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Keywords:

  • Plantago;
  • acclimation;
  • adenylate;
  • regulation;
  • respiration;
  • temperature

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

This study investigates the effect of short- and long-term changesin temperature on the regulation of root respiratory O2 uptakeby substrate supply, adenylate restriction and/or the capacityof the respiratory system. The species investigated were the lowland Plantagolanceolata L. and alpine Plantago euryphylla Briggs, Carolin& Pulley, which are inherently fast- and slow-growing, respectively. Theplants were grown hydroponically in a controlled environment (constant23 °C). The effect of long-term exposure to lowtemperature on regulation of respiration was also assessed in P.lanceolata using plants transferred to 15/10 °C(day/night) for 7 d. Exogenous glucose and uncoupler (CCCP)were used to assess the extent to which respiration rates were limitedby substrate supply and adenylates. The results suggest that adenylatesand/or substrate supply exert the greatest control overrespiration at moderate temperatures (e.g. 15–30 °C)in both species. At low temperatures (5–15 °C),CCCP and glucose had little effect on respiration, suggesting thatrespiration was limited by enzyme capacity alone. The Q10 (proportionalincrease of respiration per 10 °C) of respirationwas increased following the addition of CCCP and/or exogenousglucose. The degree of stimulation by CCCP was considerably lowerin P. euryphylla than P. lanceolata. This suggeststhat respiration rates operate much closer to the maximum capacity in P.euryphylla than P. lanceolata. When P. lanceolata wastransferred to 15 °C for 7 d, respirationacclimated to the lower growth temperature (as demonstrated by an increasein respiration rates measured at 25 °C). In addition,the Q10 was higher, and the stimulatory effectof exogenous glucose and CCCP lower, in the cold-acclimated rootsin comparison with their warm-grown counterparts. Acclimation of P.lanceolata to different day/night-time temperatureregimes was also investigated. The low night-time temperature wasfound to be the most important factor influencing acclimation. The Q10 valueswere also higher in plants exposed to the lowest night-time temperature.The results demonstrate that short- and long-term changes in temperaturealter the importance of substrate supply, adenylates and capacityof respiratory enzymes in regulating respiratory flux.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Root respiration provides plants with the carbon skeletons andenergy (i.e. ATP and reducing equivalents) that are necessary forion uptake and the synthesis and maintenance of root biomass. Respirationalso releases substantial amounts of CO2, with 8–52% ofthe CO2 fixed by photosynthesis being released back intothe atmosphere by root respiration (Lambers, Atkin& Scheurwater 1996). The rate of respiration is likelyto change with fluctuations in temperature because respiration istypically temperature sensitive. However, Q10 values(i.e. the proportional increase in respiration for every 10 °Crise in temperature) are highly variable and range between 1·4and 4·0 (Azcón-Bieto 1992; Atkin, Edwards & Loveys 2000a; Atkin, Holly & Ball 2000b; Atkin,Zhang & Wiskich 2002; Bruhn, Mikkelsen& Atkin 2002a). Although it is not known why there issuch variability, it may be caused by differences in the effectof temperature on the factors that control respiratory flux.

Control of respiratory flux can be shared between the availabilityof substrates, the concentration of adenylates (ATP and ADP) andthe capacity of the respiratory enzymes. At moderate temperatures(e.g. 25 °C) it is unlikely that the capacityof respiratory enzymes is the primary factor limiting flux. Thisis because respiration rates measured in mitochondria with saturatingsubstrates are greater than those measured in vivo (Day & Lambers 1983; Wiskich& Dry 1985). Not surprisingly, adenylates play an importantrole in the regulation of respiratory flux at moderate temperatures(Saglio & Pradet 1980; Day& Lambers 1983; Williams & Farrar1990; Lambers & Atkin 1995; Geiger, Stitt & Geigenberger 1998). Ahigh ATP : ADP ratio or low ADP concentrationwill restrict flux through phosphofructokinase, pyruvate kinase,the pyruvate dehydrogenase complex and the electron transport chain(Wiskich & Dry 1985; Hoefnagel,Atkin & Wiskich 1998; Loef, Stitt &Geigenberger, 2001). Respiration can also be stimulated bythe addition of substrates such as glucose and sucrose (Noguchi & Terashima 1997; Geiger et al.1998), with variations in respiration often correlating withvariations in carbohydrate content. This suggests that substratesupply partly controls the rate of flux through the respiratorypathway (Breeze & Elston 1978; Azcón-Bieto, Day & Lambers 1983a).However, substrate additions do not always stimulate respirationin the short term (Noguchi & Terashima 1997; Geiger et al. 1998).

