Environmental and Experimental Botany

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1 Environmental and Experimental Botany 66 (2009) Contents lists available at ScienceDirect Environmental and Experimental Botany journal homepage: Seasonal and diurnal changes in photosynthetic limitation of young sweet orange trees R.V. Ribeiro a,, E.C. Machado a, M.G. Santos b, R.F. Oliveira c a Section of Plant Physiology, Center for Research and Development in Ecophysiology and Biophysics, Agronomic Institute, P.O. Box 28, , Campinas/SP, Brazil b Department of Botany, Federal University of Pernambuco, , Recife/PE, Brazil c Department of Biological Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, P.O. Box 9, , Piracicaba/SP, Brazil article info abstract Article history: Received 1 July 2008 Received in revised form 25 February 2009 Accepted 19 March 2009 Keywords: Citrus sinensis Chlorophyll fluorescence Gas exchange Photosynthesis Seasonality This study tests the hypothesis that potted sweet orange plants show a significant variation in photosynthesis over seasonal and diurnal cycles, even in well-hydrated conditions. This hypothesis was tested by measuring diurnal variations in leaf gas exchange, chlorophyll fluorescence, leaf water potential, and the responses of CO 2 assimilation to increasing air CO 2 concentrations in 1-year-old Valência sweet orange scions grafted onto Cleopatra mandarin rootstocks during the winter and summer seasons in a subtropical climate. In addition, diurnal leaf gas exchange was evaluated under controlled conditions, with constant environmental conditions during both winter and summer. In relation to our hypothesis, a greater rate of photosynthesis is found during the summer compared to the winter. Reduced photosynthesis during winter was induced by cool night conditions, as the diurnal fluctuation of environmental conditions was not limiting. Low air and soil temperatures caused decreases in the stomatal conductance and in the rates of the biochemical reactions underlying photosynthesis (ribulose-1,5-bisphosphate (RuBP) carboxylation and RuBP regeneration) during the winter compared to the values obtained for those markers in the summer. Citrus photosynthesis during the summer was not impaired by biochemical or photochemical reactions, as CO 2 assimilation was only limited by stomatal conductance due to high leaf-to-air vapor pressure difference (VPD) during the afternoon. During the winter, the reduction in photosynthesis during the afternoon was caused by decreases in RuBP regeneration and stomatal conductance, which are both precipitated by low night temperature Elsevier B.V. All rights reserved. 1. Introduction In relation to daily changes in environmental conditions, partial stomatal closure in citrus plants is expected around midday (Veste et al., 2000; Machado et al., 2002; Medina et al., 2002; Jifon and Syvertsen, 2003; Ribeiro et al., 2009), when the leaf-to-air vapor pressure difference and the temperature reach their highest values. Reduced stomatal conductance may lead to decrease in CO 2 availability, with a resulting decrease in CO 2 assimilation (Jones, 1985; Vu, 1999; Machado et al., 2002; Medina et al., 2002; Ribeiro et al., 2009). Extreme temperatures may also disturb the biochemical reactions of photosynthesis in citrus plants. Under high temperatures, those disturbances are related to reductions in mesophyll CO 2 conductance (Vu, 1999) and in the carboxylation efficiency of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzyme (Jifon and Syvertsen, 2003). However, as Vu (1999) has stated, the biochemistry of citrus photosynthesis is poorly under- Corresponding author. Tel.: ; fax: address: rafael@iac.sp.gov.br (R.V. Ribeiro). stood, especially under natural conditions. With regard to the photochemistry, high radiation loading is harmful to citrus plants and may cause photoinhibition of photosynthesis (Veste et al., 2000; Medina et al., 2002; Jifon and Syvertsen, 2003), which is mainly observed at midday. In such situations, photochemical activity is reduced due to excessive light energy at the photosystem II (PSII) level, caused by high solar energy and/or reduced photosynthetic activity. In fact, non-stomatal factors are the main regulators of CO 2 assimilation during radiation and high temperature stresses in both potted and field-grown plants (Jifon and Syvertsen, 2003). Regarding seasonal changes, the summer is a rainy season characterized by high temperatures due to high incoming solar energy in the citrus growing areas of Brazil (Ribeiro et al., 2005, 2006). According to these thermal and water cycles, the summer season has a priori environmental characteristics that are more conducive to the photosynthetic activity of citrus plants compared to winter. However, the high energy availability in summer may not be suitable for photosynthesis in citrus plants, which show a relatively low light saturation and have low maximum photosynthetic rates (Vu, 1999; Medina et al., 2002; Jifon and Syvertsen, 2003; Machado et al., 2005). This suggests that citrus leaves may be sub /$ see front matter 2009 Elsevier B.V. All rights reserved. doi: /j.envexpbot

