Heat sensitivity of photosynthetic electron transport varies during the day due to changes in sugars and osmotic potential

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1 Variation in heat sensitivity of PSII due to sugars K. Hüve et al. Plant, Cell and Environment (2006) 29, doi: /j x Heat sensitivity of photosynthetic electron transport varies during the day due to changes in sugars and osmotic potential KATJA HÜVE, IRINA BICHELE, MARI TOBIAS & ÜLO NIINEMETS Department of Plant Physiology, University of Tartu, Riia 23, Tartu EE 51010, Estonia ABSTRACT In water-stressed leaves, accumulation of neutral osmotica enhances the heat tolerance of photosynthetic electron transport. There are large diurnal and day-to-day changes in leaf sugar content because of variations in net photosynthetic production, respiration and retranslocation. To test the hypothesis that diurnal and day-to-day variations in leaf sugar content and osmotic potential significantly modify the responses to temperature of photosynthetic electron transport rate, we studied chlorophyll fluorescence rise temperatures (i.e. critical temperatures at break-points in fluorescence versus temperature response curves, corresponding to enhanced damage of PSII centers and detachment of pigment-binding complexes) in the dark at a background of weak far-red light (T FR ) and under actinic light (T L ), and responses of foliar photosynthetic electron transport rate to temperature using gas-exchange and chlorophyll fluorescence techniques in the temperate tree Populus tremula L. Sucrose and sorbitol feeding experiments demonstrated strong increases of fluorescence rise temperatures T FR and T L with decreasing leaf osmotic potential and increasing internal sugar concentration. Similar T FR and T L changes were observed in response to natural variation in leaf sugar concentration throughout the day. Increases in leaf sugar concentration led to an overall down-regulation of the rate of photosynthetic electron transport (J), but increases in the optimum temperature (T opt ) of J. For the entire dataset, T opt varied from 33.8 C to 43 C due to natural variation in sugars and from 33.8 C to 52.6 C in the sugar feeding experiments, underscoring the importance of sugars in modifying the response of J to temperature. However, the correlations between the sugar concentration and fluorescence rise temperature varied between the days. This variation in fluorescence rise temperature was best explained by the average temperature of the preceding 5 or 6 days. In addition, there was a Correspondence: Ülo Niinemets. Fax: ; ylon@ut.ee significant year-to-year variation in heat sensitivity of photosynthetic electron transport that was associated with year-to-year differences in endogenous sugar content. Our data demonstrate a diurnal variation in leaf heat tolerance due to changes in sugar concentration, but they also show that this short-term modification in heat tolerance is superimposed by long-term changes in heat resistance driven by average temperature of preceding days. Key-words: day-to-day variation, diurnal variability, heat stress, optimum temperature, osmotic potential, photosynthetic electron transport, sugar concentration. Abbreviations: F 0, basal fluorescence yield; F m, maximum fluorescence yield; F m, light-adapted maximum fluorescence yield; F S, steady-state light-adapted fluorescence; J, photosynthetic electron transport rate; PSI, photo-system I; PSII, photosystem II; Q, photosynthetic quantum flux density; T, temperature; T D, fluorescence rise temperature (break point) in dark without far-red light; T FR, fluorescence rise temperature (break-point) in dark under far-red light; T L, fluorescence rise temperature (breakpoint) under actinic light; T opt, optimum temperature of J. INTRODUCTION Experimental studies and meta-analyses of the parameterizations of photosynthetic electron transport (J) versus temperature (J T) response curves in C 3 plants have demonstrated that the shape of the J T curve varies little over a temperature range of 10 C to 30 C among the species studied (Dreyer et al. 2001; Leuning 2002; Medlyn et al. 2002). However, there are large differences in J responses to temperature at elevated temperatures, and these variations are difficult to predict by mechanistic models (Dreyer et al. 2001; Leuning 2002; Medlyn et al. 2002). Given that the sensitivity of photosynthesis to temperature increases with increasing atmospheric CO 2 concentrations (Kirschbaum 1994; Cannell & Thornley 1998), and the globally increasing air temperatures, understanding the responses at 2005 Blackwell Publishing Ltd 212

2 Variation in heat sensitivity of PSII due to sugars 213 high-temperature is crucial to predict future changes in vegetation productivity. In natural environments, leaf temperatures vary widely during the day and from day-to-day (Singsaas et al. 1999; Jifon & Syvertsen 2003), and there is also a significant gradient in average leaf temperature from the top to the bottom of plant canopy (Niinemets, Oja & Kull 1999; Jifon & Syvertsen 2003). Photosynthetic electron transport is the functional limitation at high temperature in the field (Wise et al. 2004), and plants are commonly thought to acclimate to long-term average air temperature primarily by changes in the optimum temperature of photosynthetic electron transport (T opt ; Björkman, Badger & Armond 1980; Medlyn, Loustau & Delzon 2002; Bernacchi, Pimentel & Long 2003; Sung et al. 2003; Yamori, Noguchi & Terashima 2005). Such an acclimation to long-term temperature environment occurs through changes in the fluidity of thylakoid membranes via alteration of membrane lipid and protein composition (Björkman, Badger & Armond 1980; Santarius & Weis 1988; Sung et al. 2003) and by de-novo protein and lipid synthesis (Havaux 1993). Assuming that the adjustment of the responses of J to temperature is slow, most current canopy-level photosynthesis models use a constant response to temperature for all leaves and during the entire season (Leuning 2002; Medlyn et al. 2002). However, daily temperature maxima largely exceed the daily average and long-term average temperatures (Singsaas et al. 1999), indicating that plant leaves are frequently exposed to potentially damaging high temperatures that significantly exceed the threshold for leaf damage. In fact, there is evidence that the heat-stress resistance of photosynthesis may rapidly adjust to ambient temperature independently of long-term acclimation. Several studies show rapid enhancement of heat tolerance already after short-term exposure of leaves to