Abstract

Elevated CO2, in the dark, is sometimes reported to inhibit leaf respiration, with respiration usually measured as CO2 efflux. Oxygen uptake may be a better gauge of respiration because non‐respiratory processes can affect dark CO2 efflux in elevated CO2. Two methods of quantifying O2 uptake indicated that leaf respiration was unaffected by coincident CO2 level in the dark.

Introduction

Whether CO2 partial pressure (pCO2) in the dark directly affects higher‐plant respiration rate (R) is unresolved. Several reports indicate that R in the dark is inhibited by coincident short‐term (min to hours) changes in pCO2, but others show R to be independent of coincident pCO2 (reviewed in Amthor, 1997, 2000; Atkin et al., 2000). Usually, R is measured as CO2 release rate, but elevated pCO2 might increase dark CO2 fixation (catalysed by phosphoenolpyruvate carboxylase) which would reduce CO2 efflux from a plant, perhaps without affecting respiration itself (Amthor, 1997). When dark O2 and CO2 fluxes are measured simultaneously, a decline in respiratory quotient (RQ; CO2 efflux/O2 uptake) with pCO2 increase would be consistent with a stimulation of dark CO2 fixation. Other factors might also be important. Specifically, an increase in pCO2 inside a gas‐exchange measurement system would increase the rate of CO2 loss from within that system through any leaks in the system. This could be interpreted as a reduction in R because the rate of CO2 accumulation within the system (i.e. the measure of R) would be slowed. An increase in pCO2 within a system could also increase adsorption of CO2 onto surfaces inside the system, again resulting in a slowing of CO2 accumulation within the system airspace, at least for a time. Thus, O2 uptake may be a better measure of R when pCO2 is changed within a gas‐exchange measurement system.

To distinguish direct effects of pCO2 on R from effects on other processes involving CO2 exchange (e.g. dark CO2 fixation or CO2 losses through measurement system leaks), two independent methods were used to quantify leaf O2 uptake in response to coincident changes in pCO2 in the dark.

Materials and methods

Method 1: O2 electrode experiments

The leaf‐disc gas‐phase O2 electrode system described earlier (Björkman and Demmig, 1987) was used without the light source to measure dark O2 uptake. The method of Björkman and Demmig was modified by leaving the inlet and outlet valves of the chamber open. The outlet valve was connected to a peristaltic pump, which was connected to an infrared gas analyser, which was connected to a small Ascarite‐filled (A‐M Systems, Carlsborg, WA, USA) tube to absorb CO2, which was connected to the inlet valve. All connections were made with flexible tubing. Air was continuously circulated through the system at about 33 μl s−1. The whole system was airtight, with an internal volume of about 6.6 cm3.

Discs (1000 mm2) were collected during the day from leaves of Rumex crispus L. growing naturally outside the laboratory at the Department of Plant Biology, Carnegie Institution of Washington, Stanford, California, USA, in February, 1993. They were immediately transferred to the darkened O2 electrode chamber.

To begin an experiment, the chamber contained a fresh leaf disc positioned on a stainless steel screen above a water‐saturated filter disc. System air flow was diverted around the Ascarite. After the pCO2 in the system increased (from disc respiration) to about 60 Pa, O2 uptake was measured for 15 min, during which pCO2 increased to about 100–160 Pa. The system was then opened, the filter disc was replaced with one soaked in NaOH (to absorb CO2 in the chamber), the chamber was resealed, and air flow was directed through the Ascarite. This configuration maintained pCO2 at less than 10 Pa within the chamber. Oxygen uptake rate was then measured for 15 min. The system was then reopened, the NaOH‐soaked filter disc was replaced with a water‐saturated filter disc, the chamber was resealed, flow was diverted around the Ascarite, and O2 uptake was again measured for 15 min during which pCO2 increased from about 60 to 100–160 Pa. The chamber temperature was 25 °C.

Oxygen uptake rate (i.e. R) was calculated for the last 10 min of each 15 min measurement based on total system volume and rate of [O2] decline. The first and third measurements were averaged to obtain a single value for R in high pCO2 for each leaf disc. Nine leaf discs were used. Respiration rates in low versus high pCO2 were evaluated with a paired comparison t‐test (PROC MEANS, SAS Institute Inc., Cary, NC, USA).