2 204 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) ject to excessive light energy during both the summer and winter seasons (Ribeiro et al., 2005). Photoinhibition of photosynthesis is expected to cause reduced photochemical activity and to impair leaf CO 2 assimilation (Xu and Shen, 1999). In addition, the possibility of a down-regulation of photochemistry during stressful situations (Horton et al., 1996) cannot be ruled out for citrus plants, nor should the protective effects of dynamic photoinhibition (Medina et al., 2002; Jifon and Syvertsen, 2003; Ribeiro et al., 2009) and radiationless energy dissipation at the PSII level (Horton et al., 1996; Xu and Shen, 1999). Although citrus plants have been cultivated around the world for many centuries (Davies and Albrigo, 1994), there is little information about their physiological behavior in relation to environmental changes. As photosynthesis is a key physiological process related to plant growth and development (Goldschmidt, 1999; Vu, 1999), the understanding of how the environment affects citrus photosynthesis is essential for improving horticultural management techniques for young plants grown either in the field or under nursery conditions. This study examines the hypothesis that potted sweet orange plants show significant variations in photosynthesis rates over seasonal and diurnal cycles, even in well-hydrated plants. In comparing the winter and summer seasons, such photosynthetic differences are caused primarily by the higher energy availability in the summer, which leads to increased photosynthesis due to higher stomatal conductance and higher biochemical activity, even with the impairment of photochemistry due to excessive light energy. With regard to the daily cycle, we believe that decreases in stomatal conductance and a low ribulose-1,5-bisphosphate (RuBP) carboxylation efficiency are the main causes of reduced photosynthesis in the afternoon period during both seasons. The above hypotheses were tested by measuring leaf gas exchange, chlorophyll fluorescence, and leaf water potential in potted Valência sweet orange plants growing in a subtropical climate. 2. Materials and methods 2.1. Plant material and growth conditions One-year-old Valência sweet orange (Citrus sinensis [L.] Osb.) scions grafted onto Cleopatra mandarin (Citrus reticulata Blanco) rootstocks were grown in plastic pots (36 L) containing a mixture of soil:sand:manure (2:1:1) and fertilized according to vanraijetal. (1992). Plants were irrigated daily and treated with agrochemical (fenpropathrin 300 g L 1 and milbemectin 50 g L 1 ) to avoid interference from pathogens affecting plant growth and development. Plants were exposed to natural environmental conditions in Piracicaba, SP, Brazil (22 42 S, W, 576 m of altitude). This region has Cwa-type climate according to Köppen classification, with rainy summers and dry winters, and a mean air temperature in the warmest month higher than 22 C. Plants were grown under these conditions from January 2003 to June Air temperature (T AIR, C), photosynthetic photon flux density (PPFD, mol m 2 s 1 ), leaf temperature (T LEAF ), and leaf-to-air vapor pressure difference (VPD, kpa) were evaluated during the measurements of leaf gas exchange in both the winter and summer. These environmental- and plantrelated data were monitored with an infrared gas analyzer (LI-6400, Li-Cor, Lincoln/NE, USA). In this paper, winter refers to the measurements taken from 15 to 20 July 2003, whereas summer refers to the evaluations performed from 16 to 21 February The maximum, minimum, and mean T AIR, minimum air relative humidity (RH), and daily-integrated global solar radiation (Qg) during winter (10 to 25 July 2003) and summer (11 to 26 February 2004) were monitored by an automatic weather station about 1 km from the experimental site. Therefore, macroclimate was assessed with the weather station, whereas microclimate was monitored with the LI system. Plants were irrigated every 2 days throughout the experimental period Leaf gas exchange and chlorophyll fluorescence Measurements of leaf gas exchange and chlorophyll (chl) fluorescence were taken in sun-exposed and fully expanded leaves (around 6 months old) between 9:00 and 10:30 h (mid-morning) and between 13:30 and 15:00 h (afternoon) during the winter and summer seasons. The evaluations of leaf gas exchange and chl fluorescence were performed simultaneously on a clear day (without clouds) for each season. Leaf gas exchange was measured with an infrared gas analyzer (LI-6400), previously calibrated against standards of CO 2 and water vapor and zeroed using CO 2 - and H 2 O-free air. The diurnal changes in CO 2 assimilation (A) and stomatal conductance (gs) were evaluated. Measurements were recorded when the total coefficient of variation (CV) was less than 0.5%. The air pumped into the LI-6400 was passed through a buffering gallon (5 L) to reduce the time for measurement stabilization. As described above, environmental conditions (T AIR, VPD and PPFD) were monitored with the LI-6400 system, with T LEAF being measured on the abaxial leaf surface with a thermocouple built into the LI-6400 cuvette. Measurements were taken considering the natural fluctuation of environmental elements. PPFD was assessed at the beginning of a specific measurement time and then fixed during this evaluation time using the light sensor (LI-250, Li-Cor) and light source (LI LED, Li-Cor) of the LI-6400 system. Chl fluorescence was measured with a pulse amplitude modulation fluorometer (PAM-2000, Heinz Walz GmbH, Germany). Leafclips were used for measurements of the minimal (Fo) and maximal (Fm) fluorescence yield in dark-adapted (30 min) leaf tissues. In light-adapted leaves, steady-state (F ) and maximal (Fm ) fluorescence