elevated temperatures (Seemann, Berry & Downton 1984; Havaux 1993; Lazár & Ilík 1997). This increase in heat tolerance has been associated with conversion of xanthophyll violaxanthin to zeaxanthin, which increases the rigidity of thylakoid membranes (Havaux & Tardy 1996; Havaux et al. 1996). Data also demonstrate improved heat tolerance of leaves drought-stressed for a few hours and up to a few days (Seemann, Downton & Berry 1986; Havaux 1992; Lu & Zhang 1999) and that this rise of heat resistance is correlated with changes in leaf osmotic potential and sugar concentration (Seemann, Downton & Berry 1986; Santarius & Weis 1988). Immediate increases in heat resistance of chloroplasts suspended in sugar solutions (Santarius 1973; Seemann, Downton & Berry 1986; Tsvetkova et al. 1995) further demonstrate that changes in sugar status may provide a mechanism for rapid temperature acclimation in the field. The water contents and osmotic potentials of leaves significantly decrease from morning to mid-day due to elevated transpiration rates even in plants that are not waterstressed (e.g. Marigo & Peltier 1996; Nardini Lo Gullo & Salleo 1999), implying important non-specific diurnal modifications in heat resistance of the photosynthetic apparatus. Furthermore, sugar concentrations in leaves also vary widely throughout the day, partly because of concentration of leaf osmotica as leaf water content decreases, but to a larger degree, because of enhanced photosynthesis at midday when radiation is highest (Hendrix & Huber 1986; Kalt-Torres et al. 1987). Because sugars can be translocated both during light and dark periods, the capacity for sugar translocation is lower than the capacity for photosynthesis, explaining large mid-day and afternoon increases in leaf sugar concentrations (Hendrix & Huber 1986; Kalt- Torres et al. 1987) and further suggesting a significant potential for adjustment of leaf heat resistance during the day. A decrease and collapse of linear photosynthetic electron transport rate due to heat stress primarily results from enhanced thylakoid membrane leakiness and damage at photosystem II (PSII), while photosystem I is more stable at elevated temperatures (Björkman, Badger & Armond 1980; Thomas, Quinn & Williams 1986; Havaux 1996; Schrader et al. 2004). Irreversible damage at PSII is reflected in rapid increases in dark-adapted chlorophyll fluorescence at supra-optimal temperatures and with far-red light (Briantais et al. 1996; Yamane et al. 1997). These break-points in the fluorescence response versus temperature curves (referred to throughout as fluorescence rise temperature, T FR ) are commonly associated with the phasechange temperature of thylakoid membranes (Armond, Björkman & Staehelin 1980; Terzaghi et al. 1989). Above phase-change temperatures, thylakoid membranes are characterized by enhanced fluidity and leakiness and partial dissociation of light-harvesting complexes of PSII (Armond, Björkman & Staehelin 1980; Gounaris et al. 1984; Briantais et al. 1996). In addition, the fluorescence rise temperature also depends on the blockage of PSII reaction centers (Bukhov & Mohanty 1993; Briantais et al. 1996; Yamane et al. 1997) that probably results from the disruption of water-splitting and oxygen-evolving system (Nash, Miyao & Murata 1985; Enami et al. 1994). There is good correspondence between the fluorescence rise temperature, and the threshold temperature at which the capacity for photosynthetic CO 2 fixation and photosynthetic electron transport become temperature-unstable, i.e. they start to decrease in time at a constant temperature (Seemann, Berry & Downton 1984). The fluorescence rise temperature is also strongly correlated with development of leaf necrosis after heat stress treatments (Bilger, Schreiber & Lange 1984), suggesting that fluorescence rise temperatures provide a valuable basis to assess the overall sensitivity of photosynthetic electron transport to heat stress. We studied leaf fluorescence rise temperatures and the responses of photosynthetic electron transport to temperature in the temperate tree Populus tremula L., both in relation to feeding with sugar solutions of different concentrations and in relation to the diurnal variation in leaf sugar concentrations and osmotic potential, to test the hypothesis that fluctuations in sugar concentrations in the field modify

3 214 K. Hüve et al. the heat sensitivity of photosynthetic electron transport. We demonstrate that changes in leaf sugar concentrations may protect the leaf photosynthetic apparatus from heat stress non-specifically, and at higher sugar concentrations, significantly modify the temperature response the curves of photosynthetic electron transport. MATERIALS AND METHODS Plant material The experiments were conducted from the end of June to the beginning of August 2003, and at the end of June and beginning of July 2004 in Tartu, Estonia. We used rooted cuttings of a Populus tremula L. clone of local origin that had been planted in Spring The potted trees were grown outside in full sunlight, and by the time of experiments, the trees were 2 5 m high, and possessed leaves. The average diurnal temperature during the time of experiments was similar in 2003 (17.1 C) and 2004 (16.7 C), but the year 2004 was characterized by prolonged cool temperatures (5 10 C) during leaf growth, and as a result the fully expanded leaves were only about half the area of those in The irrigation was natural, except for drought periods, during which the plants were irrigated with tap water. The meteorological characteristics for the time span of the study were obtained from the weather station of the Institute of Environmental Physics of the University of Tartu that was 1.5 km from the plant growth location. Foliar sampling Fully exposed mature leaves of similar size were used for the experiments. Leaves were taken between 05:00 and 19:00 h to study the diurnal variability in fluorescence rise temperatures and the leaf photosynthetic electron transport responses to temperature. For experiments with artificially altered osmotic potential, the leaves were harvested between 18:00 and 19:00 h. The petiole of the selected leaf was cut under distilled water, and the leaf with the petiole placed in distilled water was immediately transported to the laboratory. In the laboratory, the leaf was either darkened at room temperature (25 C) for at least 30 min prior to the start of experimental treatments (leaves for diurnal variability study) or subjected to feeding with sugars in the dark. Artificial adjustment of leaf osmotic potential The harvested leaves