Shape of leaf photosynthetic electron transport ... - Wiley Online Library

Plant, Cell and Environment (1999) 22, 1497–1513

Shape of leaf photosynthetic electron transport versus temperature response curve is not constant along canopy light gradients in temperate deciduous trees Ü. NIINEMETS,1 V. OJA2 & O. KULL1 1

Department of Ecophysiology, Institute of Ecology, Tallinn University of Educational Sciences, Kevade 2, Tallinn EE-10137, Estonia and 2Department of Plant Physiology, Institute of Cell and Molecular Biology, University of Tartu, Soinaste 39 A, Tartu EE-50404, Estonia

ABSTRACT Responses of foliar light-saturated net assimilation rate (Amax), capacity for photosynthetic electron transport (Jmax) and mitochondrial respiration rate (Rd) to long-term canopy light and temperature environment were investigated in a temperate deciduous canopy composed of Populus tremula L. in the upper (17–28 m) and of Tilia cordata Mill. in the lower canopy layer (4–17 m). Climatic measurements indicated that seasonal average daily maximum air temperature (Tmax) was 5·5 °C (range 0·7–10·5 °C) higher in the top than in the bottom of the canopy, and strong positive correlations were observed between Tmax and seasonal average integrated quantum flux density (Qint), as well as between seasonal average daily mean temperature and Qint. Because of changes in leaf dry mass and nitrogen per unit area, Amax, Jmax, and Rd scaled positively with Qint in both species at a common leaf temperature (T). According to Jmax versus T response curves and dark chlorophyll fluorescence transients, photosynthetic electron transport was less heat resistant in P. tremula with optimum temperature of Jmax, Topt, of 33·5 ± 0·6 °C than in T. cordata with Topt of 40·7 ± 0·6 °C. This difference was suggested to manifest evolutionary adaptation of photosynthetic electron transport to cooler environments in P. tremula, the range of which extends farther north than that in T. cordata. Possibly because of acclimation to long-term canopy temperature environment, Topt was positively related to Qint in P. tremula, foliage of which was also exposed to higher irradiances and temperatures, but not in T. cordata, in the canopy of which quantum flux densities and temperatures were lower, and gradients in the environmental factors less pronounced. Parallel to changes in Topt, the activation energy for photosynthetic electron transport decreased with increasing Qint in P. tremula, indicating that Jmax of leaves acclimated to colder environment was more responsive to T in lower temperatures than that of high T acclimated leaves. Similar alterations in the activation Correspondence: Ü. Niinemets. Fax: 00372 7-383013; e-mail: [email protected] © 1999 Blackwell Science Ltd

energy for mitochondrial respiration rate were also observed, indicating that acclimation to temperature of mitochondrial and chloroplastic electron transport proceeds in a co-ordinated manner, and possibly involves longterm changes in membrane fluidity properties. We conclude that, because of correlations between temperature and light, the shapes of Jmax versus T, and Rd versus T response curves vary within tree canopies, and this needs to be taken account in modelling whole canopy photosynthesis. Key-words: Populus tremula; Tilia cordata; chlorophyll fluorescence; feedback limitations; light gradients; photosynthetic electron transport; temperature acclimation.

INTRODUCTION In temperate trees, foliar maximum photosynthesis rates (Amax) increase with long-term integrated quantum flux density (e.g. Walters & Field 1987; Ellsworth & Reich 1993; Pearcy & Sims 1994; Niinemets & Tenhunen 1997; Niinemets, Kull & Tenhunen 1998), and the light acclimation of leaf photosynthetic characters is the major factor optimizing whole canopy carbon gain (Gutschick 1988; Gutschick & Wiegel 1988; Baldocchi & Harley 1995). Although the positive scaling of Amax with irradiance improves canopy carbon gain under moderate environmental stresses that always occur in natural ecosystems, in long-term, canopy carbon accumulation is also dependent on the ability of leaves to maintain this increased capacity over periods of more severe and long-lasting stresses that may frequently accompany light gradients in tree canopies. For example, daily average integrated quantum flux density incident on the leaves (Qint) correlates positively with daily average air temperature and vapour pressure deficit (Chiariello 1984; Shuttleworth et al. 1985; Margolis & Ryan 1997) within the canopy. Because of these correlations, the leaves exposed to higher irradiance frequently also suffer from greater water (Young & Smith 1979; Ellsworth & Reich 1992; Valladares & Pearcy 1997) and heat stresses (Young & Smith 1979; Hamerlynck & Knapp 1994; He, Chee & Goh 1996) than the shaded leaves. 1497

1498 Ü. Niinemets et al. There are important interactions between environmental stresses on their influences on photosynthesis. Photosystem II (PSII) is the most susceptible component of chloroplastic electron transport chain with respect to high light and heat, and high light stress may exaggerate the influences of heat stress on whole chain linear photosynthetic electron transport rate (J), and vice versa (Bongi & Long 1987; Al-Khatib & Paulsen 1989; Chaves et al. 1992; Valladares & Pearcy 1997). At the same time, prolonged water stress may enhance the resistance of J to heat (Seemann, Downton & Berry 1986; Havaux 1992; Epron 1997; Valladares & Pearcy 1997), possibly because of accumulation of leaf osmotica that improve the heat-stability of PSII (Santarius 1973; Kaiser 1984; Seemann et al. 1986). However, water stress generally also results in stomatal closure, and accordingly, in less advanced transpiratory cooling as the result of which leaf temperatures may exceed the threshold for heat damage (e.g. Valladares & Pearcy 1997). To avoid the damage through high and low temperature stresses, foliage photosynthetic apparatus acclimates to the temperature of leaf environment (Berry & Björkman 1980; Hällgren & Öquist 1990). Often, the temperature optimum of net assimilation rate at current ambient CO2 concentrations (A350) increases with increasing Ig (Slatyer 1977; Berry & Björkman 1980; Ferrar, Slatyer & Vranjic 1989). This is primarily because of alterations of temperature dependence of photosynthetic electron transport, which belongs to most heat-sensitive partial reactions of photosynthesis (Mooney, Björkman & Collatz 1978; Berry & Björkman 1980). In the short term, the heat stability of PSII may rapidly be increased by exposing leaves to moderately elevated temperatures (Havaux 1993; Havaux & Tardy 1996; Havaux et al. 1996; Valladares & Pearcy 1997). This shift may play an important role in protecting PSII from denaturation by moderately elevated temperature during diurnal leaf temperature variations, and may be explained by conversion of xanthophyll violaxanthin to zeaxanthin in chloroplasts (xanthophyll cycle, Havaux & Tardy 1996; Havaux et al. 1996) or by enhanced isoprene (Singsaas et al. 1997) or terpene (Loreto et al. 1998) emission in emitting species. There is evidence that both zeaxanthin formation (Gruszecki & Strzalka 1991) and isoprenoid solubilization (Singsaas et al. 1997) in chloroplast membranes decrease membrane fluidity. The short-term adjustments in temperature resistance of chloroplastic electron transport reactions are superimposed by long-term (days, weeks) modifications (Valladares & Pearcy 1997) that depend on changes in lipid composition of chloroplast membranes (Raison et al. 1980). It is likely that both short-term changes in response to daily changes in leaf temperature, and longterm modifications in response to canopy temperature gradients and seasonal variability in temperature alter the heat stability of light reactions of photosynthesis in temperate deciduous canopies. Yet, current canopy photosynthesis models use a single light-saturated photosynthetic electron transport rate (Jmax) versus leaf temperature (T) curve for

