Leaf photosynthesis and Rubisco activity and kinetics ... - PubAg - USDA

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Leaf photosynthesis and Rubisco activity and kinetics of soybean, peanut and rice grown under elevated atmospheric CO 2 , supraoptimal air temperature and soil water deficit Joseph C. V. VuI2*, Leon H. Allen, Jr. 1 '2 and W. Widod02

'Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL 32608-1069, USA. 'Agronomy Department, University of Florida, Gainesville, FL 32611-0500, USA ABSTRACT Soybean (Glycine max L. Merr. cv. Bragg), peanut (Arachis hypogaea L. cv. Georgia Green) and rice (Oryza saliva L. cv. IR-72) were grown for a full season in sunlit, controlled-environment chambers at 350 (ambient) and 700 (double-ambient, elevated) imol CO2 moF' air, and under daytime maximum/nighttime minimum air temperature regimes ranging from 28/18 to 48/38°C for soybean and peanut, or soil water deficit for rice. The objectives were to characterize the interactive effects of elevated [CO2] and high air temperature, or elevated growth [CO 2] and soil water deficit, on leaf CO2 exchange rate (CER) and Rubisco activity, and to test whether elevated [CO 2], high temperature or severe drought stress would induce changes in the kinetic behavior [Km (CO 2 )] Of Rubisco. Leaf CER of soybean, peanut and rice were increased by CO 2 enrichment, but decreased by high temperature and drought. For soybean, despite the deleterious effect of high temperature on CER, CO2-enriched plants not only outperformed ambient-0O2 plants at the optimum growth temperature (32/22°C) for photosynthesis, but also *To whom correspondence should be addressed: Dr. Joseph C. V. Vu, Center for Medical, Agricultural and Veterinary Entomology, Chemistry Research Unit 1600/1700 SW 23°f Drive, Gainesville, FL 32608-1069 USA

compensated much better for the adverse effects of high temperatures on CER. In addition, the degree of enhancement induced by elevated [CO2] on soybean CER increased in a linear manner with increased growth temperature. For peanut, however, the degree of enhancement in CER by elevated [CO2] as a function of growth temperature differed from that for soybean. For rice subjected to drought stress, elevated [CO 2] delayed by one day the substantial reduction in midday leaf CER. Elevated [CO2], high temperature and drought reduced the initial (non-activated) and total (1-lCO3 7Mg2 -activated) activities as well as the activation state of midday-sampled leaf Rubisco. The Rubisco initial Km(CO 2 ) of soybean and peanut was not markedly altered by elevated [CO2] or high temperature, as neither severe drought imposed at panicle initiation changed the initial Km(CO 2 ) value of the enzyme for rice. However, moderate increases in Rubisco total K,(CO 2 ) were observed for soybean and peanut at elevated [CO2] and high temperature. The moderate increases in Rubisco total K 3(CO2) suggest that multiple generations of growth under CO2 enrichment might be required for Rubisco of the C3 species to evolve towards a more effective type enzyme. KEYWORDS: CO2 enrichment, supraoptimal growth temperature, soil water deficit, photosynthesis, Rubisco activity and kinetics

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INTRODUCTION With rapid increases in world industrial development, fossil fuel dependence and changes in land use practices, the global atmospheric CO2 concentration ([CO 2]), currently at about 380 tmol mol', is expected to double within this century [1, 2]. As a consequence, changes in global climate, including increases in air temperature, possibly as much as 4-6°C, and alterations in regional scale rainfall patterns which could result in decreased soil moisture availability in many areas of the world, may occur in the coming decades [1, 3, 4]. Atmospheric CO2 is an essential component for life on earth. Through photosynthesis, plants obtain carbon for their growth and provide sustenance for other living things, ourselves included. The ability to predict responses of the net carbon exchange and growth and yield of crop plants in response to future rising atmospheric CO2 and climate changes is critical for assessment of the potential of global warming and rainfall shortage on agricultural crop production. In plants of the C3 photosynthetic category, which comprise about 95% of terrestrial vegetation and include major agronomic crops such as soybean, rice and peanut, leaf photosynthetic CO 2 exchange rate (CER) is primarily controlled by ribulose- 1,5bisphosphate carboxylase/oxygenase (Rubisco). In addition to the carboxylation reaction that incorporates atmospheric CO 2 into plants via the photosynthetic carbon reduction cycle, Rubisco is also capable of catalyzing an energy-wasteful oxygenation reaction in which atmospheric 02, competing with CO 2 as a substrate for the enzyme, reacts with ribulose-1,5- bisphosphate to form phosphoglycerate and phosphoglycolate. The metabolism of phosphoglycolate and subsequent release of CO2, widely known as photorespiration, have an adverse effect on the photosynthetic efficiency of C3 plants [5]. The current atmospheric [CO2]:[02] ratio restricts most terrestrial vegetation to only 60-70% of its photosynthetic potential, because of kinetic constraints imposed by Rubisco [6]. Consequently, an increase in atmospheric [CO2 ] enhances leaf CER, partly because elevated [CO2] inhibits the oxygenation reaction of Rubisco and the subsequent loss of CO 2 through photorespiration. However, for a number of C3 species, long-term exposure to elevated growth

