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Tree Physiology 28, 785–795 © 2008 Heron Publishing—Victoria, Canada Impact of an exceptionally hot dry summer on photosynthetic traits in oak (Quercus pubescens) leaves P. HALDIMANN,1–3 A. GALLÉ1 and U. FELLER1 1 Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland 2 Present address: Department of Plant Molecular Biology, Biophore – Biology building, University of Lausanne, CH-1015 Lausanne, Switzerland 3 Corresponding author ([email protected]) Received June 3, 2007; accepted August 16, 2007; published online March 3, 2008 Summary Climatic constraints on diurnal variations in photosynthetic traits were investigated in oaks (Quercus pubescens Willd.) growing in the Swiss Alps. The measurement period included the summer of 2003, when central Europe experienced a record-breaking heat wave. During the summer, a combination of moderate heat and drought caused a reduction in photosynthetic CO2 assimilation rate (Pn ) by mid-morning, which increased by the afternoon. More extreme drought and heat caused a sharp day-long reduction in Pn. These effects were closely related to changes in stomatal conductance (gs ), but low gs was unaccompanied by low intercellular CO2 concentrations (Ci ). Around midday, a combination of heat and drought increased Ci, indicating metabolic limitation of photosynthesis. Chlorophyll a (Chl a) fluorescence measurements revealed reversible down-regulation of photosystem (PS) II activity during the day, which was accentuated by heat and drought and correlated with diurnal variation in zeaxanthin accumulation. A combination of heat and drought reduced leaf Chl a + b concentrations and increased ratios of total carotenoids, xanthophyll-cycle carotenoids and lutein to Chl a + b. The combination of summertime heat and drought altered the 77 K Chl fluorescence emission spectra of leaves, indicating changes in the organization of thylakoid membranes, but it had no effect on the amounts of the major light-harvesting Chl-a/b-binding protein of PSII (LHCII), Rubisco, Rubisco activase, Rubisco-binding protein (cpn-60), phosphoribulokinase and chloroplast ATP synthase. The results demonstrate that Q. pubescens can maintain photosynthetic capacity under adverse summer conditions. Keywords: chlorophyll fluorescence, chloroplast ATP synthase, drought, heat, leaf pigments, photosynthesis, Rubisco, xanthophyll cycle. Introduction Pubescent oak (Quercus pubescens Willd.) is a thermophilous tree found throughout central Europe, most abundantly in areas with a hot dry Mediterranean summer. In Switzerland, Q. pubescens is mainly found on warm and sunny south-facing slopes of the Wallis, Jura and Tessin regions, typically on rocky shallow soils with limited water-holding capacity, where it plays an important role in protecting slopes from erosion and scree-fall. Quercus pubescens is a winter-deciduous species, and summer drought and elevated temperatures limit photosynthetic yield. Such limitation may be expected to intensify in the event of continued climate warming (Luterbacher et al. 2004, Schär et al. 2004). The progressive reduction in stomatal aperture in response to water stress (i.e., negative tisssue water potential) reduces water loss but limits the influx of CO2 and, hence, net photosynthetic rate (Pn ) (Epron and Dreyer 1993a, 1993b, Damesin and Rambal 1995, Valentini et al. 1995). In addition, water stress may reduce Pn by reducing diffusion of CO2 from leaf intercellular spaces to the site of carboxlation within the mesophyll (Epron and Dreyer 1993b, Epron et al. 1995, Flexas et al. 2002). Whether water stress affects Pn primarily through limitation on transcellular diffusion of CO2 or by impairment of the carboxylation process remains subject to debate (Cornic 2000, Flexas and Medrano 2002, Lawlor 2002, Lawlor and Cornic 2002, Medrano et al. 2002, Tang et al. 2002, Flexas et al. 2004). A study with sunflower (Helianthus annuus L.) indicated that drought-dependent inhibition of Pn is largely due to a reduction in in the concentration of ribulose-1,5-bisphosphate, which is, in turn, the result of reduced ATP synthesis due to a decrease in chloroplast ATP synthase (Tezara et al. 1999). Our objective was to investigate the existence of a comparable mechanism of drought-inhibited photosynthesis in Q. pubescens. Photosynthesis is sensitive to inhibition by high temperature (Berry and Björkman 1980, Salvucci and Crafts-Brandner 2004a, Sharkey 2005), and high solar irradiance can raise leaf temperature above air temperature (Singsaas and Sharkey 1998, Leakey et al. 2003), an effect enhanced by drought, which