partial pressure of CO2 (Ci) was calculated using qH20and includes a slight correction for effects of entrainment ofCO2 molecules by high rates of transpirational H20 efflux(Eq. 6.20 of ref. 16). Dissolved leaf internal 02 concentrationswere assumed to be in equilibrium with the partial pressureof 02 in the bulk gas phase. Perturbation of the internal [02]by photosynthetic 02 evolution was neglected. Molar aqueousphase ratios of leaf internal [02]/[CO2] were computed usingtabular data on the respective solubilities of these gases inH20 at the leaf temperatures (±0.05°C) employed (7).Changes in Chl fluorescence yield were monitored using a

weak modulated measuring beam of red light (H. Walz,Effeltrich, FRG). The procedures employed are described indetail in (23). Maximal fluorescence yields (Fmax and Fm' fora fully dark-adapted leaf and during continuous actinic illu-

mination, respectively) were recorded during a saturating(7500 umol photons m-2s-' for 0.7 s) flash of white light.Initial fluorescence yields were recorded for a fully dark-adapted leaf (Fod) and during steady state photosynthesis (F.',by imposition of a 2 to 4 s dark interval). The value of Fmax(and associated Fod) were measured just prior to the beginningof the experiment. The mean variable fluorescence yieldassociated with full dark-adaptation (Fmax - Fod)/Fmax for theexperiments reported herein was 0.833 (SD = 0.010). Photo-chemical (qp) and nonphotochemical (qN) fluorescencequenching coefficients were computed as (Fm' - Fs)/(Fm'-Fo') and (Fmax- Fm')/(Fmax - Fo'), respectively (25). The F,and Fm' fluorescence yields were recorded at a measuringbeam modulation frequency of 100 kHz. All other measure-ments were performed at a modulation frequency of 1.6 kHz.

RESULTS

Figure 1 shows mean rates of net assimilation of CO2 forthe combined data obtained in the experiments performed at250 and 30°C. Three-way ANOVA indicated that the maineffects of irradiance and [02] were highly significant (P <0.001). Neither the main effect of temperature nor the tem-perature x [02] interaction were significant (P > 0.1). Theincrease in CO2 assimilation with irradiance was accompaniedby an increase in the total gas phase conductance to H20 (notshown). Mean values of qH2o increased from 132 mmol H20m-2s- (SD = 52) at the lowest irradiance level to 545 mmolH20 m-2s-' (SD = 110) at the highest. Three-way ANOVAshowed that, as with A, the main effects of irradiance and[02] were highly significant (P< 0.001). When averaged acrossall irradiance levels and both temperatures, qH2o declined from384 to 304 and 284 mmol H20 m-2s-' at 1.6, 20.5, and 42.0%02, respectively.

Effects of irradiance on fluorescence quenching parametersare shown in Figure 2. Only the effect of irradiance wassignificant (P < 0.05) in accounting for overall variation inphotochemical quenching (qp; Fig. 2, top). Nevertheless, anincrease in qp of -25% was associated with elevated 02 levelsat the highest irradiance employed. Nonphotochemical

40

Z 30

!;IE 20

en O" E 1 0

c) _

0 500 1000 1500 2000IRRADIANCE (Amol photons m-2s-1)

Figure 1. Plots of mean net CO2 assimilation rate versus incidentirradiance at three levels of gas phase [02] for the experimentsreported here with N. tabacum. The mean gas phase [CO2] was 355(SD = 8) Mbar. Each point is a mean of six determinations. Error barsindicate ±SE.

893

Dow



ovember 2021

Plant Physiol. Vol. 94, 1990

1.0

0.8q0p

0.6

0.4

0.2

0.0L1 .0

0.8(IN

0.6

0.4

0.2

0.01.0

0.8

L-

LL

0.6

0.4

0.2

0.00 500 1000 1500 2000IRRADIANCE (,Amol photons m-2s-1)

Figure 2. Changes in the photochemical fluorescence quenchingcoefficient (qp top panel), nonphotochemical quenching coefficient(qN, middle panel), and steady-state variable fluorescence yield (Fv'/Fm', bottom panel) with increasing irradiance. The data shown are forthe three experiments performed at each 02 concentration and 300C(error bars indicate ±SE). The corresponding plots for replicate ex-

periments performed at 250C were very similar.

