SCOPE 56 - Global Change: Effects on Coniferous Forests and Grasslands

7.1 INTRODUCTION

Rising atmospheric CO₂ concentrations are expected to cause important responses in photosynthesis, photorespiration and stomatal conductance, and because of this, these processes have been the subject of biochemical, physiological, agronomic and ecological studies (Allen 1990; Bazzaz 1990; Hogan et al . 1991, Morison 1990; Mott 1990). The effects of rising CO₂ and climate change on respiration may have a significant impact on whole plant and ecosystem carbon budgets, but little attention has been given to the possible effects of rising atmospheric CO₂ on this process although there has been considerable emphasis placed on the potential increase of global respiration resulting from increased temperature (review in Ryan 1991). Recent experimental evidence suggests that elevated CO₂ can reduce respiration. It appears that this effect is sufficient to increase plant growth over and above what might be expected strictly from the stimulation of photosynthesis (Reuveni and Gale 1985), and it may be large enough to counteract the expected stimulation of increased temperature of 2-3 °C on respiration.

Respiration is essential for growth and maintenance of tissues. The solar energy conserved during photosynthesis and stored as chemical energy in organic molecules is released in a regulated manner for the production of adenosine triphosphate (ATP), the universal currency of biological energy transactions. Phytomass and carbohydrate content generally increase in C₃ plants grown in elevated CO₂ levels as a result of increased photosynthetic activity (Allen 1990; Mott 1990), and when attention was first directed to the global importance of vegetation in the mediation of atmospheric CO₂, it was thought that the stimulation of photosynthesis and growth should increase dark respiration rates due to increased carbohydrate concentration. In some species, particularly young, growing tissues, increased dark respiration has been shown to accompany the stimulation of photosynthesis by elevated CO₂ (Gifford et al. 1985; Poorter et al. 1988). But not all studies in which photosynthesis has been increased have produced the expected stimulation of respiration by increased carbohydrates. The effects of photosynthesis on subsequent respiration rates were less when photosynthetic rates were increased by high CO₂ than when the rate was similarly increased by light (Ludwig et al. 1975) and many studies clearly indicate that long-term adaptation of plants to elevated atmospheric CO₂ results in significantly lower dark respiration rated (Bunce 1994). An important point is the recent rediscovery that plant respiration responds reversibly to rapid changes in atmospheric CO₂ concentration (Amthor et al. 1992).

The effects of rising atmospheric CO₂ and climate change on respiration have been reviewed by Amthor (1991), Ryan (1991), and Farrar and Williams (1991), Poorter et al. (1992) and Bunce (1994). These reviews emphasize the potential for direct effects of CO₂ on different aspects of respiration including maintenance respiration (Ryan 1991; Bunce 1994), enzymatic rates (Amthor 1991), carbon partitioning (Farrar and Williams 1991), and growth (Amthor 1991; Bunce 1994; Poorter et at. 1992). All of them indicate the considerable potential for rising CO₂ and climate change to alter the respiration of vegetation.

In many species, a direct, immediate and reversible inhibition has been demonstrated (Amthor et al. 1992; Bunce 1994). In these cases, the rate of CO₂ emission from the plant tissue is decreased within minutes after CO₂ concentration was increased and is reversed upon returning the CO₂ concentration to a lower level. The inhibition of dark respiration resulting from increasing the concentration of CO₂ in the ambient air of a plant has been reported by Kidd (1916), Nilovskaya and Razoryonova (1968), Kaplan et al. (1977), Silsbury and Stephens (1984), Reuveni and Gale (1985), Gifford et al. (1985), Bunce (1990), El Kohen et al. (1991), and Amthor et al. (1992). In all of these reports, dark respiration was reduced when the concentration of CO₂ was increased. Long-term effects are determined by comparing the rate of CO₂ emission (or O₂ assimilation) from tissues at a common CO₂ background. This class of effects represents the acclimation of tissues to higher CO₂ and involves effects on tissue composition, especially carbohydrate accumulation and reduced protein content which is reflected in the reduction of nitrogen concentration, as well as the effects of growth, response to stress, and other factors that are a consequence of the higher efficiency of key plant processes often found in high CO₂ concentration.

There are conflicting views expressed in the literature regarding the effects of elevated CO₂ on respiration (Poorter et al. 1992; Thomas et al. 1993). These differences represent unresolved conceptual problems as well as different methods of measurement and different bases for expressing the results of measurements. One view suggests that all measurements should be expressed per unit of leaf area, which would make it very difficult to compare results of different tissue types, such as stems and roots, with foliage. In addition, this approach places the emphasis on the photosynthesizing organ and the consequences of photosynthesis. We suggest that the basis of comparison should be the specific rate of respiration, expressed as the loss of carbon or the assimilation of oxygen per gram carbon (g g^-1 ). In addition, it is important that the background concentration of CO₂ during growth and during measurement be made clear.

