Risø-R-1332(EN)

Plant Respiration and Climate Change Effects

Dan Bruhn Ph.D. thesis Plant Research Department Risø National Laboratory Botanical Institute University of Copenhagen

Risø National Laboratory, Roskilde April 2002

Abstract

The ongoing climate changes can affect many plant physiological processes. In turn, these effects on plants may result in a feedback between the climate change and the vegetation.

Plant respiration is one of the key processes in terms of an understanding of plant growth and functioning in a future climate. Plant respiration can release up to about half of the assimilated carbon by photosynthesis on a daily basis. Thus potential effects of e.g. changed air temperature and atmos-pheric [CO2] are important to investigate for predictions of respiratory release of CO2 to the atmos-phere. This is further emphasised by the fact that global terrestrial respiratory CO2 release is about ten times as high as the antropogenic contribution of CO2 to the atmosphere on an annual basis. Plants therefore constitute an important component of the global carbon cycle.

Short- and long-term (direct and indirect, respectively) effects of temperature and [CO2] on plant respiration were investigated in a number of plant species. The experiments tested effects of ei-ther temperature and/or [CO2] from the level of individual respiratory enzymes, isolated mitochondria, whole-tissue (above- and below-ground tissue), and up to the whole canopy level in forests.

The short-term effects of elevated atmospheric [CO2] on plant respiration appeared to be less than suggested so far in the literature. This was true both at the tissue-level and for intact mitochon-dria. Respiratory enzymes can, however, be affected already at low [CO2]. Bicarbonate rather than CO2 was found to be the carbon species that affected one of the respiratory enzymes. These effects did not manifest itself at the tissue level, though, due to low degrees of control on the whole respiratory process exerted by the particular enzymes.

Plant respiration on the other hand was affected by long-term growth at elevated atmospheric [CO2]. The findings of the reduced plant respiration at the leaf level were consistent with the literature and potential causes are discussed.

Short-term effects of temperature on plant respiration were demonstrated to be dependent on the actual measurement temperature. Further, it is shown that mitochondrial leaf respiration in dark-ness and light differ substantially in the temperature sensitivity with the former being the far most sen-sitive. This has implications for modelling CO2 exchange between vegetation and atmosphere as dem-onstrated here, since this has so far been neglected. The underlying mechanisms per se call for further investigation as the much lower temperature sensitivity of leaf respiration in the light to a great degree may influence the daily carbon budget of plants.

Long-term effects of temperature resulted in respiratory acclimation in a number of species. Respiratory acclimation appeared not to occur to any one single type of growth temperature. The im-plications of this finding in combination with the timing of acclimation are discussed for modelling respiratory CO2 release. In addition are some new methods for calculation of the degree of acclimation proposed.

ISBN 87-550-3032-7 ISBN 87-550-3033-5 (Internet) ISSN 0106-2840

Print: Pitney Bowes Management Services Danmark A/S, 2002

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Contents

Abstract 2

Preface 4

1 Introduction 5

Climate Change in a Plant Ecophysiological Perspective (2000) In: Climate Change Research - Danish Contributions. Jørgensen A.M.K., Fenger J. & Halsnæs (eds.), DMI, Ministry of Transport, Gads Forlag.

2 Introduction to Plant Respiration 29

3 Phosphorus uptake by arbuscular mycorrhizal hyphae does not increase when the host plant grows under atmospheric CO2 enrichment 35

New Phytologist (2002) 154 In press.

4 Does the direct effect of atmospheric CO2 concentration on leaf respiration vary with temperature? Responses in two species of Plantago that differ in relative growth rate 45

Physiologia Plantarum 114: 57-64, 2002

5 Direct effects of [CO2] on enzyme activity and O2 uptake of isolated mitochondria and intact tissues: Comparing the response of Solanum tuberosum L. tubers and Glycine max L. cotyledons 53

Plant Physiology, submitted

6 Determining the temperature that leaf and root respiration acclimates to in several contrasting plant species differing in photosynthesis type and growth characteristics 69

Global Change Biology, submitted

7 Partial Respiratory Acclimation to Temperature by Plants - Discussion of Definitions, Calculations, and Applications 89

Prepared for Functional Ecology

8 The interaction of temperature and irradiance on leaf respiration is important to take into account when modeling CO2 flux between atmosphere and vegetation 101

Prepared for Tree Physiology

9 Supplementary Discussion, Conclusions, and Future Research 119

Appendix 133

Acknowledegements 140

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Preface

This Ph.D.-thesis is submitted as part of the requirements for a Ph.D. degree at University of Copenhagen, Botanical Institute, Denmark. The project period was April 23rd 1999 to April 22nd 2002. The project has been carried out at the Plant Environment Interactions Programme, Plant Research Department, Risø National Laboratory, Roskilde, Denmark and Department of Biology, The University of York, York, UK. The Danish Research Academy, Risø National Laboratory, European Science Foun-dation, and Nordic Academy of Advanced Study have financially supported this project. The outline of the thesis is: Chapter 1 is an introduction to climate change and plant ecophysiology in gen-eral. Chapter 2 gives a brief introduction to plant respiration, its control, and methodological aspects. Chapter 3 to 5 deals mainly with effects of [CO2] on plant respiration. Chapter 3 treats the indirect (or long-term) effects and Chapter 4 & 5 treat the direct effects of [CO2]. Chapter 6 to 8 is about effects of tem-perature on plant respiration (Chapter 4 does also treat temperature effects to some extent). Chapter 6 is a study about the respiratory acclimation to tempera-ture. Chapter 7 treats respiratory acclimation to temperature in a theoretical manner. Chapter 8 is about direct (short-term) effects of temperature in combi-nation with light and the only chapter where effects are scaled up to the canopy level. Finally, Chapter 9 gives a personal supplementary discussion to the dis-cussion parts of Chapter 3 to 8. Chapter 9 also contains the conclusions from my own experiments separated out into each of the four main topics of this the-sis, i.e. short- and long-term effects of [CO2] and short- and long-term effects of temperature on plant respiration. In addition is presented a personal view on future research. In the appendix are included two Danish articles about climate change research within plant ecophysiology ongoing at Risø. These two articles are written for a broader forum than plant biologist. The thesis finishes with my acknowledgements to a number of people, all of who have helped me during this Ph.D. project at various stages. Research is both investigation and publication. Thus, the changing lay-out of the different chapters throughout the thesis reflects the stage of publica-tion of the individual chapters. Chapter 1 and 3 to 8 are all meant to be pub-lished separately.