all leaves in the canopy (Harley & Baldocchi 1995; Lloyd et al. 1995; Williams et al. 1996). So far, no studies have been conducted to analyse the temperature responses of Jmax over a broad canopy gradient, but in addition to lightrelated changes in Jmax, covariation between long-term light and temperature gradients through the canopy may imply that the shape of the Jmax versus T response curve varies through the canopy because of temperature acclimation. In the current contribution, we studied the variability of Jmax versus T relationships within a deciduous temperate canopy composed of Populus tremula L. in the upper layer and Tilia cordata Mill. in the lower layer to find out how large is the variation between the shapes of Jmax versus T response curves through the canopy, and whether the optimum temperature for Jmax and initial activation energy vary with long-term canopy light environment. Both steady-state and dynamic gas-exchange and chlorophyll fluorescence techniques were used to gain detailed insight into the temperature responses of Jmax. Studies indicate that the capacity for acclimation of foliar electron transport to temperature environment varies genetically, and ecotypes of the same species (Fryer & Ledig 1972; Slatyer 1977) or different species (Berry & Björkman 1980; Ferrar et al. 1989; Read 1990) originating from diverse habitats may have developed genotypic adjustments to their evolutionary temperature environment. Both P. tremula and T. cordata are widely distributed in Europe, but T. cordata is less frost-tolerant than P. tremula (Otto 1994); the native range of P. tremula extends farther north (just beyond 70°N in northern Norway, Sokolov 1951) than that of T. cordata (rarely found beyond 65° northern latitude Pigott 1991). The southern limit of the two species is similar (cf. Sokolov 1951; Pigott 1991), but the data indicate that the species are constrained by water availability rather than by their low heat tolerance in the southern limit (Pigott 1991). In addition, P. tremula is an isoprene-emitting species, and T. cordata is a non-emitter (Rasmussen 1978). Thus, we also asked whether speciesspecific attributes alter Jmax versus T response curves in the studied canopy and whether both species respond similarly to canopy light and temperature gradients.

MATERIALS AND METHODS Research site The study was conducted during the seasons of 1995–97 in Järvselja mixed deciduous forest (58°22¢ N, 27°20¢ E, elevation 38–40 m), Estonia. The canopy with total leaf area index of about 6 m2 m–2 was dominated by P. tremula and Betula pendula Roth. in the upper layer (17–28 m), and by T. cordata in the lower layer (4–17 m). Corylus avellana L. and T. cordata were the major understory species (see Niinemets 1998; Niinemets et al. 1998 for a detailed description of the study area). The canopy was accessed from permanent scaffoldings (height 25 m) located at the site, and foliar samples were taken throughout the entire canopy.

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1497–1513

Modifications in temperature response of photosynthetic electron transport in trees 1499

Climatic measurements: estimation of daily integrated quantum flux densities (Qint) for sample locations In 1995, photosynthetically active quantum flux density (Q) was continuously monitored in 18 canopy locations with self-made quantum sensors as described in Niinemets et al. (1998), and air temperature in five canopy heights with shielded temperature sensors [K2607 thermometer adaptor (Velleman Kits NV, Gavere, Belgium) based on LM3911 thermistor (National Semiconductor, Santa Clara, CA, USA)]. Hemispherical photographs were taken just above the light and temperature sensors at weekly intervals, and from the sample locations immediately after foliage collection. These photos were used to compute the fractions of penetrating diffuse (Idif, diffuse site factor) and of potential penetrating direct solar radiation of open sky (Idir, direct site factor) as described in Niinemets & Kull (1998). To calculate mean seasonal daily integrated quantum flux densities (Qint, mol m–2 d–1) for the locations of leaf sampling as well as of temperature sensors, regression equations in the form of Qint = a ¥ Idif + b ¥ Idir were developed between the values of Qint of light sensors and their concurrent estimates of Idif and Idir. For each specific sample date, Qint values of the sensors used for computing the regressions were averaged over the period between the completion of lamina expansion-growth (approximately 3 June 1995) and the date of leaf sampling. These specific regressions (r 2 always greater than 0·98) were used to calculate Qint for each sample location from their Idif and Idir (see Niinemets et al. 1998 for further details). Since no direct measurements of Q were available in 1997, a correlation between global solar radiation and Qint was used to calculate Qint as outlined in Niinemets et al. (1998) using the global solar radiation data of Tõravere Actinometric Station (58°16¢ N, 26°28¢ E), and a conversion factor of 1·92 mol MJ–1 (e, see Niinemets et al. 1998). Using the specified value of e, the average Qint above the canopy (Qint°) was calculated between 1 June 1997 and the date of leaf sampling (e.g. Qint° = 38·2 mol m–2 d–1 for the time period between 1 June to 31 August 1997. Hemispherical photographs taken from the sample locations were used to calculate the ‘global site factor’, Isum, – the fractional penetration of solar radiation Isum = pdifIdif + (1 – pdif)Idir, where pdif is the ratio of diffuse irradiance to total irradiance above the canopy. Qint for each sample location was found as the product of Qint° and Isum.

Shoot sampling: determinations of leaf dry mass per area and foliar nitrogen Foliage net assimilation (A) versus intercellular CO2 (Ci), and A versus leaf temperature (T) response curves were measured on detached shoots. The twigs (generally two on a single day) were cut under water, transported to the laboratory within an hour of collection, recut under water and held in dim light before the measurement of assimilation parameters as described in Niinemets et al. (1998).

Leaf circumferences of all leaves on the shoot were traced by a computer digitizer (QD-1212; QTronix, Taiwan) and projected area calculated with a self-developed computer program. Petioles were removed, and each leaflet was weighed after oven-drying at 70 °C for at least 48 h. Leaf dry mass per unit area (MA) was then calculated for each leaf on the shoot. Every leaf used for gas-exchange measurements as well as a pooled sample of the rest of the leaves on the shoot was analysed for nitrogen content with an elemental analyser (CHN-O-Rapid; Foss Heraeus GmbH, Hanau, Germany).

Correction of Qint data of the gas-exchange leaves for the variability in light climate within the shoot Overall, a considerable variability in MA was observed within a single shoot (data not shown), but excellent correlations were observed between Qint, calculated by means of the fish-eye photographs taken above the shoot, and MA averaged over all leaves on the shoot (for linear regressions, r 2 = 0·89, P < 0·001 for P. tremula, and r 2 = 0·69, P < 0·001 for T. cordata). We assumed that the variability in MA along the shoot is related to differential exposure of the leaves, and used the correlation between Qint and average MA to calculate the average integrated quantum flux density for each specific leaf used in gas-exchange and chlorophyll fluorescence measurements from their MA (see Niinemets, Kull & Tenhunen 1998 for the details). Although this routine did not change any of the statistical relationships qualitatively, it resulted in higher explained variance in most of the dependencies.

