Acclimation of snow gum (Eucalyptus pauciflora) leaf respiration to seasonal and diurnal variations in temperature: the importance of changes in the capacity and temperature sensitivity of respiration

Authors


Correspondence (and present address of O.K.A.): Owen K.Atkin, Department of Biology, The University of York, PO Box 373, York, YO10 5YW, UK. Tel.: + 44 1904 432857; fax: + 44 1904 432860; e-mail: oka1@york.ac.uk

ABSTRACT

We investigated the relationship between daily and seasonal temperature variation and dark respiratory CO2 release by leaves of snow gum (Eucalyptus pauciflora Sieb. ex Spreng) that were grown in their natural habitat or under controlled-environment conditions. The open grassland field site in SE Australia was characterized by large seasonal and diurnal changes in air temperature. On each measurement day, leaf respiration rates in darkness were measured in situ at 2–3 h intervals over a 24 h period, with measurements being conducted at the ambient leaf temperature. The rate of respiration at a set measuring temperature (i.e. apparent ‘respiratory capacity’) was greater in seedlings grown under low average daily temperatures (i.e. acclimation occurred), both in the field and under controlled-environment conditions. The sensitivity of leaf respiration to diurnal changes in temperature (i.e. the Q10 of leaf respiration) exhibited little seasonal variation over much of the year. However, Q10 values were significantly greater on cold winter days (i.e. when daily average and minimum air temperatures were below 6° and –1 °C, respectively). These differences in Q10 values were not due to bias arizing from the contrasting daily temperature amplitudes in winter and summer, as the Q10 of leaf respiration was constant over a wide temperature range in short-term experiments. Due to the higher Q10 values in winter, there was less difference between winter and summer leaf respiration rates measured at 5 °C than at 25 °C. The net result of these changes was that there was relatively little difference in total daily leaf respiratory CO2 release per unit leaf dry mass in winter and summer. Under controlled-environment conditions, acclimation of respiration to growth temperature occurred in as little as 1–3 d. Acclimation was associated with a change in the concentration of soluble sugars under controlled conditions, but not in the field. Our data suggest that acclimation in the field may be associated with the onset of cold-induced photo-inhibition. We conclude that cold-acclimation of dark respiration in snow gum leaves is characterized by changes in both the temperature sensitivity and apparent ‘capacity’ of the respiratory apparatus, and that such changes will have an important impact on the carbon economy of snow gum plants.

INTRODUCTION

Leaf respiration represents a major source of CO2 release in plants. For example, up to 35% of the CO2 fixed by photosynthesis each day is released back into the atmosphere by leaf respiration at night, in plants grown under controlled-environment, constant-temperature conditions ( Van der Werf, Poorter & Lambers 1994; Atkin & Lambers 1998). However, the extent of daily leaf respiratory CO2 release may differ under natural conditions where air temperatures vary diurnally and seasonally, as respiration is very sensitive to short-term changes in temperature ( Körner & Larcher 1988). Understanding the effect of variations in temperature on respiratory CO2 loss is therefore a prerequisite for predicting plant growth in a changing global environment.

The degree to which leaf respiration changes with temperature is highly variable, with Q10 values (i.e. the proportional increase in respiration for each 10 °C rise) being as low as 1·4 and as high as 4·0 ( Azcón-Bieto 1992). Q10 values differ between species (e.g. Larigauderie & Körner 1995) and are influenced by the metabolic state of the tissue and the growth environment. For example, Q10 values are higher in plants with high concentrations of soluble carbohydrates ( Wager 1941; Breeze & Elston 1978; Berry & Raison 1981; Azcry & Raison 1983) and are often higher in winter than in summer ( Breeze & Elston 1978; Sowell & Spomer 1986; Hagihar & Hozumi 1991; Criddle et al. 1994 ; Stockfors & Linder 1998). Determining the extent of daily leaf respiratory CO2 release under natural conditions therefore requires that the Q10 under natural conditions be accurately determined.

The extent of respiratory CO2 release by leaves under natural conditions will also depend on the degree of respiratory acclimation. Leaf respiration can acclimate to changes in growth temperature, such that cold-grown plants (e.g. in winter) exhibit higher rates at a set temperature (i.e. the apparent ‘capacity’ of respiration is greater) than plants grown under warmer conditions ( Mooney 1963; Klikoff 1968; Billings & Mooney 1968; Körner & Larcher 1988; Arnone & Körner 1997; Atkin & Lambers 1998). Perfect homeostasis of respiration (e.g. Collier 1996) results in identical rates of respiration in plants grown at contrasting temperatures, when measured at their respective growth temperatures. However, the degree of acclimation varies substantially between species, with little or no acclimation occurring in some species ( Larigauderie & Körner 1995). The speed at which respiration acclimates to changes in temperature will be critical in determining the extent to which CO2 is released by leaf respiration.

