Received for publication March 29, 1988
and in revised form August 8, 1988
Plant Physiol. (1989) 89, 61-68
0032-0889/89/89/0061/08/$01 .00/0
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.)
Crassulacean acid metabolism affords a mechanism for the
temporal separation of C02-fixation and C02-reduction. During the dark period, PEP-Case' catalyses the fixation of CO2
and malate is formed. During the light period, CO2 is released
from malate decarboxylation. This CO2 is fixed by Rubisco
and assimilated in the PCR (in this paper, the term C02assimilation means C02-reduction in the PCR). Atmospheric
C02-uptake is also possible in CAM during late light period
when 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 net
atmospheric CO2 fixation (23). The precise timing and rate
of internal C02-assimilation, which cannot be determined
soley by the solely net CO2 exchange, is less well documented.
One objective of this study is to determine the rate of malatedecarboxylation-dependent C02-assimilation and of total CO2
assimilation (assimilation of external CO2 included) in the
CAM plant Ananas comosus. Net 02-evolution in the light in
plants is equivalent to the amount of CO2 reduced in the PCR
(17). Therefore, in CAM, the difference between the rates of
net 02-evolution and net light atmospheric C02-fixation
should give the rate of internal C02-assimilation. This is
supported by the results of several workers who observed that,
in CAM plants, the rate of net 02-evolution can be higher
than the rate of net light C02-fixation (2, 7, 21). Thus, in our
investigation we measured the hourly rates of net 02 and CO2
exchange in order to determine the time course of the internal
CO2-assimilation. This approach yielded new informations
about aspects of CAM often reported but rarely quantified,
such as the influence of malate decarboxylation on the pho-
ABSTRACT
Photosynthesis and light 02-uptake of the aerial portion of the
CAM plant Ananas comosus (L.) meff. were studied by C02 and
02 gas exchange measurements. The amount of C02 which was
fixed during a complete day-night cycle was equal to the amount
of total net 02 evolved. This finding justifies the assumption that
in each time interval of the light period, the difference between
the rates of net 02-evolution and of net light atmospheric C02uptake give the rates of malate-decarboxylation-dependent C02
assimilation. Based upon this hypothesis, the following photosynthetic characteristics were observed: (a) From the onset of the
light to midphase IV of CAM, the photosynthetic quotient (net 02
evolved/net C02 fixed) was higher than 1. This indicates that
malate-decarboxylation supplied C02 for the photosynthetic carbon reduction cycle during this period. (b) In phase IlIl and early
phase IV, the rate of C02 assimilation deduced from net 02evolution was 3 times higher than the maximum rate of atmospheric C02-fixation during phase IV. A conceivable explanation
for this stimulation of photosynthesis is that the intracellular C02concentration was high because of malate decarboxylation. (c)
During the final hours of the light period, the photosynthetic
quotient decreased below 1. This may be the result of C02-fixation
by phosphoenolpyruvate-carboxylase activity and malate accumulation. Based upon this hypothesis, the gas exchange data
indicates that at least 50% of the C02 fixed during the last hour
of the light period was stored as malate. Light 02-uptake determined with 1802 showed two remarkable characteristics: from the
onset of the light until midphase IV the rate of 02-uptake increased
progressively; during the following part of the light period, the
rate of 02-uptake was 3.5 times higher than the maximum rate of
C02-uptake. When malate decarboxylation was reduced or suppressed after a night in a C02-free atmosphere or in continuous
illumination, the rate of 02-uptake was higher than in the control.
This supports the hypothesis that the low rate of 02-uptake in the
first part of the light period is due to the inhibition of photorespiration by increased intracellular CO2 concentration because of
malate decarboxylation. In view of the law of gas diffusion and
the kinetic properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase, 02 and C02 gas exchange suggest that at the
end of the light period the intracellular CO2 concentration was
very low. We propose that the high ratio of 02-uptake/C02-fixation
is principally caused by the stimulation of photorespiration during
this period.