yields were assessed. The variable fluorescence yield in both dark-adapted (Fv = Fm Fo) and light-adapted (Fv = Fm Fo ) leaves was calculated. The term Fq was calculated as Fq = Fm F, representing the photochemical quenching of chl fluorescence caused by open PSII centres (Baker et al., 2007). Fm and Fm were measured after a light saturation pulse ( < 710 nm, PPFD mol m 2 s 1, 0.8 s). These chl fluorescence parameters were used to estimate the maximum quantum efficiency of the PSII photochemistry (Fv/Fm), PSII operating efficiency (Fq /Fm ), the apparent electron transport rate (ETR = Fq /Fm PPFD ), the PSII efficiency factor (Fq /Fv ) and the non-photochemical quenching [NPQ =(Fm Fm )/Fm ](Baker et al., 2007). For ETR calculation, it was assumed that quanta were evenly distributed between photosystems II and I (0.5), and leaf light absorption was considered to be 0.84 (Schreiber et al., 1998). Fo was measured using far-red light ( = 735 nm, PPFD <15Wm 2, 3.0 s) Leaf water potential Leaf water potential ( ) was measured in leaves similar to those used for leaf gas exchange and chl fluorescence measurements in both the winter and summer seasons. At pre-dawn (6:00 h) and during the afternoon (14:30 h), leaf discs (diameter of 0.6 cm) were detached and immediately placed into sample chambers (C-52, Wescor, Logan/UT, USA). Evaluations of were taken at pre-dawn and afternoon since at these times plants have a priori the highest and lowest values, respectively, representing points of high and low shoot hydration given by the water tension in the plant body (Machado et al., 2002). After a stabilization time of 60 min, was evaluated by the psycrometric method, using a microvoltmeter (HR-33T, Wescor) operating in the hygrometric dew-point mode. All sample chambers were calibrated before the experimental period using NaCl solutions ranging from 0.1 to 1.2 mol L 1.

3 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) Table 1 Environmental conditions during the experimental period, considering the variation of macroclimate between 15 and 20 July 2003 (winter) and 16 and 21 February 2004 (summer). Macroclimate refers to meteorological data recorded with an automatic weather station: global solar radiation (Qg); maximum (Tmax) and minimum (Tmin) daily air temperatures; and minimum daily relative humidity (RHmin). Microclimate refers to the environmental- and plant-related data taken during the physiological evaluations in mid-morning (9:00 to 10:30 h) and afternoon (13:30 to 15:00 h): photosynthetic photon flux density (PPFD); air (T AIR ) and leaf (T LEAF ) temperatures; and leaf-to-air vapor pressure difference (VPD). Variables Season/daytime period Winter Daily Summer Daily Macroclimate Qg (MJ m 2 d 1 ) Tmax ( C) Tmin ( C) RHmin (%) Variables Season/daytime period Winter Summer Mid-morning Afternoon Mid-morning Afternoon Microclimate * PPFD ( mol m 2 s 1 ) 885 ± 55 Bb 1250 ± 17 Ba 1550 ± 100 Ab 2035 ± 95 Aa T AIR ( C) 22.6 ± 0.3 Bb 26.5 ± 0.4 Ba 27.3 ± 0.4 Ab 31.0 ± 0.2 Aa T LEAF ( C) 22.7 ± 0.2 Bb 27.9 ± 0.3 Ba 28.2 ± 0.5 Ab 32.6 ± 0.3 Aa VPD (kpa) 1.11 ± 0.02 Bb 2.14 ± 0.09 Ba 1.61 ± 0.10 Ab 2.69 ± 0.08 Aa * Mean value of 10 replications (±S.E.). Different capital letters indicate statistical difference (P < 0.05) between seasons for the same daytime period, while different minuscule letters indicate significant differences between daytime periods for the same season A/Ci response curves Measurements of the leaf CO 2 assimilation response to increasing intercellular CO 2 concentrations (A/Ci) were carried out in the same leaves evaluated for diurnal changes of gas exchange and chl fluorescence. A/Ci curves were recorded under saturating PPFD (1200 mol m 2 s 1 ) and natural T AIR and VPD in the morning (9:00 to 10:30 h) and afternoon (13:30 to 15:00 h) for both seasons. According to Vu (1999) and Machado et al. (2005), light saturation of citrus photosynthesis (Rubisco activity and A) has already been reached at a PPFD of 1200 mol m 2 s 1. Environmental conditions during measurements of A/Ci response curves are shown in Section 3.3. The intercellular CO 2 concentration (Ci) was controlled by varying air CO 2 concentrations (Ca)from50to1500or2000 mol mol 1 in 11 steps. First, Ca was set at 400 mol mol 1, and afterwards was decreased until it reached 50 mol mol 1. Next, Ca was again increased to 400 mol mol 1, and then to 1500 mol mol 1 (winter) or 2000 mol mol 1 (summer). During the summer, a higher Ca value was necessary to achieve CO 2 saturation of A. The procedure to perform A/Ci response curves followed the recommendations given by Long and Bernacchi (2003). The A/Ci curves were used to estimate the maximum rate of RuBP carboxylation (V c,max ) and the maximum rate of electron transport driving RuBP regeneration (J max ). At high Ca, no limitation of A was observed due to the utilization of triose-phosphate (TPU), regardless of the time of day or season. The Farquhar model of leaf photosynthesis was fitted to the A/Ci curves to calculate V c,max and J max (Farquhar et al., 1980; von Caemmerer, 2000; Sharkey et al., 2007) using the least-square method in Origin 7.5 software (OriginLab Corp., Northampton/MA, USA). The values of the CO 2 compensation point in the absence of daytime respiration ( *) and the Michaelis Menten constants of the Rubisco activities for CO 2 and O 2 (Kc and Ko, respectively) at the measured leaf temperatures were calculated according to the temperature-dependent equa- Table 