Photosynthetic acclimation of young sweet orange trees to elevated growth CO 2 and temperature

Transcription

1 J. Plant Physiol (2002) Urban & Fischer Verlag Photosynthetic acclimation of young sweet orange trees to elevated growth CO 2 and temperature Joseph C. V. Vu 1, 2 *, Yoana C. Newman 2, L. Hartwell Allen, Jr. 1, 2, Maria Gallo-Meagher 2, 3, Mu-Qing Zhang 2 1 US Department of Agriculture c/o Agronomy Department, 2183 McCarty Hall A, P.O. Box , University of Florida, Gainesville, Florida , USA 2 Agronomy Department, University of Florida, Gainesville, Florida 32611, USA 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611, USA Received July 23, 2001 Accepted October 4, 2001 Summary Two-year old trees of Ambersweet orange, a hybrid of Clementine tangerine (Citrus reticulata Blanco) and Orlando tangelo (C. paradisi Macf. C. reticulata), were grown for twenty-nine months under two daytime [CO 2 ] of 360 (ambient) and 720 (elevated) µmol mol 1, and at two temperatures of 1.5 and 6.0 C above ambient temperature. The objectives were to characterize the physiology and biochemistry of citrus photosynthesis in response to both elevated [CO 2 ] and temperature, and to test if the photosynthetic capacity of sweet orange, in terms of rubisco activity and protein concentration, was down-regulated under long-term elevated growth [CO 2 ]. Both mature (old) and expanding (new) leaves of trees grown under elevated [CO 2 ] had higher photosynthetic rates, lower transpiration and conductance, and higher water-use efficiency (WUE), compared to those grown under ambient [CO 2 ]. Although leaf WUE was reduced by high temperature, elevated [CO 2 ] compensated for adverse effect of high temperature on leaf WUE. Activity and protein concentration of rubisco were down-regulated in both new and old leaves at elevated [CO 2 ]. In contrast, the amount of total leaf soluble protein was not affected by elevated [CO 2 ] and high temperature. Down-regulation of photosynthetic capacity was greater for the old leaves, although activity and protein concentration of rubisco in the new leaves were higher. Contents of soluble sugars and starch in all leaves sampled, which were higher under elevated [CO 2 ], were generally not affected by high temperature. Within each specific CO 2 -temperature treatment and leaf type, total soluble sugars remained relatively unchanged throughout the day, as did the starch content of early morning and midday samples, and only a moderate increase in starch for the old leaves at late afternoon sampling was observed. In contrast, starch content in the new leaves increased substantially at late afternoon. Activities of sucrose-p synthase and adenosine 5 -diphosphoglucose pyrophosphorylase were reduced at elevated [CO 2 ]in the old leaves, but not in the new leaves. The photosynthetic acclimation of Ambersweet orange leaves at elevated [CO 2 ] allowed an optimization of nitrogen use by reallocation/redistribution of the nitrogen resources away from rubisco. Thus, in the absence of other environmental stresses, citrus photosynthesis would perform well under rising atmospheric [CO 2 ] and temperature as predicted for this century. * corresponding author: jcvu@mail.ifas.ufl.edu /02/159/ $ 15.00/0

2 148 Joseph C. V. Vu et al. Key words: carbohydrate metabolism Citrus CO 2 enrichment high temperature photosynthetic acclimation rubisco Abbreviations: ADGP adenosine 5 -diphosphoglucose pyrophosphorylase. CER CO 2 exchange rate. EDT eastern day time. PPFD photosynthetic photon flux density. rubisco ribulose bisphosphate carboxylase-oxygenase. SPS sucrose-p synthase Introduction Global climate change appears to be inevitable in the coming decades due to a continuing rise in atmospheric CO 2 concentration ([CO 2 ]) (Rosenzweig and Hillel 1998, Morison and Lawlor 1999). The current atmospheric [CO 2 ] of 365µmolmol 1 limits the photosynthetic capability, growth and yield of many agricultural crop plants, with C 3 species showing great potential for response to rising [CO 2 ] (Bowes 1993, Kimball et al. 1993, Allen 1994, Drake et al. 1997). With the atmospheric [CO 2 ] expected to double within this century, various atmospheric general circulation models predict alterations in precipitation patterns and significant increases in global air temperatures, possibly as much as 4 to 6 C (Kattenberg et al. 1996, Morison and Lawlor 1999, Schneider 2001). Leaf photosynthetic CO 2 exchange rate (CER) is directly influenced by the activity of ribulose bisphosphate carboxylase-oxygenase (rubisco), which in turn is influenced by environmental factors, including atmospheric [CO 2 ] and air temperature. Additionally, the metabolism of primary photosynthetic products such as sucrose and starch is controlled and/ or regulated by aerial environmental conditions. In C 3 plants, rubisco activity is CO 2 -limited under present atmospheric conditions, and increased [CO 2 ] enhances CER. However, in the longer term, this initial stimulation of CER by elevated [CO 2 ] is often followed by biochemical