' Abbreviations: PEP-Case, phosphoenolpyruvate carboxylase; PN,
nocturnal net C02-uptake; PC, diurnal net C02-uptake; RO, nocturnal net 02-uptake; P0, diurnal net 02-evolution; U, light 02-uptake;
E, gross 02-evolution; A, gross dark C02-fixation; B, malate dependent 02-evolution; C, amount of net C02-uptake not reduced in the
PCR at the end of the light period; Phase I-IV, phases of net C02exchanges in CAM as described by Osmond (23); PEP, phosphoenolpyruvate; PPFD, photosynthetic photon flux density; PQ, photosynthetic quotient (02 evolved/CO2 fixed); RUBP, ribulose 1.5 bisphosphate; Rubisco, ribulose- 1 ,5-bisphosphate carboxylase/
oxygenase.
61
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62
COTE ET AL.
tosynthetic reduction cycle, the characteristics of the transition from malate-decarboxylation-dependent C02-assimilation to atmospheric CO2-fixation during phase IV, and the
contribution of PEP-Case to CO2-fixation during the light
period.
Net O-evolution in plants is the result of two opposite
fluxes: gross O2-evolution by photolysis of water and 02uptake processes, such as photorespiration. The presence of
photorespiration in CAM is well-established (2, 19). In A.
comosus, this was concluded from the results of measurements
of the postillumination burst of CO2 (11) or from the dependence of the light 02-uptake rate to the 02 and CO2 concentrations (22). However, in CAM plants, timing and rate of light
O2-uptake during the different phases of CO2 exchange is not
well known (2, 27). Using 1802 as a tracer, we determined this
time course of 02-uptake rates in A. comosus.
MATERIAL AND METHODS
Plant Material and Growth Conditions
Pineapple plants were obtained from one clone of Ananas
comosus and provided by l'Institut de Recherche sur les Fruits
et Agrumes, CIRAD Montpellier. Gas exchange measurements were performed with plants propagated by in vitro
techniques (24). When total fresh weight of the plants was 3
to 5 g, they were transferred to an inert substrate (perlite) and
placed in a growth cabinet. Environmental light/dark conditions for growth and gas exchanges measurements were as
follows: photoperiod 12h/12h; (PPFD = 600-700 Mmol m-2
s-'); temperature 28°C/22°C; RH 60 to 70-80%. Plants were
watered 6 times per d using a Hoagland-Arnon solution, pH
5 (16). Four to 5 months-old plants (total fresh weight of
leaves: 120-160 g; total leaf area 17-22 dm2; number of leaves
30 to 35) were used.
Gas Exchange Measurements
Gas exchange determinations were made in a automatic
culture chamber (C23A system) previously described in detail
(1). The complete system consisted of (a) a controlled environment chamber (volume 4-25 L) which was thermoregulated with air ventilation and radiator and illuminated with
five lamps (OSRAM HQI 400W); (b) a gas analysis system
consisting of a CO2 IR analyser (ADC MK3) and a quadripolar mass spectrometer (RIBER QMM 17); (c) a C02 regulation circuit with calibrated valves to inject or trap CO2 in
order to maintain the CO2 concentration at 340 4L L` +
10); (d) a minicomputer (Telemecanique T1600) to collect
and 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 O2
exchange was calculated from the variation of the 1602 concentration determined with the mass spectrometer. The onedirectional flow of light 02-uptake was determined by measuring the disappearance of 802 relative to that of neon, an
inert reference gas. This method has been previously described
and discussed (14). Each experimental point is the sum of the
gas exchange during 1 h and reflects the activity of the whole
shoot. This section of plant was isolated from the root using
an air tight putty joint (Terostat Teroson).
Plant Physiol. Vol. 89, 1989
Time courses of gas exchange were monitored using three
different plants and reproducible results were obtained. For
better transparency, time courses of the gas exchange of only
one plant is shown in the figures. Nevertheless, mean values
of certain parameters calculated from the results obtained
with the two other plants are presented in the paper.
RESULTS
C02-Assimilation
Net C02 Exchange
Like pineapple plants reproduced using the conventional
techniques of slips or crowns (6), the plants obtained by in
vitro multiplication showed the four typical phases of net CO2
exchange of CAM (Fig. 1). The rate of CO2 uptake during
phase I was maximal after 2 h in the dark and then decreased
until the end of the dark period. Phase II lasted for 1 h and
phase III lasted for 2 to 3 h. Phase IV began after the first
third of the light period. The precise regulation of environmental conditions of the plant resulted in a nearly identical
CO2 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 rhythmically (Fig. 1). In terms of balance, all CO2 from respiration
was refixed by the PEP-Case in the closed growth chamber
used. Therefore, assuming a respiratory quotient close to 1
during the night in pineapple like in other CAM plants (18),
the rate of RO added to that of PN is equivalent to the
amount of internal CO2 stored into malate. This gross dark
CO2 fixation, termed A, is shown shaded in Figure 1. Thirty
to 40% of the total CO2 fixed into malate during the night
originated from respiratory activity. Similar values were determined with the two other plants studied.
