We investigated the extent to which variations in the temperaturesensitivity of root respiration are associated with variations insubstrate supply and/or adenylate restriction of respiratoryflux. In general, Q10 values were higher in the presenceof uncoupler and/or exogenous glucose than their absence,particularly in warm-grown roots. This suggests that the Q10 isdependent in part on the degree of adenylate restriction and/orsubstrate availability. Our results also demonstrate that the Q10 ofroot respiration varies with measurement temperature and that itincreases following cold-acclimation to the overnight lower temperature. Variationsin physiological state, measuring temperature and/or growthtemperature may therefore explain why Q10 valuesvary so much in previous studies (James 1953; Forward 1960; Azcón-Bieto1992; Atkin et al. 2000a; Tjoelker, Oleksyn & Reich 2001; Atkin et al. 2002; Bruhn et al. 2002a).
Theregulation of respiration varies with temperature
Restriction by adenylates and substrate supply are importantmechanisms by which respiratory flux is regulated in many plantspecies. Our results suggest that the proportion of control exertedby adenylates and substrates changes with temperature. At low temperatures(i.e. below 15 °C) the addition of CCCP and/or exogenous glucose resulted in little stimulation of respiration for both P. lanceolata and P. euryphylla (Fig. 1a). This suggests that the degree of control exerted by adenylates and substrates de-creasedin the cold. We had initially hypothesized that adenylate limitation of respiratory flux would increase (i.e. stimulation by CCCP would increase) as temper-aturesdecreased because processes such as ion uptake, growth and maintenance processes (which require res-piratoryATP) are likely to slow down in the cold. Moreover, membranes becomeless fluid at low temperatures, which should result in respiration being more coupled. Why was there an apparent decrease in adenylate re-strictionat low temperatures? Increased flux via the alternative pathwaywas unlikely to be responsible for the apparent decrease in adenylaterestriction at low temperatures (see Introduction). Furthermore,respiration was unlikely to be limited by substrate availabilityat low temperatures, as the addition of glucose did notstimulate respiration in the cold (Fig. 1a& b). Rather, respiratory flux was most likely limitedby enzymatic capacity at low temperatures (resulting inlittle stimulation by CCCP or glucose).
To definitely determine whether respiratory enzyme capacity limitsrespiratory flux at low temperatures, data is needed on the maximumpotential flux of the respiratory apparatus in intact tissues atlow temperatures. This can be obtained via measurements of respirationin isolated mitochondria, in the presence of saturating substratesand ADP. Mitochondrial rates can then be scaled up to represent ratesper gram of root tissue using a mitochondrial marker enzyme suchas fumarase (e.g. Van Emmerik, Wagner & Vander Plas 1992). Unfortunately, we were unable to successfullyisolate intact mitochondria from the roots of P. lanceolata or P.euryphylla, due to the fibrous nature of the roots. However,a recent study by Atkin et al.(2002) showed that the Q10 for in vivo respiration(1·9) is lower than the Q10 for substrate-saturatedmitochondria (2·4) in soybean cotyledons. Therefore therespiratory temperature response curves for substrate-saturatedmitochondria (i.e. the maximum potential respiratory flux) and invivo respiration converge as the temperature decreases. Thus,it seems likely that respiratory enzyme capacity increasingly limitsrespiratory flux at lower temperatures.