and/or molecular changes, resulting in decreased photosynthetic capacity which is generally manifested through reductions in both activity and protein concentration of rubisco (Vu et al. 1997, Gesch et al. 1998, Moore et al. 1998). Such long-term experiments under controlled environmental conditions have focused primarily on annual species (Sage et al. 1989, Van Oosten and Besford 1995, Nie et al. 1995, Moore et al. 1998, Gesch et al. 1998, Vu et al. 1997, 1999, 2001). Physiological and biochemical studies of perennial fruit crops subjected to increases in global levels of [CO 2 ] and temperature are particularly limited. Citrus is one of the most important global fruit crops. However, limited knowledge of citrus physiology, biochemistry and molecular biology hinders classical breeding efforts, as well as genetic engineering, to achieve improved production under environmental stress conditions (Vu 1999). Therefore, characterization of the regulatory mechanisms of citrus metabolism in response to future rises in atmospheric [CO 2 ] and other predicted global climate change factors is critically needed. In this study, two-year old Ambersweet orange trees were grown for twenty-nine months under ambient or doubleambient [CO 2 ] and at two temperatures, 1.5 and 6.0 C above outdoor ambient temperature, to characterize the physiology and biochemistry of leaf photosynthetic responses to elevated growth [CO 2 ] and temperature. Our hypothesis was that photosynthetic capacity of this perennial citrus cultivar, in terms of rubisco activity and protein concentration, would be down-regulated when grown at elevated growth [CO 2 ]. We also assessed whether such acclimation for sweet orange trees occurred in both expanding and mature leaves. Materials and Methods Plant material and growth conditions Uniform-appearing, two-year old trees of Ambersweet orange, propagated on Swingle citrumelo (Citrus paradisi Macf. Poncirus trifoliata L. Raf.) rootstocks, were transplanted and continuously grown in paired companion temperature-gradient greenhouses (TGGs) (University of Florida, Gainesville, FL; August 9, 1994). Ambersweet orange resulted from crossing Clementine tangerine (C. reticulata Blanco) to Orlando tangelo (C. paradisi Macf. C. reticulata) (Hearn 1989). Temperature and CO 2 controls were based on the TGG infrastructure used by Okada et al. (1995) and the modified hardware as described by Sinclair et al. (1995) and Fritschi et al. (1999), with the following additional modifications. These TGGs, with semi-cylindrical galvanized steel arch structures, were 27.4-m long, 4.3-m wide, and 2.2-m high at the ridgepole. Each TGG was divided into a 3.6-m long entry segment to stabilize incoming flow, four sequential experimental segments, each 5.5-m long, and a 1.8-m flow convergence zone before the air was expelled (Fig. 1). A computer-controlled, variablespeed ventilation fan mounted at the south end of each TGG controlled airflow and regulated the temperature gradient, which averaged from 1.5 C above outside ambient temperature (T A ) at the airentry north end (segment 1) to 6.0 C above T A at the south end (segment 4) (Figs. 1, 2 A and B). During much of the time, heat was provided by two 1,500-watt electric heaters mounted on each side of the TGG at 5.5-m increments along the length at the beginning of segments 2, 3, and 4. Incoming solar radiation supplanted the need for continuous electrical heat during bright weather, and the heaters were turned off and on in concert with the variable speed fan to provide an average temperature gradient of 4.5 C between segments 1 and 4. The ventilation fan speed and electric heaters were controlled by mi-

3 Citrus photosynthesis at elevated growth CO 2 and temperature 149 of recent past CO 2 values. Temperature and [CO 2 ] were controlled and monitored for each TGG individually. Dewpoint temperatures were measured in the outside ambient air and at the warmest end of the TGGs. Dewpoint temperature of the outside ambient air averaged 22 C, and that of the warmest end of the TGGs averaged 24 C at night and 30 C during the day. Although not quantified, the increase in dewpoint temperature along the length of the TGG decreased the vapor pressure deficit from the high values that would have occurred if temperature only had increased from entry to exit. The [CO 2 ] was measured continuously in segment 1 to control the rate of CO 2 injection for maintaining the set point [CO 2 ], and once every 20 min in segment 4 from sampled air that was pumped from a sampling port into a 10-L mixing volume. During the nighttime period, the increase of [CO 2 ] due to tree respiration was less than 10 µmol mol 1 because of continuous slow ventilation. During the daytime period, the decrease in [CO 2 ] because of CO 2 removed by photosynthesis was typically less than 25 µmol mol 1. A Keithley-Metrabyte supervised controller/data acquisition system (SCADA) (Woburn, MA) with Intellution FIX DMACS supervisory software (Intellution, Inc., Norwood, MA) was used to measure