7.2 RESPONSE OF DARK RESPIRATION TO ATMOSPHERIC CO₂ CONCENTRATION

The inhibitory effect of elevated atmospheric CO₂ on dark CO₂ evolution and O₂ uptake rates has been reported for different tissues and organs: whole plant (Reuveni and Gale 1985; Bunce 1990), seedlings (Reuveni and Gale 1985), shoot (El Kohen et al. 1991), fruit (Kerbel et al. 1988), roots (Reuveni and Gale 1985; Nobel and Palta 1989), leaves (Amthor et al. 1992; Bunce 1990; Thomas and Griffin 1994), leaf epiderman strips (Shaish et al . 1989), tissue culture cells (Kerbel et al . 1990), plant mitochondria (Spalding et al . 1980; Palet et al. 1992), microorganisms(Koizumi et al. 1991) and even animal tissues (Palet et al. 1991). Respiration of mature leaves of Rumex crispus was reversibly inhibited by CO₂ in the range of 5-950 ppm (Amthor et al. 1992). The magnitude of the inhibition is quite variable (Poorter et al. 1992; Reuveni et al. 1993), but averages when the increase in CO₂ is from 350 to 700 µul l^-1.  

At the most fundamental level, respiration can be considered to have a dual nature: it is the source of metabolic intermediates used in the synthesis of cellular constituents as well as the source of ATP and reduced nucleotide (particularly pyridine nucleotide), the driving force of these syntheses. A variety of substrate (carbohydrates, lipids, and proteins) can be respired through pathways occurring in several cell compartments (e.g. cytosol, mitochondria, chloroplast) with the products converging on the tricarboxylic acid cycle and hence on mitochondrial oxidative phosphorylation (see Dennis and Turpin 1990). This duality of respiration, as a source of substrate as well as a source of the energy to drive the reactions, is probably reflected in the regulation of the rate by respiratory substrate and by adenosine diphosphate (ADP) availability, although it remains a goal of plant physiologists to fully establish the mechanism for this control. One of the most confounding facts about the effects of elevated CO₂ on respiration is that elevated CO₂ appears to cause both inhibition and stimulation of respiration (Amthor 1991; Azcón-Bieto 1992; Poorter et al. 1992). Stimulation of respiration by immediate exposure to elevated CO₂ has been shown in lemon fruit (Young et al. 1962), potato tubers (PerezTrejo et al. 1981), tissue cultures (Palet et al. 1991), and plant mitochondria (Gonzàlez-Meler 1995; Gonzàlez-Meler 1996). We propose that the effects of different CO₂ concentrations on the rate of CO₂ efflux are in the first instance caused by the effect of CO₂ on the activities of certain key respiratory enzymes, discussed in section 7.2.1, and we suggest that some of the apparently contradictory responses to elevated CO₂ might be explained by the interaction of the alternative and cytochrome pathways as discussed in section 7.2.2 below.

Figure 7.1 The effect of CO₂ concentration on the activity of cytochrome c oxidase in vitro

7.2.1 Effects of CO₂ concentration on enzyme activity

Several authors have suggested that CO₂ might have direct inhibitory effects on some respiratory enzymes (Shipway and Bramlage, 1973; Kerbel et al. 1988, 1990) including cytochrome c oxidase (Palet et al . 1991). We will consider the effects reported for CO₂ inhibition of respiratory enzymes of the glycolytic pathway, Krebs cycle and mitochondrial electron transport. We conclude that cytochrome c oxidase could be the main target of the direct CO₂ inhibition of respiration. 

There is evidence that atmospheric CO₂ concentration affects the activity of respiratory enzymes, although the CO₂ levels at which some of these enzymes were inhibited was much higher than projected atmospheric levels. Cytochrome c oxidase (cytox hereafter) is the final step in the mitochondrial electron transport chain and the rate of oxidative phosphorylation is tightly coupled to the activity of this enzyme. Cytox is inhibited by CO₂-bicarbonate (Miller and Evans 1956; Bendall et al. 1958; Palet et al . 1991, 1992) (Figure 7.1). Cytox activity of photomyxotrophic carnation callus homogenate was inhibited 38% by 10 mM of potassium bicarbonate (about 2.5% CO₂ in air) at pH 7.2 (Palet et al. 1991 ). The activity of purified beef heart cytox was inhibited almost 50% by 20 mm of potassium bicarbonate (about 5% CO₂ in air) at the same pH (Palet et al. 1992). Palet et al . (1991) concluded that external free CO₂ is the species responsible for the effects on cytox and suggested a carbamate formation in proteins as possible mechanism. In contrast, cytox from roots of spinach and soybean were strongly inhibited by high sodium bicarbonate concentrations at pH 8.8 suggesting a competitive model as mechanism of inhibition (Miller and Evans 1956). Cytox from different plant species have different sensitivities to inhibitors: cytox from C₄ leaves was highly sensitive to CO compared with cytox from leaves (Naik et al. 1992), suggesting that there may be a range of sensitivity of inhibition of respiration to elevated CO₂ in the air.