2 Physiological variables related to the photosynthesis of Valência sweet orange plants as affected by season (winter and summer) and daytime period (mid-morning and afternoon): CO 2 assimilation (A); stomatal conductance (gs); maximum quantum efficiency of the PSII photochemistry (Fv/Fm); PSII operating efficiency (Fq /Fm ); apparent electron transport rate (ETR); PSII efficiency factor (Fq /Fv ); non-photochemical quenching (NPQ); relationship between the apparent electron transport rate and the CO 2 assimilation (ETR/A); and leaf water potential ( ). Physiological variables * Season/daytime period Winter Summer Mid-morning Afternoon Mid-morning Afternoon A ( mol m 2 s 1 ) 5.48 ± 0.30 Ba 3.10 ± 0.30 Bb ± 0.37 Aa 7.92 ± 0.24 Ab gs (mol m 2 s 1 ) ± Ba ± Bb ± Aa ± Ab Fv/Fm ± Ba ± Bb ± Aa ± Ab Fq /Fm ± Aa ± Aa ± Aa ± Aa ETR ( mol m 2 s 1 ) 57.4 ± 4.7 Ba 67.7 ± 12.2 Ba 92.8 ± 6.9 Aa 88.5 ± 9.8 Aa Fq /Fv ± Aa ± Aa ± Aa ± Aa NPQ ± Ba ± Aa ± Aa ± Ab ETR/A ± 0.89 Ab ± 2.36 Aa 8.39 ± 0.48 Bb ± 1.23 Ba Physiological variables * Season/daytime period Winter Summer Pre-dawn Afternoon Pre-dawn Afternoon (MPa) 0.60 ± 0.05 Aa 1.84 ± 0.04 Bb 0.55 ± 0.07 Aa 1.10 ± 0.34 Ab * Mean value of 4 10 replications (±S.E.). Different capital letters indicate statistical difference (P < 0.05) between seasons for the same daytime period, while different minuscule letters indicate significant differences between daytime periods for the same season.

4 206 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) tions and parameters given by Bernacchi et al. (2001). As suggested by von Caemmerer (2000) and also put into practice by Onoda et al. (2005), daytime respiration (Rd) was assumed to be 0.01 of V c,max. The stomatal limitation of A (S L ) was estimated according to Long and Bernacchi (2003), considering the relationship between the photosynthetic rate A at Ca of 400 mol mol 1 and the hypothetical photosynthetic rate A that would be obtained when Ci = Ca: S L =(A A )/A. Additionally, the non-stomatal limitation (NS L =1 S L ) was estimated for both daytime periods (mid-morning and afternoon) and seasons (winter and summer) Leaf gas exchange and chl fluorescence under controlled conditions Diurnal courses of leaf gas exchange and chl fluorescence were also measured in plants under controlled conditions, where PPFD, T AIR, and VPD were constant in both seasons from 7:00 to 17:45 h (winter) and from 6:15 to 18:15 h (summer). These time intervals followed the natural daily cycle, i.e., sunrise and sunset. Plants were moved to laboratory conditions before sunrise on the day of measurement. Again, we evaluated the same leaves used to measure the diurnal changes of leaf gas exchange and chl fluorescence and the A/Ci curves. Measurements of leaf gas exchange were taken at 5 min intervals using the LI-6400 system, whereas chl fluorescence variables were assessed at 1 h intervals using a modulate fluorometer (FMS1, Hansatech, Norfolk, UK), as was done with the PAM-2000 in plants under natural conditions. Leaves were subjected to a PPFD of 630 ± 2 mol m 2 s 1, T AIR of 24.9 ± 0.1 C, T LEAF of 27.7 ± 0.1 C, VPD of 1.39 ± 0.02 kpa and Ca of 367 ± 1 mol mol 1 during both seasons. Before beginning illumination, Fv/Fm was evaluated to verify the physiological state of plant tissues, i.e., that the leaves were healthy. The rest of the plant was maintained at room temperature and in near darkness (PPFD <10 mol m 2 s 1 ) under laboratory conditions, according to the methods of Allen et al. (2000) Statistical analysis Data were subjected to analysis of variance (ANOVA) and mean values were compared by the Tukey test (P < 0.05) when a significant difference was detected in physiological variables due to time of day or season. Mean values were calculated from 3 (for parameters derived from the A/Ci response curves and measurements under controlled conditions), 4 (for leaf water potential) and 10 replications (for diurnal courses of the photosynthetic traits). Replications were taken from different plants. 3. Results 3.1. Environmental conditions The environmental conditions as related to the macroclimate were quite different between seasons, with Qg values reaching 15.3 and 27.0 MJ m 2 d 1 in winter and summer, respectively (Table 1). Air temperature was also higher in summer, when maximum values were greater than 32 C(Table 1). The greatest difference between winter and summer was in minimum T AIR, which reached 10.0 C in winter and 17.2 C in summer. The minimum RH was similar between seasons, with higher values being observed during summer (Table 1). Fig. 1. Diurnal and seasonal changes in the maximum rate of ribulose-1,5- bisphosphate (RuBP) carboxylation (V c,max, in (a)), the maximum rate of electron transport driving RuBP regeneration (J max, in (b)), and the relationship between J max and V c,max (in (c)) of Valência sweet orange plants during the mid-morning (9:00 to 10:30 h, white bars) and afternoon (13:30 to 15:00 h, gray bars) in the winter and summer (Piracicaba, SP, Brazil). Each bar is the mean value of three replications (±S.E.). Measurements were taken under PPFD of 1200 mol m 2 s 1. The other conditions were: T LEAF of 21.6 ± 0.3 C (mid-morning) and 27.0 ± 0.1 C (afternoon) in the winter and 35.0 ± 0.2 C (mid-morning) and 36.3 ± 0.1 C (afternoon) in the summer; VPD of 0.78 ± 0.04 kpa (mid-morning) and 1.56 ± 0.01 kpa (afternoon) in the winter and 1.65 ± 0.03 kpa (mid-morning) and 3.14 ± 0.02 kpa (afternoon) in the summer.