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1 Introduction

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2 Introduction to Plant Respiration

This is an introduction to the plant physiological process, respiration – the piv-otal point in this thesis. Plant respiration was briefly introduced in Chapter 1 as one of several plant physiological processes it is necessary to understand to an-swer questions about climate change at the plant ecophysiological level. This is in order to at higher levels of integration address ecological challenges of the ongoing global climate change. Potential short-term and long-term effects by climate changes were also discussed in Chapter 1. The following chapter is lim-ited to dark (mitochondrial) respiration in plants. Photorespiration was shortly introduced in Chapter 1. Overview of Plant Respiration

In respiration the relatively reduced organic compounds, carbohydrates, organic acids, and proteins are oxidized and some of the released energy is used for growth and maintenance. In plants the main substrate for respiration can be said to be the disaccharide, sucrose, which is the predominant sugar translocated via the phloem in most plants. The free energy released is transiently stored in ATP, which is the energy currency used for both cellular maintenance and growth. Sucrose may be fully oxidized to give CO2 and the net reaction is then:

C12H22O11 + 12 O2 → 12 CO2 + 11 H2O Respiration takes place as a multistep process. Thereby the free energy

is released in several steps preventing cellular damage. The three main stages are glycolysis, the TCA (tricarboxylic acid) cycle, and the oxidative phosphory-lation including the mitochondrial electron transport chain. The intermediates can enter the respiratory process at various points as substrates; equally can in-termediates leave the respiratory chain to function as precursors for biosynthesis of various compounds. Respiration is hereby coupled to many other metabolic pathways. Glycolysis takes place in the cytosol, but some glycolytic enzymes are also located in the plastids. Thus, substrates can also enter the respiratory proc-ess directly from both chloroplasts and amyloplasts. During glycolysis, sucrose is split into two monosaccharides and the respiratory substrates are prepared for the TCA cycle along with a small production of reducing equivalents, NADH, and possibly ATP. The end product of the glycolysis is via phosphoenolpyru-vate (PEP) one of two organic acids, pyruvate or malate. When pyruvate is the end product four molecules of ATP are produced per molecule of sucrose enter-ing the glycolysis. When malate is the end product, there is no net production of ATP. The possibility of malate as the glycolytic end product is a unique feature of plants and malate is in fact the major end product of glycolysis in plant cells. The oxidative pentose phosphate pathway is an alternative route to glycolysis of respiratory substrates. Enzymes located both in the cytosol and in the plastids carry out the pentose phosphate pathway. This route plays an important role in providing intermediates, e.g. precursors for synthesis of nucleotids. The pentose phosohate pathway accounts only for 5-20% of the use of sugar depending on developmental stage of the tissue (Taiz and Zeiger 2002). The TCA cycle takes place in the mitochondria. The substrates for the TCA cycle is the pyruvate and/or malate produced in the glycolysis. During the TCA cycle the organic acids undergo a number of decaboxylations. Concomi-tantly both reducing equivalents, NAD(P)H and succinate, and ATP are pro-duced. NAD(P)H and succinate are electron donors, and thereby substrates for

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the mitochondrial electron transport chain. In addition the TCA cycle serves as a producer of intermediates for biosynthesis. Oxidative phosphorylation, i.e. the electron transport chain and ATP synthesis takes place in the inner mitochondrial membrane. During this third part of respiration electrons from NADH and FADH2, mainly formed in the TCA cycle flow through a system of protein complexes (I-IV) in the inner mito-chondrial membrane. Some of the free energy released during this electron transfer is used to pump protons from the mitochondrial matrix across the inner membrane and out into the intermembrane space during passing of electrons through the complexes. Not all complexes pump protons, though; only complex I, III, and IV. Electrons from Complex I, II and multiple internal and external NAD(P)H dehydrogenases are flowing through the ubiquinone pool down the electron transport chain. This generates an electrochemical proton gradient. The terminal oxidase, Complex IV, reduces O2 to H2O – the cytochrome pathway. The ATP synthesis is accomplished by passage of protons back from the inter-membrane space though the ATP synthase. One molecule of ATP is produced per three protons passing though the ATP synthase as this is coupled to the con-version of ADP and Pi into ATP. However, the cytochrome pathway may be bypassed by the alternative oxidase, whereby two thirds (depending of the sub-strate) of the proton pumping is prevented and therefore less ATP is produced. The function of this alternative oxidase is not fully understood, but may func-tion as heat production in some tissues, since less energy is used in ATP forma-tion. Less ATP is also produced if NADH is oxidized by e.g. the internal rote-none-insensitive NADH dehydrogenase and complex I is thereby bypassed re-sulting in less protons pumped out. Control of Plant Respiration

The current view of on control of cellular respiration is a control exerted from the ‘bottom up’ by the cellular level of ADP (Teiz and Zeiger 2002). There ex-ists an adynelate control of the oxidative phosphorylation. If the energy demand of the cell drops and thereby the use of ATP, then less ADP can react with Pi to re-synthesize ATP and the flow of electrons though the innermembrane is re-duced. This can in turn regulate the use reducing equivalents produced in the TCA cycle, which can lead to a build up of TCA cycle intermediates. Of these can compounds such as e.g. malate inhibit the cytosolic pyruvate kinase, why cytosolic concentration of PEP increases. This affects the rate of the glycolysis. The activity of the alternative oxidase can increase as the reduction level of the ubiquinone pool increases as well as by build up of various organic acids, which act as stimulators. Several so-called control points in respiration are discussed in the literature as ‘rate-limiting steps’. Metabolic control analysis (reviewed by ap Rees and Hill 1994) is a way of dealing with the theory about the control of e.g. respiration is distributed between several components of the process. Each enzyme or part of the respiratory chain has individual flux-control coefficients depending on the flux through in the system. For the whole process the sum of control-coefficients equals 1 by definition.

Respiration in Tissues and Whole Plants

From the tissue level and to higher levels of integration, plants and canopies, respiration can be controlled both by the energy demand (the biochemistry of

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which is described above) and by the substrate supply. That is respiration can be limited by a low supply of photosynthetic assimilates, and the energy demand does not play the same role. If the substrate supply on the other hands exceeds the energy demand, then the activity of the alternative oxidase and the internal NADH dehydrogenase may play an increasingly important role and facilitate a higher rate of respiration with less production of ATP (energy).

The respiratory machinery (the amount of respiratory enzymes) is not limiting under normal conditions, since the rate of respiration can be increased by manipulation of either the energy demand or the substrate supply. However, the respiratory capacity can be changed through time to e.g. high substrate sup-ply through the transcription of genes encoding respiratory enzyme (Lambers et al. 1998).

On a daily basis about half of the carbon assimilated via photosynthesis can be respired on a whole plant basis. This fraction is species dependent and can be altered by environmental control too. A functional approach has been adapted in many studies to investigate these two factors (genetically and envi-ronmentally control) on respiration. Further, there seems to be some ontogentic control on the fraction of the daily assimilated carbon that is respired as well as the underlying causes.