In this study, we investigated the effect of daily and seasonal temperature variation on leaf respiration in snow gum (Eucalyptus pauciflora Sieb. ex Spreng) grown either in a natural field or under controlled-environment conditions. Snow gum is an ideal species for such a study, as its leaves are long-lived, thus enabling measurements on a given leaf type to be conducted over several months. A large amount of information is available on the interaction between photosynthesis and temperature in snow gum (e.g. Slatyer & Morrow 1977; Kirschbaum & Farquhar 1984; Küppers et al. 1986 ; Kirschbaum & Farquhar 1987; Ball, Hodges & Lauglin 1991; Ball 1994; Holly, Laughlin & Ball 1994; Ball et al. 1997 ; Hovenden & Warren 1998). Snow gum is common in the colder environments of south-eastern Australia and regularly experiences large seasonal changes in leaf temperature, as well as large diurnal leaf temperature changes in all seasons ( Ball et al. 1997 ).

MATERIALS AND METHODS

Field study

Snow gum (Eucalyptus pauciflora Sieb. ex Spreng) seedlings were raised from seed from a population collected in Gudgenby Valley in Namadgi National Park, south-eastern Australia (35°45′ S 148°59′ E). Seeds were vernalized at 4 °C for 6 weeks and then germinated in November 1995 on seed trays under glasshouse conditions at the Research School of Biological Sciences (RSBS), Australian National University, Canberra (600 m a.s.l.). Seedlings were transplanted to 20 cm potting tubes, fertilized with 10% Hoagland’s solution and placed outdoors. The seedlings were grown at RSBS until March 1996, reaching a height of approximately 0·1 m. The 5-month-old seedlings were then transported to an open grassland field site (north-facing, 5° slope) in the Gudgenby Valley near the seed collection site (elevation 1000 m a.s.l.) and transplanted into existing soil.

Seedlings were planted 50 cm apart on the north side of an east–west oriented, 1 m high, vertical panel of 50% shade cloth. The seedlings were 25 cm from the shade cloth. As the sun tracks across the northerly sky at this southern-hemisphere field site, plants were exposed to unshaded sun throughout the day in all seasons. The shade cloth did not have any relevance to the current study per se; rather, the seedlings were planted next to the shade cloth as part of a larger study of the effects of north, south, east and west-facing aspects on plant growth and leaf photosynthetic characteristics. The seedlings were randomly allocated to six replicate blocks of each treatment, with each block containing six plants. Each block was surrounded by a 1-m-high wire fence to protect the plants from mammalian herbivores. Regular mowing kept grass cover within each block uniform.

Measurements of air temperature at 0·2 m above the grass-covered ground (copper constantan thermocouples, 42 SWG, referenced against Pt-100 platinum resistance thermometers) were taken every 10 s, averaged for 30 min intervals and stored in a data logger (Datataker 100F; Data Electronics Melbourne, Australia). These values were used to calculate average, minimum and maximum daily values. Daily temperature ranges were typically 17–25 °C, both in winter and summer ( Table 1). In 1996, the average daily air temperature was 0 and 14 °C on the coldest (winter) and warmest (summer) measurement days, respectively ( Table 1; Fig. 1). Night temperatures in winter were typically less than –8 °C in winter. In summer, maximum day temperatures exceeded 40 °C on several days (data not shown). Plants received regular rainfall throughout the experiment, with the exception of the period March 1997 to July 1997.

Table 1.  Changes in air temperature, respiration (nmol CO2 g−1 DM s−1) versus temperature plots, and total soluble sugar concentrations (mg m−2) at the Namadgi field site. Air temperature values are 2 d averages (measuring day and the day preceding the measuring day) and were recorded at 0·2 m above the ground. Data on the temperature sensitivity of leaf respiration in darkness are shown as the slope and y-axis intercept (respiration rate at 0 °C) of the respiration (log10 scale) versus temperature plots (± SE; n = 4). These data were used to calculate the Q10 values shown in Fig. 5. Leaf samples were not collected for sugar analyses in 1996 and 1998 (n/a)
      Respiration (log10 scale) vs temperature
      parameters Soluble sugar
Measurement dayAir temperature (°C) Slope of log10Respiration at 0 °Cconcentration
Year/MonthDayAverageMaximumMinimumrespiration vs temp.(nmol CO2 g−1 DM s−1) (mg m−2)
  1. Aug

  2. 239

  3. 5·1

  4. 10·8

  5. 1·6

  6. 0·0466 ± 0·0047

  7. 1·26 ± 0·15

  8. n/a

1996 June164 2·012·7–4·90·0492 ± 0·00581·79 ± 0·35n/a
 Aug.234 0·812·6–8·60·0547 ± 0·0074 1·6 ± 0·27n/a
 Oct.29710·519·11·70·0389 ± 0·00221·98 ± 0·40n/a
 Nov.33414·325·90·80·0328 ± 0·00152·00 ± 0·21n/a
1997April107 8·223·1–4·70·0359 ± 0·00191·62 ± 0·140·76 ± 0·09
 May139 7·011·33·60·0307 ± 0·00511·93 ± 0·051·04 ± 0·15
 July206 5·112·5–2·90·0623 ± 0·00231·42 ± 0·010·65 ± 0·09
 Oct.290 9·016·7–0·70·0307 ± 0·00511·40 ± 0·080·89 ± 0·27
 Nov.31614·124·05·50·0359 ± 0·00361·61 ± 0·400·94 ± 0·28
 Dec.24915·922·37·30·0418 ± 0·00221·01 ± 0·16n/a
1998Feb5015·926·87·30·0372 ± 0·00071·22 ± 0·10n/a
 May141 7·215·68·30·0264 ± 0·00391·30 ± 0·09n/a
Figure 1.

