Plant Physiol. (1989) 89, 61-680032-0889/89/89/0061/08/$01 .00/0

Received for publication March 29, 1988and in revised form August 8, 1988

CO2 and 02 Exchanges in the CAM Plant Ananas comosus(L.) Merr.

Determination of Total and Malate-Decorboxylation-Dependent C02-Assimilation Rates;Study of Light 02-Uptake.

Fran9ois Xavier Cote*, Marcel Andre, Michel Folliot, Daniel Massimino, and Alain Daguenet

Service de Radioagronomie, Departement de Biologie, Cen Cadarache, 13108 St. Paul lez Durance, France(F.X.C., M.A., D.M., A.D.), and Laboratoire de Physiologie et Biochimie, Institut de Recherche sur les Fruits et

Agrumes Tropicaux (IRFA-CIRAD), 34032 Montpellier, France (M.F.)

ABSTRACT

Photosynthesis and light 02-uptake of the aerial portion of theCAM plant Ananas comosus (L.) meff. were studied by C02 and02 gas exchange measurements. The amount of C02 which wasfixed during a complete day-night cycle was equal to the amountof total net 02 evolved. This finding justifies the assumption thatin each time interval of the light period, the difference betweenthe rates of net 02-evolution and of net light atmospheric C02-uptake give the rates of malate-decarboxylation-dependent C02assimilation. Based upon this hypothesis, the following photosyn-thetic characteristics were observed: (a) From the onset of thelight to midphase IV of CAM, the photosynthetic quotient (net 02evolved/net C02 fixed) was higher than 1. This indicates thatmalate-decarboxylation supplied C02 for the photosynthetic car-bon reduction cycle during this period. (b) In phase IlIl and earlyphase IV, the rate of C02 assimilation deduced from net 02-evolution was 3 times higher than the maximum rate of atmos-pheric C02-fixation during phase IV. A conceivable explanationfor this stimulation of photosynthesis is that the intracellular C02-concentration was high because of malate decarboxylation. (c)During the final hours of the light period, the photosyntheticquotient decreased below 1. This may be the result of C02-fixationby phosphoenolpyruvate-carboxylase activity and malate accu-mulation. Based upon this hypothesis, the gas exchange dataindicates that at least 50% of the C02 fixed during the last hourof the light period was stored as malate. Light 02-uptake deter-mined with 1802 showed two remarkable characteristics: from theonset of the light until midphase IV the rate of 02-uptake increasedprogressively; during the following part of the light period, therate of 02-uptake was 3.5 times higher than the maximum rate ofC02-uptake. When malate decarboxylation was reduced or sup-pressed after a night in a C02-free atmosphere or in continuousillumination, the rate of 02-uptake was higher than in the control.This supports the hypothesis that the low rate of 02-uptake in thefirst part of the light period is due to the inhibition of photorespir-ation by increased intracellular CO2 concentration because ofmalate decarboxylation. In view of the law of gas diffusion andthe kinetic properties of the ribulose-1,5-bisphosphate carboxyl-ase/oxygenase, 02 and C02 gas exchange suggest that at theend of the light period the intracellular CO2 concentration wasvery low. We propose that the high ratio of 02-uptake/C02-fixationis principally caused by the stimulation of photorespiration duringthis period.

Crassulacean acid metabolism affords a mechanism for thetemporal separation ofC02-fixation and C02-reduction. Dur-ing the dark period, PEP-Case' catalyses the fixation of CO2and malate is formed. During the light period, CO2 is releasedfrom malate decarboxylation. This CO2 is fixed by Rubiscoand assimilated in the PCR (in this paper, the term C02-assimilation means C02-reduction in the PCR). AtmosphericC02-uptake is also possible in CAM during late light periodwhen stomata are open (19, 23, 30).Rhythmic patterns of net C02-exchange are well known in