Little is known about how the regulation of root respiration(i.e. substrate supply, adenylate control, and/or maximumenzyme activity) is altered following short-term changes in temperature.Nevertheless, it is known that most reactions catalysed by enzymesexhibit a lower Q10 when the substrates are limiting(Berry & Raison 1981; Atkin et al. 2002).Moreover, Azcón-Bieto, Lambers &Day (1983b) reported that the Q10 of leafrespiration is greater in leaves containing high concentrationsof soluble carbohydrates. In contrast, Breeze& Elston (1978) and Atkin et al.(2000b) reported a negative or no relationship, respectively, between Q10 valuesand soluble carbohydrate con­centrations. Thus, differencesin the availability of respi­ratory substrates do not alwaysaccount for the observed variations in Q10 values.In such cases, variations in Q10 values mightresult from variations in the effect of temperature on adenylaterestriction of respiration. Previous ­studies have suggestedthat the proportion of flux via the non-phosphorylating alternativeoxidase (AOX) pathway increases following short-term exposure tolow temperatures (Smakman & Hofstra 1982; McNulty & Cummins 1987); increases inAOX activity might decrease the extent to which respiratory fluxis limited by adenylates. However, more recent studies have suggestedthat the proportion of flux via the AOX does not always increasein the cold, at least not in the short term (GonzàlezMeler et al. 1999; Ribas-Carbo et al.2000; Atkin et al. 2002).Therefore, it cannot be assumed that adenylate restriction willdecrease in the cold as a result of increased flux via the AOX.In fact, it seems more likely that the degree of adenylate control willincrease in the cold. This is because the demand for ATP in thecold will decrease as the rate of ATP-requiring processes (i.e.growth, ion uptake and maintenance) decrease. Moreover, plant membranesare less ‘leaky’ at low temperatures (Dufour et al. 1996). As aresult, mitochondrial respiration is more tightly coupled to protontranslocation and ATP turnover at low temperatures (e.g. Atkin et al. 2002). Finally,different Q10 values could also result from variations inthe effect of temperature on maximum potential enzyme activity.Temperature is known to alter enzyme activity in most tissues, withmaximum potential activity being inhibited by both low and veryhigh temperatures.

Many plant species acclimate to long-term temperature changeswith the result that plants grown in the cold exhibit higher respirationrates than plants grown at warmer temperatures, when measured ata common temperature (Atkin et al.2000a). Plants show varying degrees of acclimation to temperature(Smakman & Hofstra 1982; Weger &Guy 1991; Gunn & Farrar 1999; Tjoelker, Oleksyn & Reich 1999a); however,the cause(s) of this variability is not known. One possibility isthat the demand for ATP increases as growth, maintenance and/orion uptake processes adjust to a new growth temperature, therebyreducing the degree of adenylate restriction on respiration (Atkin et al. 2000a). In addition,changes in substrate availability may play a role (e.g. Atkin et al.2000b). Finally, increases in enzymatic capacity may be necessaryin order for root respiration to acclimate to low temperatures [e.g. increasesin the number of mitochondria (Miroslavov & Kravkina1991) and/or mitochondrial capacity (Klikoff 1966, 1968)].

Our study investigated the effect of short- and long-term changesin temperature on the regulation of root respiration. We also determinedthe temperature to which root respiration acclimates (e.g. dailymean, daytime or night-time temperature; see Will2000), when grown under different day/night-timetemperature combinations, but where the daily mean temperaturesare kept constant. The temperature which plants acclimate to islikely to be important in determining the rate of respiratory energyproduction at any given measuring temperature. It is also likelyto play a role in determining the total amount of CO2 releasedinto the atmosphere by root respiration over long periods under conditionsof fluctuating temperature. To assess whether contrasting speciesdiffer in their respiratory temperature response, we compared theresponse of the fast-growing Plantago lanceolata and theslow-growing Plantago euryphylla. Slow-growing species exhibitunusually high root respiration rates (given their low relativegrowth rate and rates of ion uptake) due to less efficient uptakeof anions and concomitant higher demand for respiratory ATP (Poorter, Remkes & Lambers 1990; Poorter et al. 1991; Van Der Werf, Welschen & Lambers 1992; Scheurwater et al. 1998).Such differences in the demand for ATP might affect the way in whichtemperature affects the regulation of the respiratory apparatus.