limits transpiration and evaporative cooling of foliage. Under natural conditions, climatic constraints act in concert, and the combination of drought, elevated temperature and high solar irradiance may limit photosynthesis (Ludlow 1987, Havaux 1992). An acute and long-lasting combination of high temperature, drought and high solar irradiance may also affect photo- 786 HALDIMANN, GALLÉ AND FELLER synthesis by altering amounts of key photosynthetic proteins. Such effects may be reversed only slowly, so that carbon assimilation may remain low for some time following the termination of extreme conditions. Here we investigated effects of environmental stress during the summer, including the summer of 2003, the warmest in Europe in the last 500 years (Luterbacher et al. 2004, Schär et al. 2004, Ciais et al. 2005), on various photosynthetic traits of Q. pubescens in a warm dry region of the Swiss Alps. Materials and methods Study site and plant material The study site is located near Salgesch in the Wallis valley in the Swiss Alps (46°19′27″ Ν, 7°34′40″ Ε, 975 m a.s.l.) in one of the warmest and driest regions of Switzerland. It is located on a steep, south-facing oak– pine wooded slope with a shallow rocky soil with limited water-holding capacity, where Quercus pubescens trees (less than 70 years old) develop as shrubs, typically with two to four stems. Observations focused on a single tree, about 3 m tall, with three trunks with basal diameters of about 0.10 m. The tree was located 10 m from a meteorological station. Measurements were carried out on four sunny days: August 15, 2002, June 24 and July 15, 2003 and May 18, 2004. Additional measurements on the primary sample tree in late July and August 2003 were precluded by exceptionally hot weather which caused leaf mortality by the second half of July. Measurements on July 23, 2003 were therefore performed on another tree, about 3 m tall and at a distance of about 50 m from the weather station, which had leaves that remained photosynthetically active in late July. By early August 2003, none of the oaks in the stand had photosynthetically active foliage, with the exception of trees adjacent to a creek that crosses the slope 250 m north of the meteorological station. It was among these trees that measurements were made on August 6, 2003. Measurements carried out on several trees located around the meteorological station under non-stressful conditions on August 15, 2002 and May 18, 2004 indicated that Pn was similar in all trees, whether or not they were adjacent to the creek, which suggests uniformity of response to environmental constraints among trees at the site. All measurements were made on randomly selected, fully sun-exposed leaves. Microclimate, leaf temperature and leaf water potential Microclimatic data were recorded with a locally installed meteorological station (see Zweifel et al. 2005). Global irradiance, air temperature (Ta ) and vapor pressure deficit (VPD) were determined at 10-s intervals and averaged every 10 min. Daily precipitation was estimated with a conventional rain gauge. Photosynthetic photon flux (PPF) was measured with a Li-Cor radiometer (LI-250, Li-Cor, Lincoln, NE). Leaf temperature (Tl ) was determined with an infrared thermometer (Oakton TempTestr IR, Cole-Parmer International, Vernon Hills, IL). On each date, predawn (Ψpd ) and midday (Ψm ) leaf water potentials were measured on three leaves with a Scholander pressure chamber (SKPM, Skye Instruments, Powys, U.K.). Gas exchange and chlorophyll a fluorescence Photosynthetic gas exchange measurements were made on attached leaves with an infrared gas analyzer (IRGA) (CIRAS1, PP-Systems, Hitchin, U.K.) operated in open mode. Temperature and relative humidity in the leaf chamber were close to ambient values. Recorded data included Pn, stomatal conductance to water vapor (gs ), transpiration rate (E) and calculated intercellular CO2 concentration (Ci ). Measurements on four to eight leaves were made throughout the day. Chlorophyll (Chl) a fluorescence was measured with a pulse amplitude modulated fluorometer (FMS-1, Hansatech, King’s Lynn, Norfolk, U.K.). The fiber-optic was connected to a leaf-clip that shielded the sample from ambient light. Records were taken after 5 min of illumination with a PPF corresponding to that incident on the leaves, and after a subsequent 5–10 min exposure to a PPF of 500 µmol m – 2 s – 1. The following parameters were monitored: maximum quantum yield of photosystem (PS) II primary photochemistry in the dark- and lightadapted state (Fv /Fm and Fv′/Fm′, respectively); photochemical quenching (qp ), which increases