temperatures, calculate temperature gradients, adjust ventilation rates, control heaters, measure CO 2 concentration, control CO 2 injection rates, and log data. Four galvanized metal containers, 1.5-m long 0.6-m wide 0.6 m deep, were arranged in each of the four segments of the ambientand elevated-co 2 TGGs and filled with mineral top soil. Five sweet orange trees were transplanted into each container. The TGG steel framework was covered with a transparent greenhouse polyethylene plastic that transmitted 90 % of the solar photosynthetic photon flux density (PPFD) (Fig. 2 C). Soil moisture was checked daily, and additional irrigation applied as needed to ensure adequate soil moisture for tree growth. Appropriate fertilizer was applied to the soil at transplanting, and intervally thereafter, at doses recommended for commercial citrus production. Only trees grown in segments 1 (T A C) and 4 (T A C) of the TGGs were used for our studies. Leaf gas exchange measurements Figure 1. Treatment layout in a temperature-gradient greenhouse (TGG). Unidirectional arrows indicate the direction of air flow. T A, outdoor ambient temperature; T A C, average temperature of the TGG segment 1; T A C, average temperature of the TGG segment 2; T A C, average temperature of the TGG segment 3; T A C, average temperature of the TGG segment 4. croprocessor algorithm. Overhead paddle fans at the beginning of each segment mixed the heated air to minimize vertical gradients of temperature. The [CO 2 ] was maintained at ambient (360 µmol mol 1 )in one TGG, and 360 µmol mol 1 above ambient (720 µmol mol 1 ) in the other. CO 2 enrichment was implemented during daylight hours by injection of CO 2 at 1.8 m into the air-entry segment of the TGG through a predilution system that provided cross-sectional uniform CO 2 concentrations. The CO 2 was injected with a proportionally controlled fine metering valve, which was regulated by an algorithm based on ventilation fan speed, current CO 2 concentration, and a feedback integral CER, conductance, and transpiration of single-attached, fullyexpanded mature leaves of the old growth flushes, to be referred to further as old leaves, and most-expanding leaves of the new growth flushes, to be referred to further as new leaves, were measured with a LI-6200 Portable Photosynthesis System (LI-COR, Inc., Lincoln, NE) during October 15 31, These old or new leaves were selected from separate old or new growth flushes of eight different trees for each treatment. Measurements were performed at midday, between 1100 and 1400 EDT, when solar PPFD was at 1,200 1,600 µmol m 2 s 1. Leaf sampling and analyses of enzymes and carbohydrates Leaves (old and new) were sampled at midday on October 21, 1996, a clear day with solar PPFD of 1,200 1,400 µmol m 2 s 1. At each sampling, 32 leaves of each type were detached from the growth flushes of 8 different trees for each treatment and immediately immersed in liquid N 2. Sampled leaves were pooled by treatment and leaf type, ground to a fine powder in liquid N 2, and stored in liquid N 2. Subsequently, activities of rubisco, sucrose-p synthase (SPS) and adenosine 5 -diphosphoglucose pyrophosphorylase (ADGP) were assayed as previously reported (Vu et al. 1997, 2001). Rubisco activation was

4 150 Joseph C. V. Vu et al. Figure 2. Monthly averages for the year 1996 of (A) mean daily temperature in the 360 µmol CO 2 mol 1 temperature-gradient greenhouse (TGG), (B) mean daily temperature in the 720 µmol CO 2 mol 1 TGG, and (C) mean daily photosynthetic photon flux density. T A, outdoor ambient temperature; T A C, average temperature of the TGG segment 1; T A C, average temperature of the TGG segment 4. computed as the ratio of the initial to the corresponding total activity. Rubisco protein content was determined by radioimmuno-precipitation procedures (Vu et al. 2001). Additionally, 32 leaves from each treatment were also sampled at 0800, 1200 and 1600 EDT, oven-dried at 60 C, ground to a powder, and subsampled for carbohydrate measurements. Soluble sugars were extracted from approximately 100 mg of the oven-dried leaf powder with 80 % (v/v) ethanol at 85 C. Glucose, fructose and sucrose were quantified using the microtiter method (Hendrix 1993). Pellets containing starch were oven-dried overnight at 60 C. Starch in the pellet was first gelatinized by addition of 1 ml of 0.2 mol/l KOH and incubation in a boiling water bath for 30 min (Rufty and Huber 1983). After cooling, 0.2 ml of 1mol/L acetic acid was added, and the solution was incubated with 2 ml acetate buffer (ph 4.6) containing amyloglucosidase (6 units, Boehringer Mannheim) at 55 C for 1 h. The reaction was terminated in a boiling water bath, and the resulting supernatant was analyzed for glucose. Subsets of 48 leaves were also harvested from each treatment for determination of leaf biomass and area at the same time that samples were taken for enzyme and carbohydrate analyses. Statistical analyses Differences among treatment means were determined using the Duncan Multiple Range Test.