CAM; four phases have been defined with regard to the netatmospheric CO2 fixation (23). The precise timing and rateof internal C02-assimilation, which cannot be determinedsoley by the solely net CO2 exchange, is less well documented.One objective of this study is to determine the rate of malate-decarboxylation-dependent C02-assimilation and oftotal CO2assimilation (assimilation of external CO2 included) in theCAM plant Ananas comosus. Net 02-evolution in the light inplants is equivalent to the amount ofCO2 reduced in the PCR(17). Therefore, in CAM, the difference between the rates ofnet 02-evolution and net light atmospheric C02-fixationshould give the rate of internal C02-assimilation. This issupported by the results of several workers who observed that,in CAM plants, the rate of net 02-evolution can be higherthan the rate of net light C02-fixation (2, 7, 21). Thus, in ourinvestigation we measured the hourly rates of net 02 and CO2exchange in order to determine the time course of the internalCO2-assimilation. This approach yielded new informationsabout aspects of CAM often reported but rarely quantified,such as the influence of malate decarboxylation on the pho-

' Abbreviations: PEP-Case, phosphoenolpyruvate carboxylase; PN,nocturnal net C02-uptake; PC, diurnal net C02-uptake; RO, noctur-nal net 02-uptake; P0, diurnal net 02-evolution; U, light 02-uptake;E, gross 02-evolution; A, gross dark C02-fixation; B, malate depend-ent 02-evolution; C, amount of net C02-uptake not reduced in thePCR at the end of the light period; Phase I-IV, phases of net C02-exchanges in CAM as described by Osmond (23); PEP, phosphoen-olpyruvate; PPFD, photosynthetic photon flux density; PQ, photo-synthetic quotient (02 evolved/CO2 fixed); RUBP, ribulose 1.5 bis-phosphate; Rubisco, ribulose- 1 ,5-bisphosphate carboxylase/oxygenase.

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Plant Physiol. Vol. 89, 1989

tosynthetic reduction cycle, the characteristics of the transi-tion from malate-decarboxylation-dependent C02-assimila-tion to atmospheric CO2-fixation during phase IV, and thecontribution of PEP-Case to CO2-fixation during the lightperiod.Net O-evolution in plants is the result of two opposite

fluxes: gross O2-evolution by photolysis of water and 02-uptake processes, such as photorespiration. The presence ofphotorespiration in CAM is well-established (2, 19). In A.comosus, this was concluded from the results ofmeasurementsofthe postillumination burst ofCO2 (11) or from the depend-ence of the light 02-uptake rate to the 02 and CO2 concentra-tions (22). However, in CAM plants, timing and rate of lightO2-uptake during the different phases of CO2 exchange is notwell known (2, 27). Using 1802 as a tracer, we determined thistime course of 02-uptake rates in A. comosus.

MATERIAL AND METHODS

Plant Material and Growth Conditions

Pineapple plants were obtained from one clone of Ananascomosus and provided by l'Institut de Recherche sur les Fruitset Agrumes, CIRAD Montpellier. Gas exchange measure-ments were performed with plants propagated by in vitrotechniques (24). When total fresh weight of the plants was 3to 5 g, they were transferred to an inert substrate (perlite) andplaced in a growth cabinet. Environmental light/dark condi-tions for growth and gas exchanges measurements were asfollows: photoperiod 12h/12h; (PPFD = 600-700 Mmol m-2s-'); temperature 28°C/22°C; RH 60 to 70-80%. Plants werewatered 6 times per d using a Hoagland-Arnon solution, pH5 (16). Four to 5 months-old plants (total fresh weight ofleaves: 120-160 g; total leafarea 17-22 dm2; number ofleaves30 to 35) were used.

Gas Exchange Measurements

Gas exchange determinations were made in a automaticculture chamber (C23A system) previously described in detail(1). The complete system consisted of (a) a controlled envi-ronment chamber (volume 4-25 L) which was thermoregu-lated with air ventilation and radiator and illuminated withfive lamps (OSRAM HQI 400W); (b) a gas analysis systemconsisting of a CO2 IR analyser (ADC MK3) and a quadri-polar mass spectrometer (RIBER QMM 17); (c) a C02 regu-lation circuit with calibrated valves to inject or trap CO2 inorder to maintain the CO2 concentration at 340 4L L` +10); (d) a minicomputer (Telemecanique T1600) to collectand store all data and to control the system.CO2 exchange of the plant was calculated from the amount

of CO2 injected or trapped by the regulation circuit. Net O2exchange was calculated from the variation of the 1602 con-centration determined with the mass spectrometer. The one-directional flow of light 02-uptake was determined by meas-uring the disappearance of 802 relative to that of neon, aninert reference gas. This method has been previously describedand discussed (14). Each experimental point is the sum of thegas exchange during 1 h and reflects the activity of the wholeshoot. This section of plant was isolated from the root usingan air tight putty joint (Terostat Teroson).