with the fraction of open PSII reaction centers (RCs), being equal to it if no connectivity between PSII units is assumed; and quantum yield of PSII electron transport (φPSII ) (Maxwell and Johnson 2000). We measured Fv /Fm after 30 min of dark-adaptation. Measurements were performed at different times of day, with three leaves measured on each occasion. The quantum yield of PSII electron transport and Fv /Fm (n = 4–10) were measured on three dates (August 15, 2002 and June 24 and July 15, 2003) with a PAM-2000 fluorometer (Heinz Walz, Effeltrich, Germany), and φPSII was directly determined under ambient light. On completion of the physiological measurements, 1.4-cmdiameter leaf disks were excised, frozen in liquid nitrogen and stored at –80 °C for later biochemical and biophysical analyses. Low-temperature chlorophyll fluorescence emission spectra Low temperature chlorophyll fluorescence was determined by immersing a small piece of oak leaf tissue in liquid nitrogen and mounting it in an LS50 luminescence spectrophotometer (Perkin-Elmer) 1 cm above a reservoir of liquid nitrogen, thereby maintaining the sample temperature between –190 and –180 °C (Sperling et al. 1998). The emission spectra were recorded with an excitation wavelength of 440 nm. Measurements were performed on three to four leaves collected between 0530 and 0730 h local solar time (LST) and between 1330 and 1530 h. Leaf pigment analysis Leaf chlorophylls and carotenoids were extracted from the frozen leaf disks with 80% ice-cold acetone containing sodium carbonate. The extract was centrifuged at 20,000 g for 2 min and the supernatant filtered through a 0.2-µm membrane (Anatop 10, Merck, Darmstadt, Germany). Pigment separa- TREE PHYSIOLOGY VOLUME 28, 2008 IMPACT OF CLIMATIC CONSTRAINTS ON PHOTOSYNTHETIC TRAITS IN OAK tion and quantification were performed by high performance liquid chromatography by the method of Gilmore and Yamamoto (1991) as modified by Färber et al. (1997). The de-epoxidation state of the pool of xanthophyll-cycle carotenoids (V + A + Z) was calculated as the (Z + 0.5A)/(V + A + Z) molar ratio, where V, A and Z are violaxanthin, antheraxanthin and zeaxanthin, respectively. Measurements were made on samples collected at different times of day, with three to five samples analyzed at each sampling time. If the concentration of a pigment showed no diurnal variation, data were pooled (n = 21 to 33) and daily means calculated. SDS-PAGE and Western-blot analysis Proteins were extracted from the frozen leaf disks after grinding to a powder in liquid nitrogen with a Heidolph tissue homogenizer (Heidolph Instruments, Schwabach, Germany). Proteins were extracted from the powder at 4 °C in a buffer containing 20 mM sodium phosphate (pH 7.5), 1% (w/v) polyvinylpolypyrrolidone and 0.1% (v/v) 2-mercaptoethanol. Extracts were filtered through Miracloth (Calbiochem, Luzern, Switzerland) and an aliquot used to determine total soluble protein (TSP) concentration by the Bradford method (1976). The remainder of each extract was stored at –20 °C, for later protein analysis by gel electrophoresis. Measurements were performed on three samples that had been collected between 0800 and 0900 h LST and between 1330 and 1530 h LST. Results presented are means for each date, as the concentration did not change between morning and afternoon. Polypeptides were analyzed in a single extract of three leaf disks (collected between 1330 and 1530 h LST). Polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12%) according to Laemmli (1970) with a Mini Protean II Dual slab Cell (Bio-Rad, Glattbrugg, Switzerland). Immunoblot analysis of polypeptides was performed as described by Feller et al. (1998). Soluble carbohydrates Leaf water-soluble carbohydrate (WSC) concentration was 787 determined as described by Stieger and Feller (1994), with glucose as a standard. Measurements were performed on three leaves collected in the morning between 0800 and 0900 h LST and in the afternoon between 1330 and 1530 h LST, but we present only the mean WSC concentration for each date (n = 6), because concentrations did not change during the day. Statistics Although most measurements were made on leaves of the same tree, data were considered to be independent, and we performed variance analyses (ANOVAs) taking date or time within date as factors for the different studied parameters. A least-significant test (LSD; P < 0.05) was used to analyze the differences between means. Results Meteorological parameters and leaf temperature Daily precipitation and maximum Ta measured at the study site in Years 2002, 2003 and 2004 are shown in Figure 