quenching offluorescence yield showed significant (P< 0.001)effects of irradiance and [02] as shown by plots ofqN and Fv'/Fm' versus irradiance from the 30°C experiments. The higherlevels of 02 resulted in substantially lower values of qN as

light-saturation was approached (Fig. 2, middle). Likewise,elevated [02] resulted in a detectable increase in Fv'/Fm' over

the same range of irradiance levels (Fig. 2, bottom). A small,yet statistically significant main effect of temperature was

indicated by three-way ANOVA. However, partitioning ofthe sums of squares indicated that temperature accounted foronly about 2% ofthe observed variation in nonphotochemicalquenching. The temperature x [02Q interaction was not sig-nificant.

Plots of IPs (=A/I) versus the product qp X Fv'/Fm' at 30°C

for each [02] employed are presented in Figure 3. Similarresults were obtained for the experiments performed at 250except that the slopes of the linear regression fits to the data(see legend to Fig. 3) were somewhat higher in accordancewith the small effect of temperature on nonphotochemicalquenching. Clearly, elevated 02 levels result in lower valuesof 4P for any given qp X Fv'/Fm' due to diversion of a

proportion of linear photosynthetic from net fixation of CO2toward 02-dependent processes such as photorespiration.Since 02-dependent processes are likely to be suppressed at1.6% 02 the associated regression line constitutes a predictionof the quantum yield of total linear electron transport versus

qp X Fv'/Fm'.As defined previously (21, 22), the proportion of total

noncyclic electron transport which is partitioned to 02-de-pendent processes is Pdim. Furthermore, the predicted quan-

tum yield of linear electron transport at any elevated 02

concentration (4V, as CO2 equivalents) is calculated by sub-stitution ofthe associated value ofqp X Fv'/Fm' into the linearregression equation obtained at 1.6% 02 (Fig. 3). Thus, Pdiss= ('sf - tj)/4Ps' where (D is the observed A/I at the specifiedhigh [02]. Such estimates of Pdim are free of any independenteffects of changing [02] on photochemical or nonphotochem-ical quenching ofabsorbed excitation and therefore constituteunbiased estimates of allocation of photosynthetic reducingpower.The dependencies of Pdims and intercellular aqueous phase

molar [02]/[CO2] at 20.5% and 42.0% 02 upon irradianceare shown in Figure 4 (top and bottom, respectively). Meanvalues for C, of 235 (SD = 51), 271 (SD = 26), and 301 (SD =

0.10

0.08

0.06S0.04

0.02

0.0o00.00 0.25 0.50 0.75 1.00

qp Fv'/FmI

Figure 3. The relationship between cI, and the product Fv'/Fm' x qpfor the three replicate experiments performed at each 02 concentra-tion shown and 300C. Data are from Figures 1 and 2. (Note that foreach experiment a single value ofC was computed by linear regres-sion as the slope of A versus I response for the two lowest irradiancelevels examined. This was done to minimize errors due to the pres-ence of dark respiration at the low irradiances. Corresponding valuesof Fv'/Fm' x qp were based on mean values for the respectivequantities at the two irradiance levels.) The solid lines are linearregression fits to data. For the data shown the equations are: for1.6% 02, y = 0.07697x + 0.00264 (R = 0.983): for 20.5% 02, y =

0.04627x - 0.00093 (R = 0.937): for 42.0% 02, y = 0.03624x -

0.00284 (R = 0.950). The linear regression equation for the experi-ments performed at 250C and 1.6% 02 was y = 0.08983x + 0.00254(R = 0.979).

