Plant Cell Physiol. 48(1): 198–203 (2007)
doi:10.1093/pcp/pcl056, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Short Communication
Calibration of Simultaneous Measurements of Photosynthetic Carbon Dioxide
Uptake and Oxygen Evolution in Leaves
Vello Oja, Hillar Eichelmann and Agu Laisk *
Tartu Ülikooli Molekulaar- ja Rakubioloogia Instituut, Riia tn. 23, Tartu, 51010, Estonia
(Radmer and Kok 1976, Canvin et al. 1980, Kiirats 1985,
Thomas et al. 1987, Kirschbaum and Pearcy 1988, Avelange
and Rebeille 1991, Avelange et al. 1991, Laisk and Oja
1998). For example, electron flow to alternative acceptors
other than CO2—such as nitrite and oxaloacetate—may be
detected as an excess of O2 evolution over CO2 uptake
(Noctor and Foyer 2000).
Zirconium cell O2 analyzers are sensitive devices for the
measurements of the photosynthetic O2 evolution in a
flowthrough system simultaneously with the CO2 uptake.
These analyzers are especially well suited for the measurements at very low background O2 concentrations, inhibiting
all types of O2 reduction, ending up with the closed water–
water cycle (Asada 2006). Recently we reported that in a
sunflower leaf, O2 evolution exceeded CO2 uptake by 7%
when the light intensity was varied, but only 2% excess O2
evolution was observed when the CO2 concentration was
varied (Laisk et al. 2006). Since the differences between CO2
and O2 fluxes are small, both gas analyzers need precise
calibration. We report a new method for the calibration of
CO2 and O2 concentration scales for simultaneous measurements of CO2 uptake and O2 evolution during photosynthesis. The method is based on the changes of
O2 concentration in a flow of air when CO2 or O2
is added through one and the same capillary. The ratio of
O2 evolution to CO2 uptake during photosynthesis,
measured with the recalibrated instruments in mature
sunflower leaves, was very close to unity.
The gas system used is equipped with three different
sized capillaries for mixing CO2 into the flow of the carrier
gas (Laisk and Oja 1998). In the experiments reported
below, CO2 and O2 were added through the largest capillary
and later the calibrated CO2 and O2 scales were transferred
from the large capillary to the two smaller capillaries. Fig. 1
shows the original computer recording of the O2 analyzer
when CO2 inflow through the capillary was interrupted
by rapidly zeroing the pressure difference of 200 mm H2O
on the capillary (in the figures, we show the gas off transient
where the addition of CO2 or O2 was stopped, because it
was faster than the gas on transient). In order to increase
the signal/noise ratio, the CO2 and O2 on/off procedures
The stoichiometric ratio of O2 evolution to CO2 uptake
during photosynthesis reveals information about reductive
metabolism, including the reduction of alternative electron
acceptors, such as nitrite and oxaloacetate. Recently we
reported that in simultaneous measurements of CO2 uptake
and O2 evolution in a sunflower leaf, O2 evolution changed by
7% more than CO2 uptake when light intensity was varied.
Since the O2/CO2 exchange ratio is 1, small differences are
important. Thus, these gas exchange measurements need
precise calibration. In this work, we describe a new
calibration procedure for such simultaneous measurements,
based on the changes of O2 concentration caused by the
addition of pure CO2 or O2 into a flow of dry air (20.95% O2)
through one and the same capillary. The relative decrease in
O2 concentration during the addition of CO2 and the relative
increase in O2 concentration during the addition of O2
allowed us to calibrate the CO2 and O2 scales of the
measurement system with an error (relative standard
deviation, RSD) of 51%. Measurements on a sunflower leaf
resulted in an O2/CO2 ratio between 1.0 and 1.03
under different CO2 concentrations and light intensities,
in the presence of an ambient O2 concentration of
20–50 mmol mol1. This shows that the percentage use of
reductive power from photochemistry in synthesis of inorganic
or organic matter other than CO2 assimilation in the C3 cycle
is very low in mature leaves and, correspondingly,
the reduction of alternative acceptors is a weak source of
coupled ATP synthesis.
Keywords: Alternative electron transport — Carbon dioxide
— Oxygen — Photosynthesis.
Abbreviations: MDH, malate dehydrogenase; PFD, photon
flux density; PGA, phosphoglycerate.
During photosynthesis, CO2 is taken up and fixed in
the primary products of photosynthesis, while O2 is evolved
from water. Simultaneously, CO2 is evolved and O2 is taken
up during photorespiration and mitochondrial respiration
in the light. The ratio of the two gaseous fluxes carries
information about electron acceptors during photosynthesis
*Corresponding author: E-mail, [email protected]; Fax, þ372-742-0286.