Time courses of gas exchange were monitored using threedifferent plants and reproducible results were obtained. Forbetter transparency, time courses of the gas exchange of onlyone plant is shown in the figures. Nevertheless, mean valuesof certain parameters calculated from the results obtainedwith the two other plants are presented in the paper.

RESULTS

C02-Assimilation

Net C02 ExchangeLike pineapple plants reproduced using the conventional

techniques of slips or crowns (6), the plants obtained by invitro multiplication showed the four typical phases of net CO2exchange of CAM (Fig. 1). The rate of CO2 uptake duringphase I was maximal after 2 h in the dark and then decreaseduntil the end of the dark period. Phase II lasted for 1 h andphase III lasted for 2 to 3 h. Phase IV began after the firstthird of the light period. The precise regulation of environ-mental conditions of the plant resulted in a nearly identicalCO2 exchange pattern from one day to the other (Fig. 1).

Net 02-Exchange in the Dark

During the night, net 02-uptake did not change rhythmi-cally (Fig. 1). In terms of balance, all CO2 from respirationwas refixed by the PEP-Case in the closed growth chamberused. Therefore, assuming a respiratory quotient close to 1during the night in pineapple like in other CAM plants (18),the rate of RO added to that of PN is equivalent to theamount of internal CO2 stored into malate. This gross darkCO2 fixation, termed A, is shown shaded in Figure 1. Thirtyto 40% of the total CO2 fixed into malate during the nightoriginated from respiratory activity. Similar values were de-termined with the two other plants studied.

Net 02-Exchange in the Light

Assimilation of 1 mol of CO2 in the PCR requires theoxidation of 2 mol of NADPH and is accompanied by theevolution of 1 mol of oxygen (17). In accordance with thisstatement, we have determined a daily integrated PQ close to1 in Ananas comosus (Table I). Daily PQ was calculated asthe ratio of total net O2-evolved in the light to gross dark CO2fixation plus net light CO2 fixation. Other determinations ofthe daily PQ using two different plants gave the values 0.97± 0.03 and 1.02 ± 0.03 (means ofthree successive days understable growth conditions).With a daily PQ equal to 1, C02-assimilation can be con-

sidered equivalent to net 02-evolution throughout the lightperiod. With this statement, three periods ofCO2 assimilationare distinguished according to the value of the hourly PQ:

1. During phase II, III, and the beginning of phase IV, therate of net 02-evolution was higher than the rate of net C02-uptake (Fig. 1). This is explained by the simultaneous assim-ilation ofinternal CO2 released during malate decarboxylationand assimilation ofatmospheric CO2. The quantity ofinternal

62 COTE ET AL.

C02 AND 02 EXCHANGES IN CAM

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TIME(h)Figure 1. Net C02 and 02 hourly exchanges of A. comosus over 2 consecutive days. PN, noctumal C02-uptake; RO, noctumal 02-uptake; PC,diumal C02-exchange; PO, 02-evolution. Measurements were taken from the aerial portion of the plant (total leaf fresh weight and area 134 g;17.5 dM2, respectively). Night periods are delineated by a black bar. Roman numerals indicate the phases of C02 exchange of CAM as describedby Osmond (23). On d 2, shaded areas represent: A, the gross dark C02 fixation; B, the malate-dependent net 02-evolution; C, an amount ofnet C02 fixed which was not assimilated in PCR during late phase IV. PPFD was 660 Amol m-2 s-1; day/night temperature 28°C/220C.