Materialsand methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Plantgrowth

Two species were used in the experiments: Plantago ­lanceolata L.and Plantago euryphylla Briggs, Carolin & Pulley. Seedfor P. lanceolata was obtained from Emorsgate Seed   Pty   Ltd   (Norfolk,   UK).   Plantago   euryphylla  seed wascollected at a field site in Kosciusko National Park, NSW, Australiaat an altitude of 1940 m (G.R. 165687). Seeds were plantedon soil and initially grown in a ­controlled environmentchamber with an irradiance of 190 µmol m−2 s−1,  23 °C/14 °C  day/night  temperature  and 14/10 hday/night. Once the seedlings were large enough, they weretransferred into hydroponics and grown in a Conviron CMP 3023 growthchamber (Conviron, EIS, ­Winnepeg, Canada) (300 µmol m−2 s−1,14/10 h day/night, constant 23 °C).The modified Hoaglands nutrient solution [Ca(NO3)2·4H2O(0·241 m), KNO3 (0·318 m),KH2PO4 (0·076 m),MgSO4·7H2O (0·108 m),MnSO4.H2O (0·8 mm), ZnSO4·7H2O(0·34 mm), CuSO4·5H2O(0·06 mm), H3BO3 (8 mm),Na2MoO4.H2O (0·1 mm), [CH2.N(CH2.COO)2]2FeNa 16·19 mm] wasmaintained at a pH of 5·5–5·8. The nutrient solutionwas replaced weekly. Measurements using P. lanceolata weremade when the mean root fresh mass was approximately 1·1 g(0·07 g mean dry mass), typically after 11 dgrowth in hydroponics. Plantago euryphylla plants were harvestedwhen mean root fresh mass was 0·30 g (0·02 gdry mass), typically after 2–3 weeks growth in hydroponics.Preliminary experiments showed that ontogenetic changes in respirationwere limited once roots had reached these mass values (data notshown).

Rootrespiration measurements

Root respiration was measured polarographically using Clark typeO2 electrodes (Dual Digital Model 20; Rank Brothers,Cambridge, UK). The rate of O2 uptake by whole roots(which had been excised from the shoot) was ­measured inan airtight cuvette containing 30–50 mL of modifiedHoagland solution (pH 5·8). The roots were left toequilibrate for 10 min; the respiration rate was then ­measuredover the subsequent 5 min. The electrodes were maintainedat a constant measurement temperature using Lauda E100 Lauda (Lauda-Köningshofen,Germany) water baths and insulation on the tubing and cuvettes.The temperature in each cuvette was checked before measuring commenced.

Measurementsfollowing short-term changes in temperature

Plantago lanceolata control respiration rates (i.e. inthe absence of uncoupler and/or exogenous substrate) were measuredon eight replicates at 13 temperatures over the range 5–50 °C. Plantagoeuryphylla control respiration rates were measured on four replicatesat 10 temperatures over the range 5–50 °C.Uncoupled respiration was measured by placing four new excised rootsinto cuvettes containing nutrient solution plus 3 µm CCCP(from a 3 mm stock dissolvedin ethanol). The uncoupled rate was measured over a 5 minperiod after 10 min equilibration. The same method wasused to measure the effect of 50 mm glucose(from a 2 m stock) on respiration.In all cases the pH of the solution was adjusted to 5·8prior to O2 consumption being measured.