5 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) At the experimental site, i.e., the microclimate, maximum PPFD was found to occur in afternoon (Table 1). However, this environmental variable was significantly higher in the summer than in the winter. Accordingly, a higher T AIR was also found in the summer, with the highest values around 31 C being observed in afternoon (Table 1). At about this period, maximum T AIR was around 26.5 Cin the winter (Table 1). T LEAF reached 32.6 C in exposed leaves under summer conditions, while maximum T LEAF values were around 27.9 C in winter (Table 1). The VPD was higher in the summer, regardless of the time of day (Table 1) Diurnal and seasonal changes in leaf gas exhange, chlorophyll fluorescence and plant water status A significant increase in CO 2 assimilation was observed during the summer (P < 0.05), regardless of the time of day (Table 2). The highest A values were found in mid-morning in both seasons, with maximum A values in the summer being twofold as high as those found in the winter. We observed a decreasing pattern for A between mid-morning and afternoon in both seasons, accompanying gs trends (Table 2). The highest gs values were observed during mid-morning in both seasons and it was more than twofold higher in the summer compared to the winter. Although a low gs was present in the winter, a significant reduction in gs was also noticed when comparing values observed in mid-morning to those obtained in afternoon, the time at which the highest VPD occurred (Tables 1 and 2). In well-watered plants, significant differences in leaf water potential between seasons were observed only in afternoon (Table 2). Plants had higher at pre-dawn during both seasons, however, showed a significant decrease at 14:30 h. This pattern was more accentuated in the winter (3.1 times) than in the summer (2.0 times). The diurnal changes of the maximum quantum efficiency of the PSII photochemistry (Fv/Fm) were similar in summer and winter (Table 2). However, higher Fv/Fm was observed in summer season, when there is higher solar energy availability (Table 1). In both seasons, reduced Fv/Fm values were found in afternoon (Table 1), with plants showing recovery trends (data not shown). The PSII operating efficiency (Fq /Fm ) was similar in both winter and summer and no differences were observed between mid-morning and afternoon measurements (Table 2). In addition, non-significant changes were detected for the PSII efficiency factor (Fq /Fv ) when considering season and the time of the day (Table 2). Regardless evaluation time, ETR was higher in the summer than in the winter (Table 2). The non-photochemical quenching (NPQ) was affected by season and evaluation time, being observed the highest NPQ values in the mid-morning of summer season (Table 2). In this season, plants showed high NPQ in the mid-morning with decreasing trend in the afternoon. This decreasing pattern between the mid-morning and afternoon was not verified during the winter (Table 2). Higher ETR/A was noticed in the winter than in the summer season, with increasing of ETR/A in the afternoon of both seasons (Table 2). These increases were higher in Fig. 2. Stomatal (S L, white bar) and non-stomatal (NS L, gray bar) limitation to photosynthesis of Valência sweet orange plants during the mid-morning (9:00 to 10:30 h) and afternoon (13:30 to 15:00 h) in winter (in (a)) and summer (in (b)) days (Piracicaba, SP, Brazil). The relationship between S L and the leaf-to-air vapor pressure difference (VPD, in (c)) and the relationship between NS L and the leaf temperature (T LEAF, in (d)) in both seasons and times of the day are shown. Each bar (in (a) and (b)) or symbol (in (c) and (d)) is the mean value of three replications (±S.E.). Environmental conditions during the measurements are shown in the legend of Fig. 1.