Net 02-Exchange in the Light
Assimilation of 1 mol of CO2 in the PCR requires the
oxidation of 2 mol of NADPH and is accompanied by the
evolution of 1 mol of oxygen (17). In accordance with this
statement, we have determined a daily integrated PQ close to
1 in Ananas comosus (Table I). Daily PQ was calculated as
the ratio of total net O2-evolved in the light to gross dark CO2
fixation plus net light CO2 fixation. Other determinations of
the daily PQ using two different plants gave the values 0.97
± 0.03 and 1.02 ± 0.03 (means of three successive days under
stable growth conditions).
With a daily PQ equal to 1, C02-assimilation can be considered equivalent to net 02-evolution throughout the light
period. With this statement, three periods of CO2 assimilation
are distinguished according to the value of the hourly PQ:
1. During phase II, III, and the beginning of phase IV, the
rate of net 02-evolution was higher than the rate of net C02uptake (Fig. 1). This is explained by the simultaneous assimilation of internal CO2 released during malate decarboxylation
and assimilation of atmospheric CO2. The quantity ofinternal
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C02 AND 02 EXCHANGES IN CAM
63
r
F,4.0
F4..
Cu
p-,
Cu
c
Q
E
E
LLI
r
0
r
r0
CC
U)
U)
E
E
w
r- UI
x
P4
L&
I
C-)
r
cr
I
x
X
x
w
N
0
-
N
0
Iw
Z
LL
Z:
t
0
x-0
U)
Ln
U)
r-_I
-1
x
UA.
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 described
by Osmond (23). On d 2, shaded areas represent: A, the gross dark C02 fixation; B, the malate-dependent net 02-evolution; C, an amount of
net 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 02
Exchange of One Shoot of Ananas comosus Over Two Consecutive
Days
Data are the sum of hourly exchanges presented in Figure 1. PO,
the photosynthetic quotient, is the ratio of total net 02 evolved in the
light to total dark and light C02 fixed: P0 = ([PO]/[PN + (RO) +
PC]).
Cumulative net CO2 and 02 Exchange
X(PN)
D1
D2
10.6
11.1
PQ
Light
Dark
Z(RO)
Z(PC)
mmol-plant-1. 12 h-1
-6.4
-6.3
10.4
10.7
2(PO)
27.1
27.5
0.99
.0.98
the PQ was close to 1 (Fig. 1). This shows that only atmospheric C02 was assimilated during this period.
3. In the final hours of phase IV, the rate of net 02evolution was lower than that of net C02 fixation. This means
that part of the C02-uptake did not occur in the PCR and so
no 02 was evolved. This amount of C02-uptake is represented
by the hatched area C (C = PC-PO when PC>PO) in Figure
1. C amounted 9.8 to 3.4% of the net night C02-uptake
during d 1 and 2, respectively (Fig. 1). With the two other
plants studied the C phase was also present and amounted to
15.9 ± 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
CO, assimilated is represented by the hatched area B (B =
P0-PC when PO>PC) in Figure 1. The rate of C02-assimilation (deduced from the rate of net 02 evolution) in the time
interval between the second and fourth hour of the light
period was 2.9-fold higher than the maximum rate of atmospheric C02-uptake in phase IV (Fig. 1). The most likely origin
of such 3-fold stimulation of photosynthesis during phase III
and early phase IV is an increasing internal C02 concentration
which is known to occur in CAM plants during malate
decarboxylation (10, 13, 26).
2. For about 2 h in the middle of phase IV, the value of
Light 02-uptake in the shoot of A. comosus displayed the
following characteristics: (a) The rate of 02-uptake varied
throughout the light period, 02-uptake increased progressively
from the onset of the light period until the middle of phase
IV (Fig. 2). (b) During the following part of the day, the rate
of 02-uptake clearly exceeded the rate of photosynthesis, 02uptake was about 3.5 fold higher than the maximum rate of
C02-uptake (Fig. 2).