Acclimationof P. lanceolata roots to a long-term change in temperature

Plantago lanceolata seedlings were grown as describedpreviously. After 5 d growth in hydroponics at a constant23 °C, plants were transferred to another ConvironCMP 3023 cabinet set to 15/10 °C day/night(all other conditions kept constant). A separate group of controlplants was maintained at 23 °C. Two sets of measurementswere conducted: respiration measurements following 7 dof treatment and changes in the concentration of soluble sugarsover the 7 d period. Respiration in the 15/10 °C-treatedplants was measured in the absence of CCCP and glucose, in the presence ofCCCP alone and in the presence of glucose alone. To assess the impactof cold treatment on substrate availability, we harvested cold-treatedplants 2, 4 and 7 d after initial exposure to 15/10 °C.The 23 °C-maintained plants were also harvestedon days 2, 4 and 6. The concentration of glucose and sucrose ineach root sample was then measured using ground, freeze-dried rootsamples. Soluble sugars were extracted by hot extraction using 80% (v/v)ethanol. Soluble sugars (glucose, sucrose and fructose) were estimatedby three micro plate-based assays. For glucose, extracts were incubatedin the presence of hexokinase and glucose-6-phosphate dehydrogenase.The reduction of NADP to NADPH was then followed spectrophotometricallyat 340 nm (Dynatech Laboratories MRX, Guernsey, UK). Forfructose, the extracts were incubated in the presence of hexokinase,phosphoglucose isomerase and glucose-6-phosphate dehydrogenase andthe reduction of NADP to NADPH was followed. The concentration ofsucrose plus glucose was estimated via incubation of the extractwith invertase (Sigma, St Louis, MO, USA), followed by measurementof the total glucose concentration as above.

Temperatureto which respiration acclimates: daily average, daytime or night-time?

To establish the temperature at which root respiration acclimatesfor P. lanceolata, we moved the 23 °C-grownplants (conditions as described above) to five temperature regimesin which the daily average temperatures were identical (12·5 °C,with a 12/12 h day/night) but where thedaily daytime and night-time temperatures differed (18/7 °C,17/8 °C, 15/10 °C,14/11 °C and 13/12 °Cday/night; 12 h day/night in all treatments).After 7 d growth at these temperatures, the plants wereharvested and root respiration was measured at six temperaturesin the range 5–25 °C. Individual rootswere used for respiration measurements at each temperature. Twocriteria were used when analysing the data:

  • 1
    Full acclimationis often assumed to result in homeostasis; thus, if respirationacclimated to the daily mean temperature (12·5 °C),then the rate of respiration at 12·5 °C wouldbe the same for all treatments. Similarly, if respiration acclimatedto the night-time temperature, then respiration rates would be mostsimilar at the respective night-time temperatures of each treatment.
  • 2
    Even in tissues thatdo not exhibit full homeostasis, cold-acclimation invariably resultsin a higher rate of respiration measured at a common, moderate temperature(e.g. 20 °C) than in tissues experiencing highergrowth temperatures. Thus, if respiration acclimates to the night-timetemperature then the highest respiration rate measured at 20 °Cshould occur in tissues experiencing the lowest night-time temperature(i.e. 18/7 °C day/night). Conversely,the lowest respiration rate measured at 20 °C shouldoccur in tissues experiencing the highest night-time temperature,e.g. (13/12 °C day/night). Alternatively,acclimation to the daytime temperature should mean that respirationrates measured at 20 °C are highest and lowestin those tissues experiencing the lowest and highest daytime temperatures,respectively (i.e. 13/12 °C and 18/7 °Cday/night).

Statisticalanalysis

Data were analysed using analysis of variance (anova)to determine whether the factors measurement temperature, species,absence or presence of CCCP or glucose, and the growth temperatureregime affected respiration significantly (SPSS version 10; SPSSScience, Birmingham, UK). Significance was defined as at the 95% confidencelevel.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Responseof coupled and uncoupled root respiration to temperature

Figure 1 showsthe short-term temperature response of P. lanceolata and P.euryphylla root respiration, both in the absence/presenceof CCCP (Fig. 1a& b) and absence/presence of exogenous glucose(Fig. 1c& d). In all cases, root respiration was temperaturedependent. In the absence of CCCP and glucose, P. lanceolata respiration(closed circles in Fig. 1a anddashed line in Fig. 1c)increased exponentially as temperatures increased from 5 to 32 °Cand then began to decline at temperatures above 43 °C.Respiration rates for P. euryphylla (closed circles in Fig. 1b anddashed line in Fig. 1d)were similar to P. lanceolata in the range 5–32 °C.However, there were significant differences in the response of thetwo species to changes in measurement temperature (P = 0·004, F7,178 = 3·130).For example, respiration rates in the absence of CCCP and glucosewere higher in P. euryphylla (Fig. 1b)than P. lanceolata (Fig. 1a) attemperatures above 32 °C