Table I. Nocturnal and Diurnal Cumulative net C02 and 02Exchange of One Shoot of Ananas comosus Over Two ConsecutiveDays

Data are the sum of hourly exchanges presented in Figure 1. PO,the photosynthetic quotient, is the ratio of total net 02 evolved in thelight to total dark and light C02 fixed: P0 = ([PO]/[PN + (RO) +PC]).

Cumulative net CO2 and 02 Exchange

Dark Light PQ

X(PN) Z(RO) Z(PC) 2(PO)

mmol-plant-1. 12 h-1D 1 10.6 -6.4 10.4 27.1 0.99D 2 11.1 -6.3 10.7 27.5 .0.98

CO, assimilated is represented by the hatched area B (B =P0-PC when PO>PC) in Figure 1. The rate of C02-assimi-lation (deduced from the rate of net 02 evolution) in the timeinterval between the second and fourth hour of the lightperiod was 2.9-fold higher than the maximum rate of atmos-pheric C02-uptake in phase IV (Fig. 1). The most likely originof such 3-fold stimulation of photosynthesis during phase IIIand early phase IV is an increasing internal C02 concentrationwhich is known to occur in CAM plants during malatedecarboxylation (10, 13, 26).

2. For about 2 h in the middle of phase IV, the value of

the PQ was close to 1 (Fig. 1). This shows that only atmos-pheric C02 was assimilated during this period.

3. In the final hours of phase IV, the rate of net 02-evolution was lower than that ofnet C02 fixation. This meansthat part of the C02-uptake did not occur in the PCR and sono 02 was evolved. This amount ofC02-uptake is representedby the hatched area C (C = PC-PO when PC>PO) in Figure1. C amounted 9.8 to 3.4% of the net night C02-uptakeduring d 1 and 2, respectively (Fig. 1). With the two otherplants studied the C phase was also present and amounted to15.9 ± 0.9 and 10.3 ± 2.6% of the net night C02 fixation(mean of three successive days under stable growth condi-tions).

Light o2-Uptake

Light 02-uptake in the shoot of A. comosus displayed thefollowing characteristics: (a) The rate of 02-uptake variedthroughout the light period, 02-uptake increased progressivelyfrom the onset of the light period until the middle of phaseIV (Fig. 2). (b) During the following part of the day, the rateof 02-uptake clearly exceeded the rate of photosynthesis, 02-uptake was about 3.5 fold higher than the maximum rate ofC02-uptake (Fig. 2).

It is conceivable that the lower 02-uptake rate, principallyobserved during phase III, indicates a repression ofthe RUBP-oxygenase activity by the previously reported increase in

63

Plant Physiol. Vol. 89, 1989

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TIME(h)Figure 2. Light 02-uptake and net C02 and 02 exchange in A. comosus. U, light 02-uptake; PO, net 02-evolution; E, gross 02-evolution; PC,diumal net C02-exchange; RO, noctumal 02-uptake; PN, noctumal C02-uptake. For simplification in the graphical representation, both net 02-evolution and light 02-uptake have been counted positively. Measurements were taken from the aerial portion of the plant (total leaf fresh weightand area 134 g; 17.5 dM2, respectively). PPFD was 660 umol m-2 s-1; day/night temperature 28°C/22°C. Roman numerals indicate the phaseof C02-exchange of CAM as defined by Osmond (23).

intracellular CO2 concentration. In order to test this hypoth-esis, two different experiments were performed:

Light 02-Uptake After a Dark Period in a C02-FreeAtmosphere

A pineapple plant was put in a CO2-free atmosphere for a

night. During this period, only respiratory CO2 was fixed bythe PEP-Case and one can expect that a small quantity ofmalate was formed (Fig. 3). The following light period, themaximum rate of CO2 assimilation (deduced from net O2-

evolution) during phase III was lower than in the control (i.e.the phase III of the preceding light period). This suggests thatthe intracellular CO2 concentration achieved during malatedecarboxylation was lower than in the control. Concomitantlyto the lower rate of C02-assimilation and the expected de-crease in the intracellular CO2 concentration, we observed a

high rate of 02-uptake comparatively to that of the controlphase III (Fig. 3).