6 208 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) the winter than in the summer (1.94 and 1.33 times, respectively) Diurnal and seasonal variations in the carboxylation and regeneration of RuBP and stomatal and non-stomatal limitations to CO 2 assimilation Higher RuBP carboxylation (V c,max ) was noticed in the summer than in the winter (P < 0.05), regardless of the day period (Fig. 1(a)). In relation to the winter, V c,max was increased in more than 5 times during the summer season. The RuBP regeneration dependent upon electron transport (J max ) was also higher (P < 0.05) in the summer than in the winter (Fig. 1(b)). In the summer, J max was similar in the mid-morning and afternoon, whereas in the winter J max was significantly reduced (49%) in the afternoon. The balance between RuBP regeneration and carboxylation was evaluated by the relationship J max :V c,max, which is close to 1 in the summer in both the mid-morning and afternoon (Fig. 1(c)). This balance was broken during the morning in the winter, when J max :V c,max reached values higher than three. This imbalance was reduced in the afternoon (Fig. 1(c)). The stomatal limitation (S L ) of photosynthesis increased during the summer compared to the winter season (Fig. 2). The highest S L was observed during the summer in the afternoon (P < 0.05) when values of around 60% were attained (Fig. 2(b)). On the other hand, the lowest S L was noticed during the morning in the winter (P < 0.05), with values of 38.9 ± 3.9% (Fig. 2(a)). With regard to nonstomatal limitation (NS L ), the highest values were observed in the winter, varying around 60% in both the morning and the afternoon (Fig. 2(a)). This limitation was reduced in the summer, when NS L was 47.3 ± 2.6% in the morning and 36.7 ± 2.1% in the afternoon. It is noteworthy that S L increased with increasing VPD (Fig. 2(c)) and NS L decreased with increasing T LEAF (Fig. 2(d)), regardless of the season or the time of the day. A significant S L was noticed in the winter even under a VPD of 0.8 kpa (Fig. 2(c)), indicating that other environmental factors caused lower gs and A in the winter season. Under these conditions, the highest NS L was observed when T LEAF was a priori appropriate for photosynthetic activity (Fig. 2(d)) Leaf gas exchange under constant environmental conditions Significant variations in A and gs were observed during measurements under constant environmental conditions (Fig. 3). This diurnal variation was present and similar during both seasons; however, the highest values of both A and gs were found in the summer season (Fig. 3). Between 10:00 and 11:00 h, A values were around 4.7 and 3.0 mol m 2 s 1 in the summer and the winter, respectively, indicating a difference of around 50% (Fig. 3(a)). When considering gs for the same time period, the difference between seasons was around 100%, with gs values of around 0.02 and 0.04 mol m 2 s 1 in the winter and the summer, respectively (Fig. 3(b)). One interesting finding was the difference between the mid-morning and afternoon even with all environmental conditions maintained constant. Under these controlled conditions, no diurnal variations in photochemistry were observed in either season, nor were any significant differences noticed between the winter and summer. 4. Discussion 4.1. Diurnal changes in photosynthesis Fig. 3. Diurnal changes in the CO 2 assimilation (in (a)) and stomatal conductance (in (b)) of Valência sweet orange plants under constant controlled conditions during the winter (closed symbols) and the summer (open symbols), in Piracicaba, SP, Brazil. Each symbol is the mean value of three replications (±S.E.). In both seasons, PPFD = 630 ± 2 mol m 2 s 1 ; T AIR = 24.9 ± 0.1 C; T LEAF =27.7± 0.1 C; VPD = 1.39 ± 0.02 kpa; and Ca =367± 1 mol mol 1. Arrows indicate the moment at which illumination began. The similar diurnal changes of A and gs suggest that both processes are dependent, with the high photosynthetic performance in the morning being supported by high stomatal aperture (Table 2). In fact, the environmental conditions were less limiting to leaf gas exchange in the morning, when T LEAF and VPD were lower than in the afternoon (Table 1). Light availability seems to be sufficient for photosynthesis (Table 1), since citrus has low light saturation of A and gs (Vu, 1999; Machado et al., 2005). We may argue that increasing VPD (>2.0 kpa) in both seasons caused the decreasing trend of gs from morning to afternoon (Tables 1 and 2). The higher evaporative demand found in afternoon also caused a reduction in measured at 14:30 h (Table 2). The values shown in this paper are in accordance with Syvertsen and Albrigo (1980) and Machado et al. (1999), who found to vary between 0.5 MPa (at pre-dawn) and 2.0 MPa (at 14:00 h). Therefore, the reduction in A during the afternoon in both seasons was related to low gs (Table 2), which was a consequence of high VPD (Table 1) and low (Table 2). However, we cannot exclude the effects of high temperature (>31 C) and high PPFD (>1800 mol m 2 s 1 ) on the photosynthesis of potted plants in the afternoon during the summer season (Table 1). According to the

7 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) literature, light saturation of photosynthesis is already observed at a PPFD of 1200 mol m 2 s 1 (Vu, 1999; Ribeiro et al., 2003; Machado et al., 2005), and citrus photosynthesis decreases at T LEAF higher than 25 C (Ribeiro et al., 2004; Machado et al., 2005). High PPFD and T LEAF were observed in afternoon during both seasons (Table 1), when a significant reduction in photosynthesis occurred. Regarding light availability, our results show that there was no PPFD limitation to photosynthesis; rather, high PPFD could lead to photoinhibition and consequent photooxidation of leaf tissues (Osmond et al., 1997; Medina et al., 2002). Such a situation of excessive PPFD would be found in both seasons; however, plants seem to be well adapted to the contrasting light regimes of the winter and summer. This assumption is based on high Fv/Fm and NPQ values observed in both seasons (Table 2). In fact, NPQ is an important mechanism to avoid light-induced damage in plant tissues (Horton et al., 1996; Schreiber et al., 1998). The diurnal changes of the PSII operating efficiency (Fq /Fm ) maintained high ETR values during the mid-morning and afternoon (Table 2). These results indicate that the diurnal variation in A was not induced by photochemical activity, and that PPFD fluctuation did not represent an important element in regulating the photosynthesis of exposed leaves in the mid-morning and afternoon under subtropical conditions. It is noteworthy that the amount of