Figure 7.2 The relationship between respiration in whole tissues of the sedge, Scirpus olneyi, and the in vitro activity of cytochrome c oxidase from extracts of the sedge.  Plants were grown in the field and in the field exposed to normal ambient CO₂ and in elevated CO₂ (680 ppm) in open top chambers (Azcón-Bieto et al.1994)

The tight coupling between the activity of cytox and respiration has been demonstrated in plants having different levels of the enzyme. Figure 7.2 shows the linear relationship between respiration, measured by O₂ assimilation, in whole sections of green tissues from Scirpus olneyi grown in two different atmospheric CO₂ concentrations, and the activity of cytox in extracts from these tissues (Azcón-Bieto et al. 1994). In S. olneyi, the effects ofe1evated CO₂ on the activity of cytox from this species was sufficient to explain the reduction of respiration measured by gas exchange in the field.

It appears that other enzymes are also inhibited by elevated CO₂ concentrations. Amthor (1991) suggested that the dehydrogenases are inhibited by increased CO₂ concentrations. Studies of apple tissues showed that a very high CO₂ concentration Krebs cycle activity was reduced (Monning 1983). In pear fruits, a concentration of 5% CO₂ in air inhibited the activity of succinate dehydrogenase by approximately 50% in the first month of CO₂ exposure and somewhat more after six months' exposure (Frenkel and Patterson 1973).

Other enzymes of the Krebs cycle and some glycolytic enzymes are also inhibited by high concentrations of CO₂ (Amthor 1991). Both ATP: phosphofructokinase (PFK) and PPI: phosphofructokinase (PFP), were strongly (up to seven times) inhibited by 10% CO₂ in air (Kerbel et al. 1988). Carbon dioxide (CO₂/bicarbonate) decreased the activity and the affinity of the NAD (nicotinamide adenine dinucleotide) malic enzyme from the CAM (reduced nicotinamide adenine dinucleotide) plant Sedum praealtum with a decrease in decarboxylation rate of malate and mitochondrial O₂ uptake (Spalding et al. 1980). Carbon dioxide CO₂-bicarbonate) stimulated malate oxidation, but reduced NADH (crassulacea acid metabolism) oxidation in apple mitochondria (Shipway and Bramlage 1973). Amthor (1991) reviews evidence suggesting that succinate dehydrogenase is particularly sensitive to elevated CO₂.

It is clear that some enzymes can be inhibited by increasing concentrations of CO₂ (Figure 7.3) and that some of these (e.g. PFP or PFK) are sensitive to very small changes in pH, which can be affected by very high atmospheric CO₂ (Bown 1985). We propose that the inhibition of respiratory enzymes, especially cytox, by increased atmospheric CO₂ is the basis for the inhibition of CO₂ efflux or O₂ uptake by whole tissues. To apply this hypothesis to those cases in which increased CO₂ stimulates respiration it is necessary to consider the coordinated \ system of the mitochondrial electron transport chain.

7.2.2 CO₂ and the cytochrome and alternative pathways

A complication in relating the rate of respiration to the background CO₂ concentration is the partitioning of electron flow through the electron transport chain between the cytochrome oxidase pathway and the alternative, cyanide-resistant pathway (Figure 7.3). Both pathways are involved in the consumption of substrate and in CO₂ emission and O₂ uptake, but the alternative pathway is nonphosphorylating and therefore uncoupled from energy production. 

Cyanide and other compounds inhibit aerobic respiration of most organisms by inactivation of cytox. In many plant tissues, however, cyanide has a small effect on respiration determined as CO₂ efflux or O₂ assimilation and the remaining respiration in the presence of cyanide is said to be due to an alternative pathway from electron flow. Cyanide-resistant respiration is present in plants, bryophytes, several fungi, algae, a few bacteria and some animal tissues (Henry and Nyns 1975).