image

Figure 1. Temperatureresponse curves of root respiration (nmol O2 g−1 DM s−1)for the fast-growing Plantago lanceolata (a) and (c) and the slow-growing P.euryphylla (b) and (d). (a) and (b) show respirationrates in the absence (d) and presence (s) of an uncoupler (CCCP),whereas (c) and (d) show rates in the absence (dashed line showingthird-order polynomials fitted to the control data shown in (a)and (b) and presence (□) of exogenous glucose. Each respirationvalue in the absence of effectors is the mean of eight replicateswhereas each uncoupled and added substrate value is the mean offour replicates (± SE).

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Respiration in the presence of CCCP was measured to give an indicationof whether the degree of adenylate restriction changed with temperature(open circles, Fig. 1a &b). The degree to which respiration was stimulated by CCCPvaried with temperature (P < 0·001, F8,178 = 6·541) forboth species. In P. lanceolata, uncoupled respiration rapidlyincreased exponentially over the temperature range 5–40 °C(Fig. 1a).Above 40 °C the uncoupled rate declined rapidly.In P. euryphylla CCCP had less effect on respiration thanin P. lanceolata (P < 0·001, F1,178 = 30·855; Fig. 1b). Figure 2a& b illustrate how the degree of respiratory stimulationby addition of CCCP varied with measurement temperature and species.In P. lanceolata, the degree of stimulation rose from nostimulation at 5 °C to a maximum of 250% at28 °C. The percentage stimulation then declined steadilyto approximately 60% at 50 °C. In P.euryphylla, the percentage stimulation was consistently lowerover the temperature range than for P. lanceolata. The maximum stimulationin P. euryphylla was approximately 100%; this occurredbetween 20 and 23 °C (Fig. 2b).The stimulatory effect of uncoupler decreased as temperatures were increasedabove 23 °C, with no stimulation occurring at temperaturesabove 40 °C (Fig. 2b).

image

Figure 2. Stimulatoryeffect of uncoupler (a) and (b) and exogenous glucose (c) and (d) on root respirationof the fast-growing Plantago lanceolata (a) and (c) and theslow-growing P. euryphylla (b) and (d). The symbols (s) showrates in the presence of CCCP or glucose expressed as a percentageof rates in their absence. Values are for individual temperatureswhere values of respiration in the absence and presence of CCCPor glucose were measured. Zero percent indicates no stimulation.

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Effectof exogenous substrates (± uncoupler)

To determine whether substrate limitation was restricting respiration,rates were measured in the presence of 50 mm glucoseover the temperature range (open squares, Fig. 1c &d). The effect of exogenous glucose varied with temperature(P = 0·007, F8,157 = 2·763).Glucose stimulated respiration in P. lanceolata at moderatetemperatures (15–35 °C) but had littleeffect on respiration at 10 °C (Fig. 1c).The percentage stimulation by glucose was greatest at 23–27 °C (130%)(Fig. 2c).The effect of glucose on respiration in P. euryphylla wassubstantially less than that observed for P. lanceolata (P < 0·001, F1,157 = 12·766, Fig. 1d).The average stimulation was 15%, and there was little differencein the degree of stimulation across the temperature range (Fig. 2d).

To assess whether uncouplers had a greater effect on respirationin the presence of exogenous substrates, we exposed P. lanceolata rootsto a combination of CCCP and glucose (open circles, Fig. 3). Between 10and 30 °C, ­respiration rates in thepresence of CCCP + glucose were similar to respirationrates if CCCP alone was present (Fig. 3).

image

Figure 3. Effectof exogenous substrates on the temperature response of P. lanceolata rootrespiration. The solid line is a third-order polynomial fitted tothe data shown in Fig. 1 forroot respiration in the presence of CCCP alone. The symbols (s)show respiration rates in the presence of both CCCP and glucose.Each value is the mean of four replicates (± SE).