Gas exchange measurements In 1995, steady-state A versus Ci response curves were measured between 28 June and 29 August, i.e. during the period the leaf photosynthesis parameters were stable (Niinemets et al. 1999a). The open gas-exchange system was that described in Niinemets et al. (1998), except that the gasanalyser was replaced by LI-6262 (Li-Cor, Inc., Lincoln, NE, USA). Photosynthetically active quantum flux density (Q) provided by a halogen lamp (type H1; Osram GmbH, Berlin, Germany) was held at 1000 mmol m–2 s–1 in the centre of leaf cuvette, mean ± SD leaf temperature (TL) was 25·8 ± 0·4 °C, and leaf-to-air vapour pressure deficit (VD) varied from 0·013 to 0·016 mol mol–1 across all measurements. Since there are almost no stomata on the abaxial leaf surface in either species, only the gas-exchange through the adaxial leaf surface was measured. Before each change of air CO2 concentration (Ca), the leaf was stabilized at a Ca of 350 mmol mol–1 for about 20 min to keep the stomata open, and to avoid the depletion of Calvin cycle intermediates. Thus, measurements for a single CO2 response curve took on average 2–3 h. After the steady-state values of A at the highest CO2 concentration had been reached, light was switched off, CO2 concentration changed to


1500 Ü. Niinemets et al. 350 mmol mol–1, and CO2 emission was monitored for 20 min to get an estimate of leaf mitochondrial respiration rate (Rd). In 1997, A versus T response curves were measured between 11 and 31 August with a rapid response (fullresponse time of the system less than 3 s) gas-exchange apparatus, which is described in detail in Oja (1983) with modifications outlined in Laisk & Oja (1994, 1998). To obtain the best achievable control over leaf temperature, the upper side of the leaf was glued directly to the thermostatted window of the gas-exchange cuvette using starch paste. Thus, as in 1995, only the gas-exchange through the adaxial leaf side was measured. More advanced temperature control allowed us to use close to saturating light intensities during the measurements without significantly affecting leaf temperature. Quantum flux density, provided by a 150 W halogen lamp from Schott light source (KL1500; Schott Glas GmbH, Mainz, Germany), ranged from 1620 to 1680 mmol m–2 s–1 for most of the leaves, and from 600 to 630 mmol m–2 s–1 for the lower canopy leaves of T. cordata, for which this light intensity was saturating (see Niinemets et al. 1998). VD was kept close to a low level of 0·013–0·016 mol mol–1 at leaf temperatures between 20 and 40 °C, but was about 0·023 mol mol–1 at 45 °C, and 0·040 at 50 °C. Despite low VD, we had problems in keeping stomata open under high cuvette CO2 concentrations (2000 mmol mol–1) at all temperatures, and therefore, the following baseline conditions, Ca = 350 mmol mol–1 and TL = 25 °C, were used to stabilize the gas-exchange rates and keep stomata open in illuminated leaves. Net assimilation rates were measured at each temperature, starting with the lowest temperature, after the steady-state rates had been achieved at the baseline environment. Exceptionally, damage, as seen in the failure to achieve similar values of A with open stomata at baseline conditions as measured previously, was observed after transfer to higher temperatures. Therefore, the 45 °C and 50 °C temperature points were measured without switching back to stabilization conditions, and for all leaves, the readings of A were taken after the leaves had been at these high temperatures for 5 min. Two thermostats, one circulating water at 25 °C, and the other at the measurement temperature, were used, and at leaf temperatures below 40 °C a new leaf temperature was established 20–30 s after switching between the thermostats, but it took approximately 1 min at higher temperatures. At the same time with changes in leaf temperature, the cuvette CO2 concentration was switched to 2000 mmol mol–1 (Fig. 1). Leaf gas-exchange rates were followed until steady-state rates were attained, after which the cuvette was darkened for 10 min to measure leaf mitochondrial respiration rate at a given temperature (Rd). Thereafter, the leaf was illuminated again, and switched back to baseline conditions, whereas the time necessary for stabilization at low CO2 and 25 °C increased with increasing measurement temperature, being about 20 min for the 25 °C point, and approximately 50 min for the 40 °C point. Thus, the measurements for a single net assimilation versus temperature response curve took generally about 5 h. All foliar gas-

exchange calculations were performed according to von Caemmerer & Farquhar (1981). Altogether 26 leaves were measured for A versus Ci curves, and 24 for A versus T curves.

Measurements of chlorophyll fluorescence Concurrently with the measurements of net assimilation versus T response curves, chlorophyll fluorescence was excited by weak red light modulated at 100 kHz, and fluorescence yields of illuminated leaves in steady-state (Fs) and after application of a saturating pulse (8000 mmol m–2 s–1 for 1 s) of white light (Fm¢) were measured with PAM-101 (Heinz Walz GmbH, Effeltrich, Germany) to calculate the effective quantum yield of photosystem II (PSII) [FPSII = (Fm¢ – Fs)/Fm¢, see Genty, Briantais & Baker 1989]. The value of FPSII was determined both during steady-state gas-exchange as well as immediately after each leaf transfer from low (350 mmol mol–1) to high (2000 mmol mol–1) air CO2 (Fig. 1). After the leaf was darkened for Rd measurements, a 2 s pulse of far-red light provided by Schott light source equipped with an interference filter with the maximum at 720 nm and half band-width of 10 nm (see Laisk & Oja 1994) was given to fully oxidize the pool of primary electron acceptors, QA, and the true initial level of chlorophyll fluorescence (F0) was measured instantly. Intensity of dark fluorescence (Fd) was followed over the entire period the leaf was kept darkened, and pulses of far-red light were irregularly given to check for the reduction state of QA. During Fd measurements, the excitation light was modulated at 1·6 kHz to avoid possible actinic influences.

Measurements of leaf absorptance In 1997, leaf absorptance in the photosynthetically active spectral region (q), necessary for Jmax calculations, was determined using a home-made integrating sphere. In 1995, leaf chlorophyll content per unit area (K) was determined by high-pressure liquid chromatography as described in Niinemets et al. (1998), and q was determined from K by means of an empirical equation (Evans 1993). The average calculated absorptances (mean ± SD = 0·873 ± 0·009 for P. tremula and 0·847 ± 0·013 for T. cordata) in 1995 differed little from the average measured values (0·894 ± 0·008 in P. tremula and 0·880 ± 0·016 in T. cordata) in 1997. Thus, these simplifications had only minor influences on the calculations of photosynthetic electron transport rate from the gas-exchange data.

Determination of the maximum carboxylase activity of Rubisco (Vcmax), and the capacity for photosynthetic electron transport (Jmax) Maximum carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), Vcmax, was calculated from the initial slope of A versus Ci curve as described in Niinemets et al. (1999c). The estimate of the light-



Figure 1. Examples of the measurements of foliar net assimilation rates (lines marked with A) and chlorophyll fluorescence (lines marked with F ) in the laboratory in an upper canopy leaf of Populus tremula (a, b) and in a lower canopy leaf of Tilia cordata (c, d). The illuminated leaf [incident quantum flux density 1680 mmol m–2 s–1 (a–c) or 620 mmol m–2 s–1 (d)] was kept at a cuvette CO2 concentration of 350 mmol mol–1 and leaf temperature of 25 °C until stomata opened and gas-exchange rates stabilized before each change of environmental conditions. The steady-state values of A were used to calculate the capacity for photosynthetic electron transport (Jmax) in steady state (Jsmax), and the plateau values of A observed after the change in CO2 concentration and leaf temperature (parts of A curves within the rectangles) were used to calculate Jmax in transition (Jimax), i.e. in conditions when RUBP synthesis reactions did not limit Jmax. The very first peak values of A observed just after the transition, probably most severely affected by CO2 solubilization effects (see Ruuska et al. 1998), and also close to the response–time limits of the gas-exchange system, were not used in Jimax calculations.