Seasonal variation in average daily air temperature, measured at 0·2 m above the ground in (a) 1996 (b) 1997 and (c) 1998. Equipment failures during April–August 1998, necessitated us to calculate the air temperatures at the Namadgi field site using data from Canberra airport (supplied by the Bureau of Meteorology). A correction factor was established for data calculated from airport and Namadgi field site data.

Respiration measurements (measured as CO2 release) began in June 1996 (winter) when the plants were approximately 0·2 m high. Measurements were made on fully expanded leaves (the third set of leaves from the base of the plant) that were approximately 4 months old when the study began. The same set of leaves were also used for subsequent measurements in August, October and late November 1996. Further measurements using leaves of this developmental stage were not possible as they senesced in January 1997 (summer). A second set of leaves (approximately the sixth set of leaves from the base) was used for all subsequent measurements from April 1997 through to August 1998. These leaves were approximately 4 months old and near-fully expanded in April 1997. Preliminary experiments indicated that 4 months were required for leaves to be near fully expanded and before respiration rates stabilized (data not shown); prior to the 4 month stage, respiration rates declined exponentially with time.

On each measuring day, diurnal variation in leaf respiration in darkness was determined by repeatedly measuring CO2 release in intact, attached leaves. Measurements were done using a portable Infra Red Gas Analyzer (IRGA) system (LI-6400; Li-Cor Inc., Lincoln, NB, USA) with a CO2 controller. Prior to every measurement, the gas stream from the sample cuvette was matched with that of the reference.

During daylight hours, we covered leaves with blackened cloth for 30 min prior to each measurement to avoid transient post-illumination bursts of CO2 release ( Atkin, Evans & Siebke 1998a; Atkin et al. 1998b ). After each measurement, leaves were uncovered and allowed to photosynthesize for a further 1·5–2·5 h before the next set of measurements. Dark respiration rates of covered/uncovered leaves were compared with those of leaves covered for the full day, and no significant difference in rates between the two treatments were found (data not shown). All diurnal measurements were done at an atmospheric CO2 concentration of 350 μmol mol−1.

Three different approaches were used to assess diurnal and seasonal variation in leaf respiration. Firstly, temperature response curves for respiration in darkness were made for individual, attached leaves (using the temperature controller of the Li-Cor 6400) in June 1996. Leaves were allowed 5 min to adjust to each new temperature. The rest of the shoot remained at a temperature governed by the surrounding air temperature. Secondly, to assess whether there were any diurnal changes in respiration at any set temperature, leaves were exposed to 10 °C for repeated measurements over the day in June 1996. Finally, diurnal measurements were conducted at the actual ambient temperatures exhibited by leaves under natural conditions throughout the day. These ambient leaf temperature measurements formed the majority of the measurements made over the 2·5 year experimental period.

At the end of each measuring day, the measured leaves were harvested and transported in sealed containers to Canberra. The leaf area (LI-3100; Li-Cor Inc.), fresh mass and dry mass (70 °C for 2 d) of each leaf was determined. Total nonstructural carbohydrates were determined from 5 mg aliquots of ground dry sample extracted in 80% (w/v) ethanol and incubated at 80 °C for 20 min. After centrifugation, the supernatant was removed and the sample re-extracted twice. The supernatant from leaves was cleared with 20 mg activated charcoal per 600 mm3 of supernatant. Aliquots (400 mm3) of the supernatants were dried at 50 °C and resuspended in 100 mm3 of water. Total soluble sugars (sucrose, glucose and fructose) were assayed after incubation with invertase at 55 °C for 20 min by the glucose oxidase/peroxidase method (including phosphoglucose-isomerase) adapted to a titre plate assay read at 500 nm (Glucose assay kit, Megazyme, Melbourne, Australia). Total starch was determined on the pellet that remained after soluble sugar extraction, by converting it to glucose using amyloglucosidase and α-amylase (Total starch kit; Megazyme).

Controlled-environment studies

Two growth chambers (Thermoline, Wetherill Park, Australia; PAR, 400 μmol photons m−2 s−1; 16 h photoperiod; relative humidity, 60%) were used to assess the impact of short- and long-term changes in temperature on respiration rates of snow gum leaves. One of the chambers was set at a constant 20 °C and the other at a constant 10 °C.