Our results suggest that adenylates and/or substrateslimited root respiration at moderate temperatures (i.e. 15–35 °C; Fig. 1), as reportedpreviously (see Introduction). Importantly, however, our resultsdemonstrate the effect of CCCP or exogenous glucose was not constantover this temperature range (Fig. 2a–d),with maximal stimulation of respiratory flux occurring at 28 and20 °C in P. lanceolata and P. euryphylla,respectively. At these temperatures, respiration was likely to bemore limited by adenylates [(either high ATP : ADPratio or low ADP concentrations per se) (Wiskich& Dry 1985; Hoefnagel et al.1998; Loef et al. 2001)] thansubstrate availability, as the stimulatory effect of glucose waslower than that of CCCP (Fig. 2).Moreover, rates in the presence of CCCP were near maximal, as the additionof glucose in the presence of CCCP resulted in near identical ratesof respiration in P. lanceolata compared with experimentsin which CCCP was added alone (Fig. 3). Above28 or 20 °C (i.e. for P. lanceolata and P.euryphylla, respectively), the degree of stimulation by additionof CCCP was reduced (Fig. 2a& b) with absolute rates of uncoupled respiration decreasingabove 40 °C. A reduction in adenylate controlas temperature increases has been reported in potato tubers (Geigenberger, Geiger & Stitt 1998). Inthat study the PEP : pyruvate ratio increasedby 50% at high temperatures indicating that the activityof adenylate regulated enzymes converting PEP to pyruvate (PK) ormalate (PEPC) were stimulated (Geigenberger et al.1998). Metabolic control measurements on isolated mitochondriaalso suggest that as the rate of flux increases towards the maximumpossible flux, i.e. state 3, the degree of control exerted by thephosphorylation reactions decreases, whereas control exerted bythe respiratory chain increases (Diolez et al.1993). Therefore, as temperature causes an increase in fluxthrough the pathway it is possible that adenylate control decreasesdue to a decrease in the proportion of control exerted by the phosphorylationreactions. In addition, the levels of NADH are likely to rise as fluxvia the TCA cycle increases. This could potentially cause greaterflux through the rotenone-insensitive bypass, allowing unrestrictedflow around the adenylate-restricted complex I (Lambers& Atkin 1995). The decrease in uncoupled respirationabove 40 °C probably reflects the stimulatoryeffect of high temperatures on proton permeability of the mitochondrialinner membrane (Lin & Markhart 1990). Veryhigh temperatures are also likely to have a detrimental effect onthe enzyme activity (Berry & Raison 1981), andphysiologically active plant cells are likely to die at about 45–55 °C(Forward 1960).
Although our results suggest that adenylates were more limitingat moderate temperatures than substrate supply (see above), we areunable to definitively state the proportion of control exerted byadenylates versus substrate supply at moderate temperatures. Thisis because the addition of CCCP to roots has a dual effect: first,CCCP dissipates the proton motive force across plant membranes,including the inner mitochondrial membrane. This results in thecessation of ATP production and an increase in ADP availability(thus resulting in respiration not being limited by adenylates inthe presence of CCCP). Second, CCCP may result in additional fluxthrough glycolysis and concomitant increase in substrate supplyto the mitochondria. The addition of exogenous glucose will alsohave a dual effect; not only will it potentially increase substrateavailability but also it can result in a slight decrease in adenylaterestriction as ATP will be consumed for transport of glucose intothe cell (Hatzfeld & Stitt 1991). Despitethese limitations, we feel confident that the addition of CCCP andexogenous glucose provides an indication of how changes in temperaturealter the extent to which respiration is limited by adenylate restrictionand/or substrate supply. This is because, inpotato leaf protoplast cell fractions, CCCP dramatically reducesthe ATP : ADP ratio in the cytosolic and mitochondrialfractions over a range of temperatures (Covey-Crump, Bykova, Gardeström& Atkin, in preparation). Our use of CCCP and glucose to estimatetemperature-mediated changes in respiratory capacity is similarto that of Noguchi, Nakajima & Terashima(2001) who used them to estimate changes in leaf respiratorycapacity during acclimation to contrasting irradiances.
The data in Figs 1and 2 were fitted with third-order polynomials; such fitswere chosen as they allowed for decreases in the temperature coefficientof respiration (i.e. decreases in Q10 values)with increasing measuring temperature. The fits exhibited high R2 values(within the range 0·83–0·98, with themajority being above 0·96) and were likely to be theirmost accurate in the middle of the temperature range. However, cautionis needed when interpreting the shape of the polynomial fits atthe temperature extremes, as third-order polynomials are less accurateat the ends of their fit. For example, the fits suggest that rates ofroot respiration are higher at 5 °C than at 10 °C. Althoughhigher rates at low measuring temperatures are possible (other studieshave reported unusually high rates at low measuring temperatures; Forward 1960; Atkin et al. 2000b),it is equally possible that the apparent increase in O2 uptakeat 5 °C was an artefact of the third-order polynomials.Measurements below 5 °C are needed to check whetherthe polynomial fits are correct.
Howdoes the response of respiration to temperature vary between a fast-and slow-growing species?