saturated rate of photosynthetic electron transport (Jmax) in steady state (Jsmax) was calculated using the values of A obtained from CO2 or temperature response curves at high CO2 after the steady-state values of A were reached (typically approximately 4–5 min after the change in cuvette CO2 concentration, cf. Fig. 1; Niinemets et al. 1999c). Given that under high CO2 and light, ribulose-1,5bisphosphate (RUBP) regeneration is typically curbed by limited phosphate availability because of inadequate triose phosphate use capacity in sugar and starch synthesis (Sharkey 1985), and that this may lead to a downregulation of the rate of photosynthetic electron transport (Sharkey 1990), we also tried to obtain an estimate of Jmax that was not limited by RUBP formation reactions. Thus, Jmax just after the change in chamber CO2 concentration (350 mmol mol–1 Æ 2000 mmol mol–1) and leaf temperature (Jimax) was calculated from the plateau values of A of the first oscillation after the alteration of conditions as shown in Fig. 1. Although it has been demonstrated that the abrupt

increase in CO2 uptake just after the increase of CO2 in the chamber is mainly related to the rapid use by Rubisco of the RUBP pool built up in low CO2 (Laisk & Oja 1976, 1998; Ruuska et al. 1998), conditions of high chloroplastic RUBP concentration also relieve the feedback limitation of photosynthetic electron transport (Stitt 1986). Moreover, both CO2 uptake and O2 evolution respond similarly to an abrupt increase in CO2 (Laisk & Oja 1998), indicating that both carboxylation and electron transport reactions attain their potential maximum value in RUBP-saturated conditions. The data for transient CO2 uptake were not corrected for CO2 solubilization effects, which result in a slight overestimation of carbon assimilation rates just after low Æ high cuvette CO2 concentration change (Laisk & Oja 1976, 1998; Ruuska et al. 1998). We did also not take account of the delayed photorespiratory CO2 release from the glycollate pool that has not yet come to equilibrium with the changed conditions (cf. Laisk & Sumberg 1994; Pearcy, Gross & He 1997; Kirschbaum et al. 1998). Because


1502 Ü. Niinemets et al. the leaf was stabilized under photorespiratory conditions, but the measurements of Jmax were conducted at high CO2 concentration where photorespiration was suppressed, delayed decline in photorespiratory CO2 evolution may imply that Jmax, determined from the gas-exchange rates just after the change in CO2 concentration, was underestimated somewhat. In all Jmax calculations, we used the estimates of leaf absorptances found as indicated above, computed the temperature dependence of the CO2 compensation point in the absence of mitochondrial respiration, G *, using the temperature parameters of Rubisco given in Niinemets & Tenhunen (1997), and assumed that RUBP regeneration is NADPH limited, and that the mitochondrial respiration (Rd) continuing in the light is equal to the steady-state value of the respiration measured in the darkened leaves at the same temperature (Azcón-Bieto & Osmond 1983; McCashin, Cossins & Canvin 1988). Although other data suggest that mitochondrial respiration becomes partly inhibited in the light (Laisk 1977; Brooks & Farquhar 1985; Villar, Held & Merino 1995), these studies do not account for the recycling of respired CO2 in the leaf. The rates of photosynthetic electron transport were also calculated from quantum yields of PSII measured at high light and high CO2 during the steady-state gas exchange (Jf) according to Genty et al. (1989), and assuming that both photosystems absorbed equal amount of quanta.

Fitting of temperature response curves of Jmax and mitochondrial respiration (Rd) Non-linear least-squares regression (Wilkinson 1990) was used to fit the temperature dependencies of the capacities (Jsmax, Jimax, Jf) of photosynthetic electron transport by (see also Niinemets & Tenhunen 1997): c - DH a RTk

J max =

e , 1+ e( DSTk - DHd ) RTk

(1)

where c is the scaling constant, DHa (kJ mol–1) is an activation energy, DHd (kJ mol–1) is a deactivation energy, DS (kJ K–1 mol–1) is an entropy term, Tk (K) is leaf temperature and R (0·008314 kJ mol–1 K–1) the gas constant. Overall, Eqn 1 provided a function closely fitting the data, and r2 of the non-linear regressions was always greater than 0·94, and averaged 0·97 (cf. Fig. 4 for the fits). Taking the first derivative of Eqn 1, and further expressing Tk from the equation ∂Jmax/∂Tk = 0, the optimum temperature of photosynthetic electron transport, Topt, was calculated as: Topt =

DH d . DS + Rln[DH d DH a - 1]

(2)

Random omission of one or two measurement points of Jmax versus T response curve generally altered all of the parameters of Eqn 1, but had little effect on the calculated Topt value, indicating that Topt values obtained were reliable. Harley & Baldocchi (1995) have demonstrated that identical fits to the Jmax versus T response curves may be

attained by another function that substitutes two parameters of Eqn 1, the scaling constant, c, and the entropy term, DS, with two others, Topt and Jmax at Topt. Because rough estimates of Topt and Jmax at Topt may be obtained directly from the data, the expression of Harley and Baldocchi is more useful for practical purposes. However, Eqn 1 was used in our analysis because it is conceptually more informative and provides mechanistic insight into the sources of variation in Topt (Eqn 2). Initially, the temperature response curves of mitochondrial respiration (Rd) were modelled by the Arrhenius equation: Rd = ec -DHa

RTk

(3)

,

where c is the scaling constant, and DHa (kJ mol–1) the activation energy. Arrhenius equation gave excellent fits to the data with a minimum r 2 of 0·94, and an average of 0·97 (cf. Fig. 5a for the fitted curves). Yet, a wide range of the values of c and DHa was observed (data not shown) across all measured temperature response curves, and because the parameters of Eqn 3 are not fitted independently, they were strongly correlated (r 2 = 0·99 for a linear relationship), thus, making it impossible to compare the changes in the shape of the temperature response curve. To eliminate the autocorrelation, the respiration data for a leaf were standardized with respect to the respiration rate measured at 25 °C [Rd(298·2)] as in Harley & Baldocchi (1995). Given that the standardized rate, l, is equal to: l=

Rd (Tk ) ec - DHa RTk = c - DHa R◊ 298◊2 = e Rd (298 ◊ 2) e

DH a Ê 1 1 - ˆ R Ë 298◊2 Tk ¯ ,

(4)

c is eliminated, and for each standardized rate, li, we found an estimate of DHa independent of c as: 1 1 -1 DH a = R ◊ ln(li )Ê - ˆ . Ë 298◊2 Tk ¯

(5)

Further, an average of all values of DHa per respiration curve was obtained, and having fixed DHa, c was computed by a non-linear regression. In addition, Q10 values with respect to standardized rate were found as: 10