were fed through the petiole with solutions of two different carbohydrates, monosaccharide sorbitol, which cannot be metabolized, and disaccharide sucrose, which may be present in leaves in large concentrations in physiological conditions and can be metabolized. The concentrations used were 0.2, 0.4, 0.8, 1.0, and 1.2 M. These feeding solution concentrations are common in studies of the heat resistance of PSII (Seemann, Downton & Berry 1986), but in our study, the concentrations of 1.0 and 1.2 M may have resulted in non-physiological leaf sugar concentrations (see the results). The leaves were kept in specific solutions for 12 h (overnight) in the dark at room temperature (25 C). For these experiments, the controls were kept overnight in distilled water. Experimental set-up The predarkened leaf was enclosed in a circular clip-on type leaf chamber of a two-channel fast-response measurement system (Laisk et al. 2001). The upper side of the leaf was glued to the glass window of the chamber water-jacket with starch gel to enhance the temperature exchange between the leaf and thermostatted water. Leaf temperature was measured with a thin copper-constantan thermocouple attached to the lower leaf surface. In general, leaf temperatures differed from the thermostatted water temperatures by < 0.5 C. Synthetic air, mixed from pure N 2, O 2 and CO 2 passed over the lower leaf surface with a flow rate of 0.5 mmol s 1. The O 2 concentration was kept at 21% and the chamber CO 2 concentration was maintained at 360 µmol mol 1. Air humidity was adjusted to 60% in all conditions. The leaf chamber could be illuminated from three Schott KL 1500 tungsten halogen light sources (Schott A.G., Mainz, Germany) through a multiarm fiberoptic light guide. One Schott light source equipped with a heat-reflecting filter (Optical Coating Laboratory, Inc., Santa Rosa, California, USA) provided white actinic illumination, the second light source was equipped with a 720 nm narrow-band interference filter (Andover Corp., Salem, New Hampshire, USA) for far-red light (40 70 µmol m 2 s 1 ), and the third could be used for saturating pulses of white light of µmol m 2 s 1. Chlorophyll fluorescence was measured with a PAM 101 fluorometer (Heinz Walz, Effeltrich, Germany) operated at 1.6 khz pulse frequency for darkened leaves and 100 khz for illuminated leaves and during saturation pulses. Determination of the rise temperature (T R ) of chlorophyll fluorescence After enclosure in the leaf-chamber, the leaf was stabilized at 20 C and 360 µmol mol 1 ambient CO 2 until full stomatal opening and steady-state gas-exchange rates were achieved. This was observed generally 5 10 min after leaf enclosure in the experiments conducted in dark, and min after the enclosure in the experiments with actinic light. In experiments in light, the leaf was kept in darkness for the first 5 min of enclosure. After stabilization, leaf temperature was increased by 1 C min 1 as in Havaux (1993) to 56 C. The fluorescence rise temperature (T R ) was determined as the break-point in the fluorescence temperature response curve as demonstrated in Fig. 1. Specifically, T R is

4 Variation in heat sensitivity of PSII due to sugars 215 It has been demonstrated previously that the simple fluorescence parameter F S, that reflects the overall redox and energy status of light-adapted leaves in any given time, very sensitively tracks plant responses to stress (Flexas et al. 2002). The fluorescence yields, F, can be expressed as (Krause & Weis 1991): F = α Q a k F /(k F + k P + k H + k T ), (1) Figure 1. Sample temperature responses of minimum fluorescence yields in dark-adapted leaves (F 0 ) with weak far-red light (filled symbols) and without far-red light (open symbols) in temperate deciduous tree Populus tremula. Fluorescence was recorded while the leaf temperature was increased with a rate of 1 C min 1. The temperatures of dark fluorescence rise, T FR (with far-red light) and T D (without far-red light) are defined as the intersection points between the linear portions of slow and rapid fluorescence rise (T FR = 48.0 C and T D = 44.4 C). Determination of the temperature for peak fluorescence (T P ) is also shown (T P = 53.2 C). the point at which a rapid rise in chlorophyll fluorescence begins (for further details see Bilger, Schreiber & Lange 1984; Seemann, Berry & Downton 1984). Three experimental protocols were employed to determine the rise temperature of chlorophyll fluorescence. In the first two series of measurements, the experiments were conducted in darkness to monitor the temperaturedependent changes in the basal fluorescence level (F 0 ). The measurements were conducted either under far-red light to measure the true level of F 0 (first protocol, fluorescence rise temperature, T FR ; Havaux et al. 1996) or without the far-red light (the second protocol, fluorescence rise temperature T D ; Fig. 1). For both measurement protocols, saturating pulses of white light were given at 2 min intervals to monitor changes in the maximum fluorescence yield (F m ). The third series of experiments was conducted under the actinic illumination (Q) of 760 µmol m 2 s 1 to measure the temperature-dependent alterations in steady-state fluorescence yield (F S ) in light-adapted leaves. In aspen leaves, this light intensity resulted in 90% of light-saturation of photosynthetic electron transport (J) but it did not lead to any photoinhibitory decrease of J (Niinemets, Oja & Kull 1999). The fluorescence rise temperature for F S (T L ) was determined as for T FR (Fig. 1), but because of stronger fluctuations in F S level than in F 0 level, the baseline for F S was determined for the 10 temperature range before the rapid onset of F S increase. This protocol was employed to simultaneously determine the fluorescence rise temperature and the light-adapted maximum fluorescence yield (F m ) that was further used to calculate the rate of photosynthetic electron transport, as detailed below. where α is the fraction of light absorbed by PSII, Q a is the absorbed quantum flux density, k F is the rate constant for fluorescence, k P is the rate constant for the PSII photochemical reaction, k H is the rate constant for thermal energy dissipation and k T the rate constant for excitation energy transfer to non-fluorescent pigments (e.g. PSI antennae). In analyses of fluorescence parameters, the rate constant of fluorescence is assumed to be invariable in all conditions (Krause & Weis 1991). Thus, the differences in fluorescence yields reflect changes in the rate constants for photochemistry, thermal dissipation and energy