1. During the heat wave of summer 2003, Ta exceeded 30 °C on most days from the end of May to the end of August, whereas Ta values greater than 30 °C occurred infrequently in most other years. Total May–August precipitation was 377 mm in 2002, but only 201.1 and 213.7 mm in 2003 and 2004, respectively. Annual precipitation was 690, 426.6 and 500.5 mm in 2002, 2003 and 2004, respectively (MeteoSwiss, Sion, 20 km WSW of the study site). Mean annual precipitation between 1983 and 2002 was 623 mm. Figure 2 shows the diurnal courses of global irradiance (PPF), Tl, Ta and VPD on the dates of the physiological measurements. The hottest days were July 15 and August 6, 2003, when Ta was close to or higher than 35 °C for much of the afternoon. In July and August 2003, Tl was several degrees above Ta, and for several hours each day, was near or above 40 °C. In general, high Ta values correlated with high VPD values. Figure 1. Daily maximum air temperature (Ta, 䊉, 䊊) and precipitation (bars) recorded in 2002, 2003 and 2004. Different symbols were used to distinguish maximum Ta values higher (䊉) or lower (䊊) than 30 °C. Arrows indicate the dates when diurnal variations in leaf physiological traits were examined. DOY = day of year. TREE PHYSIOLOGY ONLINE at http://heronpublishing.com 788 HALDIMANN, GALLÉ AND FELLER Figure 2. Diurnal variations in (A) global irradiance (䉱) and photosynthetic photon flux (PPF, 䉭); (B) air (Ta, 䊉) and leaf (Tl, 䊊) temperatures; and (C) air vapor pressure deficit (VPD, 䉲) on each measurement date. The dotted line corresponds to 35 °C. Values of PPF and Tl are means ± SE of 4–10 replicates. Leaf water potential In May 2004, Ψpd and Ψm were within the normal range (Epron et al. 1992, Epron and Dreyer 1993a, Damesin and Rambal 1995, Valentini et al. 1995), averaging –0.34 and –1.85 MPa, respectively (Table 1). Similar values were measured on August 15, 2002 (Ψpd = –0.30 MPa; Ψm = –1.70 MPa). However, Ψpd as low as –2.50 MPa and Ψm as low as –3.34 MPa were recorded in summer 2003, when significant water deficit developed by June. Due to technical problems, Ψpd and Ψm were not determined on July 15, 2003. Leaf protein and water-soluble carbohydrate concentrations and pigment composition Total leaf soluble protein concentration did not vary greatly among dates, whereas total WSC concentration was greater in July 2003 than on the other dates (Table 1). Chlorophyll a + b concentration, despite minor differences among dates, was lower in June and July 2003 than in August 2003 and May 2004. In summer 2003, there was a large reduction in Chl concentration, and leaves acquired a yellowish appearance, unlike leaves of the tree adjacent to the creek. Variations in Chl a/b ratio were observed among dates, but there was no clear correlation between Chl a/b ratio and leaf water potential. In June and July 2003, leaves had lower total carotenoid (X + C) concentrations than in the spring, but a higher (X + C)/Chl a + b ratio (Table 1). Concentrations of lutein and xanthophyll-cycle carotenoids (V + A + Z) were greater in July 2003 than on other dates. The concentrations of the other carotenoids and Table 1. Mean ± SE predawn (Ψpd ) and midday (Ψm ) leaf water potentials (n = 3), concentrations of total leaf soluble protein (TSP; n = 6), total leaf water-soluble carbohydrates (WSCs; n = 6) and chlorophyll (Chl a + b), Chl a/b ratio and concentration of total carotenoids (X + C; n = 21 to 33) in oak (Quercus pubescens) leaves on specific dates (month/day/year). Values a column followed by different letters differ significantly at P = 0.05. Leaf water potentials on 07/15/03 were not determined due to instrument failure. Date 06/24/03 07/15/03 07/23/03 08/06/03 05/18/04 Ψpd (MPa) –2.05 ± 0.14 ab – –2.50 ± 0.24 a –1.90 ± 0.11 b –0.34 ± 0.02 c Ψm (MPa) TSP (g m –2 ) Total WSCs (mol m –2 ) Chl a + b (µmol m –2 ) Chl a/b (mol mol –1 ) X+C (µmol m –2 ) (X + C)/Chl a+ b (mmol mol –1 ) –3.34 ± 0.05 a – –2.56 ± 0.10 b –3.32 ± 0.18 a –1.85 ± 0.09 c 1.26 ± 0.10 ab 1.41 ± 0.06 a 1.28 ± 0.15 ab 1.21 ± 0.13 b 1.34 ± 0.17 ab 503.6 ± 30.8 a 672.1 ± 43.3 ab 684.6 ± 38.4 b 566.3 ± 11.8 ab 500.6 ± 72.5 a 160.3 ± 4.6 a 135.3 ± 5.9 b 173.5 ± 9.2 a 258.5 ± 9.4 c 270.9 ± 7.5 c 4.15 ± 0.03 a 3.93 ± 0.04 b 3.55 ± 0.05 c 3.76 ± 0.05 d 4.27 ± 0.05 e 71.3 ± 2.2 a 67.3 ± 2.6 a 80.0 ± 3.8 b 104.0 ± 3.0 c 109.9 ± 2.7 c 445.9 ± 7.2 a 503.6 ± 8.8 b 465.5 ± 10.1 a 407.6 ± 7.5 c 407.3 ± 4.3 c TREE PHYSIOLOGY VOLUME 28, 2008 IMPACT OF CLIMATIC CONSTRAINTS ON PHOTOSYNTHETIC TRAITS IN OAK 789 Table 2. Carotenoid composition of oak (Quercus pubescens) leaves on five measurement dates. Means ± SE of concentrations