light energy during the summer was significantly higher than in the winter (almost twofold, Table 1), and that plants did not show signs of light-induced damage, such as chronic photoinhibition (low Fv/Fm). Changes in leaf temperature during the diurnal cycle may be responsible for significant alterations in the biochemical reactions related to CO 2 uptake, mainly during the afternoon. However, no significant changes were found in RuBP carboxylation (V c,max ) between the mid-morning and afternoon in both seasons (Fig. 1(a)), when T LEAF changed from 21.6 to 26.9 C in the winter and from 34.9 to 36.3 C in the summer. In addition, no diurnal differences were found in the maximum rate of electron transport driving RuBP regeneration (J max ) in the summer (Fig. 1(b)). On the other hand, a significant reduction ( 50%) in J max occurred during the afternoon in the winter season (Fig. 1(b)) accompanied by increased ETR/A (Table 2). The low night temperature likely caused this phenomenon, as it reached values below 12 C in the winter (Table 1). In fact, low night temperature has been reported to cause a reduction in J max at midday in mango plants as well (Allen et al., 2000), and has also been reported to alter guard cell sensitivity to CO 2 (Allen et al., 2000; Allen and Ort, 2001). Changes in the photosynthesis of citrus plants caused by low temperature involve a reduction in the physical diffusion of gases in leaves due to low gs and decreases in A and chlorophyll content (Vu, 1999). This latter consequence of low temperatures may be eliminated in our paper as plants had similar total chlorophyll content and chlorophyll a/b ratio in the winter and summer seasons (data not shown). As a consequence of changes in J max, the relationship J max :V c,max was quite different between the morning and afternoon under winter conditions, with morning ratios higher than 3.0 and afternoon estimates around 1.5 (Fig. 1(c)). Usually, the ratio of J max to V c,max varies between 1.0 and 3.0 and represents the balance between RuBP carboxylation and regeneration (Wullschleger, 1993; von Caemmerer, 2000; Onoda et al., 2005). The highest J max :V c,max was found during the morning in the winter (Fig. 1(c)), providing evidence for an imbalance between RuBP regeneration and RuBP carboxylation. In comparing daytime periods, photosynthesis was biochemically limited by reduced RuBP regeneration during the afternoon in the winter, whereas plants did not show differences in the biochemistry of photosynthesis between the mid-morning and afternoon during the summer season (Fig. 1). As suggested by the diurnal changes of gs and A/Ci-derived parameters, stomatal limitation was the main factor responsible for reducing A during the afternoon in the summer season. This finding was also supported by the partitioning between stomatal (S L ) and non-stomatal (NS L ) limitations (Fig. 2). During the summer, S L reached almost 60% in the afternoon, when the highest VPD values were observed (Table 1). In fact, Sage and Kubien (2007) reported that the sensitivity of A to changes in gs was higher in warmer temperatures due to a photosynthetic biochemistry that is more sensitive to fluctuations in Ci. However, there is also a kind of endogenous regulation of citrus photosynthesis, as significant diurnal variations in A and gs were found even under constant environmental conditions (Fig. 3) Seasonal variation in photosynthesis A higher photosynthetic activity for citrus plants was observed under summer conditions when stomata showed higher aperture than in the winter (Table 2). Considering that the plants were well irrigated in late afternoon of the previous day, we may argue that such differences between A and gs relative to the seasons were caused by T AIR, PPFD, and/or VPD. During the measuring time, T AIR was more suitable for the photosynthetic activity of citrus plants under winter conditions (Ribeiro et al., 2004; Machado et al., 2005), which does not explain the lower photosynthetic performance during this season (Table 2 and Fig. 1). In addition, lower VPD values were observed during the winter season, when gs was almost halved when compared to the summer (Table 2). One could argue that the higher A under summer conditions was caused by high level of incoming radiation (given by high PPFD). In fact, significant differences in light energy availability were found between the winter and summer seasons (Table 1). However, citrus plants have reached around 90% of their photosynthetic capacity with PPFD values around 800 mol m 2 s 1 (Vu, 1999; Machado et al., 2005). An important environmental element affecting plant physiology is low temperature (Allen et al., 2000; Allen and Ort, 2001), which occurs at night in both air and soil. Minimum T AIR was around 10 C in winter, whereas it varied around 17 C during the summer season (Table 1). Citrus plants have a low temperature threshold of around 13 C, with plant metabolism being severely reduced at lower temperatures (Davies and Albrigo, 1994). Low night T AIR has also been reported to cause stomatal closure and reduced RuBP carboxylation (Allen et al., 2000). Low soil temperatures disrupt root functionality and decrease shoot hydration due to an increase in plant hydraulic resistance (Norisada et al., 2005), with citrus plants showing these symptoms at soil temperatures below 13 C(Reuther, 1977). Indeed, stomatal closure may also be a consequence of modified sap composition and ph (Wan et al., 2004) and/or of hormonal imbalances caused by low soil temperature such as increases in ABA and decreases in cytokinin content in shoots (Wan et al., 2004; Veselova et al., 2005). Therefore, we may argue that potted citrus plants showed a reduced gs and in the afternoon (Table 2) due to the low soil temperature during the winter, which reached around 9.4 C at 10 cm depth (data not shown). With regard to the photochemical reactions, the main differences between seasons were the higher Fv/Fm, ETR and NPQ values found in the summer (Table 2). While heat dissipation of excessive energy was higher under summer conditions, the alternative electron sinks (AES) were more active during the winter season, as indicated by increases in ETR/A (Table 2). According to Baker et al. (2007), the proportion of electrons driven to electron sinks other than CO 2 fixation increases under unfavorable environmental conditions, with nitrogen metabolism, photorespiration, and the water water cycle being the main sinks (Osmond et al., 1997; Ort, 2001). High AES and NPQ are protective strategies against photodamage (Xu and Shen, 1999) and have previously been reported in citrus (Medina et al., 2002; Ribeiro et al., 2003).