The functional approach (introduced in Chapter 1) assumes that we can explain what ‘the respiration is used for’. Traditionally respiration has been di-vided into respiration associated with growth, maintenance, and ion uptake. To produce new biomass, carbon skeletons, reducing equivalents (NAD(P)H), and energy (ATP) are needed. The biochemical composition of a tissue determines the construction cost of that specific tissue. This factor, the construction cost, can be standardized to the amount of glucose (respiratory substrate) needed to construct different compounds. The compound construction cost of a tissue multiplied by the relative growth rate of the tissue gives the respiration needed to produce this new tissue. Different amounts of CO2 are released, though, de-pending on the tissue composition due to the specific biosynthetic pathways of different compounds. Maintenance respiration is needed to meet the energy de-mand for repair and maintenance of existing tissue. Primarily energy for protein turnover and maintenance of ion gradients across membranes accounts for most of the energy demand associated with maintenance. However, the exact energy demands for the individual processes in vivo are not well known (Lambers et al 1998). Ion uptake requires energy for sustaining electrochemical potential gra-dients for uptake of anions. Additional costs may be associated with the reduc-tion of nitrate, once taken up from the soil. Leaf respiration varies much less than root respiration (Lambers et al. 1998) because assimilation, respiration, and biomass allocation of leaves is commonly affected by environmental changes in similar ways. Significant amounts of carbon can be respired in periods with flowering and seed produc-tion. Root respiration constitutes a large amount of the daily fixed carbon. Nev-ertheless, during growth, carbon availability for root respiration seems to be ‘what is left over from other sinks’ (Atkin et al. 2000), due to a low capacity. Fast-growing plants are characterized by larger proportions of the available car-bon used for biosynthesis than for respiration compared to inherently slow growing species. Ontogentic drift causes maintenance respiration to increase in importance relative to respiration associated with growth and ion uptake.

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Environmental Control of Plant Respiration

In addition to environmental parameters such as temperature, atmospheric [CO2], and water availability as described in chapter 1 also nutrient supply, light, pollutants, soil pH, salinity, pathogens and inter- and intra-species compe-tition influence rates of respiration by tissues and whole plants.

The control of respiration by these environmental factors can be exerted in different ways. Simple mechanisms such as direct effects of e.g. temperature on individual enzymes are important. Indirect effects comes around by e.g. lower substrate supply at low light conditions, changed chemical composition of the tissue after growth at a different [CO2], or altered water and/or nutrient availability causing changed biomass allocation within the plant as a part of ac-climation. Pollutants can impair various physiological processes and thereby affect respiration due to increased energy demand for repair and maintenance.

Methodological Aspects of Investigation of Plant Respira-tion

Approaches to measurements of rates of respiration differ between levels of in-terest. Usually respiration is measured as gas exchange, either release or uptake of CO2 or O2, respectively. Alternatively, respiration can be estimated from heat production. Irrespective of the method chosen, the investigator only achieves indirect measures of rates of respiration. (De)carboxylation and (de)oxygenation occurs throughout the plant in different biochemical reactions independent of respiration (glycolysis, TCA cycle and oxidative phosphorylation) (Amthor 1994), so CO2 and O2 exchange rates between the plant (or tissue) and the sur-rounding medium is only an approximate estimate of the rate of respiration. Likewise heat production per sucrose (or glucose) molecule can vary depending on the engagement of e.g. the alternative oxidase. The activity of individual respiratory enzymes can be measured in vitro, but the activity will not necessarily tell anything about in vivo rates. This is be-cause the enzyme activity may be completely uncoupled from restrictions im-posed in vivo by the interactions with other processes such as supply of sub-strates and use of end products as well as other factors that affect the activity of the enzymes per se. Measurements of rates of respiration by intact mitochondria (isolated from a tissue) are subject to the same kind of considerations. Mitochondrial ac-tivity is highly dependent on the type and concentration of substrate, respiratory control (availability of ADP), and oxygen concentration. Again it can be diffi-cult to tell much about in vivo rates of mitochondrial activity due to difficulties in scaling up to the whole tissue level. The capacity, at best, can be indicated by comparing concentrations of mitochondrial specific proteins in the mitochon-drial isolate with the corresponding concentration in the tissue from which the mitochondria were isolated. If effects of individual environmental factors are of interest then up-scaling of the found effects is complicated by the artificial na-ture of investigation of respiration by isolated mitochondria, since they com-monly will be held in a reaction medium suitable for optimal activity of the mi-tochondria, conditions which not necessarily are found in vivo. Effects found at this level are clearly dependent on the distribution of flux-control coefficients of both individual enzymes and thereby also by parts of the whole respiratory process. At the level of intact tissue it apparently becomes easier to estimate rates closer to actual in vivo rates of respiration. However, other considerations

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have to be taken into account by the investigator. This is e.g. the choice of time of day when measurements are conducted. Substrate supply at the tissue level changes throughout the day due to the diurnal rhythms in photosynthetic rates caused by light and temperature. Respiration of leaves are commonly measured at one or a few leaves, and the age of the leaves has to be considered in terms of what is expected with regards to up-scaling at the whole plant-level as well as potential interactions between ontogeny and effects investigated in the particu-lar study. Root respiration measurements are further complicated by the need for excluding the contribution by microorganisms in the soil. Respiration in roots is therefore often measured in a water medium or in sand. In addition, choice has to be made of what part of the root system to measure. Root tips are most active in terms of growth and ion uptake and their rates of respiration are higher than for older parts of the root system. On the other hand the older and less active part may be the far most dominant one in terms of biomass. Whole plant respiration or canopy respiration is in a few cases meas-ured by cuvettes enclosing the whole system. Alternatively, respiration rates of the plant or shoot are measured and scaled up by e.g. leaf area indexes. All the above-described considerations have to be done and this obviously complicates the procedure and potentially increases the possibilities of wrong estimates. References

Amthor JS (1994) Respiration and carbon assimilate use. In: Boote KJ, Bennett JM, Sinclair TR and Paulsen GM (eds.) Physiology and Determination of Crop Yield. ASA, CSSA, SSSA, USDA-ARS, Madison, Winsconsin, USA, pp. 221-250. ap Rees T and Hill SA (1994) Metabolic control analysis of plant metabolism. Plant, Cell and Environ 17: 587-599. Atkin OK, Edwards EJ and Loveys BR (2000) Response of root respiration to changes in temperature and its relevance to global warming. New Phy 147: 141-154. Lambers H, Chapin III FS and Pons TL (1998) Plant Physiological Ecology, Springer, New York, p. 540. Taiz L. and E. Zeiger (2002) Plant Physiology. The Benjamin/Cummings Pub-lishing Company, Inc. Redwood City, California. In press.

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9 Supplementary Discussion

In chapter 1 & 3 - 8 I have treated plant respiration at several levels. This in-cludes responses of respiration to potential climate changes per se as well as respiration measurements to support conclusions about findings concerning other processes. In the following I will supplement the discussion in chapter 3 – 8 in relation to other recent studies and viewpoints.

Direct effects of [CO2] on plant respiration

The direct inhibition of respiration by elevated concentrations of CO2 observed in many studies has been termed ‘mystifying’ (Drake et al. 1999) and ‘the lack of a consistent pattern has made it difficult to propose a unifying mechanism’ (Gonzàlez-Meler and Siedow 1999). Despite the growing number of studies on this subject, it remains controversial (Chapter 4 and 5).