Joseph C. V. Vu etal. [CO2] is followed by metabolic changes, resulting in an acclimation of photosynthesis with downregulation of leaf photosynthetic capacity, which is manifested through reductions in Rubisco activity, activation, protein concentration and transcripts [7-12]. Plants of the C4 photosynthetic category, which include economically important crops such as maize, sorghum and sugarcane, have developed unique anatomical and biochemical features to overcome the limitations of low atmospheric [CO2] and photorespiration. They are able to concentrate CO2 within the Rubisco-containing bundle sheath cells to levels of manifold higher than atmospheric CO2 [13]. In addition, C4 plants have evolved a Rubisco exhibiting a Km(CO 2 ) and K 0, (catalytic turnover rate) with values up to twofold higher than those of C 3 Rubisco [14-16]. The high Km(CO 2 ) of Rubisco from C4 species appears to be associated with the presence of the CO2 concentrating mechanism, while the low Km(CO 2 ) in C3 plants suggests that "these species have experienced the greatest pressure to adapt their Rubisco to the absolute concentration of atmospheric CO2" [ 17]. Since the ability of C3 plants to concentrate CO2 appears to be nonexistent, the affinity for external air-0O2 during photosynthesis relies heavily on the Km(CO 2) of Rubisco. C3 plants appear likely to have evolved a Rubisco with higher affinity for CO 2 than C4 plants [14]. A low affinity for CO2 in C4 plants, however, is not necessarily a disadvantage, since the C4 Rubisco is not directly dependent on air-0O 2 due to the presence of the CO 2 concentrating mechanism within the bundle sheath cells where the enzyme is located [18]. Besides, a lower affinity for CO 2 in C4 plants may also be associated with a high Kcaj for this photosynthetic category plant group [14, 15]. For C3 plants at current atmospheric [CO2], chloroplast stroma contains about 5 tM CO2, which is less than the Km(CO 2 ) values of 12 to 25 iM as being reported for the C3 Rub isco [6, 14]. The problem of low substrate concentration, together with the inhibitory effects of 02, will therefore constrain C 3 photosynthesis. In addition, the C3 Rubisco seems to be inhibited by CO2 concentrations higher than 60 tM, whereas activity of the C4 enzyme continues at CO2 concentrations in excess of 180 iiM [14]. The C3-type Rubisco is

Photosynthesis and Rubisco at rising CO 2 and climate changes therefore inefficient, and consequently C 3 plants must allocate as much as 50% of their leaf nitrogen to the Rubisco protein [191. Under present atmospheric CO 2 and 02 concentrations, photosynthesis of C 3 plants is below its maximum capacity even under lightsaturated conditions [18], and C 3 Rubisco may operate at only about 25% of its full potential capability [20]. The C4 plants, although less protein is invested in their Rubisco, are still able to achieve the CO 2-saturated photosynthetic rates of C3 plants. Thus, the C 4 Rubisco, being exposed to a much higher than ambient CO 2 concentration which provides a steady-state concentration of at least 70 iiM in the bundle-sheath cells and offsets the competitive effects of 02, appears to be a more effective catalyst for the photosynthetic pathway O f C4 plants [6, 14, 16]. As atmospheric [CO 2] is expected to double within this century, such [CO2] elevation may induce alterations in the C 3 Rubisco kinetic characteristics and could make the enzyme more efficient for C3 plants under elevated growth [CO2] conditions. Less investment into the Rubisco protein and a reallocation of nitrogen resources away from the enzyme have been reported for a number of C 3 plants exposed to long-term CO 2 enrichment [6]. In addition to atmospheric [CO2], photosynthesis Of C3 plants is influenced by light, air temperature and soil water availability, and Rubisco plays a central role in these responses. An increase in air temperature reduces the activation state of Rubisco carboxylation reaction [7, 21, 22], as well as decreases, relative to 0 2 , its solubility of CO2 and its specificity for CO 2 [23, 24]. The latter two effects favor oxygenation and result in greater losses of CO 2 to photorespiration as the ambient growth temperature increases. Consequently, a rise in atmospheric [CO2] and the concomitant inhibition of the Rubisco oxygenase reaction could partially offset the adverse effects of high air temperature on C3 photosynthesis. With regard to soil moisture availability, a decrease in CER due to drought has been also associated with reduced leaf photosynthetic capacity. However, a delay in the adverse effects of soil water deficit on leaf photosynthesis by elevated growth [CO2] has been reported for a number of C 3 plants, including rice, soybean and peanut [25].