Q10 = liTk -298◊2 ,

(6)

and averaged for each leaf. Alternatively, an estimate of Q10 was calculated from the slope of log(li) versus 10/(Tk – 298·2), allowing to estimate the goodness of data fitting by Q10, which is compatible with a simple exponential function. This routine indicated that Q10 gave occasionally poorer fits to the data with a minimum r 2 of 0·88 and an average of 0·95 than the Arrhenius equation (cf. above). To compare the initial slopes of Jmax versus temperature response curves, Arrhenius equation was also used to fit the data of Jmax measured below Topt, and the activation energies of photosynthetic electron transport for each standardized rate were calculated on the same manner as those for mitochondrial respiration (Eqns 4 and 5). Further, an



Light acclimation of foliar photosynthesis

Figure 2. Correlation between seasonal (from 3 June until 1 October 1995) average integrated quantum flux density (Qint) and average daily maximum (), mean (), and minimum () air temperature through the canopy in Järvselja mixed deciduous forest (58°22¢ N, 27°20¢ E). Average seasonal integrated quantum flux densities above each temperature sensor were found by a technique combining estimations of the fractional penetration of irradiance by hemispherical photography, and actual measurements of quantum flux density by quantum sensors as described in the Material and Methods. Average Qint above the canopy for the whole measurement period was 32·5 mol m–2 d–1. Thus, the uppermost temperature sensor was exposed to about 80% of above-canopy Qint.

average DHa was calculated for all estimates per response curve. Because Jmax values occasionally tended to fall off the exponential function at a lower temperature than Topt, values of DHa that differed more than ± 2SE from the average were discarded.

RESULTS

Because leaf dry mass per area (MA) scaled positively with seasonal average integrated quantum flux density (Qint) in both species (see Material and Methods), leaf nitrogen content per area, Na, the product of MA and N content per unit dry mass, was also strongly related to Qint (Fig. 3a), whereas neither the slopes nor the intercepts were different between 1995 and 1997 [P > 0·1 according to common slope analysis of covariance (ancova) conducted after testing for the significance of the interaction term by separate slope ancova model (P > 0·4)]. The slope of Na versus Qint dependence was higher in T. cordata, which also had greater nitrogen concentrations (mean ± SD = 27·6 ± 2·0 g kg–1), than P. tremula [23·1 ± 1·4 g kg–1, means are significantly different at P < 0·001 according to one-way analysis of variance (anova)]. Measured at a leaf temperature of 25 °C, foliar net assimilation rate at an ambient CO2 concentration of 350 mmol mol–1 (A350), and the rate of mitochondrial respiration in the dark (Rd), the maximum carboxylase activity of Rubisco (Vcmax), and the capacity for photosynthetic electron transport in steady state (Jsmax) all increased with increasing Qint in both species, but because of differences in nitrogen concentration, the slopes were always greater in T. cordata (data not shown, see Niinemets et al. 1998). When nitrogen content per unit area was used as the explaining variable, foliar gas-exchange variables fitted the same lines in both species (Figs 3b–e). The relationships of A350 versus Na, and Rd versus Na did not differ between years (P > 0·3), but the slope of Jsmax versus Na was higher in 1997 than in 1995 (P < 0·001 according to separate slope ancova). Greater slope in 1997 was attributable to a greater measurement light intensity (1680 mmol m–2 s–1 in 1997 versus 1000 mmol m–2 s–1 in 1995) rather than to different routines used to calculate leaf absorptance (q), because the values of q calculated from foliar chlorophyll content in 1995, were very similar to those measured in 1997.

Long-term variation in air temperature within the canopy

Different methods for generating temperature response curves of photosynthetic electron transport (J )

Average seasonal (3 June to 1 Oct 1995) daily maximum temperature (Tmax) and daily mean temperature (Tm) increased, while daily minimum temperature (Tmin) decreased with increasing seasonal integrated daily average quantum flux density (Qint, Fig. 2), and with increasing height in the canopy (data not shown). Over the entire season, average maximum temperature through the canopy varied by 5·5 °C, mean by 0·8 °C, and minimum by 2·1 °C (calculated from the regressions in Fig. 2). Temperature gradients similar to those depicted in Fig. 2 were always present even when temperatures were averaged over a single day, although the absolute differences within the canopy varied, e.g. across all days during the season, the difference in Tmax within the canopy varied from 0·7 to 10·5 °C, and the difference in Tm from 0·2 to 5·6 °C.

Both foliar chlorophyll fluorescence and assimilation rates sensitively responded to changes in cuvette CO2 concentration and leaf temperature (Fig. 1), indicating that foliar gas-exchange rates are closely coupled to photochemical processes. Within single leaf measurements, there was always an excellent correlation (r2 > 0·95 for linear correlation) between the capacity of J from gas-exchange rate in steady-state (Jsmax), and J calculated from chlorophyll fluorescence measurements close to saturating light (Jf; Figs 4a & b). Across all leaves, Jsmax and Jf were also strongly related (r2 = 0·92, P < 0·001). However, Jf was generally lower than Jsmax (Figs 4a & b), possibly because (1) fluorescence probe sampled a smaller area of leaf than was analysed by gas-exchange, and it is likely that there is some micro-heterogeneity in photosynthetic capacity within the


1504 Ü. Niinemets et al.

Figure 3. Dependence of leaf nitrogen content per unit area (Na) on Qint (a), and influences of Na on net assimilation rate (b) and the rate of mitochondrial respiration in the dark (c) measured at an ambient CO2 concentration of 350 mmol mol–1 and a leaf temperature of 25 °C, and on the maximum carboxylase activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (d) and the capacity for photosynthetic electron transport rate in the steady state (e) at 25 °C. Open symbols are for the measurements conducted in 1995, and closed symbols for those performed in 1997 in both P. tremula (squares) and in T. cordata (circles). The ranges of above-canopy Qint averaged over the period from the completion of lamina expansion-growth till leaf sampling were 36·4–42·2 mol m–2 d–1 in 1995 and 38·4–38·8 mol m–2 d–1 in 1997.

leaf; (2) Jsmax, but not Jf, calculations accounted for the fact that measurement light intensity was occasionally not saturating; and (3) mitochondrial respiration rate continuing in the dark was used as a substitute of the mitochondrial respiration rate in the light. Nevertheless, activation energies (DHa, cf. Figs 6c & d) and optimum temperatures (Topt ) calculated from Jf versus T, and Jsmax versus T response curves were similar (Figs 6a & b), and correlated with each other (r2 = 0·45, P < 0·001 for DHa and r2 = 0·84, P < 0·001 for Topt ).

Temperature responses of J: evidence of feedback limitations at low T Basically, the temperature responses of the capacities of J from gas-exchange rates in steady-state (Jsmax) and from rates just after the simultaneous change in cuvette CO2 concentration and leaf temperature (Jimax, Fig. 1), and the responses of J calculated from chlorophyll fluorescence measurements close to saturating light (Jf) were similar within single leaf measurements. However, values of Jimax were greater than Jsmax and Jf at all temperatures, though the differences were less at higher temperatures (Figs 4a & b). Provided the difference between Jimax and Jsmax results from the limited utilization of triose phosphates during steady-state gas-exchange at high CO2 and light (cf. Material and Methods), better correspondence of Jimax and Jsmax

at higher temperature suggests that the feedback limitations of J were less important at higher T. Examination of the gas-exchange traces just after the light flashes used to close all PSII centres for the determination of quantum yield of PSII (Fig. 1) further indicated that carbon assimilation rates were less light-limited at lower than at higher temperature: at higher temperature A increased occasionally in response to extra light in high-light grown leaves (Fig. 1b), but this was rarely evident at low temperature (Figs 1a & c). In addition, there were no further oscillations, which typically indicate feedback limitations of assimilation (Sivak & Walker 1985, 1986; Laisk & Walker 1986), after the first initial increase in A at higher T (Figs 1b & d), but oscillations were more distinctive at lower T (Fig. 1c). As for somewhat greater Jimax than Jsmax at higher T, where J was sensitive to extra light, this is probably related to CO2 solubilization effects following the increase in CO2 concentration (cf. Material and Methods), and to the fact that either no or little heat damage could occur in leaves just after the temperature was increased and a value of A for Jimax estimation sampled.