*--a 300C

(.) 1.6% 02

(o) 20.5% 02(tx) 42.0% 02

300CIZ9%

AB~~~~~~* °~~~~

E |894 PETERSON

Dow



ovember 2021


0.7

0.6C,)Cl)-6 0.50

0.41

0.3

60

0

C)

0L-

40

20p[

0

0 500 1000 1500 2000IRRADIANCE (,umol photons m-2s-1)

Figure 4. Changes in the partitioning of linear electron transport to02-dependent, dissipative processes and in the ratio of leaf internal[02]/[CO2] with increasing irradiance for the experiments of Figures1 and 2. Top panel, PdiS was calculated as (Wt' - )/4s' whereis the predicted quantum yield of linear electron transport (expressedas CO2 equivalents, i.e. 4e-:C02) based on substitution of the ob-served Fv'/Fm' x qp into the corresponding linear regression equationcalculated for data obtained at 1.6% 02 and the same leaf tempera-ture (see legend to Fig. 3). The quantity 4s ( = A/l) is the observedquantum yield of net CO2 fixation at either 20.5% or 42.0% 02 basedon gas exchange. The mean values shown (error bars indicate ±SE)are differentiated according to leaf temperature; i.e. 250C (circles)and 300C (squares). The solid lines represent predictions of Pas,based on the photosynthesis model presented by Farquhar et aL. (4)(see Table I). Bottom panel, the corresponding leaf intercellular (opensymbols) and chloroplast (closed symbols) aqueous phase molar [02]/[CO2] levels for the data in the top panel. The effect of leaf temper-ature on the respective quantities was exceedingly small so the datapoints represent means (error bars indicate ±SE) of data obtained atboth temperatures. See text for further explanation.

20) ,tbar were computed for 02 the levels of 1.6, 20.5, and42.0% in Figure 1, respectively. Aqueous phase molar ratiosof [02]/[CO2] based on estimates of C, do not precisely reflectthe respective concentration ratio of dissolved gases in thechloroplast where CO2 fixation occurs. The certain existenceofa finite mesophyll diffusive resistance will result in a smallerpartial pressure of CO2 in the chloroplast relative to theintercellular air spaces. The mesophyll resistance cannot bemeasured by conventional gas exchange procedures so thatthe accurate magnitude of this quantity is not known.Evans et al. (3) used an isotopic procedure to estimate the

mesophyll resistance in wheat. They concluded that the me-

sophyll resistance to diffusion of CO2 fell within the range of1.2 to 2.5 m2 s mol-' bar'. Thus, a value of 2.0 m2 s mol-'

bar-' was employed as an estimate ofthe mesophyll resistancein these experiments with tobacco. The partial pressure ofCO2 in the chloroplast (Cc in ,ubars) is given by C, = C, - 2.0.A. The irradiance dependencies of the aqueous phase molarratios of [02]/[CO2] in the chloroplast at 20.5 and 42.0% arealso shown in Figure 4 (bottom panel).The observed dependence of Pdis, on irradiance was com-

pared to that predicted based on the photosynthesis modelproposed by Farquhar et al. (4). According to the model,when the [RuBP] is saturating the velocities of carboxylation(vt) and oxygenation (v0) are expressed as VC,,/(C,+K,(l +Oc/Ko)) and V0O,/(O0 + Ko(1 + Cc/Kc)), respectively. Thelinear electron transport rate (J) supporting net CO2 fixation,glycolate metabolism, and NH4+ refixation is (4 + 40)vc whereX0 = VOKcOc/VcKOCc. Net CO2 assimilation (A) is vc - 0.5v.assuming that photorespiratory CO2 is released exclusivelyduring the conversion of glycine to serine in the mitochon-drion (l1). As defined previously, Pdiss = (J - 4A)/J. Thepredicted Pdi,, (Table I and Fig. 4, top) may thus be expressedin terms of the Rubisco kinetic constants (10) and the meanchloroplast 02 and CO2 concentrations,

Pdi =0 1+0.5 Ko(Cc + Kc(l + Oc/K0))

+l * Kc(Oc + Ko(l + Cc/Kc)). (2)

As an alternative means of analysis the in vivo Kp may becalculated directly using a formula derived previously (21).By again assuming that photorespiratory CO2 arises solelyduring glycine oxidation, then Pdi,,, Kp, and the dissolvedmolar [02]/[CO2] in the chloroplast are related by

Ksp = ([02]/[C02]) * (1.5 - Pdiss)/Pdiss. (3)

Changes in mean K, with irradiance for the data of Figure 4are shown in Figure 5.