To assess the effect of growth temperature on the temperature sensitivity of leaf respiration, equal numbers of 10-month-old, outdoor-established potted seedlings (approximately 0·2 m high) were transferred to the 10 and 20 °C chambers for 3 weeks prior to the measurement of leaf respiration rates. The temperature sensitivity of leaf respiration in darkness of plants exposed to the two treatments was then measured over the 10–30 °C range, using the LI-6400 portable IRGA described above. In these experiments, the temperature of the entire plant (i.e. not just the measured leaf) was controlled by placing the measured plants into a growth cabinet set at the measuring temperature.

The time taken for respiration in darkness to acclimate to changes in growth temperature was assessed by transferring plants from the 10 °C cabinet to the 20 °C cabinet (and vice versa), and repeatedly and non-destructively measuring the respiration rates at a set temperature (20 °C) in the days before and after the transfers.

The effect of root temperature on leaf respiration rates (measured at 20 °C) was determined by cooling/heating the roots of potted snow gum plants in a growth cabinet where the shoot temperature was maintained at 20 °C. Root cooling/heating was accomplished by submerging the pots in a temperature-controlled water bath.

RESULTS

Respiration of individual leaves: short-term temperature effects and measurements at set temperatures

We began our study by exposing individual leaves of field-grown plants to short-term changes in temperature while the rest of the shoot remained at a temperature governed by the surrounding air temperature. Short-term increases in temperature resulted in an exponential increase in leaf respiration ( Fig. 2a). However, the rate of respiration at any given temperature was faster in the afternoon than in the morning. Similar responses were observed in three other replicate leaves measured at different times of the day (data not shown).

Figure 2.

Temperature response of respiration in darkness per unit leaf area measured under field conditions in June 1996. (a) Short-term temperature curve for a single leaf in the late morning when air temperature was 10 °C (○) and in the afternoon when air temperature was 14 °C (●). (b). Rates of respiration at a set temperature (10 °C) plotted against the surrounding air temperature (± SE, n = 4).

To further investigate the impact of shoot temperature on the rate of leaf respiration, we measured the diurnal variation in respiration at a set temperature (10 °C) in the field-grown plants. The rate of leaf respiration at the set temperature was positively correlated with the surrounding air temperature ( Fig. 2b). Changes in air and shoot temperature therefore alter the respiration rate of individual leaves at a given temperature.

The results shown in Fig. 2b for field-grown plants may have been confounded by temporal changes in respiration that were independent of temperature (e.g. diurnal changes in irradiance). We therefore further investigated the impact of air temperature on the rate of leaf respiration at a set temperature under controlled conditions in the growth room ( Fig. 3a). Increasing the surrounding air temperature, increased rates of leaf respiration at set temperature compared with that of leaves for which the air temperature remained at 10 °C ( Fig. 3a). The Q10 was substantially greater when the plant and leaf temperatures were the same (Q10 = 2·6 ± 0·1; n = 4, ± SE) compared to when the plant was kept at 10 °C (Q10 = 2·1 ± 0·1; n = 4, ± SE).

Figure 3.

(a) Short-term temperature response of respiration in darkness measured under controlled-environment conditions and (b) effect of root temperature on leaf respiration measured at a set temperature (30 °C). In (a), temperature curves for individual leaves were generated either with the plants being kept at a constant temperature (10 °C; ●) or with the plant and leaf temperature both being altered (○). Values represent the mean of four replicate plants (± SE).

Figure 3b shows the effect of root temperature on leaf respiration rates (measured at 20 °C). Over the range of 5–35 °C, root temperature had no effect on leaf respiration rates ( Fig. 3b). Changes in shoot temperature per se therefore appeared responsible for the changes in leaf respiration rates at a set temperature. Taken together, Figs 2 and 3 highlight the danger in relying on measurements of leaf respiration at a set temperature conducted in an environment where the air temperature changes.

Variation in leaf respiration rates in the field: diurnal and seasonal variation

To more accurately predict the daily and seasonal variation in leaf respiratory CO2 release, we conducted subsequent measurements of leaf respiration at ambient temperatures, so that the leaf temperature matched that of the rest of the shoot. Leaf respiration rates were far greater in summer than in winter for much of the day, when expressed on an area basis ( Figs. 4a & b). However, the decrease in the ratio of leaf area to leaf mass (both fresh and dry) from winter to summer (data not shown) resulted in the rates in the two seasons being similar on mass basis ( Fig. 4c). The difference between summer and winter rates in 1997 was similar on an area and a mass basis, due to the limited change in the ratio of leaf area to mass ( Figs 4b & d).

Figure 4.

Example of the diurnal variation in leaf respiration in darkness in ‘summer’ (▪) and ‘winter’ (●) in 1996 and 1997 for values expressed on (a & b) a leaf area (μmol CO2 m−2 s−1) and (c & d) a leaf dry mass basis (nmol CO2 g−1 DM s−1). Rates were measured at the ambient leaf temperature exhibited by leaves at the time of measurement. On each sampling day, values represent the mean of four replicate plants (± SE) measured repeatedly at 2–3 h intervals. The ‘winter’ measurements in 1996 and 1997 were conducted in August and July, respectively, while ‘summer’ measurements took place in December in both years. Measurements were also conducted at other times of the year (not shown).