5 Citrus photosynthesis at elevated growth CO 2 and temperature 151 Table 1. CO 2 exchange rate (CER), transpiration, conductance, water-use efficiency (WUE) and chlorophyll of fully-developed leaves of the mature flushes (Old) and most-expanding leaves of the recent flushes (New) of Ambersweet orange grown under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Values are the mean and SE (parentheses) of 4 to 22 determinations. Values with different letters in the same column are significantly different at P < 0.05 in a Duncan Multiple Range Test. [CO 2 ] Leaf Temperature CER Transpiration Conductance WUE Chlorophyll (µmol mol 1 ) Status ( C) (µmol CO 2 (mmol H 2 O (mmol H 2 O (mmol CO 2 (mg m 2 ) m 2 s 1 ) m 2 s 1 ) m 2 s 1 ) mol 1 H 2 O) 360 Old T A (0.3)c 3.9 (0.2)b 121 (7)b 2.1 (0.1)c 553 (25)a T A (0.3)bc 5.7 (0.6)a 167 (17)a 1.6 (0.1)c 537 (32)a New T A (0.9)bc 2.8 (0.3)c 118 (12)b 3.4 (0.2)b 498 (21)ab T A (0.3)b 5.3 (0.5)a 169 (14)a 1.8 (0.1)c 457 (18)bc 720 Old T A (0.7)a 2.7 (0.2)c 76 (5)c 4.3 (0.2)ab 510 (24)a T A (0.6)a 3.2 (0.3)bc 121 (19)b 4.0 (0.2)b 491 (30)ab New T A (0.5)a 2.5 (0.2)c 75 (9)c 5.0 (0.2)a 431 (19)c T A (0.7)a 3.9 (0.4)b 107 (10)b 3.5 (0.3)b 430 (26)c Results Ambersweet orange trees grown and measured at 720 µmol CO 2 mol 1 had higher leaf CER than their counterparts at 360 µmol [CO 2 ] mol 1 (Table 1). Percent enhancement in CER by elevated [CO 2 ] for old and new leaves was 45 and 33 % under near-ambient growth temperature (T A C), and 42 and 38 % under high growth temperature (T A C), respectively. In contrast, stomatal conductance and transpiration were less under elevated [CO 2 ]. Transpiration rates of old leaves of the elevated-co 2 trees, compared to those of the ambient-co 2 controls, were 31 % less at near-ambient temperature and 44 % less at high temperature. Similarly, stomatal conductance of both leaf types of the elevated-co 2 trees under both growth temperatures was 28 to 37 % lower than that of their ambient-co 2 counterparts. There was also a small and consistent, although not significant, increase in CER for all leaves of both CO 2 treatments under high temperature (Table 1). Furthermore, high temperature increased transpiration by 18 to 46 % for old leaves and 57 to 89 % for new leaves, and stomatal conductance by 38 to 59 % for old leaves and 43 % for new leaves. As shown in Table 1, leaf photosynthetic water-use efficiency (WUE), the ratio of CER to transpiration rate, was higher for trees at elevated [CO 2 ] than those at ambient [CO 2 ]. Under near-ambient temperature, WUE of elevated-co 2 trees was 105 % greater for old leaves and 47 % higher for new leaves. Such increases in WUE due to elevated [CO 2 ] were even larger at high temperature: 150 and 94 % for old and new leaves, respectively. However, high temperature per se reduced leaf WUE, and such reduction was more evident in new leaves. Total chlorophyll content per unit leaf area was hardly affected by growth [CO 2 ] and temperature (Table 1). Trees at elevated growth [CO 2 ] and temperature had only slightly less chlorophyll than those at ambient [CO 2 ]. New leaves contained 10 to 15 % less chlorophyll than old leaves. Growth at elevated [CO 2 ] resulted in down-regulation of rubisco activity and protein concentration, expressed on a leaf area basis (Table 2). At the two growth temperatures, elevated [CO 2 ] reduced the total activities of rubisco by 31 to 36 % for old leaves, and 13 to 19 % for new leaves. Similarly, reductions in rubisco protein concentration by elevated [CO 2 ] were 36 to 39 % for old leaves, and 15 to 23 % for new leaves. Initial and total rubisco activities of new leaves were higher than those of their counterpart old leaves, and this difference was greater under elevated [CO 2 ]. Rubisco in new compared to old leaves was 34 to 37% higher in initial activity and 10 to 14 % higher in total activity at ambient [CO 2 ], but 60 to 81 % and 38 to 44 % greater, respectively, at elevated [CO 2 ]. Similarly, rubisco protein concentration in new leaves was 19 to 21 % higher at ambient [CO 2 ] and 52 to 57 % greater at elevated [CO 2 ] compared to old leaves. Within each CO 2 treatment, rubisco activities of both leaf types were generally not affected by high temperature, whereas rubisco protein concentration was slightly less (8 16 %) at high temperature. At both growth temperatures, there was no effect of CO 2 enrichment on the amount of total leaf soluble protein, expressed on a leaf area basis (Table 2). New leaves also contained a similar amount of soluble protein as old leaves. The rubisco protein/total soluble protein ratio, which averaged 25 and 32 % under ambient [CO 2 ], declined to 15 and 25 % under elevated [CO 2 ], respectively for old and new leaves. Rubisco activation and K cat for both leaf types were hardly affected by elevated [CO 2 ]. Total soluble sugars and starch, expressed on a leaf area basis, were higher in midday-sampled leaves of trees grown at elevated [CO 2 ] than their counterparts at ambient [CO 2 ] (Table 3). At elevated [CO 2 ], glucose (with the exception for the new leaves at T A C) was increased up to 115 %,