of individual carotenoids are expressed in mmol (mol Chl a + b) –1 (n = 21–33). Abbreviations: V + A + Z denotes the pool of xanthophyll-cycle carotenoids, where V, A and Z stand for violaxanthin, antheraxanthin and zeaxanthin, respectively; and X/C denotes the molar ratio of total xanthophylls to total carotenes. Values within a column followed by different letters differ significantly at P = 0.05. Date Neoxanthin Lutein V+A+Z α-carotene β-carotene X/C 06/24/03 07/15/03 07/23/03 08/06/03 05/18/04 34.5 ± 0.6 a 32.1 ± 1.2 b 35.8 ± 0.5 a 34.6 ± 0.5 a 32.9 ± 0.6 ab 121.4 ± 1.6 a 141.3 ± 2.8 b 135.0 ± 3.4 c 120.5 ± 1.3 a 120.1 ± 1.1 a 155.1 ± 5.2 a 196.5 ± 5.6 b 181.0 ± 7.4 b 132.7 ± 6.6 c 146.2 ± 3.2 ac 3.4 ± 0.2 a 2.3 ± 0.2 b 3.8 ± 0.2 a 6.7 ± 0.3 c 2.3 ± 0.1 b 131.5 ± 1.0 a 131.3 ± 2.1 a 109.8 ± 1.0 b 113.1 ± 1.2 b 105.8 ± 1.1 c 2.30 ± 0.04 a 2.78 ± 0.06 b 3.10 ± 0.10 c 2.41 ± 0.06 a 2.77 ± 0.04 b the ratio of total xanthophylls to total carotenes (X/C) showed only slight variation among dates (Table 2). None of these parameters showed diurnal variations. Gas exchange parameters Except when trees were subject to either drought or high temperature (August 2002 and May 2004), Pn increased early in the day as PPF increased, reaching a maximum (Pn,max ) by midmorning that was greater in August (15.4 µmol m – 2 s – 1 ) than in May (12.7 µmol m – 2 s –1 ), indicating that leaf aging had not affected CO2 assimilation capacity. After midmorning, Pn declined to a transitory plateau then declined further late in the day as photosynthesis became light-limited (Figure 3). When trees were subject to moderate drought (June and August 2003), midmorning Pn,max was reduced and fell sharply by early afternoon, when high temperature and VPD exerted additional constraints. During the severe drought with high temperatures in July 2003, Pn was suppressed throughout the day. Diurnal changes in Pn and gs correlated closely (Figure 3A). As with Pn, maximal gs (gs,max ) was highest in August 2002 and May 2004 and lowest in summer 2003. Compared with August 15, 2002, gs,max was 60% lower on August 6, 2003, whereas Pn,max was only 30% lower, indicating that drought had increased water-use efficiency. The diurnal course of E diverged from that of gs and Pn (Figures 3A and 3B), especially in the late morning and much of the afternoon, when declines in gs were unaccompanied by reductions in VPD. During periods of drought and high temperature, C i either remained constant or increased, despite a reduction in gs (Figure 3C), indicating metabolic limitation of Pn . On July 1, 2003, when the CO2 assimilation rate was negative, Ci remained high throughout the day. Chlorophyll a fluorescence parameters The Fv /Fm ratio was always high at 0800 h (around 0.80) but declined to around 0.2 by midday; the reduction being most pronounced during hot dry weather (Figure 3D). The lowest Fv /Fm ratio was recorded in the afternoon of July 23, 2003. On each date, recovery in fluorescence ratio was nearly, if not fully, complete by the end of the day, except on July 15, 2003, when Fv /Fm was about 15% lower at 1830 than at 0600 h. The daily time course of φPSII was closely related to incident PPF (cf. Figures 2A and 3D). Diurnal changes in φPSII (φPSII = (Fv′/Fm′)qP ) were associated with changes in both Fv′/Fm′ and qP (Figure 4). However, at a PPF of 500 µmol m – 2 s – 1, diurnal changes in qP, if any, did not always correlate with those in φPSII. For instance, the reduction in φPSII observed around midday was due mostly to a decrease in Fv′/Fm′ that reflected enhanced thermal dissipation of absorbed excitation energy at the PSII level. At a PPF of 500 µmol m – 2 s – 1, φPSII was lower throughout hot days during dry weather (summer 2003) than under non-stressful conditions (May 2004). Appreciable φPSII values were measured even when Pn was completely suppressed. Xanthophyll-cycle carotenoids The leaf concentration of Z + A showed large diurnal changes (Figure 5A). From a low value in the morning, Z + A concentration reached a peak around midday, which was higher during hot dry weather than during milder conditions. In July 2003, the de-epoxidation state (DEPS) of the V + A + Z pool reached 98% around noon (Figure 5B). High DEPS values were also observed in June (up to 83%) and August 2003 (up to 93%), whereas in May 2004, DEPS remained below 63%. In response to severe drought (July 2003), massive de-epoxidation occurred before 0900 h when solar irradiance was low or moderate. Low temperature fluorescence emission spectra Figure 6 shows the ~77 K chlorophyll fluorescence emission spectra of oak leaves in the morning (Figure 6A) and afternoon (Figure 6B). Emission spectra are plotted on an equal area basis to facilitate comparison within and among dates. On each date, spectra had three major peaks at 685 nm (F685 ), 695 nm (F695 ) and 735 nm (F735 ). In higher plants, the F685 and F695 peaks are due to the CP-43 and CP-47 components of PSII RCs, respectively, whereas F735 is due to PSI (Briantais et al. 1986, Dekker et al. 1995). When trees were subject to moderate drought (June 24 and August 6, 2003), low temperature emission spectra were comparable to those observed during a period of negligible plant water stress (May 18, 2004). In the morning, but not the afternoon, leaves of trees subject to severe drought (July 15 and 23, 2003) had a significantly lower F735 /F685 ratio, a measure that characterizes the fluorescence emission of PSI relative to that of PSII (e.g., Schrader et al. 2004), under severe drought (July 2003) (Figure 6C). TREE PHYSIOLOGY ONLINE at http://heronpublishing.com 790 HALDIMANN, GALLÉ AND FELLER Figure 3. Diurnal variations in (A) net photosynthetic CO2 assimilation rate (Pn, 䊉) and stomatal conductance (gs, 䊊); (B) transpiration rate (E, 䊏); (C) intercellular CO2 concentration (Ci, 䊐); and (D) the maximum quantum yield of photosystem (PS) II primary photochemistry (Fv /Fm, 䉱) and quantum yield of PSII electron transport (φPSII, 䉭) measured in oak (Quercus pubescens) leaves on measurement dates. Values are means ± SE of 3–10 replicates. Heat, drought and proteins of the photosynthetic apparatus Western blot analyses indicated that the large (RLS) and small (RSS) subunits of Rubisco, Rubisco activase (RA), phosphoribulokinase (PRK), the major light-harvesting Chl-a/b-binding protein of PSII (LHCII) and the γ-subunit of the chloroplast ATP-synthase (γ-CF1 ) differed little among dates, whereas amounts of RLS, RSS, RA and PRK were slightly lower in May 2004 than in other months (Figure 7). The cpn-60 Rubisco-binding protein (RBP) was more abundant in July 2003 than on other dates. The antibodies raised against γ-CF1 cross-reacted with β-CF1 , indicating that the amount of β-CF1 did not differ among dates (data not shown), thus providing evidence that drought and high temperature did not alter the amount of CF1. Discussion Under favorable conditions of temperature and water supply, Pn of oak leaves varied diurnally (Figure 3A), as observed in many other species (Epron et al. 1992, Damesin and Rambal 1995, Valentini et al. 1995). The higher value of Pn,max in Au- gust 2002 than in May 2004 probably reflects a difference in leaf maturity, as indicated by the increase in Rubisco, PRK and RA that occurred after May (Figure 7). Moderate heat and drought (June and August 2003) not only reduced Pn,max at midmorning, but reduced Pn (Figure 3A) from before noon, when Tl and VPD were high (Figures 2b and 2C), until sunset. These results and the inhibition of Pn during the period of severe drought and high temperature in July 2003 agree with earlier findings (Damesin and Rambal 1995). The changes in Pn within and among dates correlated with changes in gs (Figure 3A). If drought-dependent inhibition of Pn resulted primarily from a reduction in gs, reduced Ci values in stressed leaves would have been expected, but were not observed (Figure 3C). Intercellular CO2 concentration in stressed leaves is difficult to determine when Pn and gs are low (Cornic 2000, Lawlor 2002, Lawlor and Cornic 2002) and may have been overestimated. Alternatively, Ci may have remained high because of drought-induced metabolic limitations (Lawlor 2002, Cornic and Lawlor 2002, Tang et al. 2002) or a drought-dependent increase in mesophyll resistance to the diffusion of CO2 (Epron and Dreyer 1993b, Flexas et al. 2004). TREE PHYSIOLOGY VOLUME 28, 2008 IMPACT OF CLIMATIC CONSTRAINTS ON PHOTOSYNTHETIC TRAITS IN OAK 791 Figure 4. Diurnal variations in (A) quantum yield of photosystem (PS) II electron transport (φPSII), (B) maximum quantum yield of the PSII primary photochemistry in the light-adapted state (Fv′/Fm′) and (C) photochemical quenching (qP) determined in oak (Quercus pubescens) leaves on measurement dates. Measurements were performed in a photosynthetic photon flux (PPF) corresponding to that naturally incident on the leaves (closed symbols) or in a fixed PPF of 500 µmol m – 2 s – 1 (open symbols). Values are means ± SE of three replicates. Drought-induced inhibition of Pn is associated with reduced ATP synthesis (Lawlor 2002, Lawlor and Cornic 2002, Tang et al. 2002), which may be associated with low chloroplast ATP synthase activity (Tezara et al. 1999). Not all studies, however, support this