Interspecific variability in temperature responses of J and Rd The optimum temperature of photosynthetic electron transport was always higher (means were significantly dif-



Figure 4. Fitting of the temperature dependencies of photosynthetic electron transport rates (J) calculated from the measurements of leaf gas-exchange and from the measurements of quantum yield of Photosystem II (FPSII) by chlorophyll fluorescence. (a) and (b), rates of J calculated from FPSII at close to saturating light (Jf, ), and the capacities for J from steady-state gas-exchange rates (Jsmax, ) and from gas-exchange rates measured just after the simultaneous change in cuvette CO2 concentration (350 mmol mol–1 Æ 2000 mmol mol–1) and leaf temperature (Jimax, , cf. Fig. 1) in one T. cordata (a) and P. tremula (b) leaf (the same leaves as in Fig. 1). (c) and (d), all measured values of Jsmax (c) and Jf (d) standardized with respect to the value at 25 °C in T. cordata () and P. tremula (). The capacity for photosynthetic electron transport from leaf gas-exchange measurements was calculated according to Niinemets et al. (1998). Depicted are also the parameters of Eqn 1 corresponding to each curve. Optimum temperature, Topt, calculated from these parameters by Eqn 2 for all standardized data pooled (c, d) was equal to 34·5 °C (Jsmax) and 33·7 °C (Jf) in P. tremula, and 40·6 °C (Jsmax) and 40·3 °C (Jf) in T. cordata and similar values were observed when the Topt values calculated separately for each leaf were averaged (cf. Results). The fractions of explained variance (r2) in panels (c) and (d) are calculated for non-linear least squares fits. For the temperature response curves of single leaves, examples of which are depicted in (a) and (b), r2 was always greater than 0·95.

ferent at P < 0·001 according to one-way anova) in T. cordata (average ± SE = 40·9 ± 0·5 °C from chlorophyll fluorescence and 40·7 ± 0·6 °C from gas-exchange measurements), than in P. tremula (33·2 ± 0·5 °C from chlorophyll fluorescence and 33·5 ± 0·6 °C from gas-exchange measurements; Figs 4c & d). After being exposed to temperatures at and above 35 °C, the leaves did not generally reach the same level of assimilation at 25 °C as measured previously in P. tremula, but similar assimilation rates at 25 °C were observed before and after transfer to temperatures

35 °C and 40 °C in T. cordata (data not shown), indicating that the latter species was more heat resistant. The analysis of chlorophyll fluorescence in the dark further indicated that P. tremula was less heat tolerant as was evident in a rise of true F0 level (open symbols in Figs 5c & d) at a lower temperature than that observed in T. cordata. In general, dark fluorescence (Fd) tended to increase after F0 measurement (black symbols in Figs 5c & d) in both species, and this rise was more significant at higher temperatures and especially in P. tremula. However, a pulse of far-red


1506 Ü. Niinemets et al.

Figure 5. Fitting of the temperature dependencies of mitochondrial respiration rate in one T. cordata and P. tremula leaf (a, the same leaves as in Fig. 1) and the mitochondrial respiration rates standardized with respect to the value at 25 °C in relation to temperature (b, all measurements per species pooled); and changes in leaf dark fluorescence yield (Fd) with temperature in one T. cordata (c), and P. tremula leaf (d). Open circles denote the Fd level just after applying a 2 s pulse of far-red light, i.e. they correspond to the true Fo level; filled circles and arrows illustrate the time-dependent increases in Fd, which were largely reversible by far-red light.

light brought Fd commonly to the initial F0 level, indicating that the pool of primary electron acceptors, QA, became reduced in the dark, possibly because of the damage developing at PSII, and induction of PSII-independent electron transport through photosystem I (PSI) (Havaux 1996). Q10 values of mitochondrial respiration averaged 2·04 ± 0·06 in P. tremula and 2·21 ± 0·09 in T. cordata, and the activation energy of mitochondrial respiration (DHa) averaged 52·4 ± 1·1 kJ mol–1 in P. tremula and 60·9 ± 5·1 kJ mol–1 in T. cordata. Although the means were not significantly different at P > 0·1 between the species (one-way ancova with light as covariate), T. cordata tended to have greater standardized respiration rates at higher temperature (Fig. 5b), possibly because Rd was inhibited in P. tremula under heat stress.

Modifications in the shape of the temperature response curve of Jmax with canopy depth The temperature optima of Jsmax (Fig. 6a), Jimax (data not shown), and Jf (Fig. 6b) increased with increasing seasonal integrated quantum flux density in P. tremula, but they were independent of Qint in T. cordata, where the light range was also narrower, and temperature gradients less significant (Fig. 2). The activation energies for Jsmax (Fig. 6c), Jimax

(data not shown), and Jf (Fig. 6d) calculated from the data below Topt decreased with increasing Qint in P. tremula, and were not significantly light-dependent in T. cordata. Q10 values of mitochondrial respiration were negatively related to Qint in P. tremula (r2 = 0·33, P < 0·05), but not in T. cordata (r2 = 0·06, P > 0·6).The relationships with the activation energy for Rd (Eqn 5) were qualitatively similar to those with Q10 (Fig. 6e).

DISCUSSION Co-variations between air temperature and average integrated quantum flux density (Qint) Positive correlations between Qint and seasonal average daily mean and maximum air temperatures (Fig. 2) agree with patterns observed previously (cf. Introduction). Although the variation in mean daily temperature through the canopy was relatively small when averaged over the season, differences were more pronounced for seasonal average daily maximum air temperature, which gives a more efficient estimate of potential high temperature limits for the leaves (Fig. 2). High air temperatures beyond those potentially damaging to the leaves should not automatically imply that leaves



Figure 6. Effects of seasonal average integrated quantum flux density on (a, b) the temperature optimum of photosynthetic electron transport (Topt, Eqn 2), and on (c, d) the activation energy (DHa) of photosynthetic electron transport capacity (Jmax), and on (e) the activation energy of mitochondrial respiration rate (Eqn 5) in P. tremula () and T. cordata (). The activation energy of Jmax was calculated from the data points below Topt, and standardized with respect to the value of Jmax at 25 °C by Arrhenius equation (cf. Eqn 5). On all panels, the explained variances are given for linear regressions.

are subject to heat stress. Depending on leaf capacity for heat dissipation by convection or transpiration as well as on the coupling efficiency between leaves and atmosphere, leaf temperatures may substantially differ from air temperature. According to former studies, leaf characteristic dimensions, which determine the thickness of boundary layer for convection and water vapour diffusion for a common wind speed, were only weakly related to Qint (Niinemets, Kull & Tenhunen 1999), but maximum stomatal conductances increased with increasing Qint (Niinemets, Kull & Tenhunen 1998; Niinemets et al. 1999b) in both species. Thus, in con-

ditions of high wind speed and low water stress when both the boundary layer and stomatal conductances are high, leaf temperature may significantly be uncoupled from air temperature. Yet, water stress, leading to stomatal closure and increased leaf temperatures (Kappen 1981), often develops during the season and over the day, and is also more significant for the upper canopy leaves in temperate forest trees (Niinemets et al. 1999b). If leaf water stress is accompanied by low wind speeds above the canopy, leaf temperatures may closely follow or even exceed the air temperatures. Given also that air temperature gradients


1508 Ü. Niinemets et al. within the canopy are greatest in windless days because of low turbulent air mixing, leaf temperature profiles similar to air temperature may frequently be observed in tree canopies. Although few data are available, higher leaf temperatures in the upper than in the lower canopy leaves have been measured in Populus species (Roden & Pearcy 1993).