6 152 Joseph C. V. Vu et al. Table 2. Activity, activation, protein concentration and apparent catalytic turnover rate (K cat ) of rubisco, total soluble protein, and ratio of rubisco protein concentration to total soluble protein in midday-sampled, fully-developed leaves of the mature flushes (Old) and most-expanding leaves of the recent flushes (New) of Ambersweet orange grown under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Values are mean and SE (parentheses) of 6 determinations. Values with different letters in the same column are significantly different at P < 0.05 in a Duncan Multiple Range Test. [CO 2 ] Leaf Temperature (µmol Status ( C) mo 1 ) Rubisco Activity (µmol m 2 leaf area s 1 ) Activation Concentration K cat Initial Total (%) (g m 2 leaf (mol CO 2 mol 1 area) enzymes s 1 ) Tot. Sol. Protein Rubisco/Tot. (g m 2 leaf area) Sol. Protein (%) 360 Old T A (0.8)b 33.9 (1.0)bc (0.05)b 13.1 (0.9)b 5.41 (0.34)a 26.4 T A (1.0)b 32.5 (0.3)c (0.07)b 14.6 (0.4)ab 5.19 (0.18)a 23.7 New T A (0.4)a 38.5 (0.4)a (0.11)a 12.3 (0.4)b 5.05 (0.27)a 34.3 T A (1.9)a 35.6 (1.5)b (0.18)ab 13.4 (1.1)b 4.90 (0.20)a Old T A (0.1)c 21.7 (0.8)d (0.02)c 13.8 (0.6)b 5.52 (0.21)a 15.9 T A (0.6)c 22.4 (0.5)d (0.01)c 15.7 (0.7)a 5.43 (0.14)a 14.5 New T A (0.7)b 31.3 (1.6)c (0.09)b 12.9 (0.9)b 5.15 (0.28)a 26.0 T A (2.0)a 31.0 (1.4)c (0.12)b 13.7 (0.8)b 4.99 (0.38)a 24.9 Table 3. Soluble sugars, starch and total nonstructural carbohydrates (TNC) (soluble sugars + starch) in midday-sampled, fully-developed leaves of the mature flushes (Old) and most-expanding leaves of the recent flushes (New) of Ambersweet orange grown under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Values are the mean and SE (parentheses) of 4 determinations. Values with different letters in the same column are significantly different at P < 0.05 in a Duncan Multiple Range Test. [CO 2 ] Leaf Temperature Glucose Fructose Sucrose Total Soluble Starch TNC (µmol mol 1 ) Status ( C) Sugars (g m 2 leaf area) 360 Old T A (0.03)e 0.39 (0.07)c 4.07 (0.15)c 4.59 (0.24)c 3.87 (0.10)d 8.46 (0.38)d T A (0.09)d 0.36 (0.08)c 4.33 (0.06)c 5.17 (0.17)c 4.11 (0.10)d 9.28 (0.29)d New T A (0.43)ab 0.48 (0.10)c 3.79 (0.16)c 6.89 (0.34)b 3.91 (0.12)d (0.50)c T A (0.14)b 0.35 (0.08)c 3.80 (0.30)c 6.52 (0.46)b 5.79 (0.41)c (0.84)c 720 Old T A (0.05)e 0.71 (0.08)b 5.88 (0.09)ab 6.87 (0.23)b (0.36)b (0.58)b T A (0.04)d 0.95 (0.07)a 5.27 (0.06)b 6.75 (0.12)b 9.10 (0.26)b (0.43)b New T A (0.25)c 0.64 (0.08)b 6.57 (0.15)a 8.28 (0.27)a (0.38)a (0.81)a T A (0.36)a 0.48 (0.08)c 5.19 (0.20)b 9.10 (0.42)a (0.53)a (0.95)a fructose up to 164 %, sucrose up to 73 %, total soluble sugars up to 50 %, starch up to 424 %, and total nonstructural carbohydrates up to 166 %. High temperature did not affect the levels of fructose, sucrose, total soluble sugars, starch, or total nonstructural carbohydrates. However, the glucose concentration was generally greater under high growth temperature. Within each CO 2 treatment, difference in fructose or sucrose concentration between old and new leaves was only marginal. Glucose in the new leaves, however, was several-fold higher than that in the old leaves at both growth [CO 2 ], and this resulted in a 26 to 50 % increase in total soluble sugars. Differences in starch content between leaves was small under ambient [CO 2 ]. In contrast, at elevated [CO 2 ], starch content in new leaves was two-fold higher than that in old leaves. Analyses of the diurnal nonstructural carbohydrate contents showed that starch of new leaves sampled in the late afternoon (1600 EDT), versus the early morning (0800 EDT) and midday (1200 EDT) samples, was doubled at both growth temperatures of the ambient-co 2 treatment, and was 38 and 81% higher, respectively, for the high and near-ambient temperature of the elevated-co 2 treatment (Fig. 3). In contrast, except for the elevated CO 2 -high temperature treatment, there was no difference in leaf starch content for the diurnal samples of old leaves. Also, concentrations of total soluble sugars in both leaf types remained relatively similar throughout the day for each specific CO 2 and temperature treatment (data not presented) (see Table 2 for midday values of soluble sugars).