Figure 7.3 Points in metabolism and in the mitochondrial electron chain where CO₂ bicarbonate has an effect on the activity of enzymes, indicated as darkened background. The effect of CO₂ is given as + if a stimulation and - if inhibition. For other details see text

The basis of cyanide-resistant respiration is a short branch in the mitochondrial electron transport chain at a step prior to cytochrome c oxidase (Figure 7.3). This alternative branch takes electrons from the ubiquinone pool and passes them to O₂ through an oxidase (altox). Unlike the cytox pathway, there is no conservation of proton motive energy in the production of ATP by the alternative pathway and all free energy generated is lost as heat. For most plants, the physiological role of the alternative pathway is unknown, although the heat produced in this way is used in the spadix of arum lilies to volatilize attractants for insect pollination. High levels of carbohydrates can activate the alternative pathway (Azcón-Bieto et al. 1983) burning excess levels of photosynthate wastefully. The observation that excess levels of carbohydrate could be consumed through this pathway, led to the hypothesis proposed for its role in higher plants (Lambers 1982).

The system is believed to function as an electron overflow regulating the supply of energy within the cell (Lambers, 1982, 1985). The alternative pathway can act as an overflow when the cytochrome pathway has been saturated by rapid glycolysis or Krebs cycle activity (Lambers, 1985; Amthor 1989). Engagement of the alternative pathway in the consumption of carbohydrates seems to occur when the electron flow through the cytochrome phosphorylating pathway runs at 40% of its capacity (Dry et al. 1989).

Some reports cited earlier showed a stimulation of respiration in elevated CO₂ . This stimulation of CO₂ emission or O₂ uptake can be due to consumption of excess carbohydrates through the alternative pathway. Stimulation of cyanideresistant respiration by CO₂ has often been observed (Lange 1970; Day et al. 1978; Perez-Trejo et al. 1981; Palet et al. 1991 and 1992). Increase in the activity of the alternative pathway can be induced by inhibitors of components of the cytochrome pathway (Lambers et al. 1983; MØller et al. 1988). Blocking the cytochrome pathway with cyanide, sulfide, antimycin A or myxothiazol raises the activity of the alternative pathway towards its maximum capacity (Bahr and Bonner 1973).If the capacity of the alternative pathway is high enough, its stimulated activity can compensate or even increase the rate of CO₂ efflux or O₂ assimilation to a level higher than the initial rate from the uninhibited cytochrome pathway (Lambers 1985). The increase in the activity of the alternative oxidase was progressive with the partial inhibition of the saturated cytochrome pathway (Gonzàlez-Meler et al. 1992).

Carbon dioxide can stimulate activity of the alternative pathway through inhibition of components of the cytochrome pathway. In pea leaf mitochondria, CO₂-bicarbonate inhibited O₂ uptake and stimulated alternative pathway activity (Palet et al. 1992). In soybean cotyledon mitochondria, CO₂-bicarbonate stimulated O₂ assimilation via the alternative pathway, and this stimulation was inhibited by salycylhydroxamic acid (SHAM), a specific inhibitor of the alternative oxidase. Both the cytochrome pathway activity, determined as the rate of respiration in the presence of SHAM, and capacity, the rate of respiration uncoupled from the ubiquinone pool and fed by electron donors in the presence of SHAM, were also inhibited by CO₂ in the short term in culture cells (Palet et al. 1991). Thus, increased CO₂ may inhibit the activity of cytox and, simultaneously, in the presence of sufficient substrate and high capacity of altox, have the net effect of increasing CO₂ efflux.

The capacity of the alternative pathway appears to acclimate to different environmental conditions. Germination of wheat seeds under very high CO₂ concentrations stimulated the capacity of the alternative oxidase (McCaig and Hill 1977). In potato tubers, resistance to cyanide increased after incubation in 10% CO₂ in air for several hours (Day et al. 1978). Stimulation of CO₂ evolution or CO₂ uptake in potato tubers was increased by high concentrations of CO₂ in air, but this stimulation was not totally inhibited by cyanide, showing a stimulation of both pathways (Perez-Trejo et al. 1981).

It is clear that CO₂ inhibits components of the mitochondrial electron transport chain, especially cytox, because CO₂ can inhibit the rate of respiration when the enzyme is uncoupled from the supply of electrons via the ubiquinone pool. Carbon dioxide can indirectly increase the activity of the cyanide-resistant respiration. Thus, the compensating effects of CO₂ on these two pathways have to be accounted for in studies of the short-term effects of exposure to different concentrations of CO₂ .When the exposure is to very high CO₂ concentrations or if it persists for several days, the capacity of both pathways has to be determined if the effects of CO₂ are to be correctly interpreted.