Leaf respiration rates were often relatively high in measurements conducted in the hour leading up to sunrise (e.g. winter 1996; winter and summer 1997; Figs 4a & c; Fig. 5) when air temperatures were at their lowest. The extent to which the pre-sunrise leaf respiration rates were relatively high was estimated by comparing the measured rates with rates predicted from the post-sunrise Q10 values ( Fig. 5). On six of the 13 measuring days, pre-sunrise respiration rates were higher than expected from the post-sunrise Q10 value (data not shown). The average pre-sunrise rate for all 13 measuring days was 43% higher than calculated from the post-sunrise Q10. Higher than expected pre-sunrise respiration rates were observed in summer and winter.

Figure 5.

Example of leaf respiration in darkness (nmol CO2 g−1 DM s−1; log10 scale) plotted against ambient leaf temperature for ‘summer’ (▪, □) and ‘winter’ (●, O) in 1996 and 1997, using the diurnal data from Fig. 4 (n = 4, ± SE). The slope of each plot was used to calculate the Q10 of respiration ( Berry & Raison 1981). Closed symbols represent post-sunrise measurements and were used to calculate the Q10 values. The open symbols represent relatively high pre-sunrise measurements, if they occurred. The solid lines are the linear regressions used to calculate the Q10 values, with the dotted lines showing the same regressions extrapolated to higher and lower temperatures.

In leaves that exhibited relatively high pre-sunrise values, leaf respiration rates initially declined as temperatures increased after sunrise, and subsequently increased/decreased in a temperature-dependent manner later in the morning and in the afternoon ( Fig. 4). Respiration also increased/decreased in a temperature-dependent manner later in the day in leaves that did not exhibit the relatively high pre-sunrise respiration rates.

To assess whether respiration acclimated and/or whether the temperature sensitivity of respiration (i.e. the Q10) changed seasonally, we plotted respiration rates on a log10 scale against leaf temperature ( Fig. 5). In all cases, the resultant plots were linear, as long as any relatively high pre-sunrise values were excluded from the analysis. Q10 values calculated in this way did not differ between measuring days so long as the 2-day-average air temperature (Tavg; measuring day and the day preceding the measuring day) was above 6 °C ( Fig. 6; Table 1). However, significantly higher Q10 values did occur in winter when Tavg was below 6 °C ( Fig. 6; Table 1; P < 0·001).

Figure 6.

Seasonal variation in Q10 of leaf respiration. Q10 values for each measuring day are plotted against the 2-d average air temperature (measuring day and the day preceding the measuring day). Q10 values for days where the daily average air temperature were greater than 6 °C (●) and less than 6 °C (O) are shown. The Q10 values for each day were determined using data similar to those shown in Figs 4 and 5 (n = 4, ± SE).

The degree to which respiration at a given temperature was higher in winter than in summer varied depending on the given temperature that was used for the comparison ( Fig. 7). For example, when rates were compared at 25 °C, the values were nearly five times higher in winter (i.e. 196 d after January 1) than in summer (349 d after January 1) in 1997 ( Fig. 8). In contrast, the seasonal difference between rates at a given temperature was substantially less when the rates were compared at 5 °C ( Fig. 8). This difference was due to the higher Q10 values in winter ( Fig. 6).

Figure 7.

Rates of respiration per unit leaf dry mass in darkness (nmol CO2 g−1 DM s−1) at given temperatures [0 °C (●), 5 °C (○), 10 °C (▾), 15 °C (∇), 20 °C (▪) and 25 °C (□),] plotted against time (days after January 1st) for (a) 1996 (B) 1997 and (c) 1998. Rates at each given temperature for each measurement day were calculated using plots of log10 respiration versus temperature ( Figure 5) of the post-sunrise Q10 values shown in Fig. 5 (n = 4, ± SE). Values were calculated using the average respiration versus temperature regression equation for each sampling day ( Fig. 5).

Figure 8.

The effect of seasonal changes in Q10 values on the rate of respiration at a given temperature for winter and summer in 1997. Values shown represent the ratio of respiration rates in winter (i.e. when rates at a given temperature were at their highest) and summer (when rates at a given temperature were at their lowest).

The impact of acclimation to high summer temperatures (i.e. reduction in the rate of respiration at a given temperature and a decrease in the Q10 of respiration) on daily CO2 release was determined for the 1997 data. Daily leaf CO2 release was calculated as the respiration rate at the average air temperature for the measurement day, using the respiration versus temperature curves ( Fig. 5). We compared these calculated daily rates of CO2 release with the theoretical daily rates of CO2 release that would have occurred if leaves in summer had exhibited the same Q10 values and the same rates at a given temperature as leaves in winter. In the absence of any acclimation or change in Q10 values, the daily CO2 release would simply have increased as Tavg increased (dashed line in Fig. 9). Clearly, the summer values fall well below the theoretical winter line.

Figure 9.