7 Citrus photosynthesis at elevated growth CO 2 and temperature 153 Figure 3. Diurnal changes in starch concentrations of fully-developed leaves of the mature flushes (Old) and most-expanding leaves of the recent flushes (New) of Ambersweet orange grown in temperaturegradient greenhouses under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Each data point represents the mean (with SE bar) of 4 determinations. Where no bar is visible, the SE is smaller than the symbol. Irrespective of growth [CO 2 ], activities of SPS and ADGP were generally greater for new leaves (Table 4). Activities of SPS and ADGP in these leaves were 13 to 17 % and 27 to 29 % higher, respectively, under ambient growth [CO 2 ], and 52 to 54 % and 60 to 70 % greater, respectively, under elevated [CO 2 ], compared to old leaves. In terms of growth [CO 2 ], activities of the enzymes in old leaves were down-regulated at elevated [CO 2 ] (18 to 26 % less for SPS, and 22 to 28 % less for ADGP), whereas activities in new leaves were not decreased. Activities of the enzymes, except for those of ADGP at ambient [CO 2 ], were not significantly affected by high temperature. There was a small and consistent, although not always significant, increase in leaf fresh weight and dry weight by elevated [CO 2 ] (Table 5). In addition, CO 2 enrichment did not affect the area of individual leaves, but high temperature slightly enhanced it. Although growth [CO 2 ] did not affect leaf area, specific leaf weight was enhanced by elevated [CO 2 ]. At near-ambient growth temperature, elevated [CO 2 ] increased specific leaf weight 17 to 23 % for old leaves, and 19 to 49 % for new leaves. At high growth temperature, the enhancements in specific leaf weight by elevated [CO 2 ]were still evident, although to a smaller extent. Discussion Down-regulation of the rubisco protein concentration in both old and new leaves of Ambersweet orange, as mediated by elevated growth [CO 2 ], did not entail a change in the level of total soluble protein. Under normal ambient growth [CO 2 ], the concentration of rubisco protein in old leaves was about 25 % of the total soluble protein level, which is typical for citrus (Vu and Yelenosky 1988) and several other C 3 species (Seemann et al. 1984). In new leaves, the rubisco protein/total soluble protein ratio was greater ( 32 %), and this was primarily due to a higher concentration of the rubisco protein in those expanding leaves. Under elevated [CO 2 ], however, this ratio declined to about 15 % in the old and 25 % in the new leaves. The decline in rubisco protein concentration without a reduc-

8 154 Joseph C. V. Vu et al. Table 4. Activities of sucrose-p synthase (SPS) and adenosine 5 - diphosphoglucose pyrophosphorylase (ADGP) in midday-sampled, fully-developed leaves of the mature flushes (Old) and mostexpanding leaves of the recent flushes (New) of Ambersweet orange grown under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Values are the mean and SE (parentheses) of 4 to 8 determinations. Values with different letters in the same column are significantly different at P < 0.05 in a Duncan Multiple Range Test. [CO 2 ] Leaf Temperature SPS ADGP (µmol Status ( C) mol 1 (µmol m 2 leaf area s 1 ) ) 360 Old T A (0.37)a (0.80)b T A (0.16)a (0.20)c New T A (0.23)a (0.43)a T A (0.26)a (1.05)b 720 Old T A (0.14)b (0.70)d T A (0.27)b (0.80)d New T A (0.21)a (1.08)ab T A (0.31)a (1.14)b tion in total soluble protein content indicates a partial reallocation of the nitrogen resources away from rubisco in elevated CO 2 -grown sweet orange leaves. This reallocation of nitrogen also suggests an enhancement in concentrations of other proteins, at the sacrifice of rubisco, at elevated growth [CO 2 ], although such an implication must be proved. Rubisco protein concentration has been used as a biochemical indicator to evaluate leaf photosynthetic capacity at elevated growth [CO 2 ] (Vu et al. 1997, Moore et al. 1999). This study is the first to show a down-regulation in the rubisco protein concentration in citrus under long-term CO 2 enrichment. The declines in both rubisco concentration and activity, expressed on a leaf area basis, in Ambersweet orange under elevated [CO 2 ] were in agreement with a number of other reported elevated CO 2 -grown C 3 species (Sage et al. 1989, Nie et al. 1995, Van Oosten and Besford 1995, Gesch et al. 1998, Moore et al. 1998, Vu et al. 1997, 1999, 2001). For citrus, the reduction in activity and protein content of rubisco at elevated CO 2 may be cultivar-specific, since rubisco activity has been reported to be higher for leaves of Swingle citrumelo grown at twice ambient [CO 2 ], but not in leaf samples from Carrizo