Plant Physiol. (1992) 98, 757-760

Received for publication August 13, 1991 Accepted October 21, 1991

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Communication

CO2 Inhibits Respiration in Leaves of Rumex crispus L.' Jeffrey S. Amthor2*, George W. Koch, and Arnold J. Bloom Department of Agronomy and Range Science (J.S.A.) and Department of Vegetable Crops (G. W.K., A.J.B.), University of California, Davis, California 95616 ABSTRACT

was maintained between 0.1 and 0.3 mm by additions of KNO3 following daily measurements of the solution with a N03--selective electrode. The containers were submerged in refrigerated water baths located in a glasshouse in Davis. Solution temperature was 20.0 ± 0. 1C. Plants received sunlight supplemented with radiation from multi-vapor lamps maintaining a PPFD of 500 to 1800 ,umol m-2 s-' at shoot height during the 14 h photoperiod. Glasshouse air temperature varied between 25 and 35C. The ventilation system of the glasshouse provided CO2 partial pressures near the outside ambient values (about 35 Pa, or 350 ,uL L-l).

Curly dock (Rumex crispus L.) was grown from seed in a glasshouse at an ambient CO2 partial pressure of about 35 pascals. Apparent respiration rate (CO2 efflux in the dark) of expanded leaves was then measured at ambient CO2 partial pressure of 5 to 95 pascals. Calculated intercellular CO2 partial pressure was proportional to ambient CO2 partial pressure in these short-term experiments. The CO2 level strongly affected apparent respiration rate: a doubling of the partial pressure of CO2 typically inhibited respiration by 25 to 30%, whereas a decrease in CO2 elicited a corresponding increase in respiration. These responses were readily reversible. A flexible, sensitive regulatory interaction between CO2 (a byproduct of respiration) and some component(s) of heterotrophic metabolism is indicated.

Respiration Measurements

Plants of approximately 4 weeks of age were removed from the growth system at the end of the photoperiod, transferred to a flask containing 1 L of aerated nutrient solution, and kept in the dark overnight in the laboratory. The following morning, the youngest fully expanded leaf was enclosed in a temperature-controlled gas exchange cuvette similar to that described by Field et al. (7). This attached leaf and the remainder of the plant were kept in the light (PPFD = 800 Omol m-2 s-') at 35 Pa CO2 for 3 h, after which the light was turned off and the leaf cuvette and plant were covered with a light-tight box. Leaf temperatures were maintained at either 15.0 ± 0.2 or 25.0 ± 0.2°C during each experiment. The gas exchange system described by Bloom et al. (3) was used to regulate and monitor CO2 and water vapor exchange. The IRGA (Horiba VIA-SOOR) was calibrated (4) over the range of 5 to 95 Pa CO2. The CO2 partial pressure of the air entering the leaf cuvette was regulated by mixing CO2-free air with air containing 1% C02 using mass flow controllers (Tylan FC-260). Gas exchange calculations followed von Caemmerer and Farquhar (12). Leaf intercellular CO2 partial pressure was given by: Cj = Ca + P RA/gc, (1)

Several studies have shown that changes in ambient CO2 concentration during the day can have effects on subsequent nighttime respiration (reviewed in ref. 1). In addition, nighttime CO2 levels can have an immediate impact on the rate of dark CO2 efflux (e.g. 5, 6), but such data are limited and the mechanisms of action are unknown. Here, we quantified the direct, short-term effects of Ca3 and Ci on Rd in expanded curly dock leaves. MATERIALS AND METHODS Plant Culture Seeds of curly dock (Rumex crispus L.) were collected from wild populations near Davis, CA, and germinated on moist filter paper. After 4 to 5 d, seedlings were suspended by foam plugs in the lids of light-tight plastic containers holding 4 L of aerated nutrient solution (pH 6.0 ± 0.2) with a composition of: 1 mM CaSO4, 0.67 mM K2HPO4, 0.33 mM KH2PO4, 1 mM MgSO4, 0.67 mM K2SO4, 0.05 mM Fe-EDTA, and micronutrients according to Johnson et al. (9). Nitrate concentration

where P is cuvette air pressure (Pa), RA is the measured leaf CO2 flux area density (i.e. apparent respiration rate on an area basis; mol CO2 efflux m-2 s-'), and gc is leaf CO2 conductance (mol CO2 m-2 s-') estimated from leaf H20 conductance. (The measured conductance of H20 included boundary layer, stomatal, and cuticular components. Although the ratios of CO2 to water vapor conductances through stomata and the boundary layer are known [12], the relative