Method 2: differential O2 sensor experiments

The O2/CO2 gas exchange measurement system described previously (Willms et al., 1997, 1999) was used to measure leaf gas exchange in the dark with differing background pCO2. In November and December 1995, Rumex crispus and Glycine max (L.) Merr. were grown in pots in plant growth chambers (Model E8, Conviron, Winnipeg, Manitoba, Canada) at Queen's University, Kingston, Ontario, Canada. Photoperiod was set to start at 17.00 h and end at 07.00 h so that laboratory experiments could be conducted during the normal chamber night, but solar daytime, to enhance investigator effectiveness.

Plants were moved to the laboratory early in the chamber dark period and kept shaded. Intact, expanded leaves were sealed in an airtight cuvette to give 0.3–0.4 g dry mass. Experiments were begun by maintaining a pCO2 of about 1 Pa in the air entering the cuvette. Flow through the cuvette was 470 μl s−1, giving differentials in O2 and CO2 across the chamber of 15–30 Pa. After several hours, O2 and CO2 exchange rates were quantified according to Willms et al. (Willms et al., 1997). The pCO2 of air entering the cuvette was then increased to 76–86 Pa (depending on experiment) for 1–2 h. Near the end of that period, O2 and CO2 exchange rates were again quantified. The pCO2 of air entering the cuvette was then reduced to 1 Pa for another 1–2 h period (including gas exchange measurements), which was followed by another 1–2 h period at 76–86 Pa CO2 (with additional gas exchange measurements). Each experiment lasted 6–8 h. Cuvette temperature was 30 °C.

Effects of pCO2 on R (both O2 uptake and CO2 release) were separated from effects of time in the dark using the following equation (Amthor et al., 1992):

1
where C was pCO2inside the cuvette (Pa), t was time in the dark (h), and a (same units as R), b (Pa−1), and c (h−1) were fitted parameters (PROC NLIN, SAS Institute Inc.). The value of a was a hypothetical estimate of R at the start of the dark period if pCO2 was zero, and varied from leaf to leaf. The focus of this study was on b, which represented the effect of pCO2 on R. The value of b is negative if CO2 inhibits R, positive if CO2 stimulates R, and zero if R is independent of pCO2. The parameter c represented the change in R with time in the dark.

In additional experiments, dark CO2 efflux from leaves of chamber‐grown Rumex crispus and Ricinus communis L. was measured with a portable photosynthesis system (model LI‐6400, Li‐Cor Inc., Lincoln, NE, USA) in the laboratory, using the standard 2×3 cm leaf cuvette. Preliminary measurements indicated that with the low air flow rates and small changes in pCO2 within the system's cuvette that are characteristic of leaf R measurements, leaks in the cuvette (caused in part by incomplete contact between cuvette gaskets and leaf surfaces) compromised measurements of effects of changes in pCO2 on R. To increase the integrity of measurements reported herein, leaf gasket interfaces were sealed with Qubitac (Qubit Systems Inc., Kingston, Ontario, Canada) along the outside of the cuvette, and the cuvette gasket sections not in contact with leaves were sealed on the outside of the cuvette with TFE adhesive tape (S‐15, Saunders Corp., Sunnyvale, CA, USA).

Results and discussion

For method 1, elevated pCO2 in the dark did not inhibit O2 uptake rate. Rumex crispus leaf disc O2 uptake rate in low pCO2 (i.e. <10 Pa) ranged from 0.99 to 1.73 (mean=1.20) μmol O2 m−2 s−1 (n=9). With high pCO2 (i.e. 60–160 Pa), R ranged from 0.99 to 1.76 (mean=1.35) μmol O2 m−2 s−1 (n=9). On average, therefore, high pCO2increased R by 0.147 μmol O2 m−2 s−1 relative to low pCO2, but this effect was statistically insignificant (P>t=0.126, two‐tailed test).

For method 2, dark O2 uptake rate was independent of pCO2 (Fig. 1). In three experiments with Rumex crispus and two with Glycine max, estimates of b (Equation 1) with respect to O2 uptake were statistically indistinguishable from zero. With respect to CO2 efflux, b was less than zero for two Rumex crispus leaves, but indistinguishable from zero in the other three experiments. Although CO2 release was apparently slowed by elevated pCO2 in two of five experiments, the size of the effect (value of b) was more than an order of magnitude smaller than in previous Rumex crispus experiments (Amthor et al., 1992). Thus, a 30 Pa increase in pCO2 slowed CO2 efflux only 2–3% in these experiments, compared with 25–30% in the earlier experiments. The small effect of pCO2 on net CO2 efflux in two of the five cases in this study was similar in magnitude to that recently measured in leaves of several tree species (Amthor, 2000).