It is conceivable that the lower 02-uptake rate, principally
observed during phase III, indicates a repression of the RUBPoxygenase activity by the previously reported increase in
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COTE ET AL.
64
Plant Physiol. Vol. 89, 1989
m
I.'
c
m
0
8a1=
.5
°
-
E00o
3 I.5L I.5
8 E E
0~~~~~
E rE
E E
-
6
U
x
w
0
N
0
Po
4
w
I
CXD
N
CL U)
D
:D
I
N
0
-\j
0
M
C:
I
X
x
w
LL.
0
E
E
2
PN
J
0
?u
0
llJ
w
ZU
o
I
Roi
I
i
S
4
i
10
15
20
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 02evolution and light 02-uptake have been counted positively. Measurements were taken from the aerial portion of the plant (total leaf fresh weight
and 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 phase
of C02-exchange of CAM as defined by Osmond (23).
intracellular CO2 concentration. In order to test this hypothesis, two different experiments were performed:
Light 02-Uptake After a Dark Period in a C02-Free
Atmosphere
A pineapple plant was put in a CO2-free atmosphere for a
night. During this period, only respiratory CO2 was fixed by
the PEP-Case and one can expect that a small quantity of
malate was formed (Fig. 3). The following light period, the
maximum rate of CO2 assimilation (deduced from net O2evolution) during phase III was lower than in the control (i.e.
the phase III of the preceding light period). This suggests that
the intracellular CO2 concentration achieved during malate
decarboxylation was lower than in the control. Concomitantly
to the lower rate of C02-assimilation and the expected decrease in the intracellular CO2 concentration, we observed a
high rate of 02-uptake comparatively to that of the control
phase III (Fig. 3).
Light 02-Uptake Under Continuous Illumination
After subjecting a pineapple plant to continuous light, we
observed rhythmic changes of both net CO2-fixation and O2evolution which persisted at a lower amplitude for about 36
h (Fig. 4). This indicates that malate accumulation and malate
depletion 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 continuous
illumination, the rate of net CO2-fixation became fairly constant and the value of the PQ of about 1 indicates that the
malate content did not fluctuate (Fig. 4). The rate of light O2uptake was then nearly equal to that observed during the
control phase IV at the beginning of the experiment.
Therefore, when less or no malate is available for decarboxylation (after a night period in a C02-free atmosphere or
under continuous illumination, respectively), the rate of light
02-uptake is higher than in a control phase III. Assuming that
in CAM an increase in the intracellular CO2 concentration
occurs subsequent to malate decarboxylation, this supports
the hypothesis of the inhibition of photorespiration during
phase III.
DISCUSSION
C02-Assimilation
An increase in CO2 concentration following malate decarboxylation is the most conceivable explanation of the high
rate of internal CO,-assimilation observed during phase III in
Ananas comosus. For different CAM plants, intracellular
concentrations higher than 2000 ,uL L` have been reported
(10, 26). The following observation suggests that, in pineapple,
the intracellular CO2 concentration during malate decarboxylation is probably lower than this value: based on the law of
gas diffusion, the absence of net CO2 exchange during the
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C02 AND 02 EXCHANGES IN CAM
65
F4.Wc
'4.
Cu
1.5.8
p-
E
E
..
ID
E
E
.I=
E
EZ-:
Lu
T-
-
E
I
D
I
0
Lx)
G
CY
Z:
Lo
I.
J-
0
Z:
IX
I
TIME(h)
Figure 3. Light 02-uptake and net CO2 and 02-exchange in the aerial portion of A. comosus during
C02-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 between
the atmospheric and the intracellular C02 concentration. It is
possible, however, to object that during this period, stomatal
resistance is high and prevents a loss of internal C02. Nevertheless, during the fourth hour of the light period (Fig. 1, h
16, for example) a high rate of net O2-evolution was observed
simultaneously with a net atmospheric C02-uptake. This net
C02-uptake demonstrates that intracellular C02-concentration was lower than the external concentration during this
hour. Thus, considering that the rate of C02-assimilation
(deduced from the net O2-evolution) during the third and the
fourth hour of the light period was not very different, we can
expect that intracellular C02-concentration during these 2 h
was also not very different and was probably a little bit lower
(fourth of the light period) or in the order (third hour of the
light period) of the external concentration. With this assumption, if one considers the great change of the rate of C02assimilation (net 0,-evolution) from phase III to midphase
IV (ratio of about 3), one can expect that internal C02
concentration at the end of the day was probably far below
atmospheric C0.-concentration.