If CO2 acts as an inhibitor of one of more enzymes the respira-tion/[CO2]-relationship is expected to be non-linear (see Chapter 5). Thus the degree of inhibition will also depend on the [CO2]s chosen for comparison and this would further explain some of the variation of the reported results in the literature (Chapter 4). In theory there would be no dependence of the degree of the inhibitory effect of CO2 on whether data are expressed on a mass-, N-, or area-basis. However with sampling- and/or rounding errors it can appear so. As noted by Drake et al. (1999) both Amthor (1997) and Drake et al. (1997) re-ported that the indirect effect are smaller than the direct effect. This is interest-ing because then we would expect the direct effect to decrease with time of ex-posure to elevated growth [CO2]. If so this could indicate that a part of the ac-climation of dark respiration to elevated [CO2] is ‘regaining’ a stable rate of respiration to keep pace with energetic demand etc. A way of testing this is measuring the direct effect of elevated measuring [CO2] at both high and low growth [CO2].

Indeed some studies find an interaction between the direct and the indi-rect effect with the direct effect being lessened at the high growth [CO2] treat-ment (e.g. Jach and Ceulemans 2000). Can this be an artefact, which is associ-ated with the potential leak problem? Some data supports this suggestion, while other data does not. For example CO2 sensitivity is typically estimated in % as 100 × [1- (respirationlow[CO2]Reference/respirationhigh[CO2]Reference)]. Rearranging this relationship can give a plot of ([CO2]Reference–[CO2]Sample) versus [CO2]Reference where the ([CO2]Reference–[CO2]Sample)–line intersects at the [CO2]Ambient (when ignoring the typical slightly higher pressure inside the cuvette compared to am-bient). If the direct effect (dark respiration measured at high and low [CO2]Reference) are measured at a low [CO2]Ambient then the ([CO2]Reference–[CO2]Sample)–line is at a distance of a certain magnitude from the [CO2]Reference-axis at high [CO2]Reference due to the leak. In the opposite situation (at high [CO2]Ambient) the ([CO2]Ambient–[CO2]Sample)–line is at a distance from the [CO2]Reference-axis at exact the same magnitude but with an opposite sign. As an example this can result in a direct inhibition of respiration by 20% at a doubling of reference [CO2] at a low ambient [CO2] and by 16.7% at a high ambient [CO2] when calculating the degree of inhibition as described above. On the other hand, as shown in Chapter 4, comparisons of direct effects on uncorrected rates of dark respiration of a doubling in [CO2]Reference tends to give an apparent higher degree of inhibition at low rates of dark respiration compared to high rates of dark respiration. The rate of dark respiration is typically reduced at high

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growth [CO2]Ambient compared to at low growth [CO2]Ambient when measured at ambient [CO2]. This can to some extent counteract the above effect if the leak problem is the case.

The awareness of the leak-problem (Chapter 4) is also very important in studies of any kind where mitochondrial respiration in the light is of interest and estimated by the Laisk method. This is because these kind of measurements of CO2 exchange are usually conducted at sub-ambient [CO2]References. This can as demonstrated in Chapter 4 result in overestimation of the leaf CO2 uptake and consequently a displacement of Г* (Chapter 8), since also the leaf internal [CO2] may be overestimated (Chapter 6). In addition, it should be mentioned that dur-ing these kinds of measurements in the light, in the leaf part covered under the gaskets there will be no photosynthesis and only dark respiration. Further, this dark respiration will not be inhibited by light compared to that ongoing in the leaf part within the gaskets in the leaf cuvette. Thus if these kinds of measure-ments are conducted with traditional commercial portable photosynthesis appa-ratuses an inward (into the cuvette) flux of CO2 can be expected, which may change the apparent leaf CO2 uptake (Pons and Welschen 2002).

The increasing number of studies reporting very low or no effects of atmospheric [CO2] within the rage relevant to the climate change discussion (Chapter 4 & 5) does not conclusively eliminate the possibility of a direct in-hibitory effect (See Chapter 4 & 5). The low degree of direct inhibition of respi-ration by elevated [CO2] in a few recent studies has in fact let to ‘re-suggesting’ cytochrome c oxidase as a potential target for CO2 (Amthor 2000, Tjoelker et al. 2001a) but not in all (Bunce 2001).

Several possible mechanisms exist for direct inhibition of respiratory enzymatic activity. In short these include carbamylation, mimicking of sub-strates and/or products as reviewed by Amthor (1997) and Drake et al. (1999). Also increased dark CO2 uptake by PEP carboxylase at elevated measurement [CO2] could cause the apparent respiration (measured as CO2 release) to de-crease (Amthor 1997) but this may differ between leaves and roots (Amthor et al. 2001).

Indirect effects of [CO2] on plant respiration

In Chapter 3 indirect effects of [CO2] on pea leaf dark respiration was briefly treated for plants grown at combinations of atmospheric [CO2]s and levels of mycorrhizal inoculum. Since plant respiration was not directly the focus of that study I will here extend the discussion of Chapter 3 some.

Pea plants growing at elevated [CO2] compared to their counterparts at the ambient atmospheric [CO2] had significantly lowered leaf dark respiration. At first a higher rate of leaf dark respiration might have been expected due to the higher rates of assimilation (Chapter 3), which can lead to increased concen-trations of soluble carbohydrates if not used immediately by growth or stored as starch. The plant relative growth rate was increased as well as the leaf area (Chapter 3) indicating a higher use of assimilates but at the same time the spe-cific leaf area decreased. This may indicate and accumulation of soluble carbo-hydrates or starch. However, pea mesophyll cells have low starch storage ability (Opaskronkul et al. 1994). Further, when pea leaves are starved (kept in dark-ness for 48h) the levels of soluble carbohydrates drop and concomitantly the rates of leaf dark respiration, demonstrating a potential responsiveness of pea leaf respiration to soluble carbohydrates (Azcón-Bieto et al. 1983). Neverthe-less, leaf dark respiration decreased. Why was this?

Earlier studies have also shown a decreased whole-plant respiration rate of pea plants after growth at elevated [CO2] (Thibaud et al. 1995). A lowered

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need for ATP could be the case (Chapter 2) and in fact leaf mitochondrial pyru-vate dehydrogenase complex in pea is known to be inactivated by addition of ADP (Moore et al. 1993). During growth at elevated [CO2] plant tissue often accelerate the physiological age as a consequence of faster growth. The tissue concentration of nutrients is in many elevated [CO2] studies commonly reported to decrease (Brown 1991, Coleman et al. 1993, Overdieck 1993, Pettersson et al. 1993, Murray et al. 1996). During the increased assimilation of carbon the rate of nutrient uptake can either not change or only change to a less degree than the carbon assimilation (Eamus and Jarvis 1989), which would explain this. When taking ontogenetic drift (a way of dealing with the accelerated physio-logical plant age) into account some have found the nutrient concentration to be equal between ambient and elevated [CO2] (Coleman et al. 1993) whereas oth-ers find the difference to remain, even independent of non-structural carbohy-drates (Marriott et al. 2001). This is interesting since rates of respiration typi-cally are positively correlated with e.g. tissue nitrogen concentration (e.g. Lusk and Reich 2000, Osaki et al. 2001) and tissue nutrient concentration typically decrease with plant age/size (Ågren 1994). In pea tissue a linear relationship is also demonstrated between specific rates of respiration and tissue concentration of protein (Collier and Grodzinski 1996), which accounts for much of e.g. nitro-gen and phosphorus. We did in fact find a decreased concentration of phospho-rus in the shoots of pea plants grown at elevated [CO2] (Chapter 3), which to-gether with the increased relative growth rates and leaf areas indicates an accel-erated physiological plant age at elevated [CO2]. Pea leaf respiration decreases with plant age (Collier and Grodzinski 1996). Thus, to summarise, the reason for the observed decreased rates of pea leaf dark respiration at elevated growth [CO2] (Chapter 3) can be an accelerated physiological plant age. This explana-tion is perhaps too simplified as altered tissue chemistry, construction costs and maintenance costs also could play a part of the typically decreased leaf respira-tion after growth at elevated atmospheric [CO2] as reviewed by (Amthor 1991, 1994, 1995, 1997 Poorter et al. 1992, Bunce 1994, Wullschleger et al. 1994, Drake et al. 1997, 1999, Luo et al. 1999).