transfer to non-fluorescent pigments. The F 0 corresponds to a situation with a maximum k P and relaxed thermal energy quenching. In non-stressed leaves, the F 0 level corresponds to a situation with all PSII centers open, but PSII center openess may decrease during standard F 0 estimation protocols in heat-stressed leaves (Bukhov & Mohanty 1993; Yamane et al. 1997). Dark-adapted maximum fluorescence yield, F m provides the fluorescence yield in conditions of relaxed thermal energy dissipation and when all PSII centers are closed by a saturating light pulse (k P = 0), while in the lightadapted leaves the rate constant of thermal energy dissipation increases (fluoresence yield at F m level). Thus, the steady-state fluorescence yields in light-adapted leaves, F S, may vary at any specific actinic illumination, primarily due to modifications in the rate constants of photochemistry and thermal energy dissipation. Responses of dark respiration and photosynthetic electron transport to temperature Concomitantly with the fluorescence measurements, CO 2 exchange rate was measured with a LI-6251 CO 2 analyser (Li-Cor, Lincoln, NE, USA), and H 2 O exchange rate with a home-made micropsychrometer (Laisk et al. 2001) to obtain rapid (1 C min 1 ) temperature responses of dark respiration (R D ) from the measurements in dark, and net assimilation (A) from the measurements in light (Q = 760 µmol m 2 s 1 ). Foliar gas-exchange characteristics were determined according to von Caemmerer & Farquhar (1981). rate of photosynthetic electron transport (J) was calculated from net A (Brooks & Farquhar 1985) as: J = (A + R L )(4C i + 8Γ*)/(C i Γ*), (2) where R L is the rate of non-photorespiratory respiration continuing in the light (µmol m 2 s 1 ), Γ* is the CO 2 compensation point in the absence of R L, and C i is the intercel-

5 216 K. Hüve et al. lular CO 2 concentration (µmol mol 1 ). The temperature dependence of Γ* was calculated using the temperature parameters of Rubisco given in Niinemets & Tenhunen (1997). Gas-exchange measurements in the dark were used to construct the dark respiration (R D ) versus absolute temperature (T K ) response curves using non-linear regression: R e c H A RT K = -D, D where c is the scaling constant, H A (J mol 1 ) the activation energy, T K (K) the leaf temperature, and R (8.314 J mol 1 K 1 ) the gas constant. For the experiments in light, R D was measured at 20 C at the beginning of the experiment. This value, together with H A determined for the darkened leaf, was used to determine the scaling constant and, thus, to fix the response of R D to temperature for the illuminated leaf. Because the mitochondrial respiration is inhibited in the light by 50% (Villar, Held & Merino 1995), the value of R L at every temperature was taken as 0.5R D (Niinemets, Oja & Kull 1999). An alternative estimate of photosynthetic electron transport (J F ) was determined from chlorophyll fluorescence measurements (Genty, Briantais & Baker 1989) as: (3) J F = 0.5Φ PSII Θ Q, (4) where Q is the photosynthetically active quantum flux density, Θ is leaf absorbance, and Φ PSII is the effective quantum yield: Φ PSII = (F m F s )/F m. (5) This calculation procedure assumes that both photosystems absorb equal amount of light (Genty, Briantais & Baker 1989). Detailed estimation of light partitioning between the two photosystems support this assumption (Laisk et al. 2001). Leaf absorbance was measured on a leaf part that had not been enclosed in the leaf chamber, with an Ulbrichttype integrating sphere and spectroradiometer PS-2000 (Ocean Optics, Dunedin, FL, USA). Optimum temperature of photosynthetic electron transport The temperature dependencies of the estimates of photosynthetic electron transport determined by gas-exchange and fluorescence techniques were fitted by non-linear regression (Niinemets & Tenhunen 1997): c e -ST DH RT J = A K + ( D -D e H ) 1 RT K D K, where c is the scaling constant, H A (J mol 1 ) is the activation energy, H D (J mol 1 ) is the deactivation energy, and S (J K 1 mol 1 ) is the entropy term. The average explained variance (r 2 ) was for T K versus electron transport (6) rates determined from gas-exchange, and for the rates determined from fluorescence, demonstrating that this equation closely fitted the data. The optimum temperature for photosynthetic electron transport, T opt, was calculated as (Niinemets, Oja & Kull 1999): T opt DHD =. HD S+ R Ê D D ln -1 ˆ Ë DH Morphological measurements After completion of the temperature rise experiments, the leaf was immediately photographed with a Nikon Coolpix 990 digital camera (Nikon Corporation, Tokyo, Japan) to document any possible visible damage. Leaf lamina freshmass was determined directly after the measurements, and leaf area was measured using home-produced software from the images scanned with a resolution of 300 d.p.i. A leaf disk of 0.8 cm 2 was taken and frozen at 20 C for measurements of the osmotic potential, and the rest of the leaf lamina was dried at 70 C for at least 48 h, and leaf dry mass was estimated. From these data, leaf dry- to fresh-mass ratio (D W ) and dry mass per unit area (M A ) were calculated. Sugar analyses A Leaf total sugar content was determined from dry leaf material as detailed in Niinemets (1995). The sugars were extracted with 70% ethanol at 70 C, and the solubilized sugars were measured according to the anthrone method (Yemm & Willis 1954). The anthrone reaction sensitively estimates the total pool of mono- and oligosaccharides (Yemm & Willis 1954), but cannot detect amino sugars and alditols such as sorbitol. In natural conditions, Populus does not accumulate alditols (Cox & Stushnoff 2001). However, this insensitivity means that total leaf sugar content could not be determined for sorbitol-feeding experiments. The concentration of sugars in leaf water (C W ) was calculated as: (7) C W = C D D W /(1 D W ), (8) where C D is the sugar concentration in leaf dry matter. The contribution of sugars to the leaf osmotic potential, ψ S was further determined according to van t Hoff equation: ψ S = C W;M σrt K, (9) where C W;M is the sugar concentration in molar units (kmol m 3 ), and σ is water density (kg m 3 ). We assumed that all leaf sugars are present as disaccharides in these calculations. Measurement of leaf osmotic potential After freezing and thawing, leaf disks were homogenized with a pestle and mortar in 100 µl distilled water and