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Acclimationof P. lanceolata roots to a long-term change in temperature

Figure 4 showsthe short-term temperature response of P. lanceolata rootrespiration following 7 d exposure to 15/10 °C,for roots in the absence of effectors (Fig. 4a)and in the presence of CCCP (Fig. 4b)or exogenous glucose (Fig. 4c).Respiration rates were significantly greater in the 15/10 °C-acclimatedplants than in the 23 °C-grown plants, when measuredat moderate temperatures (e.g. 15–25 °C, P < 0·001, F1,104 = 13·930).This demonstrates that respiration had acclimated to the low temperaturetreatment. However, there was little difference in rates exhibitedby the two growth treatments when respiration was measured at lowtemperatures (e.g. 10 °C; Fig. 4a).As a result, Q10 values (i.e. the proportionalincrease in respiration for every 10 °C rise)between 15 and 25 °C were higher in cold-acclimatedplants than in their warm-grown counterparts (Fig. 5). At 20 °Cthe Q10 for the cold-acclimated plants was 2·11compared to 1·61 for control plants (Fig. 5). The critical temperaturefor 15/10 °C-acclimated respiration was30 °C; above this temperature respiration ratesdeclined (Fig. 4a).

image

Figure 4. Temperatureresponse curves of root respiration in warm-grown (23 °C)and 7 d cold-acclimated (15/10 °Cday/night) P. lanceolata. (a) respiration in theabsence of CCCP or exogenous substrates; dashed lines representthe third-order polynomials fitted to data from the 23 °C-grownplants (from Fig. 1),whereas the solid symbols (▴) are data for 7 dcold-acclimated plants. (b) Respirationin the presence of CCCP; the dashed line represents 23 °C-grownplants and the open symbols (▵) represent cold-acclimatedplants. (c) Respirationin the presence of exogenous glucose; the dashed line representsthe 23 °C-grown plants and the open symbols (s)represent cold-acclimated plants. Each value is the mean of fourreplicates (± SE).

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image

Figure 5. Effectof measuring temperature on the Q10 of P. lanceolata rootrespiration for warm-grown (dashed line) and 7 d cold-acclimated(solid line) P. lanceolata. The lines shown are for root respirationin the absence of CCCP and glucose. Q10 valueswere calculated using the equation Q10 = 10(10 × regressionslope). The regression slope was taken from the second-orderpolynomial fitted to log10 respiration versus temperatureplots. Second-order polynomials were then fitted to the Q10 data.

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To determine whether acclimation altered the degree to whichrespiration was limited by adenylates or substrate limitations,we measured respiration in the presence of CCCP (Fig. 4b)and exogenous glucose (Fig. 4c)in the cold-acclimated plants. Between 5 and 27 °C,there was little difference in the uncoupled rates of plants grownat 23 °C and those acclimated to 15/10 °Cparticularly in the low/moderate temperature range (Fig. 4b).At temperatures above 27 °C, uncoupled rates forthe cold-acclimated plants declined rapidly. Exogenous glucose hadno significant effect on rates of respiration in the cold-acclimatedplants. This contrasts with the plants grown at 23 °Cin which glucose stimulated respiration across the temperature range. Theconcentration of glucose, fructose and sucrose was measured in the23 °C-grown plants and in the 15/10 °C-grownplants (2, 4, 6 and 7 d after transfer from 23 °C; Table 1). The concentrationof sugars was significantly greater in the plants transferred to15/10 °C, compared with those remainingat 23 °C (P = 0·021, F1,11 = 7·228),being 50% higher in cold-acclimated roots after 4 dand 55% higher after 7 d. Interestingly, the 55% increasein total soluble sugars was caused by an increase in the glucose concentration,whereas fructose and sucrose concentrations did not change aftercold-acclimation.

Table 1.  Effectof growth temperature on the concentration of total soluble sugars(sucrose, fructose plus glucose; mg g−1 DM)in Plantago lanceolata roots that were either left in a constant23 °C growth environment or transferred to 15/10 °Con day 0
Days aftertransferSugar concentration(mg g−1 DM)
23 °C-grown plants15/10 °C-treated plants
  1. Each value is the mean of fourreplicates (±SE) withthe exception of the 15/10 °C-treatedplants on day 2a,for which only two samples were available for analysis. Due to misplacementof root samples, last-harvest analyses were only possible on days 6band 7cfor the warm-grownand cold-acclimated roots, respectively.