Table I. Calculation of Pdiss Based on the Photosynthesis Model ofFarquhar et al. (4)

Values of C, were calculated from C02 and H20 exchange meas-urements as described in the text. Each Cc value is a mean of threedeterminations on separate leaves (SD). Pdis, values were calculatedusing Equation 2 and the mean values of Cc. Rubisco kinetic con-stants (estimated for 27.50C) for the purified enzyme from spinachwere obtained from Jordan and Ogren (10): Kc = 400 Mbar, Kl = 470mbar, and V0/VC = 0.55. The partial pressures of 02 employed were210 mbar and 430 mbar at 20.5% and 42.0% 02, respectively.

Photon Flux 20.5% 02 42.0%02Rate C P C P

p.mol m 2 S -1 .bar pbar

81 312 (11) 0.359 329 (17) 0.569229 276 (19) 0.394 299 (17) 0.603331 250 (25) 0.423 286 (16) 0.620472 231 (22) 0.448 276 (14) 0.633577 224 (23) 0.457 272 (18) 0.638691 217 (17) 0.468 268 (19) 0.643907 217 (15) 0.468 264 (15) 0.6491114 217 (19) 0.468 265 (20) 0.6481880 230 (13) 0.449 277 (16) 0.631

I0

1 42.0% 02

/0 1T

20.5% 02.

i t ~~~~......i.-...........--.-.--.-.--.-.-

... E i + * i ................... ..............

* 0-o-0--o--o -o

[02] Cc Ci20.5% * 0

42.0% * 0

895

Dow



ovember 2021


DISCUSSION

A potentially important practical application of room tem-perature fluorescence yield measurements is to provide arapid, noninvasive, and economical means of assessing pho-tosynthetic efficiency in response to varying environmentalconditions. The occurrence ofa simple linear, albeit empirical,relationship between cI, and the product qp x Fv'/Fm' (Fig.3) provides support for the feasibility ofthe method. It shouldbe pointed out, however, that radiant energy utilization inPSII is likely to be influenced by numerous factors includingQA redox state, ApH-dependent thermal dissipation, photo-inhibition, PSII antennae size, PSII electron cycling, andoccurrence of inactive units. Therefore, one should not as-sume without verification that the same simple relationshipbetween photochemical and fluorescence yields is valid for allphysiological conditions including the occurrence of stress.Ultimate validation of the use of fluorescence measurementsto predict linear electron transport rates must await a clearerunderstanding of mechanisms which regulate energy parti-tioning and fluorescence emission in PSII.

Photorespiration is undoubtedly the dominant O2-depend-ent process competing with net CO2 fixation for photosyn-thetically generated NADPH and ATP in C3 leaves such astobacco at normal CO2 levels. Changes in partitioning ofelectron transport with varying [02]/[CO2] and irradiance(Fig. 4) will be discussed within the context of the Rubiscoenzyme model based on in vitro kinetics (Eq. 1). Specifically,comparison of in vivo values of K, obtained over varyingenvironmental conditions with reported in vitro values pro-vides a framework for identifying and assessing the signifi-cance of any suspected deviations from the model.

Figure 5 shows mean K., was stable at -85 for irradiancesranging from 350 to 700 umol photons m-2S-'. A value for

100

90[

80

70

60

5C0 500 1000 1500 2000IRRADIANCE (,umol photons m-2s-1)

Figure 5. Variation in the magnitude of the in vivo 02:CO2 specificityfactor (Ksp) with irradiance. The values were calculated by substitutingthe PH,, values of Figure 4 and the corresponding chloroplast [021/[CO2] ratios into Equation 3 (i.e. t = 0.5). Three-way ANOVA of themain effects of [02], irradiance, and leaf temperature indicated thatirradiance was the only significant source of variation (P < 0.01). Ifthe data from the lowest irradiance were omitted from the ANOVAthe effect of irradiance was still significant (P < 0.05). The data were

averaged across temperature and [02] at each irradiance level shown.Thus, each mean value is a mean of 12 replicates (error bars indicate±SE).