7.3 ACCLIMATION OF RESPIRATION TO LONG-TERM CO₂ ENRICHMENT

Long-term responses of respiration to elevated CO₂ measured by comparing the response of tissues grown in different CO₂ concentrations at a common CO₂ background are reported in Ludwig et al. (1975), Gifford et al. (1985), Reuveni and Gale (1985), Spencer and Bowes (1986), Allen (1989), Bunce(1990), Drake (1989), Wullschleger et al. (1991, 1992), Baker et al. (1992), Gary and Veyres (1992) and Mousseau and Saugier (1992).

Through stimulation of photosynthesis, high atmospheric CO₂ levels may modify adenylate energy charge and carbohydrate status, both of which are presumed to regulate respiration. Control of respiration by substrate and adenylate turnover is often superimposed as reflected in diurnal changes in respiration in response to light/dark cycles (Azcón-Bieto 1992). Increased substrate levels tend to raise the activity of the alternative oxidase path, rather than of the cytochrome path (Lambers 1985), which would result in increased CO₂ emission, but no increase in energy production. Plant respiration appears to respond to short-term changes in substrate availability (via photosynthesis) and in ATP demand, and also to long-term changes in substrate availability (see Azcón-Bieto 1992). The details of the mechanism by which respiratory pathways are regulated by substrate and adenylate levels have not been established, but in principle, this dual system of regulation allows the plant to respond more efficiently to short-term and long-term environmental changes, including CO₂ changes.

7.3.1 Relationship between respiration and photosynthesis

There is a direct interdependence between photosynthesis and respiration: the greater the respiration and growth, the larger the photosynthetic organ size, and higher rates of photosynthesis result in greater availability of substrate for respiration (Azcón-Bieto and Osmond 1983). There are reductions in concentrations of the major enzymatic constituents of the photosynthetic apparatus of plants growing in elevated CO₂ .The primary carboxylating enzyme, Rubisco, is often reduced in content in the foliage of plants grown in elevated CO₂ (Long and Drake 1992). Reduction of the content of this enzyme, which occupies a major fraction of the soluble protein content, may also be accompanied by the reduction in concentration of other soluble enzymes and this results in reduction of leaf level nitrogen concentration (Conroy 1992).

Reduced nitrogen concentration is commonly observed in plants grown in elevated CO₂ and it is to be expected that respiration rate would be lower in tissues having a lower nitrogen concentration because the respiratory cost of protein turnover and maintenance is relatively large. In studies of the salt marsh sedge, Scirpus olneyi, protein content was reduced 50% and the amount of Rubisco by a similar amount (Jacob et al. 1995). In this plant, Rubisco accounted for about 50% of the soluble protein. The metabolic cost for maintenance of tissues with high concentrations of protein is greater than the cost for the maintenance of tissues with low concentrations of nitrogen. Decrease in nitrogen concentration of the tissue seems to be an acclimation of plants growing at high CO₂ (Conroy 1992; Peñuelas and Matamala 1990). Thus, lower rates of respiration may result from the acclimation of the photosynthetic apparatus to elevated CO₂.

The higher level of respiration in preilluminated leaves is correlated with a high carbohydrate level and a higher CO₂ compensation point (Azcón-Bieto and Osmond 1983; Thomas et al. 1993). It has been suggested that the higher level of respiration of illuminated leaves may be associated with stimulated activity of the alternative pathway (Azcón-Bieto et al. 1983; Gardestrom and Edwards 1985; Thomas et al. 1993).

7.3.2 Long-term effects of high CO₂ on respiration in crops and native species

The general effect of increased photosynthesis by high CO₂ is to raise the levels of soluble carbohydrates in the cell (Dowes 1993). Although there are some exceptions ( e.g. Ziska and Teramura 1992) growth in elevated CO₂ seems to cause increased respiration in crop species, which are typically fast growing. In such species, increasing CO₂ levels result in excess carbohydrate concentrations and stimulation of activity of the alternative pathway. The energy overflow hypothesis (Lambers 1982) suggests that the alternative oxidase could wastefully burn carbohydrates present in excess to the demands of growth, maintenance, ion transport and other physiologic requirements.

The alternative pathway can be engaged in leaves and roots when additional carbohydrates are supplied exogenously or by higher rates of photosynthesis. Addition of exogenous sugars increased respiration in wheat leaves and part of the increased respiration was due to increased engagement of the nonphosphorylating alternative pathway (Azcón-Bieto et al. 1983). Azcón-Bieto and Osmond (1983) showed that dark respiration rate of wheat leaves correlated with leaf carbohydrate levels determined by previous photosynthetic rate. The alternative oxidase was engaged by high CO₂ in sunflower roots (Gifford et al. 1985). Increased respiration rates in soybean leaves after 2-4 weeks in 1000 ppm of CO₂ correlated with increased respiration, but were not mediated by changes in the activity of cytochrome c oxidase, suggesting that the high CO₂ treatment had stimulated the alternative pathway (Hrubec et al. 1985). Similar results were found in cotton (Thomas et al. 1993), barley (Williams et al. 1992), sunflower (Gifford et al. 1985) pea (Musgrave et al. 1988) and soybean (Thomas and Griffin 1994). Respiration in detached maize root tips was increased without concomitant increase in the energy charge (Saglio and Pradet 1980) suggesting that the increased respiration was due to the stimulation of the alternative pathway.

