Photosynthesis and Photorespiration in Whole ... - Plant Physiology

Plant Physiol. (1979) 64, 735-738
0032-0889/79/64/0735/04/$00.50/0
Photosynthesis and Photorespiration in Whole Plants of Wheat
Received for publication February 27, 1979 and in revised form May 30, 1979
ALAIN GERBAUD AND MARCEL ANDRE
Departement de Biologie, Service de Radioagronomie, C.E.N. Cadarache Bofte Postale 1, No. 13115 Saint-
Paul-Lez-Durance, France
ABSTRACT
Wheat was cultivated in a small phytotronic chamber. 1802 was used to
measure the 02 uptake by the plant, which was recorded simultaneously
with the 02 evolution, net CO2 uptake, and transpiration. At normal
abtospheric CO2 concentration, photorespiration, measured as 02 uptake,
was as important as the net photosynthesis. The level of true 02 evolution
was independent of CO2 concentration and stayed nearly equal to the sum
of net CO2 photosynthesis and 02 uptake. We conclude that at a given
light intensity, 02 and CO2 compete for the reducing power produced at
constant rate by the light reactions of photosynthesis.
Photorespiration is commonly measured using carbon isotopes.
Such methods give the CO2 efflux from the plant, but are inevitably
biased by the recycling of CO2 (21). The measurement of
photorespiratory 02 uptake does not have the same drawback
(22). Although the technique of 1802 has been used with algae (14,
19), it has seldom been applied to higher plants. Mulchi et aL (15),
Dimon (10), and Berry et aL (6) have shown an important uptake
of 1802 in various plants, but limited their scope to the CO2
compensation point. The method was also applied to C4 plants in
the presence of CO2 (1, 24), showing a respiration identical in light
and darkness at normal CO2 concentration and the increase of
light respiration at lower concentrations. The only measurements
of the 02 uptake of C3 plants in conditions of photosynthesis were
made by Volk and Jackson (23) and Ozbun et al. (16) With bean
leaves the latter found an 02 uptake which was only 9%o of the
photosynthesis; but the significance of this result is lessened by
the weakness of the illumination used (300-1,500 ft-c). Furthermore,
they did not find an increase of the rate of 02 uptake with
higher light intensities, whereas it is currently thought that the rate
of photorespiration increases with higher light intensities (8, 12,
25)
Ẇe found it useful to investigate the 02 exchange of a C3
(wheat) in conditions approaching those in the field: the plant was
intact, light intensity and photosynthetic rate were high; CO2 was
regulated first at normal, then at high and low levels.
MATERIALS AND METHODS
PLANT AND GROWTH CHAMBER
The experiment was conducted on a whole plant of wheat
(Triticum aestivum L., var. Champlein), aged 42 to 52 days. The
plant was grown from a few days after sowing a 7.7-liter growth
chamber, a reduced design of the C23A system previously described
(2). Day (14 h)/nght (10 h) temperatures were 20.5 ± 0.6
C/18.5 + 0.6 C. The visible light measured at plant midheight
(175 w m-2) was not saturating. The root compartment was
separated from the aerial part by an air-tight putty joint. The roots
dipped in a beaker containing 2.3 liters of Arnon-Hoagland No.
2 solution with iron as FeSO4 (3), which was changed every day
before lighting and was aerated by bubbling with C02-free air. A
single seed grew to a tuft of up to 25 tillers with about 20 leaves.
As the plant did not undergo the winter frosts, it did not flower
and the formation of tillers went on for 70 days and more. During
the 10 days of the experiment the photosynthesis increased by 2%
per day up to 0.5 liter CO2 per day and the relative growth rate
calculated from the cumulative CO2 uptake was 5% per day.
METHODS
Photosynthesis and dark respiration were determined by quantitative
monitoring of the CO2 concentration in the cell through
injection of pulses of CO2 or CO2 trapping and transpiration by
the uptake of water by the roots (2, 3).