198
O2/CO2 ratio in photosynthesis
were repeated, averaging the measured traces (Fig. 2).
The final results of the measurements are given in Table 1.
The addition of pure CO2 into the flow of air caused a
decrease in O2 concentration of 0.04248% points (concentrations given in v/v); the addition of pure O2 through the
same capillary caused an increase in O2 concentration of
0.11806% points (here% points are used, as in economics,
in the sense of the absolute increase/decrease in the
atmospheric O2 concentration of 20.95%). Since O2
formed only 20.95% of the carrier gas, its concentration
decrease of 0.042% points was equivalent to the total
concentration of added CO2 of 0.042/0.2095 ¼ 0.200%. This
is the calibration value for the capillary for the given
pressure difference of 200 mm H2O. However, during the
measurements of photosynthesis, different pressures are
applied with the aim of obtaining different concentrations.
The pressure–flow relationship of the capillary is non-linear
due to the ‘end-correction’ caused by the acceleration of
molecules at the narrow entrance of the capillary and due to
the changes in gas density dependent on the pressure.
In order to consider these factors, we calculated the flow
rates of CO2 and O2 through the capillary during the above
measurements, inserted these into the equation for the gas
flow through the capillary and calculated the efficient
diameter of the capillary for CO2 and O2.
The flow rates of CO2 and O2 through the capillary
were calculated from the formula
v x ¼ vg
Cg Cm
Cm Cx
ð1Þ
where vx is the flow rate of the added component (CO2 or
O2, mmol s1), vg is the flow rate of the carrier gas
(513 mmol s1), Cg is the O2 concentration in the carrier
gas (dry air, 0.2095), Cx is the O2 concentration in the added
gas (zero in CO2 and 0.9984 in O2) and Cm is the measured
O2 concentration. The actual measurement results were
as follows:
vO2 ¼ 513
3000
199
0:11806
¼ 0:769 mmol s1 ð2Þ
ð20:95 þ 0:11806Þ 99:84
O2 analyzer output (bits)
and
2500
2000
1500
−5
vCO2 ¼ 513
−CO2
0
5
10
15
Table 1
p21 p22 8lv2
2 v2
¼ 2 4 þ 1:39 2 24
2p2
r
r
ð4Þ
where is the gas viscosity, l is the length and r is the radius
of the capillary, v is volume flow rate, the pressures p1 and
p2 are upstream and downstream of the capillary, respectively, and 2 is the gas density downstream. Since the
Measurements of changes of O2 concentration in the flow of dry air
Added gas
O2
CO2
ð3Þ
As expected, the flow rate of O2 is slower than that of
CO2 because the lighter gas molecule causes greater flow
viscosity. These molar flow rates were converted into the
volume flow rates considering the atmospheric pressure and
temperature (the gas mixer was held at 508C), and inserted
into the general equation for gas flow through the capillary
(Laisk and Oja 1998)
Time (s)
Fig. 1 The original computer recording of the response of the O2
analyzer to the removal of CO2 from the flow of dry air. At time ¼ 0,
pressure (200 mm H2O) on the capillary through which CO2 was
added into the air flow was removed. The response of the O2
analyzer is delayed due to the movement of the gas through the
tubing. The data plotting interval was 0.2 s; the calibration readings
were taken averaging data points shown by open symbols.
0:04248
¼ 1:042 mmol s1
20:95 0:04248
Average deflection (A/D units)
2422.3 1.9
871.6 1.1
n
14
42
Relative error
3
0.78 10
1.3 103
Change in O2 concentration (% points)
þ0.11806
0.04248
Either O2 or CO2 was added into the flow of air through one and the same capillary under a pressure difference of 200 mm H2O. The
addition of O2 caused an increase, and the addition of CO2 a decrease in the O2 concentration of the air, indicated in analog–digital
converter units (A/D units) and recalculated in percentage points.
O2/CO2 ratio in photosynthesis
length of the capillary was known, the radius r of the
capillary was calculated.
The radius r in Equation 4 is not the geometric radius,
but one calculated assuming the theoretical profile of
velocities, approaching zero velocity at the wall. In practice,
gas molecules keep moving in the flow direction even right
at the wall. Thus, calculated from Equation 4, r is a little
longer than the physical radius and is expected to be
different for CO2 and O2, though both were forced to flow
through one and the same capillary. Correspondingly, the
diameter of the capillary for O2 is 2rO2 ¼ 117.44 mm 2.1 cm
length and for CO2 it is 2rCO2 ¼ 117.28 mm 2.1 cm length.