Ksp of 85 is representative of those reported for purifiedRubisco preparations from C3 plants (1, 10). The calculatedmean in vivo Ksp in Figure 5 declined, however, to 68 at lowirradiance and to 72 at the highest irradiance tested. Thephotosynthesis model proposed by Farquhar et al. (4) providesadditional support for these conclusions. Based on the model,predicted values of Pdis, (t = 0.5) agreed well with observedvalues at intermediate photon flux rates but were significantlylower than those observed at the lowest (with the exceptionof 42% 02 and 30°C, Fig. 4) and highest irradiances tested.The kinetic constants employed (see ref. 10) are consistentwith an in vitro K, of 82 (Eq. 1) based on dissolved molarconcentrations of 02 and CO2.

Various explanations for the observed variation in in vivoKsp will be considered. If it may be assumed that the kineticconstants (VO, Vc, Ko, Kc) for Rubisco from a particular sourceare invariant then a decline in the apparent in vivo K, couldindicate a diversion ofreducing equivalents from CO2 fixationto intermediary and secondary metabolism or to reduction ofalternate acceptors such as NO3-, 02, and S042-. Directphotoreduction of 02 by PSI (19) at very high irradiancewould increase PdisS for a given vo/v, and chloroplast [021/[CO2] so that the apparent in vivo Kp would decline (Eq. 2).Recent work by Weis and collaborators (15) raises some doubtas to the feasibility of this hypothesis. They reported that atrate-limiting [CO2] electron donation from reduced plasto-quinone to P700+ is regulated by the transthylakoid pHgradient. Thus, both the donor side of PSI and the NADP/NADPH pool become more oxidized with increasing irradi-ance. Since it is generally assumed that PSI-mediated photo-reduction of 02 requires an excess of reducing equivalents onthe acceptor side, the evidence does not unequivocally supportenhanced Mehler reaction activity as leading to the decline inK,p at very high irradiance. As an alternative to PSI-mediatedpseudocyclic electron flow, reduced quinones of the intersys-tem electron transport chain have been shown to donateelectrons to 02 (18). Also, evidence for synthesis of glycolatefrom externally provided pyruvate in a pathway involving theenzyme isocitrate lyase has been reported for tobacco leaftissue (29). The quantitative significance of these latter proc-esses relative to photorespiration resulting from Rubisco ac-tivity is not known, however.The foregoing discussion and the results of Figure 5 are

based on the premise that photorespiratory CO2 arises onlyduring the glycine -- seine conversion so that t = 0.5 (11).This may not always be true. Oxidative decarboxylation of a-ketoacids by H202 produced during photorespiration has beenfrequently suggested to contribute to CO2 evolution (6, 22,28). Formate and CO2 are produced by reaction of hydrogenperoxide with glyoxylate. Formate dehydrogenase is normallypresent at low levels in leaf tissue (20) so that the probablefate of any formate synthesized is entry into the C, poolfollowing ATP-dependent activation by formyltetrahydrofol-ate synthetase (27). Methylene tetrahydrofolate is condensedwith glycine to form seine as catalyzed by seine hydroxy-methyltransferase. Thus, glyoxylate peroxidation need notresult in t > 0.5 in vivo. Peroxidation of hydroxypyruvate toproduce glycolate and CO2 during photorespiration couldtheoretically result in quantitative conversion of the carbon

. T T T1 *....

I.:~~~~~~~T: *-~~~~~~~~~~~~~

t..

I I

w s -

896 PETERSON

_.0

Dow



ovember 2021


atoms ofglycolate-P to CO2 (t = 2.0) (22). Recycling ofNH4'during peroxidation of hydroxypyruvate would occur obliga-torily as it does when glycine decarboxylation is the solesource of C02 (i.e. t = 0.5). Thus, the proposed peroxidationof hydroxypyruvate does not conflict with evidence based onbiochemical (11) and genetic (26) studies which indicate rapidturnover of NH4' during photorespiration in plant cells.