interpretation (Wise et al. 1990, Ortiz-Lopez et al. 1991). Moreover, we observed no change in ATPase content in Q. pubescens during inhibition of Pn by drought (Figure 7). Reduced E (Figure 3B), due to a reduced gs , raised Tl several degrees above Ta (Figure 2B), thus intensifying temperaturedependent suppression of Pn. High-temperature inhibition of Pn in Q. pubescens is associated with the reversible inactivation of Rubisco (Haldimann and Feller 2004), which was likely the reason for the increase in Ci on hot days during dry weather when gs was low (Figures 3A and 3C), although increased mesophyll resistance may also have been a factor (Bernacchi et al. 2002). That φPSII remained high (Figures 3D and 4A) when Pn was inhibited indicates that electrons were flowing to alternative sinks, among which photorespiration was probably the most important (Valentini et al. 1995, Ort and Baker 2002). However, even when Pn was zero, carboxylation likely served as a sink for photosynthetic electron flow in the fixation of CO2 produced by mitochondrial respiration and photorespiration. The variability in Fv′/Fm′ within and among dates (Figure 4B) Figure 5. Diurnal variations in (A) total concentration of zeaxanthin and antheraxanthin (Z + A) and in (B) the de-epoxidation state (DEPS) of the pool of xanthophyll cycle carotenoids determined in oak (Quercus pubescens) leaves between June 2003 and May 2004. Values are means ± SE of 3–5 replicates. TREE PHYSIOLOGY ONLINE at http://heronpublishing.com 792 HALDIMANN, GALLÉ AND FELLER Figure 7. Western blot analysis showing cpn-60 Rubisco-binding protein (RBP), the large (RLS) and small (RSS) subunits of Rubisco, Rubisco activase (RA), phosphoribulokinase (PRK), the major lightharvesting chlorophyll a/b binding protein of photosystem II (LHCII) and the γ-subunit of the chloroplast ATP-synthase (γ-CF1 ) determined in extracts of oak (Quercus pubescens) leaves. Lanes within the same blot were loaded with extracts from equal leaf areas. Figure 6. Low temperature (77 K) chlorophyll fluorescence emission spectra of oak (Quercus pubescens) leaves collected in the (A) morning between 0600 and 0800 h local solar time (LST) or in the (B) afternoon between 1300 and 1500 h LST. Samples were collected: June 24, 2003 (dashes); July 15, 2003 (dash, dot, dot); July 23, 2003 (dots); August 6, 2003 (dash dot); and May 18, 2004 (solid). Each curve is the mean spectrum of three to four leaves. (C) The fluorescence emission of photosystem (PS) I relative to that of PSII (F735 /F685 ratio) was determined for samples collected in the morning (open bars) and in the afternoon (filled bars). Values are means + SE of 3–4 replicates. Values followed by different letters differ significantly at P = 0.05. reflects changes in the thermal dissipation of excitation energy at the PSII level. It follows that carboxylation activity, electron transfer to alternative sinks and thermal energy dissipation allowed leaves to keep a large fraction of their PSII RCs in an open configuration during periods of drought and high temperature (Figure 4). Thermal energy dissipation leading to down-regulation of PSII activity was most pronounced during periods of drought and high temperature and was associated with diurnal variations in the accumulation of zeaxanthin (Figure 5), which plays a central role in thermal energy dissi- pation and in protecting thylakoid membranes against photooxidative damage (Demmig-Adams and Adams 1996, Havaux and Niyogi 1999, Niyogi 1999, Demmig-Adams and Adams 2000). Zeaxanthin may also have improved the thermal stability of the thylakoid membranes (Havaux 1998). Unlike other species (Barker et al. 2002), Q. pubescens leaves subject to high temperature and drought stress do not retain large quantities of zeaxanthin overnight (Figure 5A). Leaves subject to drought and high temperature had reduced Chl a + b concentrations (Table 1), an effect unaccompanied by a reduction in LHCII (Figure 7). Stressed leaves displayed an increased (X + C)/Chl a + b ratio (Table 1), which correlated with both a higher (V + A + Z)/Chl a + b ratio and a higher lutein/Chl a + b ratio (Table 2). This reflects enhanced photoprotection, because both the V + A + Z pool and lutein (Niyogi 1999, Baroli et al. 2004) protect the photosynthetic apparatus against photo-oxidative damage. That Fv /Fm reached values around 0.80 in the morning on all dates (Figure 3D) shows that PSII was well protected against photoinactivation or thermal damage, or both, in agreement with earlier findings (Epron et al. 1992, Damesin and Rambal 1995). The diurnal depression in Fv /Fm probably reflects a slow