he findings of Chapter 3 need to be treated with some caution, however, as the indirect (long-term) effects of [CO2] on plant respiration were only a mi-nor component of that study. Nevertheless, the use of respiration measurements did demonstrate that mycchoriza functioning was not carbon limited. Had the mycorrhiza been carbon limited, we would have expected a higher sink strength (Chapter 1) in terms of carbon. If this had been the case, then we might have expected a decrease in rate of e.g. leaf dark respiration with increasing levels of mycorrhiza inoculum when grown at high atmospheric [CO2] as less substrates would be available for leaf respiration. However, no evidence was found to support this suggestion.

Direct effects of temperature on plant respiration

Plant respiration is temperature dependent and the change in rates to increasing temperature is commonly described by exponential functions. Such functions, may, however, be a too simplified way of describing the respiration/temperature relationship. The temperature sensitivity (Q10, the proportional change in the rate of respiration with a 10°C change in temperature) tends to decrease with increasing temperature when the temperature approaches the temperature opti-mum and further exceeds the temperature optimum (Tjoelker et al. 2001b, Chapter 4, 6 & 7). An exponential function can be a sufficient description of the respiration/temperature relationship as long as the range of temperatures is kept

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Temperature (oC)

10 20 30 40

Res

pira

tion

(µm

ol C

O2

m-2

s-1

)

0

1

2

3

4

well below the temperature optimum. It should then be kept in mind that ex-trapolations are very difficult, if not impossible. Alternatively, a changing Q10 can be incorporated in the respiration/temperature relationship (Tjoelker et al. 2001b), where the decreasing temperature sensitivity with increasing tempera-ture is taken into account. Even in a range of temperatures below the temperature optimum an ex-ponential fit can be insufficient. An example is shown in Figure 1.

Figure 1. Temperature sensitivity of respiration. Dark respiratory CO2-release by leaves of Plantago lanceolata L. at 350 µmol mol-1 CO2. Values are mean of three replicate plants. The line is fitted with a sigmoidal function; R=3.76/(1+exp(-(x-26.47)/6.19)), where R is respiration and x is temperature. The corresponding Q10-values are shown in Chapter 4. (Bruhn D, Mikkelsen TN & Atkin OK, unpublished).

It is clear from the example in Figure 1 that even below the temperature optimum an exponential fit can be too simplified as a description of the respira-tion/temperature-relationship. The respiration/temperaturerelationship takes a more or less sigmoidal shape. Others have found the same on ground area basis (Rice ecosystem, Jeffrey Baker pers. comm.), unit whole plant dry weight basis (Ziska and Bunce 1998), and at leaf level (Miller et al 2001). As a consequence we may get Q10/temperature relationships as in Tjoelker et al. 2001b and Chap-ter 4, 6 & 7. Figure 2 shows the temperature sensitivity of four dehydrogenases in the potato tuber mitochondrial electron transport chain. Q10 for diffusion rates of O2 and CO2 is about 1.1 (in water; Salisbury and Ross 1992) and to a great ex-tent temperature responses of enzymatic activity may therefore explain the tem-perature dependent Q10 of respiration at the whole-tissue level.

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Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Q10

0.0

0.5

1.0

1.5

2.0

2.5ex. NADH

ex. NADPH

int. NADH

int. NADPH

Figure 2. Temperature sensitivity of four potato tuber mitochondrial NAD(P)H dehydrogenases vs. temperature. The internal NAD(P)H dehydrogenases are rotenone insensitive and not to be confused with complex I. Calculated Q10-values are plotted for each of the four dehydrogenases vs. temperature. Q10 is calculated as exp( dln(R)/dT × 10), where dln(R)/dT is the ln-transformed rates of O2 uptake by the individual dehydrogenases. The dashed line indicates Q10 = 1 indicating the temperature optimum. A Q10-value above the dashed line indi-cate increasing O2 uptake with increasing temperature, whereas a Q10-value below the dashed line indicate decreasing O2 uptake with increasing tempera-ture. The dotted line represents a constant Q10-value of 2, which commonly is reported for respiratory activity. (Bruhn D & Møller IM, unpublished).

Another way of illustrating the changing temperature sensitivity of res-piration with measurement temperature is by expressing the respira-tion/temperature relationships in the so-called Arrhenius plots, where the log-transformed rates of respiration is plotted vs. the reciprocal of the absolute tem-perature in Kelvin [Log (rate of respiration) = a – b × (1/temperature)]. When doing so, linear regressions to various parts of the respiration/temperature-relationship can be fitted. Potential breaks between the linear regressions will indicate a change in the temperature sensitivity of the respiration. By this method Crawford and Palin (1981) demonstrated changes in the temperature sensitivity at inter-specific breakpoints of root respiration in a few species. Con-sequently, one is assuming a constant Q10 between each of the breaks. The Ar-rhenius plot has been applied in investigation of numerous plant physiological processes. It is possible to derive the activation energy for the process under investigation from the slopes of the Arrhenius plots (the minimal energy re-quired for the process to occur; Salibury and Ross 1992). The problem with applying Arrhenius plots is that the log-transformed rates have to be maximum activities. This can be difficult to ensure in intact mi-tochondria let alone experiments at the tissue level. In addition it can be difficult to interpret derived activation energies for a process such as respiration at the whole tissue or organ level.