It has been reported that 02 may accumulate in leaves of
CAM-plants to a concentration of up to 40% during phase III
(26). We calculated the internal 0,-concentration in A. comosus 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 water
vapor diffusion (150 s cm-' for pineapple leaves without water
a control
photoperiod and after
a
night in
stress, see Ref. [5]); 1.4 the ratio of the diffusivities of water
vapor and 02 in air. With this value we estimated that the 0,
concentration was no more than 0.5% higher in leaves than
in 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 to
malate storage occurs during late phase IV in A. comosus.
The synthesis of 1 mol of malate by PEP-Case requires the
oxidation of 1 mol of reducing equivalent (for oxaloacetate
reduction into malate). Therefore, C02 fixation via f3-carboxylation and the following storage of malate is accompanied in
the light by theoretically half as much net 02-evolution than
C02 fixation via Rubisco. Moreover, if the required PEP for
light-f,-carboxylation is supplied as in the dark by the glycolysis, this pathway would produce an amount of reducing
power (in the glyceraldehyde-3-P oxidation 1 to 1,3-diphosphoglycerate) stoichiometrically equivalent to that required
for CO2 fixation. Thus, in terms of balance, light C02-fixation
via PEP-Case (leading to malate storage) would be achieved
without reducing equivalent consumption and, consequently,
without net 02-evolution. Therefore, C would probably represent the amount of CO2 stored into malate. PEP-Case has
been 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. With
the above statement, data reported in Figure 1 and determined
with the two other plants studied indicates that at least 50%
of the total C02 fixed during the last hour of the light period
was stored into malate.
To summarize, these data show that in the CAM plant A.
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COTE ET AL.
66
Plant Physiol. Vol. 89, 1989
_
(U
c
_
Qa
Sims
6
_
_
a
1=
.=
E E
E E
0
E
E
4
w
w
cr.
I
L-)
X:
x
w
N
0-
.n
-j,
0
2
0
llJ
w
ID
4j1
0
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 bar
indicates the night period before continuous light. The plant was maintained as in Figure 1.
comosus, steady state conditions of photosynthesis were never
achieved during the light period: malate decarboxylation supply 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 substantially throughout the light period. It is stimulated during phase
III and early phase IV, probably by an increase in intracellular
C02-concentration; a C3-photosynthetic phase using exclusively atmospheric CO2 is limited to 1 to 2 h during the
middle of phase IV; late phase IV is characterized by competition between Rubisco and PEP-Case for CO2 fixation.
Light 02-Uptake
The lowest rate of light 02-uptake was observed in pineapple plants during phase III when stomatal resistance is known
to be the highest in CAM (19, 23, 30). One can suggest that
because of this high resistance to gas diffusion, the low rate
of 02-uptake is caused by the recycling of photosynthetic
evolved-oxygen into photorespiration. The extent of this recycling can be calculated with the equation proposed by
Gerbaud and Andre (15). We determined from the data of
Figure 2 that the 'true' value of 02-uptake during phase III
was underestimated by only about 3% when the value of
stomatal resistance is taken to equal 150 s cm-' (5). Thus, the
underestimation of '802-uptake due to oxygen recycling could
not account for the low 02-uptake rate measured during the
beginning of the day, and an inhibition of photorespiration
due to the previously reported increase in the intracellular
C02-concentration is the most probable explanation for this
low rate of 02-uptake.
Based on leaf area, the maximum rate of 02-uptake observed during phase IV was in the order of 0.3 mmol dm-2
h-' (Fig. 2). Under similar growth conditions, Canvin et al.
(9) and Badger and Canvin (4) reported rates of 02-uptake of
0.3 to 0.6 mmol dm-2 h-' for leaves of several C3 plants. The
smaller concentration of Rubisco per leaf area in CAM plants
relative to C3 plants (31) may account for this difference.