6 Variation in heat sensitivity of PSII due to sugars 217 washed sand. The osmolarity of the diluted sap was then measured in two to three repetitions with a vapor pressure osmometer (VAPRO 5520, Schlag, Bergisch Gladbach, Germany), and averages were calculated. Leaf discs for osmotic potential (Ψ S ) estimations were taken at the end of the temperature rise experiment. To estimate leaf initial osmotic potential at the beginning of the experiments, the leaves harvested in 2004 were weighed before, and immediately after, the experiments. A comparison indicated that during the experiments, they had lost an average of 0.6% of their fresh weight. The correlations between the osmotic potential and heat stress sensitivity of PSII were qualitatively the same with measured Ψ S and with the Ψ S estimate corrected for water loss during the experiment, and therefore only the relationships with measured Ψ S values are presented. Data analyses Diurnal variations were measured on four different days in dark-adapted leaves, with 3 8 leaves per day and on four different days for light-adapted leaves, with 6 7 leaves per day (two diurnal courses in both light and dark in both years). Altogether, the responses of 28 leaves fed with sucrose were measured, 14 in light-adapted and 14 in darkadapted state, and of 28 leaves fed with sorbitol, 14 in lightadapted and 14 in dark-adapted state were measured (for all treatments, 10 leaves were measured in 2004 and 4 leaves in 2003). The relationships were qualitatively identical in both years, but due to differences in endogenous sugar contents and initial osmotic potentials, the quantitative patterns were different in the two years. Therefore, we show the overall effect of sucrose and sorbitol feeding on Figure 2. Sample temperature dependencies of F 0 fluorescence yields under far-red light in four P. tremula leaves that were fed overnight with various concentrations of sucrose (a), and correlations between fluorescence rise temperature and leaf osmotic potential for leaves fed either with various concentrations of sucrose (b) or sorbitol (c) measured in the dark with far-red light (fluorescence rise temperature, T FR, filled symbols, n = 10 for (b) and n = 4 for (c)) or under actinic illumination of 760 µmol m 2 s 1 (fluorescence rise temperature, T L, open symbols, n = 10 for both (b) and (c)). Panel (d) demonstrates the correlations between fluorescence rise temperature with leaf sugar content for sucrose-fed leaves (same leaves as in (b)). Leaf sugar contents after sugar feeding were determined by anthrone assay. Because this method does not detect alditols such as sorbitol, only the data for sucrose feeding experiments are plotted in (d). Data were fitted by linear regressions separately for T FR and T L in panels (b) to (d). Determination of fluorescence rise temperatures is demonstrated in Figure 1. All experiments were conducted in 2004.

7 218 K. Hüve et al. fluorescence rise temperatures with the 2004 data, as more measurements were available. The data concerning the effect of diurnal variations in endogenous sugars and osmotic potentials, and average air temperatures are presented separately for 2003 and RESULTS Rise temperature of minimum and steady-state chlorophyll fluorescence Increase of leaf temperature above a certain threshold resulted in a sharp rise in the dark-adapted fluorescence yield, F 0 (Fig. 1) that was accompanied by decreases in the dark-adapted maximum fluorescence, F m, level. When farred light was not present during the temperature rise, rapid increases in fluorescence were obtained at lower temperatures. Typically, the break-point in dark fluorescence temperature response curves was observed at temperatures 3 lower without far-red light (T D ) than those with far-red light (T FR, Fig. 1). In the experiments without far-red light, the high F 0 yields at the apparent break-point (T D ) were still reversible when far-red light was switched on, demonstrating that the temperature of dark fluorescence rise corresponds to accumulation of reduced electron acceptors between the two photosystems, and does not reflect the true temperature sensitivity of PSII. Therefore, in the following, only the results of the dark measurements with far-red light are presented. Temperature response curves of steady-state lightadapted fluorescence (F S ) measured at a quantum flux density of 760 µmol m 2 s 1 exhibited break-points (rise temperatures) similar to those for dark-adapted minimum fluorescence. In general, the rise temperatures of F S were similar to those of F 0 fluorescence (far-red light) of leaves sampled at the same time, and the experimental treatments affected F 0 and F S rise temperatures in the same manner. Nevertheless, the rise temperature of F S often exceeded that of F 0 slightly, at most by C. For an independent assessment, the data of temperature rises of F S and F 0 are depicted with different symbols and fitted separately in the figures. After reaching the peak value, F 0 fluorescence decreased again (e.g. at 53.2 C in Fig. 1). The temperatures of peak fluorescence have also been associated with the heat tolerance of PSII (Bilger, Schreiber & Lange 1984). The peak in chlorophyll fluorescence was not always present, even at the maximum measurement temperature of 56 C (i.e. F 0 was still increasing), but whenever it was, visible leaf damage could be observed at the end of the experiment. The extent of damage ranged from small brownish dots to large necrotic areas. Effects of feeding with osmotic solutions on chlorophyll fluorescence rise temperature With increasing the concentration of osmotica in the feeding solution, the fluorescence rise temperature in the dark with far-red light (T FR ; Fig. 2a) and in the light (T L ; data not shown) were shifted towards higher temperatures. Due to leaf-to-leaf differences in stomatal opening and water transport to the leaf during feeding, changes in the fluorescence rise temperature did not strictly depend on the sugar concentration in the feeding solution (e.g. fluorescence temperature response curves for 0.8 M and 1.0 M sucrose solutions in Fig. 2a). However, there were consistent large Figure 3. Typical diurnal variations in the fluorescence rise temperature (a) and the relationship between the fluorescence rise temperature and leaf sugar content in leaf water during the same days (b). Responses of F 0 rise to temperature were measured in the dark with far-red light on two different days (T FR, filled symbols: July 15, 2003; open symbols: July 18, 2004). Inset in (b) demonstrates corresponding changes in leaf sugar contents during the day. Each data point represents a different leaf. Data were fitted by linear regressions. Fluorescence rise temperatures are calculated as shown in Figure 1.