To regulate 02 pressure in the chamber at 20%Yo, one should
dilute every volume of 02 produced by the plant by four volumes
of N2. Practically we dilute the injected CO2 in about four (exactly
k = 3.44) volumes of N2. In fact, during the night, 02 concentration
diminishes as a consequence of dark respiration; during the day,
it goes up again toward an equilibrium concentration. These
oscillations do not exceed 1%.
Gas concentrations were measured every 20 min by a quadripolar
mass spectrometer Riber QMM 17; CO2 concentration was
continuously measured by an URAS II A infrared analyzer. All
raw data were recorded and processed in real time by a Telemecanique
T 1600 computer which gave them back as files of hourly
and daily means and curves. The computer also controlled the
injections of CO2 into the chamber (or CO2 trapping at night) to
regulate its level at any present level.
02 uptake was measured according to the following principle:
a small quantity of nearly pure 1802 is introduced into the air-tight
chamber. The concentration of 1'802 diminishes afterwards due to
two causes: (a) the plant takes up 1802 as well as 1602 in the
processes of photorespiration and respiration. (b) the injections of
CO2 and N2 result in a nearly equal volume of the gas in the
chamber being swept out. This second phenomenon is measured
by the progressive dilution of a reference gas. We chose Neon
(isotope 20), as it is neutral to the plant and is present only in
trace amounts in the atmosphere. We use the following symbols:
V: volume of the chamber, dv: volume swept out of the chamber
in time dt; u: rate of 02 uptake; U: daily mean rate of 02 uptake
= (l/T) fT u.dt; o, T: initial and final time of day; pc: rate of net
photosynthesis (CO2); P,: daily mean rate of net photosynthesis
(CO2) = (I/T) fT pc dt; PO daily mean rate of net photosynthesis
(02)-
The volume dv of gas going out of the cell in time dt contains
[Nel. dv of neon gas, so the decrease of Ne is
V.d[Ne] = - [Nel . dv or d[Ne] / [Ne] = - dv/V (1)
The decrease of 1802 is due to both displacement, and plant
uptake u.dt:
735
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736
[180
V. d[ 180 = [180 . dv - 2 u. dt
2 2 [16Q is
I 02] + I 0 ]
dl[ 180 -dv u.dt/V
[180] V 1[1602] + [180 ]
By subtracting equation I from equation 3:
GERBAUD AND ANDRE Plant Physiol. Vol. 64, 1979
The comparison of the P, derived from this formula with the value
(2) already known is a test of the validity of our measures.
PHYSIOLOGICAL VALUE OF THE MEASUREMENTS
The measure of the true rate of 02 uptake can be biased by two
(3) causes. (a) If the 02 from photosynthesis is taken up as well as the
external 02 in the process of photorespiration, then our measurement
will be less than the true rate. This sort of recycling seems to
be neligible, due to the high relative pressure of the outer 02 (22).
d[ 80 1 d[Ne] - u.dt/V
1 2] [Ne] 160 ] + [ 180
During the course of the day, the total 02 concentration [02] stays
in the range 21 to 22%, so that we may consider it as a constant in
the integration of equation 4:
(4)
z
0
z
8
Trd[Ne] d[ 1802 2
1 T
=Fu.dt
INe 118 O
Jo [Ne] [ °2]- [02].V 0
Hence:
[02] *V I[0210 [Ne]1
U = in TJ
[[118021 [NeIT
(5)
(6)
The net 02 evolution P0 was calculated from the increase of 02
concentration in the chamber. In the unit time the plant assimilates
a volume Pc of CO2, (k+ 1) Pc of the mixture (CO2 + kN2) is
injected into the chamber and the plant produced P0 of 02;
therefore the following volume must be expelled from the chamber:
Lv
=
(k+1)Pc
+
P0 - Pc
or
Av
=
k.P c
+P
(7)
containing an amount 11602] Av of 1602.
The plant produces only 1602, and in our case 1802 is about 50
times less concentrated in the cell than 1602, so that we shall
equate here the uptake of 1602 with the total 02 uptake U. Hence
the increase of 1602 in the chamber is:
V A[ 16 ] 1
T
0 2
1 [ + k p [16
T
1160
L
2
'r 64
(8) .f
-E
z
(9) o
w
A
HOURS
C02
0 x
D
a
a
k.