When comparing the response of respiration to temperature inthe faster-growing P. lanceolata versus the slower growing P.euryphylla, it might be expected that the respiration ratesfor the slow grower would be less due to a lower demand for ATPfor growth. However, respiration was similar for both species overthe temperature range. This similarity may be due to greater specificrespiratory costs associated with ion uptake in the slow grower(Scheurwater et al. 1998).The stimulation of respiration by the addition of CCCP or glucosedid however, vary between the species. Respiration in P. euryphylla wasstimulated to a lesser extent after the addition of CCCP or glucosecompared to P. lanceolata (Fig. 2a–d).This suggests that flux through the respiratory pathway in P.euryphylla may be operating closer to the maximum capacity comparedwith P. lanceolata. However, decreased stimulation by addition ofan uncoupler may also reflect the fact that the slow-growing specieshas a greater relative requirement for ATP (e.g. to meetthe demand for an inefficient ion uptake system; Scheurwater et al.1998, 1999) and is therefore less adenylate restricted.
Despite the difference in the absolute effect of CCCP betweenthe species, the pattern of adenylate/substrate controlwas generally similar, i.e. respiration was strongly regulated byadenylates and/or substrate supply at moderate temperatures,but at low and higher temperatures restrictions by adenylates/substratesupply decreased. Conversely, respiratory capacity appearedto be the primary factor limiting respiratory flux atlow and high temperatures.
Acclimationof respiration to a new growth temperature
We have demonstrated that P. lanceolata root respiration acclimatedto 7 d exposure to a lower growth temperature and thatregulation of respiration differed between the cold-acclimated and23 °C-grown plants. Respiration rates in the cold(e.g. measured at 5–10 °C) were similarin both the 23 °C- and 15/10 °C-grownplants (Fig. 4a).However, exposure to higher temperatures had a greater stimulatory effecton respiratory flux in the cold-acclimated plants than in theirwarm-grown counterparts, resulting in higher Q10 valuesin the 15–25 °C range (Fig. 5). A similarpattern was also seen in the study measuring soil and root respiration overa year in warmed and unwarmed soil plots (Luo et al. 2001).This suggests that in many cases, acclimation to a new temperatureregime resulted in no change in the respiration rates at low temperatures,but higher Q10 values for plants experiencingcolder growth temperatures during the diurnal cycle.
The higher rates of respiration exhibited by the cold-acclimatedroots (when measured at moderate temperatures) could potentiallyhave been due to a decrease in the degree of adenylate restrictionin the cold-acclimated plants, possibly due to a higher demand forATP. Alternatively, the higher rates may have been due to a higherlevel of carbohydrates present in the cold-acclimated tissues (Table 1). Exposureto a low growth temperature commonly results in an accumulationof higher levels of soluble carbohydrates (e.g. Atkin et al.2000a). Moreover, Q10 values are higherin leaves that exhibit higher levels of sugars than in low-sugarleaves (Azcón-Bieto & Osmond 1983c). Supportfor the role that changes in sugar concentrations play in the acclimationresponse also comes from the fact that the addition of exogenousglucose to warm-grown roots resulted in a near identical temperatureresponse curve to that exhibited by the cold-acclimated roots (Fig. 4a &c). In addition, respiration was no longer stimulated by theaddition of glucose after cold-acclimation (Fig. 4c).We therefore suggest that increased substrate availability plays arole in determining the extent of cold-acclimation of respirationin P. lanceolata roots.
Over much of the temperature range used in our experiments, respirationin the presence of CCCP was similar in cold-acclimated and warm-grownplants (Fig. 4b).This suggests that the capacity of the respiratory system was unalteredafter 7 d growth at the lower temperature and that respirationin the cold was still limited by enzyme capacity in the cold-acclimatedplants. Further support for this suggestion comes from the factthat respiration rates measured in the cold (i.e. 5–10 °C)were similar in warm-grown and cold-acclimated roots. This conclusionappears to contrast with the results of previous studies which reportedthat cold acclimation is associated with increased number of mitochondriaand/or increased rates of respiration per unit mitochondrialprotein (Klikoff 1966, 1968; Miroslavov & Kravkina 1991). Moreover,protein levels and tissue nitrogen concentrations often increasein cold-acclimated plants (Graham & Patterson1982; Tjoelker, Reich & Oleksyn 1999b).Respiratory acclimation to changes in irradiance is also associatedwith changes in the abundance of some respiratory components (Noguchi et al. 2001). A possible explanationfor why we failed to observe evidence of an increase in enzyme capacityis that 7 d at the colder growth temperature was not longenough for changes in enzyme capacity to occur in the pre-existingroot tissues. Alternatively, pre-existing roots may be incapableof increasing the capacity of the respiratory system (as measuredin detached, whole roots) when challenged with a low growth temperature.Rather, the synthesis of new root tissues with enhanced respiratorycapacity may be needed. Work is currently under way in our laboratoryto assess whether newly developed, cold-grown tissues do in factexhibit higher respiratory capacity than warm-grown tissuesthat cold acclimate.