These dimensions were then used in Equation 4 to calculate
the actual CO2 and O2 flow rates at different pre-set
pressure differences p1 p2. With this, the whole scale of
pressures on the capillary was calibrated in terms of CO2
and O2 concentrations in the gas flow. The obtained
concentration scale was transferred to the two smaller
capillaries by comparing the flow rates at different pressure
differences and calculating the effective radius for CO2 and
O2 of the smaller capillaries. Such involvement of the radius
of the capillary in the calculation algorithm may seem an
unnecessary complication, but it is a mathematically correct
way to consider the non-linear components of the gas flow
over the whole range of applied pressures.
Having calibrated the CO2 scales of our gas exchange
measurement system, we compared them with a factorycalibrated brand new LI 7000 gas analyzer (LiCor, Lincoln,
NE, USA; S/N: IRG4-0570). The difference in the
calibration of scales remained 51% over the ranges of low
0.15
Change in O2 (% points)
−O2
0.1
0.05
20.95%
and high CO2 concentrations (Fig. 3). This coincidence
proves the reliability of such a calibration method for the
CO2 scale, as well as for the O2 scale.
The recalibrated gas system was used for the simultaneous measurement of the photosynthetic CO2 uptake and
O2 evolution in a mature sunflower leaf, taken from a plant
growing outside. The light response curve was measured at
a CO2 concentration of 300 mmol mol1, decreasing the photon
flux density (PFD) stepwise, and waiting for the stabilization
of photosynthesis at each PFD. The CO2 response curve was
measured at a PFD of 750 mmol quanta m2 s1, decreasing the
CO2 concentration stepwise from 300 to 0 mmol mol1. Since
the O2 concentration was 20–50 mmol mol1 during the
measurements, leaves were exposed for 5 min under 2% O2
at each CO2 concentration in order to reverse the consequences
of anaerobiosis.
Fig. 4 demonstrates an example of transients in the
CO2 uptake and O2 evolution recorded after the CO2
concentration was decreased from 200 to 50 mmol mol1.
There was an oscillatory type transient in the O2 evolution,
2500
A
y = 1.0089x − 2.9638
2000
1500
1000
Fast-Est CO2 scale (µmol mol−1)
200
500
0
0
150
500
1000
1500
2000
2500
B
y = 1.0014x − 0.8592
100
O2
0
50
−0.05
−CO2
0
2
4
6
8
Time (s)
Fig. 2 Comparative measurements of CO2 and O2 flow through
the capillary. As in the single measurement shown in Fig. 1,
pressure on the capillary was removed at time ¼ 0, but either CO2
(open data points) or O2 (filled data points) was added through the
capillary. The traces were averaged over 42 (with CO2) or 14
repeated transients, and the reference line without additions was
also measured.
0
0
50
100
150
LiCor CO2 scale (µmol mol−1)
Fig. 3 Comparison of the calibrated CO2 scale with the factory
calibration of a LI-7000 gas analyzer. The CO2 concentration was
pre-set by changing the pressure on the wide-scale (A) or narrowscale (B) capillary of the Fast-Est gas system, and the corresponding
reading of the LI-7000 was recorded.
O2/CO2 ratio in photosynthesis
40
35
400
O2 evolution (µmol m−2s−1)
O2 and CO2 signals (A/D units)
600
201
O2 evolution
200
CO2 uptake
30
Light curve
y = 0.9969x + 2.2576
R2 = 0.9999
25
20
15
CO2 curve
10
y = 1.0321x + 1.0865
R2 = 0.9999
5
0
0
50
100
150
Time (s)
Fig. 4 Transients in the CO2 uptake and O2 evolution of a
sunflower leaf following a decrease of the CO2 concentration from
200 to 50 mmol mol1 at time ¼ 0. The dotted line indicates the
initial O2 evolution rate at 200 mmol CO2 mol1.
damped after 100 s, but not in the CO2 uptake.
The stabilization time was critical in order to obtain the
true steady-state values of both fluxes, but the length of the
exposure under anaerobiosis imposed another constraint.
It was somewhat difficult to find the optimum stabilization
time during the measurements, so it could be that very small
differences in fluxes were still caused by the non-steadystate situation. In the sunflower leaf, the CO2 uptake and
O2 evolution rates thus measured were linearly correlated
(R2 ¼ 0.9999) during the CO2 and light responses, but the
slope and offset values were slightly different (Fig. 5).
During the light response curve, changes in CO2 and O2
fluxes were equal (slope 0.997), but a slight excess of O2
evolution was observed during the CO2 response (slope
1.032). The difference seemed to be caused by different CO2
evolution from respiration, which extrapolated to a slower
rate at the end of the measurement of the CO2 response
curve in the light than at the end of the measurement of the
light response curve in the dark.