Light 02-Uptake Under Continuous Illumination

After subjecting a pineapple plant to continuous light, weobserved rhythmic changes of both net CO2-fixation and O2-evolution which persisted at a lower amplitude for about 36h (Fig. 4). This indicates that malate accumulation and malatedepletion were still occurring in continuous light. The same

conclusions were drawn from the results of experiments with

Kalanchoe blossfeldiana (8). After about 50 h of continuousillumination, the rate of net CO2-fixation became fairly con-

stant and the value of the PQ of about 1 indicates that themalate content did not fluctuate (Fig. 4). The rate of light O2-uptake was then nearly equal to that observed during thecontrol phase IV at the beginning of the experiment.

Therefore, when less or no malate is available for decarbox-ylation (after a night period in a C02-free atmosphere or

under continuous illumination, respectively), the rate of light02-uptake is higher than in a control phase III. Assuming thatin CAM an increase in the intracellular CO2 concentrationoccurs subsequent to malate decarboxylation, this supportsthe hypothesis of the inhibition of photorespiration duringphase III.

DISCUSSION

C02-Assimilation

An increase in CO2 concentration following malate decar-boxylation is the most conceivable explanation of the highrate of internal CO,-assimilation observed during phase III inAnanas comosus. For different CAM plants, intracellularconcentrations higher than 2000 ,uL L` have been reported(10, 26). The following observation suggests that, in pineapple,the intracellular CO2 concentration during malate decarbox-ylation is probably lower than this value: based on the law ofgas diffusion, the absence of net CO2 exchange during the

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64 COTE ET AL.

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TIME(h)Figure 3. Light 02-uptake and net CO2 and 02-exchange in the aerial portion of A. comosus during a control photoperiod and after a night inC02-free atmosphere. The plant was maintained as in Figure 1 except PPFD was 600 elmol m-2 s-1.

third hour of the light period indicates equilibrium betweenthe atmospheric and the intracellular C02 concentration. It ispossible, however, to object that during this period, stomatalresistance is high and prevents a loss of internal C02. Never-theless, during the fourth hour of the light period (Fig. 1, h16, for example) a high rate of net O2-evolution was observedsimultaneously with a net atmospheric C02-uptake. This netC02-uptake demonstrates that intracellular C02-concentra-tion was lower than the external concentration during thishour. Thus, considering that the rate of C02-assimilation(deduced from the net O2-evolution) during the third and thefourth hour of the light period was not very different, we can

expect that intracellular C02-concentration during these 2 hwas also not very different and was probably a little bit lower(fourth of the light period) or in the order (third hour of thelight period) ofthe external concentration. With this assump-tion, if one considers the great change of the rate of C02-assimilation (net 0,-evolution) from phase III to midphaseIV (ratio of about 3), one can expect that internal C02concentration at the end of the day was probably far belowatmospheric C0.-concentration.

It has been reported that 02 may accumulate in leaves ofCAM-plants to a concentration ofup to 40% during phase III(26). We calculated the internal 0,-concentration in A. com-osus with the 02 exchange of Figure 1 and the equation (15):

Ci-Ce = r x 1.4 x P0

where Ci and Ce are the intercellular or atmospheric 02

concentration, respectively; r the stomatal resistance to watervapor diffusion (150 s cm-' for pineapple leaves without water

stress, see Ref. [5]); 1.4 the ratio of the diffusivities of watervapor and 02 in air. With this value we estimated that the 0,concentration was no more than 0.5% higher in leaves thanin the atmosphere during phase III.A conceivable explanation for a PQ smaller than 1 at the