Consideration of stoichiometries of reductant utilizationduring CO2 fixation and photorespiration has resulted in thefollowing general formula (22) which applies when CO2 pro-duction during the glycine -- serine conversion is accom-panied by peroxidation of hydroxypyruvate

Ksp = [02]/[CO2][(t + 1) - Pdiss]/Pdi,,. (4)

Clearly, if t were underestimated at any irradiance of Figure5 then the calculated in vivo Ksp (Eq. 3) would also be toolow. Indeed, substitution into Equation 4 of mean values ofPdi,, and chloroplast [O2]/[CO2] values for Ksp = 85 at thehighest irradiance shown in Figure 5 is consistent with anincrease in t to a mean value of 0.68 from a value of 0.50which may be assumed to be in effect over the irradiancerange 350 to 700 ,umol photons m-2S-'. Again using Equation4, such an increase in t represents a 36% increase in photo-respiratory CO2 evolution and a 12% increase in electronallocation to photorespiration assuming that Ksp remains con-stant at 85.

Similar, yet not identical, results to those presented herewere reported earlier based on CO2 exchange and fluorescenceyield measurements with tobacco in which [CO2] was variedat two levels of irradiance (21). The calculated in vivo Ksp was95 and the postulated increase in t was observed only whenboth irradiance and [02] were high (i.e. 2000 gtmol photonsm-2s-' and 42%, respectively). It should be pointed out thatthe in vivo values of Ksp reported here and previously (21)represent minimal estimates of the true C02, 02 specificityfor Rubisco in the chloroplast. This is because the in vivo Kspvalues observed over a wide range of conditions (assuming t= 0.5) are compatible with values reported in the literaturefor the isolated enzyme in vitro. If t were to actually exceed0.5 under these conditions then Ksp would be underestimated(Eq. 4).A great deal of evidence has accumulated recently for

controlled dissipation of excess absorbed excitation in PSII(4, 9, 23). This is to match rates of generation of ATP andNADPH by the light reactions to the capacity of the darkreactions to utilize these substrates while avoiding potentiallydamaging overexcitation of PSII (8). Krause et al. (12) sug-gested that interaction of 02 with carbon metabolism in theplant cell constitutes a photochemical means of dissipatingexcess absorbed light. The results presented here indicate that02-dependent photochemical dissipation of energy is aug-mented at very high irradiance and this may involve a modi-fication in the mechanism of photorespiration. Enhancedphotorespiratory energy dissipation may become importantwhen other protective processes such as thermal dissipationof excitation in the antennae pigment complex reach satura-tion. Further work on interactive effects of irradiance, [CO2],and CO2 assimilation capacity should enable critical testingof this hypothesis.

ACKNOWLEDGMENTS

I wish to thank Nancy Burns for skillful technical assistance andIsrael Zelitch for helpful comments on the manuscript.

LITERATURE CITED

1. Brooks A, Farquhar GD (1985) Effect of temperature on theC02/02 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light. Planta 165:397-406

2. Dietz K-J, Schreiber U, Heber U (1985) The relationship be-tween the redox state of QA and photosynthesis in leaves atvarious carbon-dioxide, oxygen and light regimes. Planta 166:219-226

3. Evans JR, Sharkey TD, Berry JA, Farquhar GD (1986) Carbonisotope discrimination measured concurrently with gas ex-change to investigate CO2 diffusion in leaves of higher plants.Aust J Plant Physiol 13: 281-292

4. Farquhar GD, von Caemmerer S, Berry JA (1980) A biochemicalmodel of photosynthetic CO2 assimilation in leaves of C3species. Planta 149: 78-90

5. Genty B, Briantais J-M, Baker NR (1989) The relationshipbetween the quantum yield of photosynthetic electron trans-port and quenching of chlorophyll fluorescence. Biochim Bio-phys Acta 990: 87-92

6. Hanson KR, Peterson RB (1985) The stoichiometry of photores-piration during C3-photosynthesis is not fixed: evidence fromcombined physical and stereochemical methods. ArchBiochem Biophys 237: 300-313

7. Hodgman CD, Lange NA (1931) Solubility of gases in water. InHandbook ofChemistry and Physics, Ed 16, Chemical RubberPublishing Co, Cleveland, pp 570-571

8. Horton P (1987) Interplay between environmental and metabolicfactors in the regulation of electron transport in higher plants.In J Biggins, ed, Progress in Photosynthesis Research, Vol. 2.Martinus Nijhoff, Dordrecht, pp 681-688

9. Horton P, Hague A (1988) Studies ofthe induction ofchlorophyllfluorescence in isolated barley protoplasts IV. Resolution ofnon-photochemical quenching. Biochim Biophys Acta 932:107-115