8 Variation in heat sensitivity of PSII due to sugars 219 increases of T FR and T L with decreasing leaf osmotic potential measured at the end of experiment in both sucrose-fed (Fig. 2b) and sorbitol-fed (Fig. 2c) leaves. Greater heat stability of leaves fed with osmotica was also evident from the lack of the high-temperature peaks in chlorophyll fluorescence and the lower F 0 values at the same high temperature (Fig. 2a), and from the lack of visible damage in sugar-fed leaves directly after the completion of the experiment, even at the highest temperature of 56 C. Feeding of leaves with sugars led to large increases in leaf sugar concentration (Fig. 2d). According to calculations (Eqn 9), increases in sugar concentration provided the primary explanation for changes in leaf osmotic potential from about 1.5 MPa in leaves fed with water up to 7 MPa in leaves fed with sugars. Consistent with the role of sugars in the modification of osmotic potential, the fluorescence rise temperatures increased with increasing concentrations of sugars in leaf water (Fig. 2d). Apart from modifications of the fluorescence rise temperature, feeding of osmotic solutions with the largest sugar concentrations led occasionally to an increase in the values of F S, and sometimes in F 0 (data not shown) at lower leaf temperatures as well, possibly manifesting down-regulation of the rate of PSII photochemistry. Diurnal variation in fluorescence rise temperature In general, the fluorescence rise temperature measured in dark with far-red light (T FR ; Fig. 3a) and the fluorescence rise temperature measured in illuminated leaves (T L ; data not shown) increased during the day monotonically or exhibited a maximum at mid-day. As illustrated in this figure, the daily time-course of fluorescence rise temperature varied from day-to-day (e.g. Fig. 3a). The overall field variation in the fluorescence rise temperatures was 44.7 C to 50.7 C. This range resulted from diurnal as well as day-to day and year-to-year variability. Diurnal changes in fluorescence rise temperatures were accompanied by daily variation in leaf sugar concentration (inset in Fig. 3b). Fluorescence rise temperature and leaf sugar concentration were significantly correlated (Fig. 3b, r 2 = 0.52, P < for T L versus sugar concentration for all data pooled, n = 26 leaves from four diurnal time-courses). The average leaf sugar concentration was higher in 2004 (average ± SD = ± kg kg 1 ) than in 2003 (0.047 ± kg kg 1, P < for the differences between the means according to ANOVA), partly explaining year-to-year differences in the diurnal courses of fluorescence. General role of sugars and osmotic potential in determining the fluorescence rise temperature Combining all the fluorescence rise experiments conducted with feeding sugars, or monitoring the natural variation in sugar concentration and osmotic potential in the field, yielded a general correlation between the fluorescence rise Figure 4. Effects of leaf osmotic potential on (a) the fluorescence rise temperature (measurements under far-red light; T FR ) and on (b) the rate of F 0 increase after T FR has been reached (as illustrated in the inset, the slope of F 0 versus leaf temperature). All experiments with leaves fed with osmotica as well as from daily time-courses are pooled, each data point represents one leaf. Panel (c) shows the correlation between the osmotic potential and sugar concentration. Figure 1 provides the definition of fluorescence rise temperature. temperature, T FR, and leaf osmotic potential (Fig. 4a). Simultaneously with the increasing fluorescence rise temperatures, the slope of fluorescence increase became less steep after reaching T FR (Fig. 4b). This demonstrates that the fluorescence increased more slowly with rising temperature in leaves with greater heat-stress resistance. Similar correlations with leaf osmotic potential were also observed with the fluorescence rise temperature determined under actinic illumination (T L ; data not shown).

9 220 K. Hüve et al. Figure 5. Effects of feeding with sucrose (a, c and e) and sorbitol (b, d and f) on the response of steady state fluorescence yield to temperature in light (F S (a) (b)), and on the photosynthetic electron transport rates calculated from chlorophyll fluorescence (Eqns 4 5) of the same leaves ((c) (d)). Optimum temperature (T opt ) of photosynthetic electron transport rate (Eqn 7) in relation to the concentration of osmotica in the feeding solution is shown in (e) and (f), and in relation to leaf osmotic potential in the inset of (f) (r 2 = 0.95, P < for both sucrose-fed (filled diamonds) and sorbitol-fed (open squares) leaves). The leaves were fed overnight with sucrose or sorbitol solutions of 0.2 (open diamonds), 0.4 (filled triangles), 0.8 M (open squares) or 1 M (data not shown) or kept in distilled water (filled diamonds, each dataset corresponds to one leaf). The response to temperature was measured from 20 C to 56 C by heating the leaves at a rate of 1 C min 1 under actinic illumination of 760 µmol m 2 s 1. The fluorescence rise temperatures (T L ), the concentrations of the feeding solution and corresponding leaf osmotic potentials are reported in the insets of (a) and (b). The electron transport rates in (c) and (d) were normalized with respect to the value (J opt ) observed at the optimum temperature (Eqn 7). Actual electron transport rates of the same leaves (µmol m 2 s 1 ) are shown in the insets of (c) and (d).