Finally, some crops species showed decreased respiration. In the cases of alfalfa (Dunce and Caulfield 1991), tomato (Bunce 1990) and soybean (Bunce 1990) elevated CO₂ increased biomass production, but not growth rate. 

Thus, in crop species, the general pattern seems to be that elevated CO₂ increases both carbohydrate levels and perhaps the alternative oxidase activity leading to greater efflux of CO₂. In the few cases where it has been examined, it does not appear that increased CO₂ efflux necessarily leads to increased energy production.

Native herbaceous species grown in elevated CO₂ usually show an increase in photosynthesis (Drake and Leadley 1991; Long and Drake 1992), an increase in biomass (Curtis et al. 1989; Hunt et al. 1991), a reduction in nitrogen concentration (Curtis et al. 1989; Peñuelas and Matamala 1990; Drake 1992a, b) and a decrease in dark respiration. We will now examine the effects of these changes on respiration.

Most reports of respiration in tree species show a decrease in respiration rates under elevated CO₂. Generally, experiments with trees were run for several months or even years and the magnitude of the response of respiration to high CO₂ often varies throughout the season. Chestnut seedlings grown for two years at 700 ppm CO₂ decreased respiration by about 50% of the controls at normal ambient CO₂ at the beginning and end of the growing season (El Kohen et al. 1991). Orange trees had decreased respiration by about 20% after four years of exposure to 700 ppm of CO₂ (Idso and Kimball 1992). White oak leaves and yellow-poplar leaves grown under 650 ppm CO₂ for two months had decreased respiration rate of 59 and 48% on a dry weight basis and 50 and 37% on an area basis and starch content was increased, but not sucrose levels (Wullschleger et al. 1991). After three years in high CO₂, white oak showed a decrease in young leaf respiration of 20% (Wullschlegerand Norby 1992). Three years of exposure in 650 ppm of CO₂ decreased respiration rate (dry weight basis) by 45% without changes in specific growth rate (Wullschleger et al. 1992). Similar results were obtained in leaf respiration rates (area basis) in two species of maple (35 and 52% reduction), oak (45%) and apple (29%) during one month of growth in 700 ppm of CO₂ (Dunce 1992). Respiration was also decreased in the tropical trees Acacia mangium (30%), Psychotria limonensis (25%) and Ficus obtusifolia (20%) when grown at high CO₂ (Drake 1989). Seedling of loblolly pine increased photosynthesis, carbohydrates and biomass production, but there were no differences in dark CO₂ evolution after 100 days of growth at 650 ppm of CO₂ (Griffin et al. 1993).

The effects of elevated CO₂ on native herbaceous species is variable. Respiration increased with increased growth rate in Plantago major grown in elevated CO₂ (Poorter et al. 1988). In cases in which there was no significant change in tissue composition, as for example in Lolium and clover (Ryle et al. 1992a and b), and Dactylus glomerata (Bunce and Caulfield 1991), elevated CO₂ had no effect on whole plant respiration. .

Reduced respiration is often observed in herbaceous plants grown in elevated CO₂. Respiration (biomass basis) of Lolium (Bunce and Caulfield 1991) and orchard grass were reduced in plants grown in elevated CO₂ (Ziska and Dunce 1993). Oxygen uptake decreased in the sedge, Scirpus olneyi, grown in high CO₂ for seven years. This effect occurred whether results were expressed on a dry weight or area basis and correlated with reduced activity and capacity of the cytochrome pathway and to the amount of the complex III and IV enzymes of the mitochondrial electron transport chain (Azcón-Bieto et al. 1994). These results also showed that respiration was tightly coupled to the activity of the cytochrome c oxidase and there was no change in the activity of the alternative pathway (Figure 7.2).

In summary, while there are exceptions in both cases, higher respiration rates of crop species and lower respiration rates of perennial native species grown in high CO₂ appear to be the pattern. Moreover, it appears that the stimulation of the alternative pathway accompanies increased respiration rates of annual, fast growing crops, but not slower growing, perennial species. In this survey, no effort was made to examine the influence of small rooting volume on these effects, but there may be an artifact of this effect in the results because many of the annual crop studies were done in controlled environments in which small pots may have been used. If there is an artifact in these data, the results may not apply to the vast majority of species growing in native environments, but it would emphasize our point that increased carbohydrates, especially soluble sugars, accompany stimulation of the alternative pathway reflecting the overflow of additional photosynthate.