However, determinations of 02-uptake in different species of
CAM-plants are necessary to conclude that 02-uptake per leaf
area 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 gross
02-evolution in plants is equivalent to the flow of electrons
transport in the thylakoids membranes (28), these data indicates that, in pineapple, 60 to 80% of the reducing power
produced (E} was used in 02-consuming processes (U) rather
than for CO2 assimilation (PO) (Fig. 2). For comparison, in
attached leaves or shoots of C3 plants, 45 to 55% of the
reducing-equivalents produced are used in 02-uptake processes (4, 9, 14). This high hourly rate of 02-uptake relative to
photosynthesis during phase IV in A. comosus is consistent
with the high daily U/PO ratio which was observed in several
CAM plants (2, 27, 28) and with the high quantum requirement for photosynthesis determined after malate pool deple-
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C02 AND 02 EXCHANGES IN CAM
tion in Sedum praealtum (25). Possible origins of this high
rate of 02-uptake are discussed in the following.
1. The respiratory activity of nonchlorophyllous tissues of
the plant contributes to the high ratio of 02-uptake/CO2
assimilation. However, the contribution of this respiration to
the total light 02-uptake is probably low: the mean rate of
respiration in the dark (including chlorophyllous tissue) is
only about one-ninth of that of 02-uptake during phase IV
(Fig. 2). This indicates that U is mainly a light-dependent
process.
2. In CAM plants, even if most of the triose compounds
formed from malate decarboxylation are used for gluconeogenesis, it is possible that a portion of this triose is oxidized
in the tricarboxylic acid cycle. Such oxidation is a potential
02-consuming process. However, the following observations
suggest that this oxidation is not involved in the high rate of
02-uptake determined. The maximum rate of 02-uptake was
observed in midphase IV after the malate pool was already
completely depleted (as indicated by the value of PQ equal to
1 during this period). Under continuous illumination, when
no malate decarboxylation occurred (i.e. when the hourly PQ
was close to 1), the rate of 02-uptake was also high relative to
photosynthesis.
3. The high 02-uptake/C02-assimilation ratio during phase
IV could be a consequence of the kinetic properties of RUBPoxygenase activity in A. comosus. This hypothesis is unlikely,
however, because the Rubisco of the CAM plant Kalanchoe
daigremontiana shows nearly identical carboxylase and oxygenase in vitro activity that those of C3 plants show (3).
4. It is conceivable that the high rate of 02-uptake during
the end of the light period is the result of a stimulation of the
RUBP-oxygenase activity due to a low intracellular C02
concentration. Based upon the gas exchanges data, such a low
intracellular C02-concentration during the second part of
phase IV is, as demonstrated above, probable. According to
this hypothesis, Winter (29) has determined a substomatal
CO2 concentration in the range of 170 to 200 ,L L' in
Kalanchoe pinnata during the late phase IV or a prolonged
light period. For comparison, this concentration is usually
near 230 uL L-' for C3 plants (12). Winter suggested that
PEP-Case activity during the light period accounts for this
low intercellular CO2 concentration due to the high affinity
of PEP-Carboxylase for C02. However, a decrease in the
intracellular C02-concentration due to PEP-Case activity cannot be the origin of the high U/PO ratio during phase IV in
A. comosus because we have observed a high rate of 02-uptake
even when no malate synthesis occurred (for example, when
the PQ was equal to 1 in midphase IV or under continuous
illumination). A low internal CO2 concentration could also
be the result of a considerable stomatal resistance which is
known to be high in CAM plants compared to C3 plants,
even during phase IV (19). Mesophyll resistance to gas diffusion would also contribute to increase the C02-gradient between the atmosphere and the cells. The mesophyll resistance
in crassulacean plants has not been reported.
In order to determine whether a low intra cellular C02
concentration which results in alow photorespiration alone
accounts for the high ratio of 02-uptake/C02-assimilation
during phase IV, it is necessary to determine the total resist-
67
ance for CO2 diffusion in the leaf and investigate the influence
of 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 the
agrophysiology laboratory C. Deweirt, J. Massimino, C. Richaud for
support. One of us (F. X. C.) acknowledges a fellowship from le
Ministere de la Recherche and l'Institut de recherche sur les Fruits et
Agrumes IRFA-CIRAD.
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