10 Variation in heat sensitivity of PSII due to sugars 221 Leaf osmotic potential and leaf sugar concentration were strongly correlated (Fig. 4c), and the correlations demonstrated in Fig. 4a and b were qualitatively the same if leaf sugar concentration was used as the explaining variable (r 2 = 0.41, P < for Fig. 4a and r 2 = 0.26, P < 0.01 for Fig. 4b). A large part of the scatter in these general relationships was due to day-to-day variability in the heat sensitivity of PSII (see below). because of greater sucrose uptake and/or sucrose hydrolysis in the leaf leading to lower osmotic potential in sucrosefed leaves at a common feeding concentration (inset in Fig. 5f). Yet, at the two highest sucrose feeding concentrations, T opt was larger at common leaf osmotic potential in sucrose-fed than in sorbitol-fed leaves. Changes in photosynthetic electron transport (J) vs. temperature response curves due to sugar feeding In separate experiments, we investigated the effect of sugar feeding on the shape of the temperature response curve of whole chain electron transport rate (J T curve). As illustrated in Fig. 5, modification of the fluorescence rise temperatures by sucrose (Fig. 5a) and sorbitol (Fig. 5b) feeding was accompanied by significant shifts in the optimum temperature (T opt ; Eqn 7) of whole-chain photosynthetic electron transport (Fig. 5c & d). The shift in optimum temperature due to sugar feeding was >10 C across the treatments (Fig. 5e & f). For instance, T opt, calculated for electron transport rates measured from fluorescence, increased from 32.7 C in the leaves fed with water and to 44.2 C in leaves fed with 0.8 M sucrose (Fig. 5c & e). Feeding with sucrose or sorbitol led to an overall decline in the rate of photosynthetic electron transport. While the decrease was moderate for lower feeding solution concentrations, the maximum rates of photosynthetic electron transport rate measured at T opt in the leaves fed with 0.8 M sucrose were only 25% of that in the leaves fed with water (see Fig. 5c, inset). However, the differences were significantly less at temperatures above T opt, where the absolute photosynthetic electron transport rates and net assimilation rates of sucrose- and sorbitol-fed leaves reached similar values or exceeded (above 40 C) those of water-fed leaves (Fig. 5c & d). For instance, the maximum rate of electron transport of 184 µmol m 2 s 1 was achieved at 34.2 C in the water-fed leaf, and the corresponding maximum of 154 µmol m 2 s 1 was achieved at 38.2 C in the 0.2 M sucrose-fed leaf, but at a temperature of 40.3 C, the electron transport rate was 124 µmol m 2 s 1 (corresponding net assimilation rate, A, was 1.56 µmol m 2 s 1 ) in the water-fed leaf and 137 µmol m 2 s 1 (A = 2.47 mmol m 2 1 s 1 ) in the sucrose-fed leaf. The superior performance of sucrose-fed leaves was even more pronounced at higher temperatures. Overall, feeding with sucrose and sorbitol had similar effects on fluorescence temperature response curves (cf. Fig. 5a & b) and on whole chain electron transport rate (cf. Fig. 5c & d). However, the shifts in break-points and in T opt values of electron transport were somewhat smaller than in sucrose-fed leaves. Similarly, the shift in optimum temperature of electron transport (T opt ) due to sorbitol feeding was less pronounced (Fig. 5d & f), with T opt increasing from 32.7 C in the leaves fed with water to 37.3 C in leaves fed with 0.8 M sorbitol (Fig. 5f). This difference was partly Figure 6. Diurnal variation in the temperature responses of steady state fluorescence yield (F S (a)) photosynthetic electron transport rate computed from chlorophyll fluorescence (Eqns 4 5 (b)), and stomatal conductance to water vapor (c) for three different leaves on the same day. The measurements were conducted under actinic irradiance of 760 µmol m 2 s 1. The corresponding fluorescence rise temperatures, T L, are also shown in (a). As in Fig. 5b & c, the rates of photosynthetic electron transport were normalized with respect to the value at the optimum temperature (J opt = µmol m 2 s 1 at 8 : 30, J opt = µmol m 2 s 1 at 10 : 30, and J opt = 80.6 µmol m 2 s 1 at 16 : 30). The lines in (b) correspond to non-linear regressions (Eqn 6).

11 222 K. Hüve et al. Diurnal variation in the shape of J T response curves Examination of the covariation of the fluorescence rise temperatures (Fig. 6a) and optimum temperatures for photosynthetic electron transport (Fig. 6b) during the day suggested that while the heat resistance of PSII increased in time (Fig. 6a), the optimum temperature of photosynthetic electron transport varied less during the day (Fig. 6b). This discrepancy between T L estimates and optimum temperatures in the field possibly reflects the rapid stomatal closure with rising temperature in the afternoon (Fig. 6c). Extensive decrease in stomatal conductance may have led to decreases in the rate of photosynthetic electron transport due to low internal CO 2 concentrations. In addition, leaf sugar concentrations were not always largest in the afternoon (inset in Fig. 3b), partly because of enhanced respiration rates and decreased photosynthesis rates in leaves with closed stomata. Nevertheless, the rate of photosynthetic electron transport at damaging temperatures (> 50 C) was consistently higher in the afternoon than in the morning (e.g. Fig. 6b), further confirming the importance of sugars in the heat sensitivity of photosynthetic electron transport. The T opt values were strongly correlated with leaf sugar concentrations (Fig. 7), demonstrating the major effect of the variation in leaf sugars on the optimum temperature for photosynthetic electron transport rate. This strong correlation was observed in both years (closed and open symbols in Fig. 7), although natural and artificially altered leaf sugar contents were very different. Figure 8. Variation in average daily air temperature and the fluorescence rise temperatures during the experiment in 2003 and 2004 (a) and the correlation between the fluorescence rise temperatures with average air temperature of six (2003) or five (2004) days prior to the measurements (b). Filled symbols denote the fluorescence rise temperatures derived from measurements in dark with far-red light (T FR ), and open symbols the fluorescence rise temperatures determined under actinic illumination (T L ). Data were fitted by linear regressions. Day-to-day variability of the heat sensitivity of PSII Figure 7. Optimum temperature (T opt ) of photosynthetic electron transport rate calculated from fluorescence measurements (Eqns 4 5) as a function of leaf sugar concentration (kg kg 1 leaf water). The leaves were heated at a rate of 1 C min 1 under actinic illumination of 760 µmol m 2 s 1 and the optimum temperature was calculated using Eqn 7. The data were fitted by linear regression. Filled symbols: leaves from diurnal variation experiments in 2003; open symbols: sucrose-fed leaves from 2004 experiments (same leaves as in Figure 5). Variations in diurnal time-courses of fluorescence rise temperature (Fig. 3a) as well as in the correlations between fluorescence rise temperature and leaf sugar concentration (Fig. 3b) demonstrate that not all the variation in fluorescence rise temperatures can be explained by leaf sugar concentration and osmotic potential. During the experiments, there was large day-to-day variation in daily average temperature (Fig. 8a). Yet, the correlations with the date (r 2 = 0.12, P >0.2 for 2003 and r 2 = 0.31, P >0.1 for 2004) and with the average temperature during the day of sampling (Fig. 8a, r 2 = 0.13, P >0.2 for 2003; r 2 = 0.14, P >0.3 for 2004) or with the average temperature a day preceding the sampling (data not shown) were not significant. In fact, the daily average temperature and fluorescence rise temperature were apparently shifted, suggesting that the leaf response to temperature may adjust with a delay to the