10. Jordan DB, Ogren WL (1981) Species variation in the specificityof ribulose biphosphate carboxylase/oxygenase. Nature 291:513-515

1 1. Keys AJ, Bird IF, Cornelius MJ, Lea PJ, Wallsgrove RM, MiflinBJ (1978) Photorespiratory nitrogen cycle. Nature 275: 741-743

12. Krause GH, Lorimer GH, Heber U, Kirk MR (1977) Photores-piratory energy dissipation in leaves and chloroplasts. Proceed-ings of the IVth International Congress on Photosynthesis, pp299-310

13. Krause GH, Vernotte C, Briantais J-M (1982) Photoinducedquenching of chlorophyll fluorescence in intact chloroplastsand algae. Resolution into two components. Biochim BiophysActa 679: 116-124

14. Laing WA, Ogren WL, Hageman RH (1974) Regulation ofsoybean net photosynthetic CO2 fixation by the interaction ofC02, 02, and ribulose- 1,5-diphosphate carboxylase. PlantPhysiol 54: 678-685

15. Lechtenberg D, Voss B, Weis E (1990) Regulation of photosyn-thesis: photosynthetic control and thioredoxin-dependent en-zyme regulation. Proceedings of the VIIth International Con-gress on Photosynthesis (in press)

16. Long SP, Hallgren, J-E (1985). Measurement of CO2 assimila-tion by plants in the field and the laboratory. In Techniquesin Bioproductivity and Photosynthesis, Ed 2. Pergamon Press,Oxford, pp 62-94

17. Lorimer GH, Andrews TJ, Tolbert NE (1973) Ribulose diphos-phate oxygenase. II. Further proof of reaction products andmechanism of action. Biochemistry 12: 18-23

18. McCauley SW, Melis A (1986) Quantitation of plastoquinonephotoreduction in spinach chloroplasts. Photosyn Res 8: 3-16

19. Mehler AH (1951) Studies on reactions of illuminated chloro-

897

Dow



ovember 2021


plasts. I. Mechanism of the reduction of oxygen and other Hillreagents. Arch Biochem Biophys 33: 65-77

20. Oliver DJ (1981) Formate oxidation and oxygen reduction byleaf mitochondria. Plant Physiol 68: 703-705

21. Peterson RB (1989) Partitioning of noncyclic photosyntheticelectron transport to 02-dependent dissipative processes asprobed by fluorescence and CO2 exchange. Plant Physiol 90:1322-1328

22. Peterson RB (1990) The RuBP carboxylase/oxygenase modeland photorespiration in C3 leaves. In I Zelitch, ed, Perspectivesin Biochemical and Genetic Regulation of Photosynthesis.Wiley-Liss, New York, pp 285-299

23. Peterson RB (1990) Effects of water vapor pressure deficit onphotochemical and fluorescence yields in tobacco leaf tissue.Plant Physiol 92: 608-614

24. Quick WP, Horton P (1984) Studies on the induction of chlo-

rophyll fluorescence in barley protoplasts. II. Resolution offluorescence quenching by redox state and the transthylakoidpH gradient. Proc R Soc Lond B 220: 371-382

25. Schreiber U, Bilger W, Schliwa U (1986) Continuous recordingof photochemical and non-photochemical quenching with anew type of modulation fluorometer. Photosynth Res 10: 51-62

26. Somerville CR, OgrenWL (1981) Photorespiration-deficient mu-tants of Arabidopsis thaliana lacking mitochondrial serinetranshydroxymethylase activity. Plant Physiol 67: 666-671

27. Tolbert NE (1955) Formic acid metabolism in barley leaves. JBiol Chem 215: 27-34

28. Zelitch I (1959) The relationship of glycolic acid to respirationand photosynthesis in tobacco leaves. J Biol Chem 234: 3077-3081

29. Zelitch I(1988) Synthesis ofglycolate from pyruvate via isocitratelyase by tobacco leaves in light. Plant Physiol 86: 463-468

898 PETERSON

Dow



ovember 2021

Documents

Effects Irradiance thein Vivo Specificity Factor Tobacco