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In this study, soybean, peanut and rice were grown for a full season at ambient and double-ambient [CO2] and under different temperature regimes and/or episodic soil water deficit treatments. The objectives are to characterize the interactive effects of elevated [CO2] and high air temperature, or elevated [CO2] and soil water deficit, on leaf CER and Rubisco activity. We also want to test whether season-long growth of soybean, peanut and rice at elevated [CO2], and at elevated [CO2] interacting with high air temperature or severe soil water deficit, could induce change in the kinetic behavior [Km(CO2)} of Rubisco. MATERIALS AND METHODS Plant material and growth conditions Soybean, peanut and rice were grown from seed sowing to plant maturity in eight sunlit, controlled-environment growth chambers (also known as Soil-Plant-Atmosphere-Research, or SPAR, units), located outdoors at the Plant and Soil Science Field Teaching Laboratory of the University of Florida in Gainesville (29038'N, 82 0 22'W), FL, USA, as previously reported [7, 26-30]. The aboveground chambers, 2 m x I m in cross section and 1 .5 m high, were constructed of aluminum frame and covered with transparent polyethylene telephtalate "Sixlight" film (Taiyo Kogyo Co., Tokyo, Japan) so that plants received direct, natural sunlight. These chambers transmitted an average of 90% of the solar photosynthetic photon flux density (PPFD). Soybean was grown over two consecutive years, from August to October of the first year and February to May of the second year. Peanut and rice were grown during the months of June to October of the years thereafter. Plants were grown throughout their life cycle at two daytime [CO 2] of 350 (ambient) and 700 (double-ambient, elevated) l.tmol mol' air. Nighttime [CO2] in each chamber was controlled to near ambient through a venting procedure [26, 271. Shade cloths made of black, densely woven polypropylene fibers were maintained around the canopy at canopy height to provide a light environment similar to that created by border rows in a field crop. For soybean, seeds were first inoculated with Rhizobium and then planted in 32-cm rows. Plants

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were periodically thinned, resulting in a total of 40 plants m 2 at mid-growth season. Daytime maximum/nighttime minimum air temperatures in the first year of the soybean experiment were controlled at 28/18, 32/22, 36/26 and 40/30°C for both ambient and elevated [CO,] treatments. In the second year of the soybean experiment, daytime maximum/nighttime minimum air temperatures were controlled at 28/18 and 40/30°C for both ambient and elevated [CO 2] treatments, and at 32/22, 36/26, 44/34 and 48/38°C for the elevated [CO2] treatment. Likewise, daytime maximum/ nighttime minimum dew point temperatures were maintained at 12/10, 16/12, 20/14, 24/16, 28/18 and 32/20°C that corresponded to the six air temperature patterns. For peanut, seeds were inoculated prior to sowing with Bradyrhizobium and planted in 24-cm rows, and seedlings were thinned to a population of 20 plants m 2 . Daytime maximum/nighttime minimum air temperatures were controlled at 32/22, 36/26, 40/30 and 44/34°C for both ambient and elevated [CO 2] peanut treatments, and corresponding daytime maximum/ nighttime minimum dew point temperatures were maintained at 27/17, 31/21, 35/25 and 39/29°C. Since only eight SPAR units were available, replication could not be made for each [CO2}temperature treatment for the soybean and peanut experiments. However, in the second year of the soybean study, growth temperature regimes of 28/18 and 40/30°C were repeated for both ambient and elevated [CO 2] treatments, and those of 32/22 and 36/26°C were repeated for the elevated [CO2] treatment. For rice, each chamber top was attached to an aluminum vat (2 m x 1 m in area and 0.6 m deep), which provided a water-tight, flooded root environment for growing rice in paddy culture. The vats were filled with a Kendrick fine sand soil to a depth of 0.5 m, and seeds were planted into 18-cm apart rows. The chamber air temperatures were controlled at 28/21°C (day/night) for both ambient and elevated [CO2] treatments, and dew point temperatures were controlled at 18/12°C (day/night). Seedlings were thinned to 200 plants M-2 five days after planting (DAP), and the vats were then flooded with water that was maintained at 5 cm above the soil surface. Plants were thinned periodically during the growth season, resulting in