12 Variation in heat sensitivity of PSII due to sugars 223 temperature signal. To test this hypothesis, we correlated the fluorescence rise temperatures with the daily average temperatures at various days preceding leaf sampling. The best correlation was found with the daily average temperature measured 4 days before the specific temperature rise experiment (r 2 = 0.41, P < 0.01 for 2003 and r 2 = 0.69, P < for 2004), partly supporting the hypothesis. Alternatively, leaves may track the average temperature environment. We observed the best correlation between the fluorescence rise temperature and the daily mean temperature averaged for 6 days before measurements in 2003 and for five days before measurements in 2004 (Fig. 8b). Given the larger degree of explained variance, these relationships suggest that leaf temperature acclimation is more strongly driven by the long-term average temperature environment than by a specific daily temperature signal. At a common average air temperature, fluorescence rise temperatures were larger in 2004 than in 2003, possibly reflecting the about two-fold higher internal sugar concentrations in 2004 (see above). We did not observe any significant correlations between leaf sugar content and the average temperature of the previous 5 10 d (data not shown), indicating that short- and long-term modifications in fluorescence rise temperature are independent, and that day-to-day variations do not necessarily depend on sugar concentration. DISCUSSION Fluorescence rise temperatures in the dark and light Rise temperature of chlorophyll fluorescence measured in dark with weak far-red light (F 0 ; T FR, Fig. 1) is a sensitive indicator of the heat tolerance of PSII. Early studies have provided evidence that the F 0 rise is due to increased lipid fluidity that modifies lipid protein interactions and leads to disruption of the supramolecular organization of PSII (Armond, Björkman & Staehelin 1980). Further work has, however, demonstrated that the mechanism of the F 0 rise is more complex. In addition to dissociation of certain pigment-binding complexes, the F 0 rise is partly due to accumulation of inactive reaction centers of PS II (Briantais et al. 1996; Yamane et al. 1997) that are unable to reduce the primary electron acceptor Q A (Bukhov & Mohanty 1993; Lu & Zhang 1999). Both the impairment of pigmentbinding complexes and closure of PSII centers lead to decreases in the rate constant for photochemistry, k P (Eqn 1) and collapse of the whole-chain photosynthetic electron transport rate. Therefore, the rise temperature T FR provides an excellent measure of the decline of CO 2 uptake rates (Björkman, Badger & Armond 1980; Seemann, Berry & Downton 1984; Werner et al. 2001), PS II (Björkman, Badger & Armond 1980; Havaux 1992) and whole chain (Werner et al. 2001) quantum yields, and overall leaf damage by heat stress (Bilger, Schreiber & Lange 1984). As previous work (Bilger, Schreiber & Lange 1984; Bukhov, Sabat & Mohanty 1990; Niinemets, Oja & Kull 1999), and our study demonstrate (Fig. 1), it is important to use far-red light in the determination of the true F 0 rise temperature because of accumulation of reduced Q A in the dark at higher temperature. The F 0 level commonly starts to increase at lower temperature when far-red light is not present than with the far-red light (Fig. 1). Such increases can be effectively reversed by far-red light, which selectively excites photosystem I, removing the electrons from the PS II acceptor side (Havaux 1996). The temperaturedriven accumulation of Q A in the dark has been suggested to occur because of heat-induced cyclic electron flow around PSI that is fed by stromal reductants (Havaux, Greppin & Strasser 1991; Havaux 1996; Bukhov et al. 1999). Although the heat-stress resistance has been generally assessed from the fluorescence rise temperatures measured in the dark, steady-state chlorophyll fluorescence yields (F S ) also sensitively track the environmental stresses (Flexas et al. 2002). Like F 0, the F S level rapidly increases at supra-optimal temperatures (Bukhov & Mohanty 1993; Georgieva et al. 2000; Lu & Zhang 2000). Although changes in F S may be due to both modifications in the rate of thermal energy dissipation and photochemistry (Eqn 1), the rate constant for thermal energy dissipation actually increases with increasing temperature in intact leaves (Bilger & Björkman 1991) as well as in isolated pigmentbinding complexes of PSII (Wentworth, Ruban & Horton 2003). Thus, as with F 0, the rise temperature of F S should primarily reflect the decrease in the rate constant of photochemistry (Eqn 1), possibly due to accumulation of closed PSII centers and irreversible damage (Lu & Zhang 2000). Effects of osmotic potential and sugars on the heat stability of PSII We found an overall shift in fluorescence rise temperatures after feeding leaves with sugar solutions of various concentrations (Fig. 2a) and that the increase in fluorescence rise temperatures due to sugar feeding was correlated with leaf osmotic potential (Fig. 2b & c) and sugar concentration (Fig. 2d). These results agree with previous observations demonstrating close to linear increase in heat-stress resistance of PSII in isolated chloroplasts with increasing the suspension medium sugar concentration (Santarius 1973; Seemann, Downton & Berry 1986; Tsvetkova et al. 1995). In our study, the enhanced heat tolerance in leaves with lower leaf osmotic potential and higher sugar concentration was further supported by increases in the temperature for the peak of dark fluorescence (Figs 1 & 2a; Bilger, Schreiber & Lange 1984 for discussion), and decreases in the rate of heat-induced fluorescence rise (Fig. 4b). In our study, visible leaf damage was only observed at and beyond the temperatures of peak fluorescence. Under comparable osmotic potentials and sugar concentrations, the rise temperature of F S was C higher than that of F 0 (Fig. 2b d). This observation agrees with previous reports demonstrating increases in leaf heat tolerance after