end of the light period is that PEP-Case activity leading tomalate storage occurs during late phase IV in A. comosus.The synthesis of 1 mol of malate by PEP-Case requires theoxidation of 1 mol of reducing equivalent (for oxaloacetatereduction into malate). Therefore, C02 fixation via f3-carbox-ylation and the following storage of malate is accompanied inthe light by theoretically half as much net 02-evolution thanC02 fixation via Rubisco. Moreover, if the required PEP forlight-f,-carboxylation is supplied as in the dark by the glycol-ysis, this pathway would produce an amount of reducingpower (in the glyceraldehyde-3-P oxidation 1 to 1,3-diphos-phoglycerate) stoichiometrically equivalent to that requiredfor CO2 fixation. Thus, in terms of balance, light C02-fixationvia PEP-Case (leading to malate storage) would be achievedwithout reducing equivalent consumption and, consequently,without net 02-evolution. Therefore, C would probably rep-resent the amount of CO2 stored into malate. PEP-Case hasbeen reported to be active during late phase IV in CAM plants(20). The finding of a PQ lower than 1 suggests, moreover,that this fixation leads to malate storage in A. comosus. Withthe above statement, data reported in Figure 1 and determinedwith the two other plants studied indicates that at least 50%of the total C02 fixed during the last hour of the light periodwas stored into malate.To summarize, these data show that in the CAM plant A.

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Plant Physiol. Vol. 89, 1989

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TIME(h)Figure 4. Light 02-uptake and net CO2 and 02-exchange in the aerial portion of A. comosus subjected to continuous illumination. The black barindicates the night period before continuous light. The plant was maintained as in Figure 1.

comosus, steady state conditions of photosynthesis were neverachieved during the light period: malate decarboxylation sup-ply CO2 for the PCR activity during most of the light period(from phase II to the middle of phase IV); the PCR activity(deduced from the rate of net O2-evolution) change substan-tially throughout the light period. It is stimulated during phaseIII and early phase IV, probably by an increase in intracellularC02-concentration; a C3-photosynthetic phase using exclu-sively atmospheric CO2 is limited to 1 to 2 h during themiddle of phase IV; late phase IV is characterized by compe-tition between Rubisco and PEP-Case for CO2 fixation.

Light 02-UptakeThe lowest rate of light 02-uptake was observed in pineap-

ple plants during phase III when stomatal resistance is knownto be the highest in CAM (19, 23, 30). One can suggest thatbecause of this high resistance to gas diffusion, the low rateof 02-uptake is caused by the recycling of photosyntheticevolved-oxygen into photorespiration. The extent of this re-cycling can be calculated with the equation proposed byGerbaud and Andre (15). We determined from the data ofFigure 2 that the 'true' value of 02-uptake during phase IIIwas underestimated by only about 3% when the value ofstomatal resistance is taken to equal 150 s cm-' (5). Thus, theunderestimation of '802-uptake due to oxygen recycling couldnot account for the low 02-uptake rate measured during thebeginning of the day, and an inhibition of photorespiration

due to the previously reported increase in the intracellularC02-concentration is the most probable explanation for thislow rate of 02-uptake.

Based on leaf area, the maximum rate of 02-uptake ob-served during phase IV was in the order of 0.3 mmol dm-2h-' (Fig. 2). Under similar growth conditions, Canvin et al.(9) and Badger and Canvin (4) reported rates of 02-uptake of0.3 to 0.6 mmol dm-2 h-' for leaves of several C3 plants. Thesmaller concentration of Rubisco per leaf area in CAM plantsrelative to C3 plants (31) may account for this difference.However, determinations of 02-uptake in different species ofCAM-plants are necessary to conclude that 02-uptake per leafarea is lower in CAM-plants than in C3-plants.During phase IV, we observed a high rate of 02-uptake

relative to that of CO2 assimilation. Assuming that the gross02-evolution in plants is equivalent to the flow of electronstransport in the thylakoids membranes (28), these data indi-cates that, in pineapple, 60 to 80% of the reducing powerproduced (E} was used in 02-consuming processes (U) ratherthan for CO2 assimilation (PO) (Fig. 2). For comparison, inattached leaves or shoots of C3 plants, 45 to 55% of thereducing-equivalents produced are used in 02-uptake proc-esses (4, 9, 14). This high hourly rate of 02-uptake relative tophotosynthesis during phase IV in A. comosus is consistentwith the high daily U/PO ratio which was observed in severalCAM plants (2, 27, 28) and with the high quantum require-ment for photosynthesis determined after malate pool deple-

66 COTE ET AL.

C02 AND 02 EXCHANGES IN CAM

tion in Sedum praealtum (25). Possible origins of this highrate of 02-uptake are discussed in the following.