The RQ was independent of pCO2 in three of the five experiments, and modestly reduced in the other two experiments (due to minor reductions in CO2 efflux). Note that the first leaf used on 6 December (Fig. 1) had an RQ of about 0.82, indicating oxidation of proteins and/or fats in addition to carbohydrates. The other leaves had RQs of 1.03–1.05, presumably reflecting mainly carbohydrate oxidation.

Using the LI‐6400 portable photosynthesis system, dark CO2 efflux from Rumex crispus and Ricinus communis L. leaves during 1995 at Queen's University was independent of coincident pCO2 in the range 10–80 Pa (not shown). Taken together, the 1995 experiments using the O2/CO2 exchange measurement system and the LI‐6400 indicated no effect of pCO2 on respiratory O2 exchange and no more than a minor effect on net CO2 release (i.e. no change or only a small decline in RQ).

A few measurements of dark O2 uptake as a function of pCO2 were previously reported. In the CAM species Bryophyllum daigremontianum Berger, night‐time O2 uptake slowed about 20% as pCO2 inside the leaf increased about 40 Pa (Kaplan et al., 1977), but time in the dark was positively related to pCO2, so time in the dark may have caused part of the reduction in O2 uptake. (Only C3 species were used in the present experiments.) Using a system similar to method 1 herein, Reuveni et al. reported 15–25% slower O2 uptake by Lemna gibba L. fronds, and Orobanche aegyptiaca Pers and Lactuca sativa L. seedlings, when pCO2 was increased from about 0 to 100 Pa (Reuveni et al., 1993). They reported a statistically insignificant 1–5% decline in RQ when pCO2 was increased from 0 to 100 Pa, consistent with no more than a weak effect of pCO2 on dark CO2 fixation. In later Lemna gibba experiments, O2 uptake slowed 25% and CO2 efflux slowed 35% when pCO2 was increased from 0 to 100 Pa (Reuveni et al., 1995), indicating a significant decline in R and a modest increase in dark CO2 fixation with elevated pCO2. In contrast, the present experiments found no effect of pCO2 in the dark on coincident O2 uptake. The differences between the present results and other studies may be related to species differences, tissue differences (e.g. leaves versus whole seedlings), or differences in gas exchange measurement systems or approaches.

Dark CO2 fixation in hydroponically grown Lycopersicon esculentum (L.) Mill. roots was stimulated when CO2 (and

$${\mathrm{HCO}_{3}^{{-}}}$$
) in the root solution was increased from the equivalent of 0 to ≥200 Pa in air for plants assimilating
$${\mathrm{NO}_{3}^{{-}}}$$
or
$${\mathrm{NH}_{4}^{{+}}}$$
(van der Westhuizen and Cramer, 1998). The differences between those results for roots and the present results for leaves might be due to inherent differences in dark CO2 fixation in leaves and roots, lack of dark N assimilation in this study's leaves (i.e. leaf N assimilation was perhaps limited to periods of photosynthesis), or the larger pCO2 changes used by van der Westhuizen and Cramer. Theoretical stoichiometries of CO2 exchange associated with dark N assimilation were outlined in Pate and Layzell, and indicated significant variation in RQ associated with different sources and products of N assimilation (Pate and Layzell, 1990). How changes in pCO2 might alter N assimilation and the associated RQ is unclear.

In conclusion, leaf O2 uptake rate in the dark was independent of coincident short‐term changes in pCO2 over the range about 10–100 Pa, indicating that R was unaffected by pCO2. This contrasts with several earlier reports (reviewed in Amthor, 1997), but is generally consistent with some recent reports of a lack of (or only a minor) effect of pCO2 in the dark on coincident CO2 efflux (Amthor, 2000; Tjoelker et al., 2001). When increased pCO2 in the dark did slow CO2 efflux in earlier studies, that result may have been due to small leaks in the gas exchange measurement systems and/or adsorption of CO2 on to surfaces in those systems. Results of this study indicate that rising atmospheric pCO2 will not significantly affect the carbon balance of the terrestrial biosphere through direct effects on plant R. This is important because global annual terrestrial plant respiration exceeds the releases of CO2 from fossil fuel burning by about an order of magnitude (Amthor, 1995). Nonetheless, it is expected that elevated daytime pCO2 will affect R indirectly through changes in plant growth rate, size, and tissue composition (Gifford and Morison, 1993; Amthor, 1997).