Seasonal variation in the daily respiratory CO2 release per unit leaf dry mass (closed symbols) in 1997. Daily CO2 release was calculated as the respiration rate at the two-day average air temperature (measuring day and the day preceding the measuring day), using the respiration versus temperature curves ( Fig. 5). To assess the degree of seasonal acclimation on daily CO2 release, we compared these daily rates of leaf respiration (●) with predicted values of daily respiratory CO2 release on each measuring day (dashed curve). The predicted values were calculated using the Q10 values and respiration rates exhibited by leaves on the coldest measured day and by assuming that respiration did not acclimate to the increase in daily average temperature as summer approached. Daily leaf respiratory CO2 release values are plotted against the 2-d average air temperature.

Response of leaf respiration to temperature changes under controlled conditions

To further assess whether changes in temperature per se were responsible for the seasonal changes in Q10 and respiration rates at a given temperature, we exposed potted snow gum plants to constant 10 °C or 20 °C treatments for several weeks under controlled-environment conditions. When measured at a set temperature (20 °C), respiration rates were considerably higher in the cold-treated plants compared with plants exposed to the warmer treatment ( Fig. 10). Moreover, the Q10 values exhibited by the cold-treated plants were considerably higher than those of the warm-treated plants (2·7 and 2·2, respectively).

Figure 10.

Rates of leaf respiration per unit leaf area in darkness (always measured at 20 °C) versus time for plants growing under controlled-environment conditions. Air temperature in the cabinets was either a constant 10 °C or a constant 20 °C. Plants were transferred from their initial temperature treatment to the other treatment on day 14. Respiration rates of individual leaves were repeatedly measured over the 24-d experimental period. Values represent the average of four replicate plants in each treatment (± SE).

By transferring plants from one temperature to the other, we were able to assess the time taken for leaf respiration to acclimate to the change in growth temperature. Figure 10 demonstrates that substantial acclimation occurred within 1 day, with near full acclimation 3 to 7 d after the change in growth temperature.

Relationship between acclimation and the soluble sugar concentration

When plants were transferred from 10 to 20 °C, the concentration of soluble carbohydrates decreased rapidly over a 3 d period (data not shown), whereas the concentration increased when plants were transferred from 20 to 10 °C. The increase in respiration rates at a set temperature in plants transferred from 20 to 10 °C was associated with an accumulation of sugars ( Fig. 11; open circles). Conversely, both the sugar concentration and respiration rate at a set temperature decreased when plants were transferred from 10 °C to 20 °C ( Fig. 11; closed circles). Maximum respiration rates occurred when the concentration of soluble sugars was 0·6–1·0 g m−2 ( Fig. 11).

Figure 11.

Respiration rates in darkness per unit leaf area (always measured at 20 °C) for individual leaves plotted against the corresponding concentration of soluble sugars (sucrose + glucose + fructose) for plants transferred from 20 °C to 10 °C (○) and 10 °C to 20 °C (●) growth treatments. Respiration rates and sugar concentrations of individual leaves were sampled destructively following 0, 1, 2 and 7 d after the transfers.

The correlation between the concentration of soluble sugars and respiratory acclimation under controlled conditions raised the question as to whether the seasonal acclimation under field conditions was also associated with changes in the sugar concentration. Despite some seasonal variation, there was no correlation between leaf respiration rates at a set temperature and the concentration of total soluble sugars ( Table 1). Moreover, the concentration of sugars in the field-grown plants was always high (compared with the controlled-environment-plants). Respiratory acclimation under field conditions was therefore not associated with changes in the sugar concentration.

DISCUSSION

Our study has demonstrated that leaf respiration of snow gum varies diurnally and seasonally in response to short- and long-term changes in temperature. We have also shown that acclimation to low temperature results in an increase in the apparent respiratory ‘capacity’ (i.e. respiration rate at a given temperature; Table 1; Figs 2, 6 & 10) and the sensitivity of respiration to short-term changes in temperature (i.e. it increases the Q10; Fig. 6). Changes in Q10 therefore need to be taken into account when considering the impact of acclimation on leaf respiration. The net result of these changes is that there is relatively little seasonal variation in total daily leaf respiratory CO2 release per unit leaf dry mass ( Fig. 9).

Importance of conducting respiration measurements at the ambient air temperature

Our study has highlighted the importance of estimating total daily leaf respiration using diurnal measurements carried out at ambient leaf temperatures throughout the day, rather than by simply exposing individual leaves to short-term changes in temperature, as is commonly done (e.g. Larigauderie & Körner 1995; Diemer & Körner 1996). Leaf respiration rates of individual leaves at a set temperature were considerably faster when the temperature of the remaining shoot was higher ( Figs 2 & 3). As a result, respiration–temperature curves obtained from short-term measurements in which the shoot remains at one temperature are unlikely to provide an accurate estimate of the rates of CO2 release by leaves at different times of the day. The mechanism by which shoot temperature influences the respiration rate of individual leaves is unclear. Nevertheless, we feel that it is important that diurnal studies of respiration should avoid using short-term temperature response curves on individual leaves to estimate the extent of daily respiratory CO2 release under variable temperature conditions.