1. The respiratory activity of nonchlorophyllous tissues ofthe plant contributes to the high ratio of 02-uptake/CO2assimilation. However, the contribution of this respiration tothe total light 02-uptake is probably low: the mean rate ofrespiration in the dark (including chlorophyllous tissue) isonly about one-ninth of that of 02-uptake during phase IV(Fig. 2). This indicates that U is mainly a light-dependentprocess.

2. In CAM plants, even if most of the triose compoundsformed from malate decarboxylation are used for gluconeo-genesis, it is possible that a portion of this triose is oxidizedin the tricarboxylic acid cycle. Such oxidation is a potential02-consuming process. However, the following observationssuggest that this oxidation is not involved in the high rate of02-uptake determined. The maximum rate of 02-uptake wasobserved in midphase IV after the malate pool was alreadycompletely depleted (as indicated by the value ofPQ equal to1 during this period). Under continuous illumination, whenno malate decarboxylation occurred (i.e. when the hourly PQwas close to 1), the rate of 02-uptake was also high relative tophotosynthesis.

3. The high 02-uptake/C02-assimilation ratio during phaseIV could be a consequence of the kinetic properties ofRUBP-oxygenase activity in A. comosus. This hypothesis is unlikely,however, because the Rubisco of the CAM plant Kalanchoedaigremontiana shows nearly identical carboxylase and oxy-genase in vitro activity that those ofC3 plants show (3).

4. It is conceivable that the high rate of 02-uptake duringthe end of the light period is the result of a stimulation of theRUBP-oxygenase activity due to a low intracellular C02concentration. Based upon the gas exchanges data, such a lowintracellular C02-concentration during the second part ofphase IV is, as demonstrated above, probable. According tothis hypothesis, Winter (29) has determined a substomatalCO2 concentration in the range of 170 to 200 ,L L' inKalanchoe pinnata during the late phase IV or a prolongedlight period. For comparison, this concentration is usuallynear 230 uL L-' for C3 plants (12). Winter suggested thatPEP-Case activity during the light period accounts for thislow intercellular CO2 concentration due to the high affinityof PEP-Carboxylase for C02. However, a decrease in theintracellular C02-concentration due to PEP-Case activity can-not be the origin of the high U/PO ratio during phase IV inA. comosus because we have observed a high rate of02-uptakeeven when no malate synthesis occurred (for example, whenthe PQ was equal to 1 in midphase IV or under continuousillumination). A low internal CO2 concentration could alsobe the result of a considerable stomatal resistance which isknown to be high in CAM plants compared to C3 plants,even during phase IV (19). Mesophyll resistance to gas diffu-sion would also contribute to increase the C02-gradient be-tween the atmosphere and the cells. The mesophyll resistancein crassulacean plants has not been reported.

In order to determine whether a low intra cellular C02concentration which results in alow photorespiration aloneaccounts for the high ratio of 02-uptake/C02-assimilationduring phase IV, it is necessary to determine the total resist-

ance for CO2 diffusion in the leafand investigate the influenceof the atmospheric CO2 level on 02 and CO2-uptake.

Acknowledgments

The authors gratefully thank A. Gerbaud, T. Betsche, and C.Wilson for useful comments on this manuscript and the staff of theagrophysiology laboratory C. Deweirt, J. Massimino, C. Richaud forsupport. One of us (F. X. C.) acknowledges a fellowship from leMinistere de la Recherche and l'Institut de recherche sur les Fruits etAgrumes IRFA-CIRAD.

LITERATURE CITED

1. Andre M, Daguenet A, Massimino D, Vivoli J, Richaud C (1979)Le laboratoire C23A. Un outil au service de la physiologie dela plante entiere I. Les chambres de culture et les systemes demesures associes. Ann Agron 30: 135-151

2. Andre M, Thomas DA, Von Willert DJ, Gerbaud A (1979)Oxygen and carbon dioxide exchanges in crassulacean-acidmetabolism plants. Planta 147: 141-144

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