Joseph C. V. Vu et al. a plant population of 170 plants m 2 at 58 DAP (panicle initiation growth stage). Drought stress was imposed at 59 DAP for both ambient and elevated [CO2] plants by shutting off the paddy water supply and draining the soil by opening ports at the base of the vats of the designated drought-treatment chambers, while the control chambers were maintained continuously flooded. Drought was terminated and water was added back in the afternoons of 76 DAP to the ambient-0O 2 -stredplan7DAPtohelvad-0O2 stressed plants, as their midday canopy net photosynthetic rates sharply declined to near zero, respectively. Each [CO2]-water treatment combination was duplicated for the rice experiment. Air temperatures and dew point temperatures of the growth chambers in the soybean and peanut experiments followed a modified sinusoidal control set point that varied continuously between maximum (at 1500 eastern standard time or EST) and minimum (at 0700 EST) values. The detailed chamber characteristics, specific methods for chamber environmental controls for temperature and [CO 2 ], and the quality of these controls have been described [26, 27, 31, 321. Leaf photosynthesis measurements Photosynthetic CER of single, attached, uppermost fully expanded leaves or leaflets was measured at midday on clear sunny days, between 1100 and 1400 EST, when solar PPFD was 14002000 tmol mol' m 2, using the LI-6200 Portable Photosynthesis System (LI-COR, Inc., Lincoln, NE) and leaf chambers of 4 dm' for soybean and 0.25 dm 3 for peanut and rice, as previously reported [7, 29]. Leaf CER was determined at the [CO2 ] and temperature used for growth. Leaf sampling for Rubisco analysis Samplings of single uppermost fully expanded leaves for each [CO2] and temperature or drought treatment were performed at clear midday conditions, between 1130 EST and 1400 EST, with solar PPFD of 1500 - 2000 jimol m 2 s'. For soybean, 48 to 53 DAP, 10 leaflets were sampled from 10 different plants for each [CO 2] treatment. For peanut, 36 leaflets were sampled from 9 plants of the two [CO2] treatments at 56 DAP. For rice, 15 leaves were sampled from 15 plants for the

Photosynthesis and Rubisco at rising CO 2 and climate changes control and drought treatments of the ambient [CO2] treatment at 76 DAP, and of the elevated [CO2] treatment at 77 DAP. At each sampling time, leaves were quickly detached from plants and immediately immersed in liquid N 2 . They were then pooled by treatment, ground to a fine powder in liquid N 2 with a mortar and pestle, and stored in liquid N 2 until analysis. Extraction and assay for Rubisco The extraction and assay for Rubisco initial (nonactivated) and total (HCO37Mg2-activated) carboxylation activities were performed in a manner similar to those described previously [33, 34]. A portion of the frozen leaf powder, about 0.3 g, was transferred to a pre-chilled Pyrex Ten Broeck tissue grinder (Fisher Scientific) and was ground at 2°C in 4 mL of an extraction medium, which consisted of 50mM Tricine buffer (pH 8.0) (prepared CO2-free and flushed with N 2) containing 10 mM MgCl2, 0.1 mM EDTA, 5 mM DTT, 10 mM isoascorbate and 2% (w/v) PVP40. The homogenate was microcentriguged at 12000 g for 45 s at 2°C, and the supernatant was immediately used for assay. Measurements for Rubisco initial and total activity, by determining the incorporation of '4CO2 into acid-stable products, were performed at 30°C in a total volume of 0.5 mL. The reaction mixture (0.4 mL) consisted of 50 MM CO2-free Tricine buffer (pH 8.0), 10 MM MgCl 2, 0.1 mM EDTA, 5 mM DTT, 0.5 mM RuBP, and NaH' 4CO3 with concentrations varying from 0 to 10 mM. The concentration of HCO at 10 mM is ratesaturating, but not inhibitory, for Rubisco of soybean, peanut and rice (data not shown). For assay of initial Rubisco activity, the reaction was started by injecting 0.1 mL of the supernatant into the assay mixture, and was terminated after 45 s with 0.1 mL of 6N HCI. For determination of total Rubisco activity, an aliquot of the supernatant was incubated with 10 mM NaHCO 3 for 5 min at room temperature prior to assay to activate the enzyme. The reaction was then started by injecting 0.1 mL of the activated enzyme into the reaction mixture and was terminated after 45 s at 30°C with 0. I mL of 6N HCI. The mixtures were then dried at 60°C and the acid-stable radioactivity was determined by liquid scintillation spectrometry. Rubisco initial