Several studies investigating diurnal patterns in leaf respiration have compared respiration rates at different times using measurements made at a set temperature. For example, Collier, Cummins & Villar (1991) reported no diurnal variation in dark respiration rates of leaf discs taken from naturally growing tree species. Similarly, Küppers et al. (1986) found that in illuminated snow gum leaves, respiration via the ‘dark’ respiratory pathways (i.e. non-photorespiratory mitochondrial CO2 release in the light) did not vary during the day when measured at a set temperature. Neither of these studies established the diurnal variation in leaf respiration at the ambient leaf temperatures exhibited by leaves at different times of the day. Our results suggest that diurnal variations in respiration rates in darkness do occur when measured at the actual temperatures experienced by the leaves during the day rather than a set temperature.

The cause(s) of the relatively high pre-dawn respiration rates are unknown. Nevertheless, it is clear that leaf respiration does not necessarily decline during the night. This contrasts with previous controlled-environment studies in which plants were exposed to constant night temperatures (e.g. Azcón-Bieto & Osmond 1983; Azcón-Bieto, Lambers & Day 1983; Averill & ap Rees 1995).

Do single Q10 values describe the temperature response of respiration on each measurement day?

The conclusion that the Q10 is greater in winter than in summer ( Fig. 6) relies on the assumption that, on each measurement day, a single Q10 applies over a range that allows rates in winter and summer to be compared at set temperatures ( Fig. 7). If this assumption were not correct, then the seasonal differences in Q10 may have been due to bias arising from the contrasting daily temperature amplitudes in winter and summer. Single Q10 values did apply over the range of temperatures experienced by the leaves in winter and summer, as plots of log10 of respiration rate versus leaf temperature on each measurement day were linear on all measurement days ( Fig. 5). However, there was substantial seasonal change in the temperatures experienced by the snow gum; in 1996 the average minimum and maximum air temperatures (at 0·2 m above the ground) in the coldest period in winter were – 4 °C and 12 °C, respectively, whereas in the warmest period in summer they were 12 °C and 33 °C. If the Q10 changed with short-term changes in temperature, then the lower Q10 values in summer may reflect the higher daily temperatures in summer. In some studies the Q10 does change with measurement temperature. For example, Kirschbaum & Farquhar (1984) reported that the Q10 of leaf respiration in darkness was 2·6 over the range 15–20 °C, but declined steadily with increasing temperature to reach 1·9 over the 30–35 °C range. In contrast, the Q10 of leaf respiration of snow gum did not change over the 10–22 °C range under our controlled-environment conditions ( Fig. 3a). Moreover, the Q10 did not change over the 5–30 °C range in snow gum plants that were grown hydroponically at constant temperatures of 10 and 20 °C (data not shown). We are therefore confident that the winter and summer temperature response curves obtained at the field site can be compared over a broad temperature range, and that the Q10 is indeed higher in winter than it is in summer.

The speed of respiratory acclimation

Snow gum leaf respiration acclimated rapidly to changes in temperature, with substantial changes in respiration rates at a given temperature occurring within 1 d ( Fig. 10). To our knowledge, only one other study has investigated the time taken for leaf respiration to acclimate to changes in temperature ( Billings et al. 1971 ). Billings et al. (1971) found that exposure of 25°/20 °C-grown Oxyria digyna (a circumpolar alpine/Arctic herb) plants to 32 °C decreased respiration rates measured at a set temperature by one-third within 10 h. The ability to acclimate rapidly to medium-term (i.e. days) changes in average temperature means that leaf respiration rates at a set temperature and the temperature sensitivity of leaf respiration may be constantly changing in response to changes in weather, both in winter and summer. This may have important implications for the proportion of daily fixed carbon that will be respired by leaves under variable temperature, field conditions.

Mechanisms responsible for respiratory acclimation

The mechanisms of respiratory acclimation are unclear. Acclimation is likely to involve increases in respiratory capacity [either increases in capacity per mitochondrion ( Klikoff 1966, 1968) or an increase in the number of mitochondria ( Miroslavov & Kravkina 1991)]. Concomitantly, acclimation may also involve changes in substrate supply to the mitochondria and/or changes in the demand for respiratory energy associated with cellular maintenance ( Atkin & Lambers 1998).

Under controlled-environment conditions, we found that acclimation to low temperatures was associated with an increase in the concentration of soluble carbohydrates ( Fig. 11). High substrate levels are associated with increased respiration rates in several plant species (e.g. Breeze & Elston 1978; Azcón-Bicto & Osmond 1983). In addition, seasonal changes in sugar concentrations are associated with respiratory acclimation in field-grown Norway spruce and lodgepole pine ( Ögren, Nilsson & Sunblad 1997). Moreover, respiratory gene expression is increased in tissues that accumulate carbohydrates ( Williams & Farrar 1990; McDonnell & Farrar 1992). These results suggest that acclimation under controlled-environment conditions is associated with changes in the concentration of soluble sugars.