Recently we reported simultaneous measurements of
CO2 uptake and O2 evolution in sunflower leaves, showing
that O2 evolution exceeded the CO2 uptake by 7% during
the light response, but only by 2% during the CO2 response
curves (Laisk et al. 2006). From these measurements, we
concluded that the non-Mehler type alternative electron
flow—mainly nitrite and oxaloacetate reduction—formed
no more than 7% of the phosphoglycerate (PGA) reduction
rate. These measurements were based on the calibration of
the CO2/O2 scales by forcing CO2 and air to flow through
one and the same capillary, but the ratio of the flow rates
was calculated on the basis of handbook information about
the viscosity of both gases. Since the CO2 and O2 exchange
rates are very close during photosynthesis, in this work we
0
−5
0
5
10
15
20
CO2 uptake (µmol
25
30
35
40
m−2s−1)
Fig. 5 Comparison of O2 evolution and CO2 uptake in a
sunflower leaf. Leaf photosynthesis was adjusted to steady state
at a CO2 concentration of 300 mmol mol1 and an O2 concentration of 20 mmol mol1, light intensity of 1,500 mmol quanta
m2 s1 and leaf temperature of 22.58C. Then O2 concentration
was decreased to 20 mmol mol1 and the light response curve was
measured by decreasing the light intensity stepwise to zero. Then
the initial state was restored and the CO2 response was measured
by decreasing the CO2 concentration stepwise to zero. The leaf
was exposed at 20 mmol O2 mol1 between changes in CO2
concentration and light intensity. The recorded O2 evolution rate
was plotted against the CO2 uptake rate during the light response
curve (filled data points) and during the CO2 response curve
(open data points).
calibrated both with a high precision in order to determine
the difference correctly.
We used a new calibration method of the CO2 and O2
scales, both based on the measurements of O2 concentration. CO2 and O2 were forced to flow through one and the
same capillary under the same pressure difference into
a known flow of CO2-free dry air containing 20.95% O2.
The increase in O2 concentration due to the addition of
O2 and the decrease in O2 concentration due to the addition
of CO2 were measured. The results were recalculated into
CO2 and O2 flow rates through the capillary, creating the
complete calibration function for CO2 and O2 concentrations over the whole range of pressures applied on the
capillary. The CO2 scales calibrated in this way exactly
coincided with the factory calibration of a LI-7000 CO2
analyzer, proving the correctness of the calibration procedure. Since only the relative change of O2 concentration
was important (the absolute % points were presented in
Fig. 2 only for orientation), we emphasize that metrologically both scales are based on the known concentration
of O2 in the atmosphere, not on the calibration of the
O2 analyzer.
202
O2/CO2 ratio in photosynthesis
Measurements of the CO2 uptake and O2 evolution
rates in a sunflower leaf during light and CO2 responses,
carried out with the recalibrated system, showed even
smaller differences between the two fluxes than before
(Laisk et al. 2006). During the light response curve, the two
fluxes changed in an exactly equal manner, while during the
CO2 response, O2 evolution changed 3% faster than CO2
uptake. Since O2 uptake was greatly suppressed, but CO2
evolution, e.g. anaerobic glycolysis, continued from respiration even at the very low ambient O2 concentration of about
20–50 mmol mol1, there was an offset in favor of CO2
evolution, greater in the dark at the end of the light
response measurement and smaller in the light at the end of
the CO2 response measurement. Due to this, the small
difference in the O2/CO2 exchange ratio between the light
and CO2 responses could be induced by the gradual change
of the anaerobic CO2 evolution during the experiment
(the CO2 response was measured after the light response).
Another critical compromise was the optimum exposure time under the very low O2 concentration that had
to be sufficient to damp the oscillation in the O2 evolution
(Fig. 4). Such oscillation is a typical process of readjustment
of the ATP/NADPH production ratio to changing environmental conditions, earlier recorded in the CO2 assimilation
at saturating CO2 concentrations (Laisk et al. 1991, Walker
1992). At a low CO2 concentration, like in the experiment
shown in Fig. 4, oscillations in CO2 uptake were damped by
accumulated ribulose 1,5-bisphosphate, the acceptor of
CO2, but were still pronounced in O2 evolution. Due to
these kinetic phenomena, some small difference in the
O2/CO2 ratio could be apparent, induced by the incomplete
establishment of the electron/proton transport ratio.