- 2C
From P. we calculate the photosynthetic ratio po/PC. The gross 02
evolution E was calculated from the net evolution plus the uptake:
E = PO + U.
Test of Photosynthesis. There is another way of measuring
photosynthesis than counting the pulses of injected CO2: the
decrease of neon concentration is related to volume dv, (eq. 1),
which is proportional to the photosynthesis, because AV = k
Pc+P0 in equation 7 can be replaced within a good approximation
by (k+ 1) Pc, that is, set in the differential form: dv = (k+ 1) Pcdt,
hence:
d[Ne] / [Ne] = - (k+1) pC.dt / V (10)
1 T dN 1 [me]0
= 1T kV1 d{Ne] = 1 V n (11)
P =
_ln ---
c T k+1 o [Ne] T k+1 [HeiT
0
-
y 2( to -10DO
Y.
R
I-
- T
n n n
of I I 1- 1 a I I I I I I 0
Q, I - - - - - "
2 4 6 8 10 12 1x 16 18 20 22 24
HOURS
FIG. 1. Recorded data during a typical day of a 46-day-old plant of
wheat (day/night temperature 20.3 C/18.4 C, PAR irradiance 175 w m-2,
02= 21%, CO2 = 335 ,l/1). The two parts show in parallel the time course
of the gas exchanges (A), and the isotopic concentrations (B). PR: net
photosynthesis (C02); R: night respiration; T: transpiration. The decrease
of 1802 traces the 02 traces the 02 uptake by the plant, using Ne as an
internal standard. The slight variations of 1602 correspond to the photosynthesis
or respiration. Initial concentrations were [18021 = 2970,l/1, [Ne]
= 3,160 ul/l; isotopes were added before the beginning of the light periods
when their concentrations went under 0.5%.
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(b) If the absorbed 02 is recycled through the plant into the
atmosphere, our measure will also be underestimated.
Dimon (10) has shown in short term experiments (1 h) that
plants do not discriminate between isotopes of 02 and the photosynthetic
02 is not enriched in 1802. We have checked that no 1802
is released from the plant even after several days of 1802 consumption.
As the evolved 02 comes from water, which has only
one atom of 02, any evolved 180 would be predominantly in
18016O molecules. As we detect no enrichment of the evolved 02
in 18016O, we consider that there is no recycling of the photorespiratory
02-
From these two points we conclude that the disappearance of
1802 from the atmosphere is a good measure of the true flux of 02
in photorespiration.
RESULTS AND DISCUSSION
Normal CO2 Level. Figure 1 shows the data obtained with a
plant of wheat during the course of a typical day at normal CO2
level (average 335 t,ll). Net photosynthesis P, calculated from the
number of pulses of injected CO2 is 43 ml/h. As a verification, net
photosynthesis, as seen by neon (equation 11) is 45 ml/h. From
the curves of Ne and 1802 we calculate U = 43 ml/h, that is, as
much as Pc. From the increase of 1602 concentration (from 20.6 to
21.5%), we calculate the net 02 evolution PO = 46 ml/h and the
gross 02 evolution E = 88 ml/h. The photosynthetic ratio P./PC
is 1.07. Other experiments in the same conditions give U/P, =
1.02 ± 0.05 (four measures) and P./P, = 1.05 ± 0.02. We also
found the same P./P, at other CO2 concentrations and the same
U/Pc at half-light intensity (experiment not described here).
PHOTORESPIRATION IN WHEAT
Plant Physiol. Vol. 64, 1979 737
Our estimation of the importance of photorespiration is much
larger than the most frequently found values: many authors
estimate the CO2 evolution in the light from one-sixth to one-half
of the apparent photosynthesis (8, 12, 25). These differences arise
primarily from the difficulty in measuring the CO2 evolution (12).