relaxation kinetic of the down-regulation of PSII activity. The finding that PSII was resistant to inactivation by heat accords with previous studies showing that both water stress (Havaux 1992, Epron 1997, Ladjal et al. 2000) and pre-accli- TREE PHYSIOLOGY VOLUME 28, 2008 IMPACT OF CLIMATIC CONSTRAINTS ON PHOTOSYNTHETIC TRAITS IN OAK mation to high temperature (Süss and Yordanov 1986, Haldimann and Feller 2005) occur under natural conditions, significantly improving the thermotolerance of PSII and increasing the thermal stability of the thylakoid membranes (Ghouil et al. 2003, Haldimann and Feller 2005). In July 2003, leaves displayed 77 K Chl fluorescence emission spectra in the morning that differed from those measured on other dates (Figure 6A), indicating that severe drought changed the organization of the thylakoid membranes, thus altering fluorescence emission of PSI relative to that of PSII (Figure 6C). Emission spectra are influenced by fluorescence reabsorption, which is most pronounced at short wavelengths, the extent of reabsorption is dependent, in part, on Chl concentration (Krause and Weis 1984, Weis 1985). Differences in thylakoid membrane organization rather than Chl concentration appear to have been responsible for the changes in the emission spectra observed in July 2003, as spectra of leaves with a reduced Chl concentration in June 2003 were similar to those of leaves with a high Chl concentration in August 2003 and May 2004. Diurnal variations in emission spectra in July 2003 (Figure 6) may be due to high temperature-induced State-1 to State-2 transitions (Mohanty et al. 2002, Schrader et al. 2004) or to other high temperature-induced changes that affected energy distribution between PSI and PSII. In oak, however, these postulated temperature-dependent changes occurred only in severely water-stressed leaves, as they were not seen on the hot day of August 6, 2003. That extreme heat and drought did not affect amounts of PRK and Rubisco (Figure 7) contrasts with studies of other species where drought reduced Rubisco concentration (Parry et al. 2002, Tezara et al. 2002). The increased accumulation of cnp-60 RBP observed in severely stressed leaves (Figure 7) may have prevented stress-dependent inhibition of Rubisco accumulation, because cpn-60 RBP is known to be involved in Rubisco assembly (Spreitzer 1999, Roy and Andrews 2000). However, drought may have reduced Rubisco activity, which could in turn limit Pn (Parry et al. 2002, Tezara et al. 2002). Our finding that drought and heat did not alter the amount of RA (Figure 7), a protein that plays an essential role in Rubisco activation (Portis 2003, Salvucci and Crafts-Brandner 2004a, 2004b) and is sensitive to thermal denaturation (Feller et al. 1998, Salvucci et al. 2001), shows that RA of oak leaves is resistant to, or protected against, heat-dependent denaturation. The finding that the TSP concentration was similar on all dates (Table 1) indicates that drought and high temperature did not inhibit protein synthesis, in contrast with their effects in sunflower (Tezara et al. 2002). In conclusion, we found that drought and high temperatures strongly suppressed Pn in oak leaves, without reducing leaf content of key photosynthetic proteins. The inhibition of Pn by drought was unaccompanied by a reduction in chloroplast ATP synthase. We found that severe drought changed the organization of thylakoid membranes and that leaves were protected against photo-oxidative damage by the accumulation of protective carotenoids, a reduction in chlorophyll concentration, down-regulation of PSII and the dissipation of excess ex- 793 citation energy through alternative pathways. Quercus pubescens is able to preserve the functional capacity of the photosynthetic apparatus during periods of drought and high temperature and solar irradiance, thus, ensuring rapid recovery of Pn upon alleviation of stress. Nevertheless, the exceptionally hot dry summer of 2003 resulted in premature leaf mortality in July, the first time summer leaf fall has occurred in the Swiss Alps in 70 years. Acknowledgments We thank Dr. S.J. Crafts-Brandner and Dr. M.E. Salvucci, WCRL, USDA/ARS, Phoenix, AZ (PRK and RA), Prof. K. DemirevskaKepova, Bulgarian Academy of Sciences, Sofia, Bulgaria (RBP), PD Dr. G. Groth, Heinrich-Heine-University, Düsseldorf, Germany (γ-CF1 ) and Prof. S. Gepstein, Technion Israel Institute of Technology, Haifa, Israel (RLS, RSS and LHCII) for the primary antibodies used in this study. We thank Prof. P. Jahns (Heinrich-Heine-University, Düsseldorf, Germany) and Prof. K. 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