These recalibrated measurements leave very little room
for any alternative reductions not coupled with changes in
the CO2 flux during photosynthesis, indicating that 3% or
even less of the photosynthetic linear electron flow was
directed to nitrite plus oxaloacetate reduction in the
sunflower leaf. Such a slow rate of N reduction is not
unexpected, because the process is known to be dependent
on the species and is retarded in mature leaves (Noctor and
Foyer 1998), but the situation with oxaloacetate reduction
seems to be more complicated.
Since chloroplast malate dehydrogenase (MDH) has
been shown to be active in leaves (Backhausen et al. 1998,
Backhausen et al. 2000), we cannot conclude that it did not
function in our sunflower leaf. Rather, these results show
that although the enzyme was activated, the actual reaction
rate of NADP-MDH was slow, being strictly limited by the
availability of oxaloacetate (Fridlyand et al. 1998). This
substrate is produced in mitochondria as a result of malate
oxidation and it serves as the acceptor of the 2C acetyl
moiety from cofactor A for further oxidation in the Krebs
cycle. Assuming that oxaloacetate was competitively
exported from mitochondria, the rate of the acetyl moieties
entering into the Krebs cycle could be suppressed in the
light, as well as the subsequent CO2 evolution from these
(Brooks and Farquhar 1985). In order to explain the
measured precise equality of O2/CO2 fluxes, we suggest that
the alternative electron flow to reduce oxaloacetate was still
active in chloroplasts of leaves, but the light-induced
reduction of oxaloacetate inhibited the respiratory
CO2 evolution to about the same extent as these same
electrons could have increased the CO2 uptake, if they were
used for the reduction of PGA. In other words, under the
conditions of our experiment, changes in the net CO2
uptake were independent of the electron acceptor, whether
it was PGA or oxaloacetate.
This model accounts for the measured equality of
changes in net CO2 uptake and O2 evolution, leaving the
definite role to oxaloacetate as the alternative electron
acceptor. However, in the framework of this model, the rate
of the ‘malate valve’ can be no faster than the respiratory
CO2 evolution; more precisely, it is as fast as the rate at
which respiratory CO2 evolution is suppressed in the light.
Another process that could mask oxaloacetate reduction could be the reoxidation of the produced malate by
photosynthetically generated oxygen, a kind of photosynthetic pseudocycle involving mitochondrial oxidases
(remember that external oxygen was close to zero in our
experiments). Experiments under way in our laboratory are
showing that although the affinity of mitochondrial
oxidases for oxygen is relatively high and their K0.5(O2) is
a fraction of micromolar values (Millar et al. 1994), still
their catalytic efficiency (their conductance for O2) is much
smaller than the diffusional conductance of O2 out of the
mesophyll cells. As a result of this, only a few per cent of the
photosynthetically generated O2 may be reassimilated in
mitochondria. To the same extent, the activity of the malate
valve could be masked by the reoxidation of malate in our
experiments.
In conclusion, thanks to the high sensitivity of the
zirconium cell O2 analyzer and to the new calibration
method, the precision of the simultaneous measurements of
O2 evolution and CO2 uptake during photosynthesis will
enable future studies on alternative electron sinks. These
preliminary experiments have already indicated tight
cooperation between chloroplasts and mitochondria
during phtosynthesis in leaves, where mitochondria neutralize the small disturbances in ATP/NADPH fluxes
occurring in chloroplasts (Noctor and Foyer 2000).
Materials and Methods
Sunflower (Helianthus annuus L.) plants were grown outside
on an experimental lot near the laboratory in Tartu. Full-grown
mature leaves were cut and, with the petiole in water, were taken to
the laboratory in August 2006.
O2/CO2 ratio in photosynthesis
The gas system GS-3 (Fast-Est, Tartu, Estonia) used was
based on capillary gas mixers (Laisk and Oja 1998). Orifice-type
resistances operating under a constant pressure difference determined the flow rate of the carrier gas through the gas mixer, while
capillary-type resistances operating under controllable pressure
difference determined the rate of CO2 (or O2) flow addition to the
carrier gas. The flow rate of the carrier gas (CO2-free dry air)
through the mixer was 513 mmol s1. Though normally the carrier
gas is mixed together from pressure-bottled N2 and O2, in these
experiments the system was fed by external air, passed through a
column of potassium hydroxide absorbing CO2 and water vapor.
O2 concentration was measured with an S-3A zirconium cell
analyzer (Ametek, Pittsburgh, PA, USA), equipped with an offset
device to zero the output signal at 21% O2 in the 0–100% scale.
The small changes of the output signal created by the addition of
CO2 or O2 to the air flow were amplified 1,000 times and recorded
by computer using an ADIO-1600 card (ICS Advent, San Diego,
CA, USA).
Acknowledgments
This work was supported by grants 6607 and 6611 from the
Estonian Science Foundation.
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