'Supported in part by National Science Foundation grants BSR8821255 and DCB-89-16637 and U.S. Department of Agriculture grant 88-37264-3857. 2Present address: The Woods Hole Research Center, P.O. Box 296, Woods Hole, MA 02543. 3 Abbreviations: Ca, ambient partial pressure of C02; Ci, intercellular partial pressure of CO2; Rd, apparent respiration rate (CO2 efflux rate in the dark per unit dry mass).

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importance of transport through the cuticle is not known, so the derivation of gc from measured H20 conductance is inexact. The CO2 levels at the sites of respiration are also affected by transport properties of the cell wall and plasmalemma.) Apparent respiration rate was later expressed on a leaf dry mass basis (mol CO2 g9' s-') by multiplying RA by measured leaf area mass density (m2 g-') determined after oven drying the tissue enclosed in the cuvette.

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RESULTS AND DISCUSSION Altemating C. Experiments In the R. crispus leaves, & always decreased when CO2 was increased from 35 to 65 Pa and then increased when CO2 was returned to 35 Pa and vice versa (Fig. 1). That is, the inhibition or stimulation of CO2 efflux was readily reversed by restoring Ca to its previous level. This was true at both 15 and 25C and was still distinct after more than 20 h in the dark, although respiration rate was then slower (Fig. 1). Similar results were obtained for Ludwigia uruguayensis (data not shown). In addition to the effect of CO2 level, Rd declined with time in the dark (Fig. 1). For every R. crispus leaf used in the alternating Ca experiments, the CO2 effiux rate at a Ca of 35 Pa was compared with that of the following measurement at a C. of 65 Pa for each repeated cycle of CO2 level. The mean ratio of Rd at 65 Pa CO2 to Rd at 35 Pa CO2 was 0.76 at 15C and 0.80 at 25°C. These ratios did not differ according to a t test. These experimental results show a striking relationship between C. and Rd in darkened R. crispus leaves. As such, they are counter to general notions that small changes in Ca do not influence Rd, but are nonetheless consistent with the results of the few earlier studies of the effects of CO2 partial pressures of 100 Pa and less on Rd. For example, Gale (8) reported that an increase in C. from 32 to 84 Pa inhibited Xanthium strumarium leaf Rd about 36%. Bunce (5) observed that Rd was slowed when C. was switched from 35 to 70 Pa for whole plants and leaves of three species. El Kohen et al. (6) found that Rd was about 50% lower in Castanea sativa seedlings measured at 70 versus 35 Pa; this was so for plants

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CO2 Treatments Individual leaves were exposed to one of three CO2 regimens during the dark period, which lasted up to 25 h. (a) Periods of a C. of 35 Pa were alternated with periods of 65 Pa for at least four complete cycles. (b) Beginning at 35 Pa, the C. was increased by steps of 15 or 20 Pa to 80 or 95 Pa, returned to 35 Pa, decreased to 20 and 5 Pa, and returned again to 35 Pa. (c) A series similar to (b), but the C. was decreased from 35 to 20 and 5 Pa, returned to 35 Pa, increased to 80 or 95 Pa, and returned again to 35 Pa. Leaves were exposed to each CO2 level for at least 30 min, although 15 min was sufficient to attain steady-state values of leaf CO2 efflux after a change in Ca. Between measurements in regimen (a), C. was held at 35 Pa. Regimen (a) is referred to as the alternating Ca experiments and regimens (b) and (c) are together referred to as the multi-level C. experiments.