citrange (Koch et al. 1986). However, the rubisco activity for these two citrus rootstocks was expressed on a leaf chlorophyll basis. Claims that rubisco is modulated by growth at elevated [CO 2 ] requires further careful evaluation, as the basis on which activity and/or protein concentration of the enzyme are expressed may vary or nullify observations (Bowes 1993). Several plant species expressing a decrease in photosynthetic capacity (i.e., reduced rubisco activity and/or protein concentration) at elevated CO 2 also show reductions in other components of the photosynthetic apparatus, including chlorophyll (Webber et al. 1994, Gesch et al. 1998, Moore et al. 1998, Vu et al. 1999, 2001). However, there was substantial variation from one plant species to the other in the influence of elevated growth [CO 2 ] on rubisco protein and chlorophyll concentrations, and one cannot predict the photosynthetic acclimation based on leaf chlorophyll content response. In Ambersweet orange, there was a general trend toward a small decline, although not statistically significant, in leaf chlorophyll content at elevated [CO 2 ] (Table 1). Of the sixteen elevated CO 2 -grown plant species examined by Moore et al. (1998), eleven showed declines in both rubisco protein and chlorophyll levels, while the others showed declines in chlorophyll with no reduction in rubisco protein. In soybean, leaf chlorophyll content increased while rubisco protein concentration decreased under a doubling of growth [CO 2 ] (Vu et al. 2001). In addition to rubisco, there are also reports that long-term elevated growth [CO 2 ] affects the regulation of SPS, acid invertase and ADGP activities (Moore et al. 1998, Hussain et al. 1999, Vu et al. 2001). However, these catalytic regulatory responses to high [CO 2 ] are species-specific. As for rubisco, Table 5. Biomass, area and specific leaf weight of fully-developed leaves of the mature flushes (Old) and most-expanding leaves of the recent flushes (New) of Ambersweet orange grown under 360 and 720 µmol CO 2 mol 1 and at average temperatures of 1.5 and 6.0 C above outdoor ambient temperature (T A ). Values are mean and SE (in parentheses) of 48 leaves. Values with different letters in the same column are significantly different at P < 0.05 in a Duncan Multiple Range Test. [CO 2 ] Leaf Temperature Fresh Wt. Dry Wt. Area Fresh Wt./Area Dry Wt./Area (µmol mol 1 ) Status ( C) (mg leaf 1 ) (mg leaf 1 ) (cm 2 leaf 1 ) (mg cm 2 ) (mg cm 2 ) 360 Old T A (11)b 191 (7)c 22.1 (1.4)a 24.0 (1.3)b 8.7 (0.4)cd T A (20)b 195 (13)c 22.7 (1.2)a 24.7 (1.2)b 8.6 (0.3)d New T A (24)b 171 (6)d 20.8 (1.4)a 25.5 (1.5)ab 8.2 (0.4)d T A (32)ab 198 (11)c 23.4 (1.6)a 25.3 (1.5)ab 8.4 (0.4)d 720 Old T A (30)b 216 (14)bc 20.1 (1.0)a 28.0 (1.2)ab 10.7 (0.5)ab T A (44)ab 212 (18)bc 22.0 (1.6)a 26.7 (1.4)ab 9.7 (0.4)bc New T A (45)ab 235 (19)ab 19.3 (1.1)a 30.3 (1.2)a 12.2 (0.6)a T A (46)a 259 (16)a 24.3 (1.4)a 28.4 (1.3)a 10.6 (0.5)ab

9 Citrus photosynthesis at elevated growth CO 2 and temperature 155 declines in SPS and ADGP activities in old leaves of Ambersweet orange at elevated [CO 2 ] also might be due to a reduction in their protein levels. Such a conclusion, however, cannot be firmly drawn without direct quantification of the protein concentrations of the enzymes. Leaf CER of Ambersweet orange was enhanced by elevated [CO 2 ] (Table 1), as was observed for a variety of other citrus cultivars (Downton et al. 1987, Idso and Kimball 1992, Brakke and Allen 1995). Ambersweet orange responded as did other reported herbaceous and woody species to elevated [CO 2 ] with a decrease in stomatal conductance. However, the magnitude of stomatal response to elevated [CO 2 ]is species-specific, and is smaller in trees than in annual crops. For mature leaves, elevated [CO 2 ] causes an average reduction in stomatal conductance of % in herbaceous plants, compared to 20 27% for woody species (Field et al. 1995, Saxe et al. 1998, Norby et al. 1999). In Ambersweet orange, stomatal conductance of fully-expanded mature (old) leaves of trees grown for more than two years at elevated [CO 2 ] was about 32 % lower than their counterparts at ambient [CO 2 ]. The reduced stomatal conductance was most likely the result of a direct CO 2 enrichment effect on stomatal aperture, resulting in a reduction in leaf transpiration and consequently an improvement in WUE (Table 1) and tissue water status (Drake et al. 1997, Jarvis et al. 1999). Under elevated growth [CO 2 ], the increase in WUE may be more important than the increase in CER, especially when soil moisture becomes the limiting factor (Chaves and Pereira 1992). In C 3 plants, leaf CER is affected by temperature, and this