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and total activities were presented as LineweaverBurk or Woolf plots as a function of HCO3 concentrations. These plots were used for determinations of the Vma and K,(CO2) values for carboxylase activity. The Vm , which is obtained through extrapolation, would ideally reflect the true potential activity of Rubisco [16]. The CO2 concentration was calculated from the pH and HCO3 concentration using the HandersonHasselbach equation and pK value of 6.348 at 30°C as previously reported [34]. The activation state of Rubisco, or percent activation, was determined as the ratio of initial V,, to total RESULTS Table I shows midday leaf CO 2 exchange rate (CER) of soybean grown for a full season (from seed sowing to plant maturity) in two consecutive years under ambient and double-ambient [CO2] and at six temperature regimes. In terms of photosynthesis responses to increasing growth Table I. CO2 exchange rates (CER) of single attached, uppermost fully expanded leaflets of soybean plants grown for two seasons at 350 and 700 imol mor' CO 2 and under six daytime maximum/nighttime minimum temperature regimes. Measurements were made at midday (1500 - 2000 jimol m 2 s' PPFD), 50 to 60 days after planting. Values are the mean for two growth seasons + SE of 4 to 10 determinations. NA, treatment not available Temperature 1CO21 Leaf CER (°C) (imoI mor) (imol m2 s) 28/18 350 25.5±4.4 700 34.5 ± 1.3 32/22 350 27.5 ± 1.5 700 41.7±6.8 36/26 350 23.5 ± 1.0 700 40.3 ± 3.8 40/30 350 18.2± 1.8 700 35.8 + 5.4 44/34 350 NA 700 30.3±3.1 48/38 350 NA 700 24.3 ± 5.3

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Joseph C. V. Vu et al.

temperature, leaf CER of both ambient and elevated [CO2] soybean plants showed an optimum at 32/22°C. However, soybean leaf CER was substantially enhanced by elevated [CO 2] at all growth temperature regimes used in this study, while higher temperatures were more deleterious to CER of the ambient [CO2] plants. Leaf CER at 36/26 and 40/30°C was 85 and 66% of the optimum rate at 32/22T for the ambient [CO2] plants, compared to 97 and 86%, respectively, for the elevated [CO 2] plants. In addition, even at the extremely high temperature growth regime at 48/38°C, leaf CER of the elevated CO2 plants still functioned at about 58% of the optimum rate at 32/22°C, although few plants survived this treatment and biomass per plant was low [35]. With increasing growth temperature, the degree of enhancement by elevated [CO 2 ] rose from 35% at 28/18°C to 97% at 40/30°C..

The effects of double-ambient growth [CO 2] and high air temperature on leaf CER for peanut in this study have been presented in a previous report [29], showing that elevated [CO 2] enhanced peanut leaf CER by 27%, but there was no significant impact of high temperature or interaction between temperature and [CO2] on peanut leaf photosynthetic rate. Table 2 shows midday leaf CER for the control (soil-flooded) and drought-stressed rice plants at ambient and double-ambient growth [CO 2]. Under well-watered (soil-flooded) conditions, rice plants grown at elevated [CO 2] had higher midday leaf CER than those at ambient [CO 2]. At 17 days after withdrawing the water (76 DAP), midday leaf CER of the ambient-0O2 drought-imposed plants was near zero, whereas unwatered plants at elevated growth [CO,] were showing only moderate drought stress, with leaf CER still at 43% of the

Table 2. Midday CO 2 exchange rates (CER) for single, attached, fully expanded leaves of control (soil-flooded) and drought-imposed rice plants grown at 350 and 700 imoI mol' CO2. Photosynthetic measurements were made between 1100 and 1400 EST (1200 - 1600 imol m 2 s ' PPFD). A drought cycle was initiated at 59 days after planting (DAP) by drainage of flooded soil. Flood water was restored after leaf sampling at 76 DAP for the 350 imol mol' CO2 drought treatment and 77 DAP for the 700 iimol moE' CO2 drought treatment. Values are the mean ± SE of 5 to 8 determinations DAP [CO' (tmol mor') 74 350 700 76 350 700 77 350 700 84 350 700

Water Regime

Leaf CER (tmol m 2 s')

Control Drought Control Drought

25.1 ± 2.3 21.8+ 1.9 35.6 ± 2.4 27.1 ± 1.1


23.2± 1.2 2.4± 1.1 30.7± 1.8 12.6 + 3.4


20.8± 1.5 13.0 ± 2.1 28.0+ 1.8 3.3 ± 2.2


23.1 ± 1.7 17.6± 1.7 32.4± 1.5 30.1 ± 1.9

Photosynthesis and Rubisco at rising CO 2 and climate changes soil-flooded controls. For the drought-imposed elevated-0O 2 plants, it was not until the following day (77 DAP) that midday leaf CER declined to near zero. As water was added back to the ambient [CO 2 ]-stressed plants at 76 DAP and to the elevated [CO 2 ]-stressed plants at 77 DAP, midday leaf CER determined at 84 DAP for the ambient [CO 2 ]-stressed plants was still only about 75% that of the ambient [CO 2 ]-control plants, while midday leaf CER of the elevated [CO 2 ]-stressed plants was nearly that of the elevated [CO 2 ]-control plants. The initial and total extractable activities of Rubisco, determined from leaves of plants grown at ambient and elevated [CO 2 ] and under various air temperature regimes or soil water treatments and expressed on a leaf fresh weight basis, are shown in Figs. I & 2 for soybean, 3 & 4 for peanut, and 5 & 6 for rice. These figures were used in conjunction with the presentation and discussion of the and Km(CO 2 ) parameters for Rubisco shown in Table 3 for each specific crop