Variability in foliar photosynthetic characteristics through the canopy Increasing net assimilation rate at an ambient CO2 concentration of 350 mmol mol–1 (A350, Fig. 3b), maximum Rubisco carboxylase activity (Vcmax, Fig. 3d), and the capacity for photosynthetic electron transport (Jmax, Fig. 3e) with increasing seasonal average daily integrated quantum flux density (Qint) is the common acclimation response invariably observed in temperate tree canopies (Ellsworth & Reich 1993; Kull & Niinemets 1998; Niinemets, Kull & Tenhunen 1998; Niinemets et al. 1998, 1999a). As previous studies have demonstrated, the increase in leaf dry mass per unit area (MA) with irradiance (and accordingly, leaf nitrogen content per area, Fig. 3a) is the major factor responsible for the scaling of foliar photosynthetic potentials with Qint (Niinemets & Tenhunen 1997; Niinemets, Kull & Tenhunen 1998; Niinemets et al. 1999a). Because MA is stable after leaf growth has ceased, Jmax and Vcmax are relatively invariable in fully developed leaves (Niinemets et al. 1998, 1999a). Nevertheless, the current study indicates that superimposed by the light-related acclimation in photosynthetic characteristics, there is considerable variability (Figs 4c & d) in Jmax that is linked to the temperature environment in the canopy.

The value of Jimax is intricate to interpret. There is a general agreement that transition CO2 uptake proceeds in RUBP-saturated conditions (Laisk & Oja 1998; Ruuska et al. 1998). Under such circumstances, CO2 uptake should be limited by Rubisco carboxylase activity (Laisk & Oja 1998; Ruuska et al. 1998). Yet, when steady-state photosynthesis is feedback-limited because of low turnover of phosphate within chloroplasts and between chloroplastic and cytoplasmic pools (Sharkey 1990), both Rubisco activity and that of photosynthetic electron transport are down-regulated (Pammenter, Loreto & Sharkey 1993). Feedback limitations are often induced at high light and air CO2 concentration, and are more significant at lower temperatures (Labate & Leegood 1988; Labate, Adcock & Leegood 1990). In addition, Stitt (1986) has demonstrated that the potential capacity for electron transport is higher than the actual steady-state rate at high CO2 and light. Thus, we interpret the differences between Jsmax and Jimax as primarily indicative of feedback limitations of electron transport during steady-state photosynthesis. Studies using mutants deficient in several enzymes responsible for the turnover of triose phosphates demonstrate that the difference between transient and steady-state assimilation rates is larger when feedback-limitations occur (Ruuska et al. 1998). Overall, measurements of steady-state and transient electron transport rates indicate that the current standard methodology for estimating Jmax from steady-state assimilation rates at high CO2 and light may underestimate the actual capacity in low and moderate leaf temperatures because of limited circulation of triose phosphates under these conditions (Figs 4a & b).

Species differences in Jmax versus T response curves

Temperature responses of photosynthetic electron transport The shape of the temperature response curve of steadystate light-saturated photosynthetic electron transport rate (Jsmax, open circles in Figs 4a & b), characterized by high initial slope (large activation energy) and abrupt decrease beyond the optimum temperature (Topt), is very similar to that used in current canopy photosynthesis models (Harley & Baldocchi 1995; Lloyd et al. 1995; Williams et al. 1996), because the calculation of Jmax from steady-state gasexchange measurements at high light and high intercellular CO2 concentrations is a standard routine in parameterization of ecophysiological photosynthesis models. Yet, the initial slope of Jmax versus T response curve tends to be lower for uncoupled photosynthetic electron transport in isolated chloroplasts than that observed for whole leaf measurements in steady state (e.g. Nolan & Smillie 1976; see Kirschbaum & Farquhar 1984; Niinemets & Tenhunen 1997 for a comparison). In the current study, the initial slope was also much lower for transient values of photosynthetic electron transport measured just after the change in air CO2 concentration (Jimax, closed circles in Figs 4a & b), and was similar to that observed for in vitro measurements in chloroplasts.

Although T. cordata was exposed to lower irradiances, both the measurements of Jmax versus T (Figs 4c & d) as well as dark chlorophyll fluorescence measurements (Figs 5c & d) indicated that chloroplastic electron transport is more heatsensitive in P. tremula, the range of which extends farther north than that in T. cordata. This is in general agreement with studies demonstrating lower optimum temperatures in species evolutionarily adapted to cooler environments (cf. Introduction). Across a wide range of species, foliar frost resistance and optimum temperature for photosynthesis are inversely correlated (Read & Hope 1989), and it seems that species are unable to optimize the performance in both hot and cold environments. From another perspective, P. tremula possesses leaves that flutter, and has also greater stomatal conductances than T. cordata (Niinemets et al. 1999a,b). Given that at a common wind speed the temperature of fluttering leaves is lower than that of non-fluttering leaves (Roden & Pearcy 1993), and higher transpiration rates imply a higher capacity for latent heat loss, species differences in Topt may also result from possibly lower leaf temperatures in P. tremula. However, given that average maximum air temperature was more than 5 °C lower in the bottom than in the top of


Modifications in temperature response of photosynthetic electron transport in trees 1509 the canopy, and that Topt was approximately 7 °C higher in the lower canopy species T. cordata, this seems unlikely.

Acclimation of Jmax versus T responses to canopy temperature environment Large variability in standardized Jmax versus T responses (Figs 4c & d) indicates that modelling canopy photosynthesis using a single response curve for all leaves in the canopy may lead to significant errors. Both the optimum temperature of Jmax (Figs 6a & b) as well as the initial activation energy (Figs 6c & d) were related to Qint in P. tremula, possibly as the result of acclimation to temperature gradients accompanying light gradients (Fig. 2). Photosynthetic electron transport versus T response curves have not been studied extensively through the canopy, but previous works indicate that Topt for A350 tends to increase with increasing canopy height in other temperate trees (Kusumoto 1978; Jurik, Weber & Gates 1988). Given that upper canopy leaves generally function at lower intercellular CO2 concentrations (Kull & Niinemets 1998; Niinemets, Kull & Tenhunen 1999), mainly because of greater stomatal closure (Niinemets et al. 1999b), they are expected to possess lower rather than higher Topt for A350 (see Nobel, Longstreth & Hartsock 1978) at higher irradiance. Thus, this positive correlation is also indicative of acclimation. We did not observe significant correlations between Qint, and Topt and initial activation energy for Jmax in T. cordata (Figs 6a–d). Smaller temperature gradient observed within the canopy of T. cordata may provide an explanation for these non-correlations, but electron transport may also be less plastic in this species than that in P. tremula. The potential for temperature acclimation differs widely between species (Berry & Björkman 1980; Ferrar et al. 1989). The pool of xanthophyll cycle carotenoids also increases with increasing Qint (Logan et al. 1996; Niinemets et al. 1998), possibly providing further protection against heat stress for the upper canopy leaves (cf. Introduction). In the current work, measurement of temperature response curves was started from the lowest temperature, and continued at each temperature long enough to allow sufficient time for short-term alterations in heat resistance of PSII. Thus, we suggest that changes observed in Topt and activation energy reflect modifications at the level of thylakoid membrane lipid composition. Acclimation to higher temperature involves increases in saturation state of chloroplast membrane lipids (Raison et al. 1980; Quinn & Williams 1985; Süss & Yordanov 1986) that decrease membrane fluidity and increase membrane stability at higher temperatures.