effect is primarily exerted through rubisco. An increase in temperature favors oxygenation by decreasing, relative to O 2, both the solubility of CO 2 and the specificity of rubisco for CO 2, and this results in greater losses of CO 2 to photorespiration as the temperature rises (Long 1991). Consequently, a doubling of atmospheric [CO 2 ], and the concomitant inhibition of the rubisco oxygenase reaction, should moderate the adverse effects of high temperature on C 3 photosynthesis and result in even greater enhancement of leaf CER by elevated [CO 2 ] as growth temperature increases (Long 1991). However, the data in this regard are equivocal (Farrar and Williams 1991). At any given temperature, the degree of leaf CER enhancement by a doubling of growth [CO 2 ] appears to be influenced by the temperature optimum for the species, the extent to which rubisco is down-regulated, and by as yet unidentified species-specific differences (Vu et al. 1997). This may explain some of the literature reports of species variation in CO 2 -enrichment response as a function of temperature (Farrar and Williams 1991). Sour orange grown at air enriched with an extra 300 µmol mol 1 [CO 2 ] has leaf CER enhanced by 75 % at a leaf temperature of 31 C, 100 % at 35 C, and 200 % at 42 C, although the summer daytime temperature range in Phoenix, Arizona is above the optimum for CER of this citrus cultivar (Idso et al. 1995). Similar scenarios, although to a lesser extent, have been reported for soybean. The enhancement effect on soybean leaf CER due to doubling the growth [CO 2 ] increases linearly from % with increasing day temperatures from C, whereas for rice it stays relatively constant at 60 % from C (Vu et al. 1997). Our Ambersweet orange study showed that the percentage enhancement in leaf CER due to doubling the growth [CO 2 ] was 33 % for new leaves and 42 % for old leaves at near-ambient growth temperature (T A C), and increased to 38 and 45 %, respectively, at T A C. Ambersweet orange also had a greater percentage enhancement in WUE under both elevated growth [CO 2 ] and temperature. Our results show a photosynthetic acclimation for both new and old leaves of Ambersweet orange to elevated [CO 2 ]. This photosynthetic acclimation was expressed through down-regulation of rubisco protein concentration and activity, and was correlated with high accumulation of starch and sucrose. There is evidence that such photosynthetic acclimation to elevated [CO 2 ] varies with leaf development, but the response is also species-specific. Some plants show an acclimation occurring in young leaves (Xu et al. 1994), while others show either no decreases in photosynthetic capacity in similar staged leaves (Nie et al. 1995), or decreases which are not observed until the leaves are more than 60 % expanded (Van Oosten and Besford 1995). The new leaves of Ambersweet orange, which contained % less chlorophyll than old leaves, were presumedly % expanded. A possibility exists that the photosynthetic acclimation of Ambersweet orange under elevated [CO 2 ] could occur at a much earlier stage during leaf ontogeny, but such information is not available. In this study, however, the so-called new leaves acclimated very well to elevated [CO 2 ], compared to old leaves, in terms of gas exchange parameters, photosynthetic capacity and sucrose synthesis. In addition, starch accumulation in new leaves during the day was much higher than in old leaves under elevated [CO 2 ] (Fig. 3). The photosynthetic acclimation of both young and mature leaves of Ambersweet orange to a future rise in atmospheric [CO 2 ] would allow an optimization of plant nitrogen use, either by reallocating the nitrogen resources away from rubisco to other catalytic or structural proteins within the leaves, or redistributing nitrogen from the photosynthetic proteins of source leaves to sink tissues (Stitt 1991, Bowes 1993). Also, the optimization of inorganic carbon acquisition and greater accumulation of the primary photosynthetic products would be beneficial for citrus vegetative growth. Thus, in the absence of other environmental stresses, citrus photosynthesis would perform well under the rising atmospheric [CO 2 ] and temperatures predicted for this century. Acknowledgements. We thank Ms. Joan Anderson for her skillful laboratory assistance. We also thank Mr. Wayne Wynn for construction and assembly of the TGG sensors and activators, and Mr. Doug Heuer for assembly of the Keithley-Metrabyte Controller/Data Acquisition System and programming the Intellution FIX DMACS software controller. Florida Agricultural Experiment Station Journal Series No. R

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