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plant and treatment. Such parameters were calculated from the plots for initial and total activity of the enzyme as shown in Figs. 1-6, and were designated as (I)Vmax, (T)Vmrj, (I)Krn(CO2) and (T)K,(CO 2 ) for Rubisco initial (1) and total

(T) activity assays for each specific treatment. For soybean, both long-term [CO 2 ] enrichment and high temperature reduced the initial and total extractable activities of Rubisco, and such reductions were higher for the initial activities (Figs. 1, 2 & Table 3). At 32/22°C growth temperature, (1)V, and (T)Vmax of plants at elevated [CO 2 ] were 77 and 82%, respectively, of the corresponding values for their counterparts at ambient [CO 2 ]. The (I)V,, and (T)Vm for elevated CO 2 plants grown at the extreme temperature of 48/38°C were 58 and 82%, respectively, of those for elevated CO 2 plants grown at 32/22°C, and were only 44 and 67% of the values for ambient CO 2 plants grown at 32/22°C.

125

100

E) 350 pmol mol 1 CO2 (32/22 C) o 700 pmol mor1 CO2 (32122 °C) • 700 pmol mott CO2 (48/38 C)

•

L >0 o: 0.

75

50 z

25

4 (HCO 3 ]

(mM)1

Figure 1. Lineweaver-Burk plots of initial Rubisco activity as a function of [HCO 3] in leaf extracts from soybean grown at 32/22°C under ambient (o; Y = 6.555X + 5.203, r = 0.995) and double-ambient (0; Y = I0.255X + 6.787, r = 0.996) [CO2], and at 48/38°C under double-ambient [CO2] (.;Y = 17.208X + 11.724, r = 0.998).

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Joseph C. V. Vu el al.

100

4.:-. 0g

2 2 700 pmol mar' CO 2 (48)38 'C)

LI

350 pmol mar' CO (32)22 'C)

o

700 pmol mor' CO (32)22 'C)

•

75

E

0• to

E

50

0 I25

.1 0 1 2 3 4 5 IHCO3

1

(mM)1

Figure 2. Lineweaver-Burk plots of total (l-1CO 3 iMg2 -activated) Rubisco activity as a function of [HCO3 ] in leaf extracts from soybean grown at 32/22°C under ambient (o; Y = 9.713X + 3.809, r = 0.999) and doubleambient (a; Y = 15.530X + 4.660. r = 0.997) [CO2 1, and at 48/38°C under double ambient (.; Y = 21.559X + 5.652, r = 0.998) [CO2]. Table 3. Vma and Km(CO 2) of Rubisco of soybean, peanut and rice grown at 350 (ambient) and 700 (double-ambient) iimol mor' CO 2 and under various daytime maximum/nighttime minimum temperature or soil water treatment regimes Plant Treatment Initial Activit y Total Activity Activation (%) Km(CO2) Vmax Km(COi) (AM) (tmol g fwt' h') (piM) (pimol g fwt' h') Soybean

Peanut

Rice

350-32/22 700-32/22 700-48/38

1,922 8.9 2,625 18.1 1,473 10.7 2,146 23.6 853 10.4 1,769 26.9

73.2 68.6 48.2

350-32/22 350-40/30 700-32/22 700-40/30

805 6.8 882 22.2 685 8.5 815 24.4 619 7.4 705 24.8 395 6.8 590 26.9

91.3 84.0 87.8 66.9

350 - Control 350 - Drought 700 - Control 700 - Drought

1,005 7.5 1,060 13.5 260 8.2 418 16.4 732 7.6 829 14.3 345 7.4 588 12.9

94.8 62.2 88.3 58.7

Photosynthesis and Rubisco at rising CO 2 and climate changes

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Soybean Rubisco (1)K,(CO2) was not markedly altered by elevated [CO 2] or extreme growth temperature, and averaged 10.0 ± 1.0 iiM across the CO2-temperature treatments (Fig. 1 & Table 3). At elevated [CO,] and high temperature, the (T)K,(CO2) increased, although moderately, i.e., from 18.1 tM for ambient [CO2] plants at 32/22°C to 23.6 and 26.9 piM for elevated CO2 plants at 32/22 and 48/38°C, respectively. Rubisco activation, the ratio of (1)V,0 to (T)Vm U, declined however with increasing growth [CO 2] and temperature (Table 3). Compared with ambient-0O 2 plants grown at 32/22°C, there was 6 and 34 % less in Rubisco activation state for the elevated [CO2] plants grown at 32/22 and 48/38°C, respectively.