The temperature sensitivity of respiration below the temperature opti-mum is expected to show at least one change well below the temperature opti-mum for many tissues. This is because as Johnson and Crawford (1981 in press;

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cited in Crawford and Palin 1981) suggested, a break in the temperature sensi-tivity can be caused by conformational changes in enzyme structures due to the minimum strength of the peptide bond at 18°C. Likewise is their also a potential physiological explanation for the upper breakpoints as they may be related to protein denaturation. Thus, we may expect that the respiration/temperature-relationship at whole tissue level or at higher levels of integration more or less takes a sigmoidal shape below the temperature optimum, after which it will take a steep decline. Atkin et al. (2000) discuss that the control of the rate of respiration at moderate temperatures is mostly due to adenylate control and that at low or high temperatures the enzymatic capacity has a greater role. This is supported by work on root respiration by Covey-Crump et al. (2002) and data in Figure 2. The short-term temperature response of respiration at the tissue level will de-pend on ‘changes in the degree of control exerted by individual steps in the res-piratory apparatus’ (Atkin et al. 2002). The fluidity of the inner mitochondrial membrane may constitute an important flux control of the overall respiratory flux in mitochondria (Miquel Gonzàlez-Meler pers. comm.). Gonzàlez-Meler et al. (1999) investigated the temperature sensitivity of the Cyt pathway and the Alt pathway. Exactly where the regulation takes place is difficult to say as ‘ef-fects upstream of the UQ pool are just as likely to be involved as effects on the alternative oxidase’ (Gonzàlez-Meler et al. 1999). Atkin et al. (2002) found that the Q10 for the Cyt pathway and the Alt pathway varies with UQr/UQt (without adenylate restrictions) and at low UQr/UQt, a greater proprtion of control over electron flow is likely to be exerted by the UQ reducing pathways (e.g. Com-plex I, the above mentioned- and the succinate dehydrogenases) than by the UQ oxidizing pathways.

Indirect effects of temperature on plant respiration

Here indirect effects of temperature on plant respiration are meant as long-term effects of growth at a changed growth temperature. The reason for this termi-nology is to keep it similar to that of the discussion of CO2 effects (sensu Am-thor 1997). Thus, respiratory acclimation to temperature is here treated as an indi-rect effect of temperature. Investigation of respiratory acclimation to tempera-ture may be interesting either for predictions about plant respiration in a future climate and/or for an understanding of the mechanisms. In both cases it will be useful to be able to determine the degree of acclimation and in Chapter 7 differ-ent new methods for calculation of such is discussed. In both cases it would be helpful to know about the timing; that is to make relationships between the de-gree of acclimation and time. In Chapter 6 are further discussed the different hypothetical types of growth temperatures and their influence on modelling plant respiration to climatic data, as well as the timing of acclimation.

Before commencement of any experiment concerning respiratory ac-climation to changed growth temperatures it is therefore necessary to make clear exactly what is of interest for further conclusions. As such it may be necessary to firstly determine the type of growth temperature to which respiration accli-mates for the particular species under investigation (see Chapter 6). This is be-cause as investigator one has the option as well as the ‘load’ of choosing the diurnal variation in temperature (if any), which then constitute the treatment in the particular experiment. As an example nothing is achieved if the treatments are changes in one type of growth temperature when the studied plant species is actually acclimating to another type of growth temperature. One could argue

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that in either case the potential respiratory acclimation is investigated no matter the response. This is, however, not true simply due to the fact that, as mentioned above, the investigator has to make a choice of the diurnal variation in the ap-plied temperature during the treatment and therefore a clear idea of the potential outcome of the experiment. It may not always be possible to do, but the investi-gator then has to be aware of the limited possibilities of any type of extrapola-tions from the results achieved.

Since both atmospheric- and soil temperature fluctuates throughout the day, it may be of limited use to investigate the types of growth temperature as done in Chapter 6. Nevertheless, the different types of growth temperatures can still be estimated; it just gets increasingly more complicated. Other types of growth temperatures can be hypothesised; e.g. the difference between mini-mum- and maximum temperature or the difference between the daily mean- and nightly mean temperature etc.

In Chapter 6 it was proposed that respiratory acclimation may depend-ent on the balance between assimilation and use of carbon and the respective temperature responses. Criddle et al. (1997) elaborated this and found a good correlation between the temperature response of growth and respiration, which again depended on the temperature response of the carbon conversion effi-ciency, a measure of the metabolic efficiency. Dewar et al. (1999) proposed a substrate-based model where the temperature response of the rate of respiration follows that of photosynthesis, resulting in a relatively temperature insensitivity of the respiration/photosynthesis-relationship. This is caused by a steady-state behaviour of the pools of non-structural carbohydrates and proteins (Dewar et al. 1999). This relatively temperature insensitivity of the respira-tion/photosynthesis-relationship has also been reported by others (Gifford 1995, Ziska and Bunce 1998), with increasing changes in temperature just giving slightly longer instability of the ratio of respiration to photosynthesis (Gifford 1995). It has also been shown that within a 24h period both soil temperature and photosynthesis (substrate supply) apparently caused the diurnal variation in root + soil respiration for beech seedlings growing in four different growth tempera-tures relative to ambient conditions (Leverenz et al. 1999). All together this may help explaining the relatively damped responses in relative growth rates of plants to different temperature regimes relative to ambient conditions (Bruhn et al. 2000).

Respiratory acclimation to temperature may also be influenced by other environmental factors. As examples are the interaction with soil water content (Bryla et al. 2001), management (e.g. clipping of grass prairie; Luo et al. 2001), and atmospheric [CO2] (Leverenz et al. 1999) all of which make predictions of respiratory release of CO2 in a future climate difficult. The method used to differentiate between the hypothesised growth temperatures in Chapter 6 can to a great degree be influenced by the timing of the measure-ments. We have in Chapter 6 chosen to let plants ‘adjust’ for 1h at the meas-urement temperature before commencement of measurements of dark respira-tion in the leaves. It is possible that the results would have differed some if this period was changed as the short-term temperature response can change within hours for some species after a change to another surrounding air temperature (Gale 1982, Lawrence and Holaday 2000). In addition, it should be kept in mind that the measurements reported in Chapter 6 is specific rates of respiration on a leaf area basis and different temperature responses may be expected at the whole-canopy level (Xiong et al. 2000). Further complications arises if a sea-sonal component of respiratory acclimation is incorporated (Pilon and San-tamaría 2001) and lastly, some species does not necessarily exhibit an increased rate of respiration after growth at low temperatures (Gonzàlez-Meler et al. 1999).

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The above-mentioned points do not, however, change the major conclu-sions in Chapter 6; i.e. that all plants do not necessarily acclimate to the mean daily temperature and this in combination with the speed of acclimation is im-portant for modelling plant respiration to climatic data. Equally do they not change the conclusion that the relatively temperature insensitivity of the above-mentioned respiration/photosynthesis-relationship may be caused by different degrees of control in different species/tissues at various points in the allocation of assimilates.