290 ± 9117a
499 ± 2149 ± 34
6 b/7 c83 ± 5b129 ± 3c

Theimpact of varying daily maximum and minimum temperatures on respiration

Figure 6a showsthe temperature response curves of P. lanceolata root respirationfor 23 °C-grown plants exposed to 18/7,17/8, 15/10, 14/11 and 13/12 °Cfor 7 d (mean daily temperature was 12·5 °Cthroughout). In general, respiration was more temperature sensitivein the roots exposed to 18/7 and 17/8 °Cthan roots exposed to 13/12 °C (Fig. 7), as shownby the significant interaction between log10 respirationand growth treatment in a two-way anova (P = 0·020, F15,51 = 2·175).The temperature response curves shown in Fig. 6 indicatethat there was no common respiration rate at the daily mean temperature(12·5 °C); this suggests that P. lanceolata rootrespiration did not acclimate to the daily mean temperature (seeCriteria 1 in Materials and Methods).

image

Figure 6. Short-termtemperature response curves of root respiration (nmol O2 g−1 DM s−1)for P. lanceolata grown in several environments that differin daily high (daytime) and low (night-time) temperatures, but inwhich the daily mean temperature was always 12·5 °C. (a) Shows values forall measured temperatures (n = 3, ± SE)for 23 °C-grown plants that were exposed to thefollowing temperature regimes for 7 d: 18/7 °C(s), 17/8 °C (□), 15/10 °C(e), 14/11 °C (d), and 13/12 °C(▵). The solid lines represent second-order polynomialsthat were fitted to each plot. (b) Shows thesame second-order polynomials that were fitted to each plot shownin (a) but also shows mean respiration rates at the low night-time(□) temperature of each treatment and at the high daytime temperature(d). In both (a) and (b), rates at the daily mean temperature (12·5 °C)and a set temperature of 20 °C are shown by theintersection of the dotted vertical lines and the individual treatmentsecond-order polynomials.

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image

Figure 7. Q10 valuesderived from data shown in Fig. 6 versusthe minimum growth temperature in each of the five temperature treatmentsused in the experiment to establish the temperature that respirationacclimates to.

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To more clearly show respiration rates at the low night-timeand higher daytime temperature for each regime, we re-plotted thedata as shown in Fig. 6b.Homeostasis was not observed either at the daily high or daily lowtemperatures. However, the rates at the low night-time temperature weremore similar than the rates at the high daytime temperature. Thus,in terms of the degree of homeostasis, ­respiration appearsto acclimate to the low night-time temperature. This suggestionis supported by the fact that the highest rates of respiration ata common measurement temperature (e.g. 20 °C)were highest in the treatments with the lowest night-timetemperature (18/7 and 17/8 °Cday/night), and lowest in the treatment with thehighest night-time temperature (13/12 °Cday/night) (see Criteria 2 in Materials and Methods). Weconclude therefore that under our growth conditions, P. lanceolata rootrespiration acclimates to the low night-time temperature.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

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.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Our study has demonstrated that the proportion of control exertedby adenylates, substrates and enzyme capacity on respiratory fluxchanges with temperature. We suggest that respiration is limitedby enzyme activity at very low and very high temperatures, withadenylates and/or substrate supply limiting at moderatetemperatures. The Q10 values appear to be lowestwhere respiration is limited by adenylates and/or substratesupply. The overall pattern of how temperature affects the regulationof respiration appears to be similar in the fast-growing P. lanceolata andthe slow-growing P. euryphylla. However, our results suggestthat a greater proportion of maximum potential respiratory flux isused in P. euryphylla than in P. lanceolata. We havedemonstrated that P. lanceolata acclimates to a low night-time temperatureand that the acclimation is associated with a change in the Q10 andunderlying factors that regulate ­respiratory flux, particularlythe availability of respiratory substrates.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

This work is funded in part by a BBSRC studentship to E.M.C.-C.and by a UK Natural Environment Research Council grant to O.K.A.(GR3/11898). We are grateful to Dr Calvin Dytham who advisedon which statistical tests were required and to David Sherlock forhis expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materialsand methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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Received 5 March 2002;received inrevisedform 3 June 2002;accepted for publication 6 June 2002