double-ambient [CO2} plants at 40/30°C was also IS and 51% less in and 8 and 33% less in (T)Vrna, respectively, as compared to ambient [CO2] plants at 32/22°C. In addition, Rubisco activation state declined for peanut plants grown at elevated [CO2] and high temperature (Table 3). When compared with the ambient-0O 2 plants at 32/22°C, Rubisco activation state for plants at 40/30°C was 92% under ambient growth CO 2 and 73% under elevated growth CO2. The (1)K,(CO2) averaged 7.4 ± 0.8 1iM across the CO2-temperature treatments, whereas the (T)K,(CO2) of plants at elevated growth [CO 2] and high temperature was 10-20% higher than that of ambient CO2 plants at 32/22°C.

For peanut, responses of Rubisco to elevated [CO2] and high air temperature were somewhat similar to those for soybean (Figs. 3, 4 & Table 3). At 32/22°C, Rubisco of plants grown at double-ambient [CO 2] was 23 and 20% less in (1) V,,,,, and (T) V..., respectively, than plants grown at ambient [CO 2]. Rubisco of ambient and

In rice, both elevated growth [CO 2] and drought stress reduced the activities of Rubisco (Figs. 5, 6 & Table 3). In terms of CO2 enrichment effect, (I)V.,,, and (T)Vma, of the ambient CO2 control plants were 37 and 28% higher than those of the elevated CO2 control plants. For the ambient-0O2 stressed plants, at 76 DAP when midday leaf CER

D 350 pmol mor' CO2 (32122 C( • 350 pmol MOO CO, (40130 C) o 700 pmoi mor' CO2 (32122 C( • 700 pmol mol' CO2 (40/30 C) 100 4-

oZ U. 0,

_j O 4E Fe 50

-1 IHCO

31

(MM)1

Figure 3. Lineweaver-Burk plots of initial Rubisco activity as a function of [HCO3]

in leaf extracts from peanut grown at 32/22°C under ambient (o; Y = 1. 199X + 1.242, r = 0.998) and double-ambient (0; Y = 1.699X + 1.616. r 0.999) [CO 2], and at 40/30°C under ambient (.; Y = 1.757X + 1.460, r = 0.994) and double-ambient (.; Y = 2.439X + 2.529. r = 0.999) [CO,].

Joseph C. V. Vu

36

o

2 (32/22 C)

350 pmol mo4' CO

• 350 noI mol 1 CO2 (40/30 C) o 7OOijmoImor'C01(32122C) • 700 pI mor' CO2 (40/30 ) e00t 0 . I

U . I

I m" t

El

rioot

-1

0

1 p4CO31 (mM)

2

3

4

Figure 4. Lineweaver-Burk plots of total (HCO 3 iMg2 -activated) Rubisco activity as a function of (HCO 3 ] in leaf extracts of peanut grown at 3 2/22°C under ambient (o; Y = 3.554X + 1.133, r = 0.992) and double-ambient (0; Y = 4.958X + 1.418) [CO2], and at 40/30°C under ambient (.; Y = 4.235X + 1.227, r = 0.999) and double-ambient (.; Y = 6.450X + 1.696, r = 0.999) [CO2]. 300,

0 350 Jmo mof' CO2 Control • 350 unit mof' CO2 Drought 2 o oo h id mof' COControl • 700 pmol mor' CO2 Drought > 'o 200

r

4

too4. .1 o 4E I

.2 .1 0 1 2 3 4 5 IHCO31 (-M)1

Figure 5. Lineweaver-Burk plots of initial Rubisco activity as a function of[HCO31 in leaf extracts from rice for the control (soil-flooded) (o; Y = 10.587X + 9.952, r = 0.980) and drought-stressed (.; Y = 44.744X + 38.436, r = 0.983) plants of the ambient [CO 2 1 treatment at 76 days after planting (DAP), and for the control (soil-flooded) (0; Y = 14.739X + 13.664, r = 0.981) and drought-stressed (.; Y = 27.688X + 29.008, r = 0.998) plants of the double-ambient [CO2] treatment at 77 DAP.

el al.