Trade-offs between acclimation to high temperature and photosynthesis under low temperature Decreases in membrane fluidity not only increase the heat resistance of Jmax as seen in greater Topt values (Figs 6a & b), but also modify the whole Jmax versus T response curve,

and result in lower initial activation energy (Figs 6c & d), and lower Jmax values at low temperatures. In general, the in vivo specific activity of membrane-bound enzymes decreases with decreasing membrane fluidity (Shinitzky 1984), and adaptation of membranes to higher temperature results in decreased fluidity (Cossins & Sinensky 1984). Artificial saturation of membrane lipids leads to greater heat tolerance of chloroplastic electron transport, but also to a considerable decrease in whole chain electron transport rate (Horváth et al. 1986; Gombos et al. 1988). Given that in desaturase deficient mutants energy transfer is inhibited between PSII and PSI (Apostolova, Busheva & Tsvetkova 1998), it is likely that increased membrane saturation state results in lower plastoquinone turnover due to lower diffusion rates in the membranes (but see also Gombos et al. 1988).This signifies that protein requirements for construction of photosynthetic machinery with the same Jmax at a common low temperature are greater for chloroplasts with more rigid membranes. Thus, foliar acclimation improving leaf heat tolerance may bring about enhanced protein costs for supporting electron transport at lower temperatures. Photosynthesis rates of leaves acclimated to certain temperature conditions may be similar at optimum temperature, but are often considerably lower for high-T acclimated leaves, even if high-T and low-T acclimated leaves are measured at the Topt of high-T leaves (Berry & Björkman 1980). This may be related to enhanced N requirements for construction of photosynthetic machinery of high-T acclimated leaves. Optimum temperature for Jmax was correlated with seasonal average integrated quantum flux density (Figs 6a & b) in our study. Yet, daily average air and leaf temperatures change across the season, and this may also bring about alterations in the shape of Jmax versus T response curve. Seasonal variability in the temperature optima of net assimilation and Jmax has been demonstrated in a number of investigations (Mooney et al. 1978; Mooney 1980; Skillman, Strain & Osmond 1996) and further studies are necessary to identify the kinetic constants of temperature acclimation.

Acclimation of mitochondrial respiration to temperature Respiration rates are generally higher at a common temperature (Pearcy & Harrison 1974; McNulty & Cummins 1987; Larigauderie & Körner 1995; Arnone & Körner 1997) in plants grown under lower temperatures. This modification is compatible with an increase in the scaling constant of respiration rate versus T response curve (Eqn 3), and may possibly occur because the saturation state of mitochondrial membrane lipids decreases as the result of acclimation to low temperatures (Miller, de la Roche & Pomeroy 1974), bringing about greater membrane fluidity. Furthermore, as in the case of Jmax, changes in the activation energy for mitochondrial electron transport rate (Eqn 3) may also be related to the stability of mitochondrial membranes (Lyons & Raison 1970; Raison, Lyons &


1510 Ü. Niinemets et al. Thomson 1971). Thus, respiration rates may increase more quickly with increasing temperature in plants acclimated to low temperature (Pearcy & Harrison 1974), implying increases in the activation energy for Rd (DHa, Eqn 3) with increasing growth T. A negative correlation between Qint and DHa was observed in the current study (Fig. 6e), and was also evident in several Japanese forest species (recalculated from Kusumoto 1978), further supporting the argument that DHa increases as a result of acclimation to lower T. Although the patterns may be somewhat variable in other works (Miller et al. 1974; McNulty & Cummins 1987), we conclude that acclimation in response to long-term temperature variation occurred in a similar manner in chloroplast and mitochondrial membranes, and was possibly related to alterations in membrane stability via changes in their lipid composition.

Conclusions Scaling of foliar photosynthetic capacities with long-term canopy light environment is the way efficient utilization of quanta is achieved in forest species inherently possessing extensive light gradients from canopy top to the bottom. Although higher light along with greater photosynthetic capacities increases long-term foliar light use-efficiency, leaf exposition to higher irradiances may potentially exaggerate the influence of environmental stresses on carbon gain, because of the correlations between environmental variables and the interactions between leaf acclimation state and leaf response to stress. The current study indicates that superimposed by changes in foliage photosynthetic potentials resulting from the variability in canopy light climate, the shapes of photosynthetic electron transport (Jmax) and mitochondrial respiration versus temperature (T) response curves acclimate to the temperature gradient accompanying the light profile in the forest canopy to alleviate potential heat stress influences; but also that the shapes of these response curves may significantly depend on speciesspecific attributes reflecting evolutionary adaptation to species temperature environment. Phenotypic and genotypic adjustments in the Jmax versus T responses possibly result from changes in the fluidity of thylakoid membranes and may bring about varying nitrogen requirements to attain a certain leaf photosynthetic capacity at a common leaf temperature. Because of the large inter- and intraspecific variability, we conclude that modelling whole canopy photosynthesis using a single Jmax versus T response curve is not appropriate. So far, it is a common practice in parameterizing ecophysiological canopy models to use gas-exchange measurements under high CO2 concentrations to estimate Jmax. However, another important conclusion arising from the conducted detailed measurements combining gas-exchange and fluorescence techniques under steady-state and dynamic conditions, is that high CO2 generally induces feedback limitations of photosynthetic electron transport such that steady-state gas-exchange rates may significantly underestimate ‘true’ Jmax, especially when leaf tempera-

tures are low. Potential capacity of photosynthetic electron transport may more effectively be estimated from rapid gas-exchange transients after altering cuvette CO2 concentration, and the shapes of Jmax versus T response curves estimated this way are very similar to those measured for uncoupled chloroplastic electron transport.

ACKNOWLEDGMENTS We thank Peter C. Harley (Atmospheric Chemistry Division, National Center for Atmospheric Research, Boulder, CO, USA) for insightful comments on the study, Lea Hallik (Department of Applied Ecology, University of Tartu, Estonia) for skilled assistance in the laboratory, Anne Jõeveer (Tõravere Actinometric Station, Estonia) for providing unpublished results for global solar radiation during the study period, and Heino Kasesalu (Järvselja Experimental Forest Station, Estonian Agricultural University) for allowing us to conduct the research at Järvselja. This work was supported by Research Grants (2048, 3235, 3907) from the Estonian Science Foundation.

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