It is known that all measures using '3C or "C, even corrected for
the biochemical recycling of the assimilated carbon, are still
underestimated because photorespired CO2 is recycled into photosynthesis,
to an extent depending on the different diffusion
resistances in the pathway of CO2 (21). This recycling could even
be complete, preventing any release of CO2 (4) at normal CO2
level. On the other hand, the present results are in agreement with
the observed stimulating effect of low 02 pressures on plant
growth: + 90%o for dry weight in Mimulus cardinalis (7), + 103%
in soybean (18), + 90 to 106% in Phaseolus vulgaris (17). Parkinson
et al. (17) observed that such an effect corresponds to an increase
of the photosynthetic efficiency of up to 100/o at 5% 02 concentration.
The order of magnitude of the flux of 02 raises the problem of
its pathway(s). We know that atmospheric 1802 labels glycolate
and further metabolites of the glycolate pathway, as well as the
evolved CO2 (5, 6, 11), but it is not established if all of the
absorbed 02 goes into the glycolate pathway, which should then
be as active as the Calvin cycle (13). Otherwise, other reactions,
such as the pure Mehler reaction (pseudocycic electron flow),
have to occur concurrently (10).
Response to Various CO2 Levels. For 10 days we subjected the
plant to low (200 A1/1), normal (300-400 1il/l), and high (680-
12,000 p1/l) CO2 levels; each level was maintained for I or 2 days
-1 I I I I I
41 42 43 44 45 46 47 48 49 50 51 52
DAYS AFTER SOWING
FIG. 2. Gas exchange of a plant of wheat at various CO2 levels. U: photorespiration (02 uptake in light); E: gross 02 evolution: Pc + U: photosynthesis
+ photorespiration. Other symbols and conditions, except CO2, are as in Figure 1.
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738
GERBAUD AND ANDRE Plant Physiol. Vol. 64, 1979
-c 75
A a E 1/ A A 4
z
0
P 50
_
IC
A
.-I
0
w
0
w 25
a-
/~~~~
/~~~~~~~~~
n
0 200 400 600 800 12000
CO2 (p.l-1)
FIG. 3. Gas exchange of a plant of wheat as a function of CO2 concentration. Data were comparable by correcting them for the size of the plant,
supposing a uniform growth throughout the experiment. Abbreviations as in Figures I and 2.
(Fig. 2). 02 concentration was always normal. Diurnal transpiration
rates were nearly constant, indicating that stomata can be
considered open throughout the light periods.
As expected, photosynthetic rates follow the concentration of
CO2 and respiration varies as Pc but to a smaller extent: it
diminishes only 20%o when P is halved.
Photorespiration varies oppositely to photosynthesis, so that
their sum P, + U depends only on the age and size of the plant.
At high concentrations of CO2, where it is admitted that photorespiration
is inhibited, there remains some 02 uptake, about 10%
of Pc; we suppose this is due to the continuation of night respiration
in the light, which appears also in maize (1).
Pc + U corresponds to the utilization of reducing power in the
leaves of the plant; its production is measured by the 02 evolution
E. We found E always slightly superior to Pc + U showing that
some reducing power is also consumed in the roots (nitrate reduction).
If we abstract the influence of plant growth, E appears to be
independent of CO2 (Fig. 3) and P, and U follow symmetrical
curves. The mirror effect between Pc and U was observed by
Radmer and Kok (19) with algae during the induction phase of
photosynthesis at constant CO2 level and, with a lag time, after
CO2 exhaustion by photosynthesis (20). The main result of the
present work is the constancy of the gross evolution E under
various conditions of CO2, in spite of wide variations of apparent
photosynthesis and in long steady-state experiments, which shows
that it is not a transient effect but a permanent mechanism. The
permanency of this balance suggests that the electron transport
chain turns over at a constant rate, controlled by the light intensity
and independent of the nature of the final electron acceptor.
When CO2 concentrations are low the excess of reducing power
must be trapped by 02 reduction. If the mechanism is blocked for
example by the simultaneous absence of 02 and CO2, inhibition
or damage occurs (9) because unwanted compounds have to be
reduced instead of 02 or CO2.
Acknowledgments-We are grateful to Mr. A. Daguenet and Mrs. J. Massimino for their very
valuable contribution to the experiments.
LITERATURE CITED
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