7.4 INTERACTION BETWEEN GROWTH, RESPIRATION AND CO₂

McCree (1970) fitted an empirical equation in which a substantial portion of respiration of white clover plants was proportional to the daily net carbon gain by photosynthesis or growth. The remainder of the respiration was proportional to dry weight of living material in the plant, and was assumed to reflect maintenance cost because it is apparently linked to the maintenance of plant life functions. This division of respiration has provided a useful construct within which to assess the role of various environmental and internal factors on the relationship between growth and respiration. Considerable experimental evidence agrees with the essential basis of this respiration model (see Amthor 1989), but from the strictly biochemical point of view, there is no distinction between growth and maintenance respiration. The relationship between these two components of respiration and the existence of nonphosphorylating pathways in plant mitochondria, which consume carbohydrate, but produce no useful energy, is also not clear at the moment.

High atmospheric CO₂ usually results in decreased nitrogen concentration and higher C/N ratios. It has been shown that the C/N ratio and the proportion of photosynthate invested in respiration increases with decreasing nitrogen supply (van der Werf et al. 1992). However, in plants growing in elevated CO₂ the ratio of respiration to photosynthesis decreases and it is therefore difficult to interpret the reduced nitrogen concentration in these plants as limiting plant growth. Higher C/N ratios can represent lower maintenance respiration (Wullschleger and Norby 1992; van der Werf et al. 1992; Bunce 1994) and lower costs in the growth of new tissue (Wullschleger et al. 1992; Wullschleger and Norby 1992; Griffin et al. 1993).

Wullschleger and Norby (1992) determined the effect of elevated CO₂ on growth and maintenance of leaves of white oak seedlings grown in open top chambers in the field in native, unfertilized soil. Specific respiration rates for leaves of this species were 56% lower in elevated CO₂ than in normal ambient CO₂ .The relative growth was reduced 31% and the maintenance coefficient 45%. These effects were attributed to the reduced cost of maintaining tissues ) having reduced nitrogen concentrations. Similar results were obtained in leaves of yellow poplar (Wullschleger et al. 1991). However, Ziska and Bunce (1993) report instances in which maintenance respiration was reduced for plants grown in elevated CO₂ even though these plants had no decrease in protein or nitrogen content. Thomas et al. (1993) found increased dark respiration resulting from enhanced maintenance respiration in cotton leaves growing at high CO₂ .The authors related the increase in maintenance respiration to carbohydrate metabolism rather than to changes in leaf nitrogen concentration.

Plant respiration can decrease at high CO₂ even when there is no change in relative growth rate. A decrease of 30-40% in respiration at high CO₂ occurred even though growth rates were the same as for plants grown at normal CO₂ (Bunce and Caulfield 1991). Similar results were found in some C₃ crops (Bunce 1990). An increase in 50% in relative growth rate in plants grown under high CO₂ concentrations was supported without changes in CO₂ evolution (Bunce and Caulfield 1991). The lower respiration rates with constant relative growth rate seems to be related to changes in construction costs of the new tissue. High nitrogen availability increased the costs of leaf construction by 7%, but these costs were 3.5% lower in high CO₂ grown seedlings (Griffin et al. 1993).

 Rising atmospheric CO₂ also appears to affect carbon use efficiency (CUE). CUE is the fraction of carbon taken up during the day (U) after accounting for nighttime emissions (E):

CUE = (U-E)/U

Gifford ( 1991) found that growth in elevated CO₂ either had no effect or increased CUE with an overall average of 4.4% for nine species. There was a significant correlation between the increased CUE and decreased growth respiration (respiratory cost per unit of growth). On the other hand, the results for the effects of growth in elevated CO₂ on maintenance respiration were less clear with some species showing a decline in maintenance cost and others an increase (Gifford 1991). In short-term experiments, lasting up to 12 hours, Gifford found no significant effect of elevated CO₂ on whole plant respiration. Nevertheless, his data do show that when respiration of plants grown and tested in their respective CO₂ concentrations was compared, the plants in elevated CO₂ had lower rates of respiration than the controls in normal ambient CO₂ .

7.5 CO₂ AND SOIL RESPIRATION

Energy requirements of the roots vis-à-vis the supply of assimilated carbon to the roots is an important factor determining the net carbon balance of whole plants. Some 30-60% of the daily photosynthetic carbon production is lost in respiration and a significant portion of this loss is from roots (Lambers 1985).