The acclimation of snow gum leaf respiration under field conditions was not, however, associated with a change in the concentration of soluble sugars ( Table 1). Despite some variation from month to month, soluble sugar concentrations in the field-grown leaves remained high throughout the year. Moreover, there was no correlation between the level of soluble sugars and the respiration rate at a set temperature (e.g. 25 °C). Factors other than leaf sugar concentrations must therefore have been responsible for the acclimation of leaf respiration under the environmental conditions at our field site.

In addition to changes in substrate supply, acclimation may also reflect changes in the demand for respiratory energy associated with maintenance (e.g. maintenance of solute gradients, membrane transport and repair of degraded proteins) ( Lambers, Chapin & Pons 1998) or phloem loading ( Bouma et al. 1995 ) in mature, fully expanded tissues. For example, the cold-induced photo-inhibition that occurs in snow gum ( Ball et al. 1991 ; Ball et al. 1997 ; Blennow et al. 1998 ; Hovenden & Warren 1998) may increase the need for a high respiratory capacity in mature leaves. Several studies have suggested that respiration helps ameliorate the deleterious effects of cold, bright conditions by oxidizing excess photosynthetic reducing equivalents ( Saradadevi & Raghavendra 1992; Raghavendra, Padmasree & Saradadevi 1994; Hurry et al. 1995 ). Respiration may also provide ATP to help repair proteins degraded following photo-inhibition (in particular the D1 protein of photosystem II). Respiration may also prepare the chloroplast for optimal photosynthetic activity when cold leaves are exposed to bright light at sunrise. For example, recent studies have suggested that respiration can poise the PQ pool in a partly reduced state (i.e. via transfer of mitochondrial-reducing equivalents to the chloroplast) and/or maintain the proton gradient across the thylakoid membrane (via reverse operation of the chloroplast ATPase using respiratory-derived ATP) in near-darkness ( Hoefnagel, Atkin & Wiskich 1998; and references cited therein). Maintenance of a proton gradient is necessary for dissipation of excess radiation by the xanthophyll cycle upon re-illumination ( Gilmore & Yamamoto 1992). The ATP that is required to maintain the chloroplast proton gradient prior to sunrise might be supplied by the mitochondria via a shuttle on the chloroplast membrane that uses a phosphate translocator to exchange DHAP for inorganic phosphate or 3-PGA ( Hoefnagel et al. 1998 ). An increased demand for maintenance respiration for one or more of the these processes under cold, bright conditions may partly explain why the respiratory capacity was higher in snow gum leaves in winter under field conditions.

Our data suggests that under field conditions, respiration only exhibits greater temperature sensitivity and higher rates at a set temperature when the average daily temperature is below a critical temperature (6 °C). Minimum daily air temperatures (Tmin) were typically – 1·1 °C on days when the average daily temperature (Tavg) was 6 °C (i.e. Tmin = – 6·09 + 0·836 ×Tavg, r2 = 0·65; 1996–98 combined data). Why did the respiratory apparatus increase its capacity and temperature sensitivity at these low temperatures? The changes were clearly not associated with an increase in soluble sugar concentration in the field ( Table 1). One possibility is that Tmin values below – 1 °C predispose snow gum leaves to photo-inhibition, with the resultant requirement for an increase in respiratory capacity and temperature sensitivity. Indeed, Tmin values less than –1 °C are required before field-grown snow gum seedlings experience substantial photo-inhibition ( Blennow et al. 1998 ). Increasing the respiratory capacity and temperature sensitivity of respiration would probably result in greater rates of respiratory ATP synthesis at a given temperature (assuming constant engagement of the phosphorylating cytochrome pathway of mitochondrial electron transport). This may enable the proteins degraded by photo-inhibition (e.g. D1 protein of photosystem II) to be repaired at a greater rate in the post-sunrise hours as air temperatures increase. Further work is necessary to test the hypothesis that acclimation of leaf respiration occurs in response to photo-inhibition in field-grown snow gum seedlings.

Concluding statements

In conclusion, our results demonstrate that leaf respiration of snow gum is sensitive to diurnal changes in temperature. Seasonal changes in respiratory ‘capacity’ and temperature sensitivity result in total daily leaf respiratory CO2 release being similar in winter and summer. We propose that acclimation to low temperatures is associated with an increase in the concentration of soluble carbohydrates, when the concentration of carbohydrates at the higher growth temperature is initially low (e.g. under controlled-environment conditions). However, in field-grown leaves that always exhibit high concentrations of soluble carbohydrates, acclimation in winter is unlikely to be associated with changes in substrate availability; rather, it is likely to be associated with an increased demand for respiratory energy. This increase in demand for respiratory energy may be associated with cold-induced photo-inhibition and other processes contributing to tolerance of recurrent freeze/thaw conditions.

ACKNOWLEDGMENTS

We thank Hans Lambers for providing detailed comments on a previous version of this manuscript. The Australian Capital Territory Parks and Conservation Service is thanked for providing access to the field site in Namadgi National Park. We acknowledge the expert technical assistance of Nola McFarlane and Jack Egerton. This work was partly funded by an Australian Research Council Post-Doctoral Fellowship to O.K.A.

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