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Figure 1. Time course of apparent respiration rate in young, fully expanded R. crispus L. leaves during the four experiments in which Ca was alternated between 35 and 65 Pa several times. Each group of symbols, i.e. squares (0, U), circles (0, 0), triangles (A, A), and inverted triangles (V, V), represent an experiment conducted with a leaf from a different plant. The open symbols show measurements made when Ca was 35 Pa and the filled symbols show measurements made when Ca was 65 Pa. The two lower sets of data (0, 0, A, A) are from leaves at 150C, whereas the two upper sets of data (01, *, V, V) are from leaves at 250C.

raised at 35 and 70 Pa CO2. Our study is unique in that the reversibility of nighttime CO2 effects on Rd was clearly shown. Multi-Level C. Experiments

As was the case in the alternating C. experiments, Rd declined with time in the dark irrespective of CO2 level during the multi-level C. experiments. Estimated leaf conductance averaged about 0.04 mol CO2 m-2 s-' in the dark. In some cases, conductance varied inversely with CO2 level. Estimated leaf intercellular CO2 partial pressure was on average about 2.9 Pa greater than the ambient level. Measured CO2 efflux rates were fitted (Proc NLIN, SAS Institute, Cary, NC) to a double exponential equation to distinguish the influences of Ci from time in the dark on Rd:

Rd= a ei e-Ct (2) where t is the time elapsed since darkening the plant (h), a is the hypothetical value of Rd at Ci = 0 and t = 0 in the absence of photorespiratory CO2 release, b is a fitted parameter (Pa-'), and c is a rate constant (h-'). The term ew,i scales a for the inhibition of Rd by Ci, whereas eC1 describes the influence of time in the dark. The sensitivity of Rd to Ci (i.e. the parameter b) was similar at leaf temperatures of 15 and 25°C (Table I). The mean value of b across the seven multi-level C. experiments was 0.0087

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C02 INHIBITS RESPIRATION IN LEAVES OF RUMEX CRISPUS L.

Pa'. Thus, on average, Rd declined about 9% for each 10 Pa increase in C1 (Fig. 2). This is the first quantification of a relationship between C1 and Rd over a range of CO2 levels in

Table 1. Parameters Describing Respiration Rate as a Function of the Intercellular CO2 Partial Pressure and Time in the Dark Parameter values (estimate ± asymptotic SE; Proc NLIN [SAS Institute, Cary, NC]) of the equation describing apparent respiration rate as a function of C and time in the dark (t) for fully expanded R. crispus L. leaves in each of the seven multi-level Ca experiments: Rd = a e-bcie. (See also Fig. 2.)

Equation Parameter

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nmol C02 9g' s' 37 ± 1.5 27 ± 4.0 37 ± 2.5 28 ± 1.8 85 ± 13 68 ± 3.8 120 ± 3.0

Pa-' 0.0085 ± 0.0013 0.0089 ± 0.0023 0.0095 ± 0.0013 0.0071 ± 0.0008 0.011 ± 0.0036 0.0086 ± 0.0006 0.0070 ± 0.0006

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0.042 ± 0.010 0.048 ± 0.014 0.067 ± 0.014 0.062 ± 0.007 0.027 ± 0.015 0.034 ± 0.006 0.071 ± 0.007

0.97 0.77 0.95 0.95 0.78 0.98 0.99

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Figure 2. Apparent respiration rate of fully expanded R. crispus L. experiments for which C8 was varied between 5 and 95 Pa. Each symbol type represents a single experiment conducted with a leaf from a different plant. The open symbols (EJ, 0, A, V) show data from leaves at 150C, whereas the filled symbols (HO. A) show data from leaves at 250C. The effect