Respiration of roots of wheat, mung bean or sunflower was unchanged or slightly decreased by high CO₂ , but the engagement of the alternative respiration was inconsistent (decrease, no effect, and increase, respectively) in plants grown at high CO₂ compared with plants grown at normal CO₂ (Gifford et al. 1985). Root respiration was higher in the first weeks of growth in roots of Plantago major growing at 700 ppm CO₂ (Poorter et al. 1988) and this did not correlate with shoot respiration.

Root respiration is rapidly affected by changes in aeration in the rhizosphere (Leshuk and Saltveit 1991;Drew 1992). The CO₂ tension in the soil can inhibit root (Palta and Nobel 1989) and soil microbial respiration (Koizumi et al. 1991). 

There have been no studies of the effects of long-term exposure to elevated CO₂ on soil respiration. The microbial activity of the soil accounts for an average of 70% of the CO₂ efflux from the soil (Raich and Schlesinger 1992). Soil activity can produce a high CO₂ turnover: temperate forests have an estimated 680 g Cm^-2 yr^-1 and tropical forests 1260 g C m^-2 yr^-1 soil respiration. Thus, soil microbial respiration is a major player in the global carbon budget and changes in the rate of respiration of these organisms, either through direct effects of CO₂ or indirectly through plant growth or climate change, can therefore affect atmospheric CO₂ (Koizumi et al. 1993; Wullschleger and Norby 1992).

In summary, not enough is known about the response of soil and root respiration to high atmospheric CO₂ in natural ecosystems where changes mediated by CO₂ in shoot growth requirements, biomass composition and decomposition, and changes in symbiotic associations are important components of the root + microbial CO₂ balance. Moreover, it is unclear how a change of 350 PPM CO₂ in air could influence the CO₂ in the soil, which can be of the order of 100 00 ppm. Priority should be given to the effects of elevated CO₂ on belowground processes, both of roots and microbes.

7.6 CONCLUSIONS

There is now abundant evidence that dark respiration in foliage and possibly in other plant tissues is reduced for a doubling of atmospheric CO₂ .The primary effect is immediate and reversible and this effect appears to be altered by changes in tissue composition in some species. Elevated CO₂ inhibits in vitro activity of cytochrome c oxidase, succinate dehydrogenase and perhaps other enzymes of the mitochondrial electron transport system. Studies with cell-free extracts of enzymes, mitochondria, tissue cultures and of whole tissues all show that increased CO₂ reversibly inhibits both CO₂ efflux and O₂ assimilation consistent with the hypothesis that there is a direct effect of CO₂ on enzyme activity. In some cases the effect of changes in tissue composition caused by growth of plants in elevated CO₂ is to further decrease respiration and in others to cause a net increase of CO₂ efflux. In rapidly expanding whole leaves or in fast growing species exposed to elevated CO₂ , high concentrations of carbohydrates stimulate activity of the alternate respiratory pathway. Stimulation of the alternative pathway is not accompanied by higher production of ATP.

In slow growing woody perennial species in which soluble carbohydrates do not tend to accumulate, respiration appears to be inhibited and there does not seem to be the level of engagement of the alternative oxidase that occurs in fast growing species including crops.

Although there is evidence that the inhibitory effect of elevated CO₂ on respiration extends to nonphotosynthetic microbes involved in decomposition, this would only have practical meaning for a high CO₂ world if these decomposers were exposed to near atmospheric levels of CO₂. It is hard to imagine that the high concentrations of CO₂ found in soil could be substantially altered by doubling of atmospheric CO₂ .Thus, the rates of soil respiration are not likely to be influenced by increasing atmospheric CO₂ except as related to changes in composition of decomposing plant tissues.

If increasing CO₂ concentrations reduce the rates of respiration in all plant tissues and in microbes and fungi as well, then it should also be expected that there would be a significant increase in the carbon accumulating in ecosystems as a result of the combined effects of stimulation of photosynthesis and reduction in respiration.

While the results of the effect of elevated CO₂ on respiration have been found to vary widely, even small effects to reduce respiration would have a significant impact on global carbon budgets. For this reason, additional study of the role of increasing atmospheric CO₂ in respiration should be given a high priority for research.

7.7 ACKNOWLEDGEMENTS

The authors are grateful to Drs Robert Gifford and James Bunce for useful comments in previous versions of the manuscript. This work was supported by US Department of Energy, the Smithsonian Institution and by fellowships programs of the Smithsonian Institution and Ministerio de Educación y Ciencia (FPI and FPU, Spanish Government) to M.A. G.-M.

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