8 210 R.V. Ribeiro et al. / Environmental and Experimental Botany 66 (2009) However, one would expect lower AES activity during the winter than during the summer season since T AIR is higher in the latter (Tables 1 and 2). Allen and Ort (2001) reported that chilling temperatures negatively affect plant metabolism and cause high AES due to increases in Rubisco oxygenase activity (photorespiration) and the Mehler reaction (water water cycle). As carboxylase and oxygenase are competing reactions, a probable decrease in RuBP carboxylation is expected during the winter, which we verified here (Fig. 1(a)). During the winter, both V c,max and J max were markedly lower than in the summer, regardless of the time of the day (Fig. 1). Since (i) plants were well fertilized, (ii) leaves were around 6 months old in both seasons and (iii) no visual symptoms of nutritional deficiency were found in the winter, we may argue that the reduction in the biochemical activity of photosynthesis was caused by low night temperatures. Another line of evidence for this supposition is that T LEAF at the time in which A/Ci curves were generated in winter was close to the optimum for citrus photosynthesis (Ribeiro et al., 2004; Machado et al., 2005). Citrus plants also showed plasticity of J max :V c,max between seasons (Fig. 1(c)), which has been described in other species (Onoda et al., 2005). In general, plant species tend to increase the ratio J max :V c,max when growing in low temperature regimes (Onoda et al., 2005; Yamori et al., 2005). The leaf gas exchange rhythm under controlled conditions also suggests that there was an endogenous regulation of A (Fig. 3(a)). Even under the same environmental conditions, plants showed higher photosynthesis and stomatal aperture in the summer season. An important aspect to consider in the seasonal regulation of photosynthesis is the source sink relationship (Iglesias et al., 2002). Sweet orange plants show higher shoot growth and canopy development (sinks) during the summer season. In addition, low temperatures have the potential to inhibit starch mobilization during the night and to disrupt sucrose and nitrogen metabolism (Allen et al., 2000; Allen and Ort, 2001). In turn, higher leaf carbohydrate contents may exert an inhibitory effect on photosynthesis (Syvertsen, 1994; Iglesias et al., 2002; Syvertsen et al., 2003). Therefore, a possible mechanism of regulation is the source sink relationship, in which a higher demand for leaf reserves may enhance citrus photosynthesis during the growth season (summer). This aspect should be addressed in future research to build a more complete picture of the seasonal variation of photosynthesis in sweet orange plants. 5. Conclusion With regard to our hypothesis, higher photosynthesis is found during the summer season compared to the winter. Reduced photosynthesis during the winter was induced by the cool night conditions, as the diurnal fluctuation of environmental conditions was not limiting. Low air and soil temperatures caused decreases in stomatal conductance and in the biochemistry of photosynthesis (V c,max and J max ) during the winter compared to the summer season. Citrus photosynthesis during the summer was not impaired by biochemistry or photochemistry, as CO 2 assimilation was only limited by stomatal conductance during the afternoon due to high VPD. During the winter, the reduction in photosynthesis in the afternoon was caused by decreases in RuBP regeneration and stomatal conductance, both of which were promoted by low night temperature. Acknowledgments The authors gratefully acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), the Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp, Brazil) and the Comissão de Aperfeiçoamento de Pessoal de Nível Superior (Capes, Brazil) for the fellowship (RVR and ECM) and the scholarships granted (RVR and MGS). This research was supported by the Fapesp (grant no. 02/ ) and the authors are also indebted to Dr. Camilo L. Medina (Conplant, Brazil) for providing plant material. References Allen, D.J., Ort, D.R., Impacts of chilling temperatures on photosynthesis in warm-climate plants. 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