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Conclusions

The direct inhibitory effect of atmospheric [CO2] on plant respiration in the range relevant to the climate change discussion may have been overestimated in previous studies if the potential CO2-leakage problem between cuvette and sur-rounding air has been prevailing. This applies for measurements of both whole-plant respiration and that of organs/tissues. Neither do the direct inhibitory ef-fects of [CO2] on respiratory activity by intact mitochondria seem to be a gen-eral phenomenon at levels relevant to the climate change discussion. It can, however, differ to a great extent between species and tissues. Also, the respira-tory enzymes differ in their susceptibility to [CO2] between species and tissues. As known for succinate dehydrogenase, it appears also to be bicarbonate rather than [CO2] per se that affects the activity of cytochrome c oxidase in the mito-chondrial electron transport chain. The indirect inhibitory effect of atmospheric [CO2] on plant respiration reported in many studies has been confirmed in this project. The indirect effect is likely to be simply the result of accelerated growth and thereby accelerated ontogeny. Several other possible mechanisms, may, however, play a role; these include altered tissue chemistry and thereby construction costs. A further under-standing of the indirect effects of [CO2] clearly awaits more process-based in-vestigations. The direct effects of temperature on plant respiration are well known, but perhaps too simplified described till now in some cases. The temperature sensi-tivity of respiration can depend on measurement temperature, species and/or growth conditions. Even below the temperature optimum for respiration may an exponential fit be too simplified a description of the temperature sensitivity and there appears to be plausible underlying physiological/biophysical mechanisms for a more or less sigmoidal shaped respiration/temperature-relationship. In ad-dition, there is now a need for investigation of the underlying mechanism(s) of the apparently less temperature sensitivity of mitochondrial respiration in the light compared to that in the dark. This reduced temperature sensitivity of mito-chondrial respiration in the light is furthermore clearly necessary to take into account when e.g. modelling exchange of CO2 between vegetation and the at-mosphere. The indirect effect of temperature on plant respiration can result in a respi-ratory acclimation to a changed growth temperature. Respiration in darkness does not, in all plant species/tissues, acclimate to a daily mean temperature and leaf respiration in the light may acclimate more likely to the warmer types of growth temperatures due to different mechanisms than respiration in darkness. This has important implications for modelling of respiratory responses to cli-mate change as well as for the understanding of lab experiments; equally does the timing of acclimation. Estimating the timing of acclimation requires an un-equivocal way of calculation for comparison between studies. New such meth-ods of calculations are here proposed depending on the use; it makes sense to distinguish between the methods for investigation of underlying mechanisms and methods for studies with modelling climate change responses in view.

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Pons TL and Welschen RAM (2002) Overestimation of respiration rates in commercially available clamp-on leaf chambers. Complications with measure-ment of net photosynthesis. Plant, Cell and Environ In press. Poorter H, Gifford RM, Kriedemann PE and Wong SC (1992) A quantitative analysis of dark respiration and carbon content as factors in the growth response of plants to elevated CO2. Aust J Bot 40: 501-513. Salisbury Fb and Ross CW (1992) Plant Physiology, 4th ed. Wadsworth Publish-ing Company, Belmont, California, 682 p. Thibaud M-C, Cortez N, Rivière H and Betsche T (1995) Photorespiration and related enzymes in Pea (Pisum sativum) grown in high CO2. J Plant Physiol 146: 596-603. Tjoelker MG, Oleksyn J & Reich PB (2001) Modelling respiration of vegeta-tion: evidence for a general temperature-dependent Q10. Glob Chan Biol 7, 223-230. Tjoelker MG, Oleksyn J, Lee TD and Reich PB (2001) Direct inhibition of leaf dark respiration by elevated CO2 is minor in 12 grassland species. New Phytol 150: 419-424. Wullschleger SD, Ziska LH and Bunce JA (1994) Respiratory responses of higher plants to atmospheric CO2 enrichment. Phys Plant 90: 221-229. Xiong FS, Mueller EC and Day TA (2000) Photosynthetic and respiratory ac-climation and growth responses of Antarctic vascular plants to contrasting tem-perature regimes. Am J Bot 87: 700-710. Ziska LH and Bunce JA (1998) The influence of increasing growth temperature and CO2 concentration on the ratio of respiration to photosynthesis in soybean seedlings. Glob Chan Biol 4: 637-642. Ågren GI (1994) The interaction between CO2 and plant nutrient: comments on a paper by Coleman, McConnaughay and Bazzaz. (1994). Oecol 98: 239-240.

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Future Research

The conclusions from the studies on potential climate change effects on plant respiration in this Ph.D. project clearly reveals that the goal is far from reached, i.e. a full understanding of the underlying mechanisms and tools to predict plant respiratory CO2 release in a future climate. Merely the control of plant respiration obviously needs more investiga-tion. This includes respiration in both short- and long-term scenarios of climate change. In the short-term scenarios the mechanisms investigated may further contribute to the understanding of control of respiration in general. In long-term scenarios respiration has to be investigated along with a number of associated changes; photosynthesis, growth, and ontogenetic changes has to be followed concomitantly. This is in order to understand the function of plants in a future climate. Elaboration of models of plant respiration is therefore necessary and process based investigations of plant respiratory responses to climate changes may be a way. For future climate change studies of plant respiration four considera-tions has to be made. 1) The climate change per se – how does the climate change influence diurnal and seasonal fluctuations of e.g. temperature and at-mospheric [CO2]. Already at the present climate do fluctuations of temperature and atmospheric [CO2] change plant physiological processes. 2) Underlying mechanisms – we may not get very far if not great efforts are done to track a range of potential associated changes in other processes during the time of the experiment and the timing per se may be fruitful to incorporate. 3) Climatic fac-tors as selective agents – the intra- and interspecific competition may com-pletely change the responses found in the laboratory if e.g. temperature and at-mospheric [CO2] are important selective agents. 4) Up-scaling - FACE experi-ments and other attempts to upscale plant responses may be a route to test the importance of the many potential effects found in the laboratory compared to other environmental factors. Nevertheless, predictions of e.g. plant respiratory CO2 release in a future climate is complicated by the lack of knowledge about how species and/or ecosystems react to the relatively slow climate changes in nature compared to that imposed in the lab.

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Acknowledgements

Despite the very strong solitary feeling during the work of this PhD dissertation I am very grateful to numerous people: Firstly I wish to thank my two supervisors Teis N. Mikkelsen and Helge Ro-Poulsen who have taken care of many of the tedious administrative jobs throughout the course of this project. Teis and Helge have also contributed as co-authors and/or reviewers on some of the chapters in this thesis. They have more or less let me choose my own paths, which has been liberating but cer-tainly also challenging. In the very beginning of my PhD project also Lennart Rasmussen played a great part in the administration.

Secondly I wish to thank Owen K. Atkin whom during most of the time has acted as my distant mentor and taught me a lot about plant respiration. After I initially contacted Owen about potential PhD courses he fearlessly invited me to York (UK) to do research in his lab. We agreed on a three months stay; now I have been there four times for a total of half a year (most of it in 2001). Owen has contributed as co-author on several of the chapters in this thesis; he im-proved the English as well as the scientific parts to a degree that hurts to admit. Owen is far and away my most used recipient of e-mails to this date.

Owen, his colleagues, students, post docs, and family have all been wonderful hosts during my stays in York. In the lab I got help in particular by David J. Sherlock (whom in fact came to Risø to help me out with the project, which resulted in Chapter 5), Elizabeth M. Covey-Crump, Beth R. Loveys, and Everard J. Edwards. Outside the lab, everybody helped me enjoying the life in England.