Fig. 1.

Leaf dark O2 uptake and CO2 efflux rates for three Rumex crispus plants, two measured 6 December 1995 (top panel) and one measured 10 December 1995 (bottom panel). The pCO2 of air in the cuvette was alternated between about 20–25 and 85–95 Pa.

Fig. 1.

Leaf dark O2 uptake and CO2 efflux rates for three Rumex crispus plants, two measured 6 December 1995 (top panel) and one measured 10 December 1995 (bottom panel). The pCO2 of air in the cuvette was alternated between about 20–25 and 85–95 Pa.

4

Present address: Medical School, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

5

Present address and to whom correspondence should be sent: US Department of Energy, SC‐74, 19901 Germantown Road, Germantown, Maryland 20874‐1290, USA. E‐mail: Amthor@aya.Yale.edu

We thank Olle Björkman for use of his laboratory and equipment. This work was supported by funding to JSA from the US Department of Energy's Office of Biological and Environmental Research, by US National Science Foundation grant IBN‐9514061 to GWK, and by a research grant to DBL from the Natural Sciences and Engineering Research Council (Canada).

References

Amthor JS.
1995
. Terrestrial higher‐plant response to increasing atmospheric [CO2] in relation to the global carbon cycle.
Global Change Biology

1
,
243
–274.
Amthor JS.
1997
. Plant respiratory responses to elevated carbon dioxide partial pressure. In: Allen LH, Kirkham MB, Olszyk DM, Whitman CE, eds.
Advances in carbon dioxide effects research
. Madison, WI: American Society of Agronomy,
35
–77.
Amthor JS.
2000
. Direct effect of elevated CO2 on nocturnal in situ leaf respiration in nine temperate deciduous tree species is small.
Tree Physiology

20
,
139
–144.
Amthor JS, Koch GW, Bloom AJ.
1992
. CO2 inhibits respiration in leaves of Rumex crispus L.
Plant Physiology

98
,
757
–760.
Atkin OK, Evans JR, Ball MC, Lambers H, Pons TL.
2000
. Leaf respiration of snow gum in the light and dark. Interactions between temperature and irradiance.
Plant Physiology

122
,
915
–923.
Björkman O, Demmig B.
1987
. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins.
Planta

170
,
489
–504.
Gifford RM, Morison JIL.
1993
. Crop responses to the global increase in atmospheric carbon dioxide concentration. In: Buxton DR, Shibles R, Forsberg RA, Blad BL, Asay KH, Paulsen GM, Wilson RF, eds.
International crop science
, I. Madison, WI: Crop Science Society of America,
325
–331.
Kaplan A, Gale J, Poljakoff‐Mayber A.
1977
. Effect of oxygen and carbon dioxide concentrations on gross dark CO2 fixation and dark respiration in Bryophyllum daigremontianum.
Australian Journal of Plant Physiology

4
,
745
–752.
Pate JS, Layzell DB.
1990
. Energetics and biological costs of nitrogen assimilation. In: Miflin BJ, ed.
The biochemistry of plants
, Vol. 16. Intermediary nitrogen metabolism. San Diego: Academic Press,
1
–42.
Reuveni J, Gale J, Mayer AM.
1993
. Reduction of respiration by high ambient CO2 and the resulting error in measurements of respiration made with O2 electrodes.
Annals of Botany

72
,
129
–131.
Reuveni J, Mayer AM, Gale J.
1995
. High ambient carbon dioxide does not affect respiration by suppressing the alternative, cyanide‐resistant pathway.
Annals of Botany

76
,
291
–295.
Tjoelker MG, Oleksyn J, Lee TD, Reich PB.
2001
. Direct inhibition of leaf dark respiration by elevated carbon dioxide is minor in 12 grassland species.
New Phytologist

150
,
419
–424.
Van der Westhuizen MM, Cramer MD.
1998
. The influence of elevated rhizosphere dissolved inorganic carbon concentrations on respiratory O2 and CO2 flux in tomato roots.
Journal of Experimental Botany

49
,
1977
–1985.
Willms JR, Dowling AN, Doug ZM, Hunt S, Shelp BJ, Layzell DB.
1997
. The simultaneous measurement of low rates of CO2 and O2 exchange in biological systems.
Analytical Biochemistry

254
,
272
–282.
Willms JR, Salon C, Layzell DB.
1999
. Evidence for light‐stimulated fatty acid synthesis in soybean fruit.
Plant Physiology

120
,
1117
–1127.