a non-CAM species. We conjecture that the uppermost data of Figure 2 (filled squares) are from a leaf in which secondary cell wall and organelle growth were still active, resulting in rapid respiration, even though the leaf was no longer increasing in area. Mechanisms underlying the inhibition of Rd by near ambient CO2 levels in the dark are unknown, although several possibilities have been considered (1). For example, dark CO2 fixation could increase as a result of increased CO2 level. Without commensurate change in actual respiration (i.e. glycolysis, the oxidative pentose phosphate network, the TCA cycle, mitochondrial electron transport, and oxidative phosphorylation), this would result in a decrease in apparent respiration. Gale's (8) results, however, suggest that CO2 inhibits actual respiration or processes supported by respiration. Other mechanisms by which Rd might be altered include pH changes (but this is not likely at Ca between 5 and 95 Pa) and direct effects of CO2 on membranes and enzymes (10). At a fundamental level, it is not known whether CO2 at near ambient levels affects processes consuming respiratory products and thus alters respiration through respiratory control mechanisms (2), or whether CO2 affects respiratory reactions directly, or both. We cannot distinguish between these possibilities from our data, nor from the data of previous reports. A combination of responses is possible, and perhaps even

likely. It is noteworthy that our data on leaves showed a marked response of Rd to CO2 between 0 and 100 Pa, because other organs and tissues appear to be insensitive to CO2 over the same partial pressure range; for example, in wheat apices, which are normally tightly enclosed by several leaf sheaths and probably subjected to high C02, Rd was not affected by an increase in Ca from about 30 to 500 Pa (11). Similarly, other organs such as roots, rhizomes, and fruits may be insensitive to CO2 at and below concentrations normally experienced by those organs (1). The present data and related reports intimate that some type of end product inhibition of respiration may exist with respect to CO2. Such a state of affairs has important implications for the control of metabolism. Moreover, effects of CO2 on Rd are likely to affect the productivity of vegetation and, hence, the global carbon budget, as atmospheric CO2 continues to increase. A fuller characterization of the effects of nighttime CO2 on apparent respiration rate is warranted.

leaves held in the dark in the seven

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of time in the dark has been "removed" from the data. The lines summarize the effect of Ci on Rd independent of time in the dark and are given by the equation Rd = a e-'c, where a and b are from Table I.

LITERATURE CITED 1. Amthor JS (1991) Respiration in a future, higher-CO2 world. Plant Cell Environ 14: 13-20 2. Beevers H (1974) Conceptual developments in metabolic control, 1924-1974. Plant Physiol 54: 437-442 3. Bloom AJ, Caldwell RM, Finazzo J, Warner RL, Weissbart J (1989) Oxygen and carbon dioxide fluxes from barley shoots depend on nitrate assimilation. Plant Physiol 91: 352-356 4. Bloom AJ, Mooney HA, Bjorkman 0, Berry J (1980) Materials and methods for carbon dioxide and water exchange analysis. Plant Cell Environ 3: 371-376 5. Bunce JA (1990) Short- and long-term inhibition of respiratory

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carbon dioxide efflux by elevated carbon dioxide. Ann Bot 65: 637-642 6. El Kohen A, Pontailler J-Y, Mousseau M (1991) Effet d'un doublement du CO2 atmospherique sur la respiration a l'obscurite des parties aeriennes de jeunes chataigniers (Castanea sativa Mill.). C R Acad Sci Ser III 312: 477-481 7. Field C, Berry JA, Mooney HA (1982) A portable system for measuring carbon dioxide and water vapour exchanges of leaves. Plant Cell Environ 5: 179-186 8. Gale J (1982) Evidence for essential maintenance respiration of leaves of Xanthium strumarium at high temperature. J Exp Bot 33: 471-476

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9. Johnson CM, Stout PR, Broyer TC, Carlton AB (1957) Comparative chlorine requirements of different plant species. Plant Soil 8: 337-353 10. Mitz MA (1979) CO2 biodynamics: a new concept of cellular control. J Theor Biol 80: 537-551 11. Pheloung P, Barlow EWR (1981) Respiration and carbohydrate accumulation in the water-stressed wheat apex. J Exp Bot 32: 921-931 12. von Caemmerer S, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376-387