After the start of my PhD project April 1999 I visited Hans Lambers in Utrecht (The Netherlands) for a week in May 1999. Hans devoted some time at this early stage of my project to discuss plant respiration in general and briefly introduced me to equipment used in the respiration research. Some of Hans’ students continued this by telling me about their own projects. Hans made Tjeerd Bouma send me a copy of his PhD thesis and this initiated a contact for some time where Tjeerd tirelessly answered many of my questions regarding process-based investigation of climate change effects on plant respiration. This helped me very much during my first own experiment at Risø; this project un-fortunately suffered some significant technical problems why it was discarded afterwards. Tjeerd also invited me to his lab to do some research, but due to my fumbling time management I never did visited Tjeerd. During the same time Hendrik Poorter kindly helped me answering several theoretical questions on respiration related matters. For this first (unfortunately discarded) experimental project at Risø I borrowed much equipment from Carl-Otto Ottosen and Eva Rosenqvist (Åslev, DK) as well as for a later project. Carl-Otto and Eva has al-ways kindly hosted me at several visits at Årslev and commenting on my ideas as well as telling me about their own projects.

During the autumn 1999 Mayra E. Gavito and Iver Jakobsen kindly in-vited me to participate in their excellent planned project, which resulted in Chapter 3. For this I am very grateful as the first experimental experience with plant respiration was gathered in then. Mayra also contributed to Chapter 1 as co-author.

Late 2000 Jes Fenger invited my to write a chapter to the book, Climate Change Research – Danish Contributions, which he edited together with Anne Mette K. Jørgensen and Kirsten Halsnæs. For this opportunity I wish to thank the editors and also for the co-operation with the co-authors who are Teis N.

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Mikkelsen, Kim Pilegaard, Mayra E. Gavito, and Henrik Saxe. Helge-Ro Poul-sen and Lise Bolt Jørgensen I thank for a critical review and light-microscopy photos to the text, respectively.

Ian Max Møller invited me to Lund (Sweden) for two weeks in Decem-ber 2000, where I took part of a project with Max and some of his PhD students. The project was later discarded in its original form. However, I wish to thank for this opportunity and some of the preliminary results from that project are included in Chapter 9. I also wish to thank Max for the opportunity of discus-sions of many plant respiration matters on a daily basis after Max moved from Lund to Risø as well as his critical review of Chapter 2 and parts of Chapter 9.

During my second stay in York I met Joseph T. Wiskich who taught me quite a few tricks in mitochondrial isolation methods and various assays. Joe has put up with many questions from me about work with isolated mitochondria. Later Joe and Sally Box agreed on sharing of results from their related projects on direct effects of [CO2] on mitochondrial respiration and this resulted in Chapter 5. This has been a great opportunity despite the fact that I have never met Sally. I am also grateful to Henning F. Christensen for discussion about CO2-treatments.

During the summer of 2001 Ebba Dellwik spend much time with me at the Sorø EUROFLUX field site in Lille Bøgeskov where we harvested beech branches for the experiment, which are described in Chapter 8. We also made use of data obtained by Teis N. Mikkelsen, Helge-Ro Poulsen, Kim Pilegaard, Mathias Herbst, and Werner L. Kutsch. Mathias and Werner are yet another two co-authors whom I still have to meet. Also Stephen P. Long, Carl J. Bernacchi, Eva Falge and Bernard Genty helped me by answering specific questions about gas exchange measurements and calculations for this project.

During the autumn of 2001 David S. Sherlock, Abir U. Igamberdiev, and Teis N. Mikkelsen helped me carrying out the experiment, which resulted in Chapter 8 together with modeling by Richard G. Attwood and Owen K. Atkin. Richard is yet another co-author whom I have never met. Ib Michael Skovgaard and Cherry Nielsen helped me with some statistics and by applying seeds, re-spectively.

January 2002 I visited five respiration minded labs in the USA. During these two weeks I was hosted by Miquel A. Gonzàlez-Meler, David S. Ells-worth, James A. Bunce, Lewis H. Ziska, Jeffrey T. Baker, Michael G. Ryan, and Mark G. Tjoelker, respectively. At the five labs I visited I got the opportu-nity of giving a seminar about my findings during this PhD project and discuss my results with all the hosts as well as Jeffrey S. Amthor and Bert G. Drake whom both kindly showed up at my seminar at Beltsville. All of the host, their students and families made me have a great time during those two weeks, which was very inspiring at the beginning of the period of writing up the thesis back in DK.

I also wish to thank everybody at PLE-PRD Risø for a great time during the past three years. This is especially Vagn A.T. Nielsen, René Petersen, and Poul T. Sørensen for technical assistance with operating growth chambers and sowing of plants and Claus Beier, Per Ambus, Teis N. Mikkelsen, and Kim Pilegaard for the shared authorship of the two articles in the Appendix.

Lastly I wish to thank friends and family for occasionally pretending an understanding of my ideas and interest in my projects.

Bibliographic Data Sheet Risø-R-1332 (EN) Title and authors

Plant Respiration and Climate Change Effects By Dan Bruhn ISBN ISSN 87-550-3032-7; 87-550-3033-5 (Internet) 0106-2840 Department or group Date

Plant Research Department Plant Environment Interactions Programme 22 April 2002 Groups own reg. number(s) Project/contract No(s)

Sponsorship

The Danish Research Academy, European Science Foundation, and Nordic Academy of Advanced Study Pages Tables Illustrations References 141 10 51 258 Abstract (max. 2000 characters)

Plant respiration is one of the key processes in terms of an understanding of plant growth and functioning in a future climate. Short- and long-term effects of temperature and [CO2] on plant respiration were investigated in a number of plant species. The ex-periments tested effects of either temperature and/or [CO2] from the level of individual respiratory enzymes, isolated mitochondria, whole-tissue, and up to the whole canopy level. The short-term effects of elevated atmospheric [CO2] on plant respiration ap-peared to be less than suggested so far in the literature. This was true both at the tissue-level and for intact mitochondria. Respiratory enzymes can, however, be affected al-ready at low [CO2]. These effects did not manifest itself at the tissue level, though, due to low degrees of control on the whole respiratory process exerted by the particular en-zymes. Plant respiration on the other hand was affected by long-term growth at elevated atmospheric [CO2]. The findings of the reduced plant respiration at the leaf level were consistent with the literature and potential causes are discussed. Short-term effects of temperature on plant respiration were demonstrated to be dependent on the actual meas-urement temperature. Further, it is shown that mitochondrial leaf respiration in darkness and light differ substantially in the temperature sensitivity with the former being the far most sensitive. This has implications for modelling CO2 exchange between vegetation and atmosphere as demonstrated here, since this has so far been neglected. Long-term effects of temperature resulted in respiratory acclimation in a number of species. Respi-ratory acclimation appeared not to occur to any one single type of growth temperature. The implications of this finding in combination with the timing of acclimation are dis-cussed for modelling respiratory CO2 release. Descriptors INIS/EDB AMBIENT TEMPERATURE; CANOPIES; CARBON DIOXIDE; CLIMATIC CHANGE; ENZYMES; MITOCHONDRIA; PLANT TISSUES; PLANTS; RESPIRATION; TEMPERATURE DEPENDENCE Available on request from Information Service Department, Risø National Laboratory, (Afdelingen for Informationsservice, Forskningscenter Risø), P.O.Box 49, DK-4000 Roskilde, Denmark. Telephone +45 4677 4004, Telefax +45 4677 4013

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