Q10valuesincrease following cold-acclimation
Our results suggest that the Q10 of respirationis greater in cold-acclimated roots than in their warm-grown counterpartswhen compared at moderate measurement temperatures (Fig. 5). This contrastswith Tjoelker et al. (2001)who concluded that the response of the Q10 ofleaf respiration to measurement temperature is consistent acrossa wide range of species (i.e. tropical to arctic) regardless oftheir growth temperature. Two factors may explain why our resultsdiffer from those of Tjoelker et al.(2001). First, the response of root respiration (our study)may differ from that of leaf respiration (Tjoelker et al.2001). Second, we compared the Q10 of 23 °C-grownand maintained roots with that of warm-grown roots moved to 15/10 °Cfor 7 d. In contrast, Tjoelker et al.(2001) compared respiration of tissues developed under differenttemperature environments. Thus, whereas the Q10 oftissues developed under contrasting conditions is often similar(Tjoelker et al. 2001), differencescan occur when pre-existing tissues acclimate to a new thermal regime.Constant and variable Q10 values would reflectmaintenance and changes, respectively, in the balance between substrateavailability, respiratory capacity and adenylate restriction inthe tissues developed under the contrasting temperatures.
Theimpact of different temperature regimes on acclimation
Our results suggest that P. lanceolata root respirationacclimates to the low night-time temperature and not the daily meanor high daytime temperature (Fig. 6),and that as part of the acclimation response, the Q10 increaseswhenever night-time temperatures are low (even when the daily averagetemperature remains the same). Conversely, the Q10 is lowerwhenever the night-time temperature increases (Fig. 7). If such adynamic response of the Q10 to changes in minimumtemperature occurs in nature, then such changes need to be consideredby global carbon exchange models. Failure to take into account acclimationand/or variations in the Q10 can resultin an over-estimate of the effects of global warming on respiratoryCO2 release over long periods (Atkin et al.2000a; Luo et al. 2001),particularly in models that assume a positive feedback of global warmingon respiration rates.
Why might respiration acclimate to the night-time temperaturesused in this study (Fig. 6)?One possibility is that the accumulation of soluble sugars is inverselycorrelated with the low night-time temperature (i.e. exposure tolow temperatures results in the accumulation of sugars). Indeed,our results demonstrate that cold treatment does result in an increasein the availability of respiratory substrates in P. lanceolata roots(Table 1). Presumably,the accumulation of sugars at low temperatures is not balanced outby exposure of roots to higher temperatures during the day (whichshould result in a decline in sugar concentration). Moreover, theproduction of additional sugars by photosynthesis is likely to beenhanced in those treatments with the higher daytime temperature(and thus lower overnight temperature). As a result, the steady-stateconcentration of sugars would be higher in the treatments with the highestdaytime and lowest night-time temperature.
Can our study be used to make general predictions for other species,tissues (i.e. leaves) and growth temperature regimes? Will(2000) found that root respiration did not acclimate to thedaily lowest temperature (or average). Moreover, a recent studyinvestigating a larger number of species and involving a numberof different temperature regimes, showed that respiratory acclimationand the acclimated Q10 values are highly variableamong and within species (Bruhn et al.2002b). Thus, whereas respiration clearly does not invariablyacclimate to the daily mean temperature, not all tissues acclimateto the daily lowest temperature. This is not surprising as our resultssuggest that acclimation is in part due to changes in substratesupply and ATP turnover and these are likely to differ within andamong species when exposed to different temperature regimes.