Photosynthesis and Rubisco at rising CO 2 and climate changes

37

30

o 25

350 pmol m011 CO2 Control

• 350 pnlol mot' CO 2 Drought

o 700 pmot mor' CO 2 Control • 700 pmol m011 CO2 Drought

0 U

20

I---

15

U

10

4

-2

10

[HCO31 (mM)

1Mg2+-activated) Rubisco activity as a Figure 6. Woolf plots of total (1-1CO3-

function of [HCO 31 in leaf extracts from rice for the control (soil-flooded) (o; Y = 0.944X + 1.797, r = 0.999) and drought-stressed (.; Y = 2.391X + 5.545, r = 0.997) plants of the ambient [CO2] treatment at 76 days after planting (DAP), and for the control (soil-flooded) (0: Y = 1.206X + 2.441. r = 0.999) and droughtstressed (.; Y = 1.699X + 3.089, r = 0.997) plants of the double-ambient [CO2] treatment at 77 DAP. was 2.4 iimol m 2 S 1 , ( I) Vmar and (T)V, of Rubisco were only 26 and 39%, respectively, of those of the ambient-CO 2 control plants. In contrast, for the elevated-0O2 stressed plants, the (I)V,. and (T)V,,,, at 77 DAP, when midday leaf CER was 3.3 .imol m 2 s, were still 47 and 71% of those of the elevated-0O2 control plants. Across all treatments, the (l)K,(CO 2) and (T)Km(CO2) for rice Rubisco averaged 7.7 + 0.4 and 14.3 ± 1.5 jiM, respectively. DISCUSSION

A comparison of soybean leaf CER at the two high temperature regimes of 36/26 and 40/30°C to that at the optimum photosynthesis performance

temperature of 32/22°C showed that the reductions in net photosynthetic rates by high temperatures were IS and 34% for the ambient CO 2 plants, compared to only 3 and 14% for the elevated CO2 plants, respectively. Even at 44/34°C such reduction in leaf CER was still only 27% for the elevated CO2 plants. Despite the deleterious effect of high-temperature on photosynthesis, the CO 2enriched soybean plants not only outperformed their ambient CO2-grown counterparts at the optimum growth temperature for photosynthesis, but also compensated much better for the adverse effects of high temperatures on net photosynthetic rate. In addition, the degree of enhancement induced by elevated [CO2] on soybean leaf CER

38

increased in a progressive manner with increased growth temperature. The percent enhancement in soybean leaf CER due to a doubling in growth [CO2] rose almost linearly from 35% at 28/18°C to 97% at 40/30°C. This was similar, although to a lesser extent, to the predicted response of an idealized C3 plant under an elevation of growth [CO2] from 350 to 650 tmol moE' in conjunction with a rise in air temperature from 28 to 40°C [24]. The degree of enhancement in peanut leaf photosynthetic rate by double-ambient CO 2 as a function of growth temperature somewhat differed from that of soybean. From the leaf CER data previously reported for peanut of this study [29], the percent enhancement on photosynthetic rate by elevated CO2 can be calculated for the range of the growth temperature regimes used. A doubling of the ambient [CO2] resulted in 29, 37, 24 and 18% enhancement of peanut leaf CER at growth temperatures of 32/22, 36/26, 40/30 and 44/34°C, respectively. Similar enhancement was also observed in a second study with peanut grown in temperature-gradient greenhouses under ambient and double-ambient [CO 2] and at daytime temperatures of 1.5 and 6.0°C above ambient temperature, in which the percent enhancement in peanut leaf CER by double-ambient [CO2] was 35 and 17% at 1.5 and 6.0°C above ambient temperature, respectively [36]. Thus, for peanut, the compensation effect of long-term CO2 enrichment on the enhancement of leaf photosynthetic rate at high temperatures appeared not to rise linearly over a range of supraoptimal growth temperatures as being observed for soybean, but declined once the optimum growth temperature was reached. This would indicate species-specific differences in the responses of leaf photosynthesis among C3 plants as a result of rising atmospheric [CO 2] and air temperature. For rice subjected to drought stress, elevated [CO2] delayed by one day the substantial reduction in midday leaf CER. The ability of the CO 2 -enrichdplatsominhgCERuder severe soil water deficit conditions is primarily due to the reduction in leaf stomata[ conductance [27, 28], which is also commonly observed in

Joseph C. V. Vu et al. other drought-imposed plant species at elevated growth [CO 2 ] [37]. Consequently, elevated growth [CO2], through the reductions in leaf conductance and evapotranspiration and an enhancement in leaf CER, would improve leaf water-use efficiency, thus making the CO2-enriched rice plants capable to partially compensate for and delay the adverse effects of drought stress [27, 28]. There is indication that atmospheric CO 2 enrichment may actually prevent plants from succumbing to the rigors of environmental stresses and enable them to maintain essential growth processes [38]. Prolongations of the photosynthesis period during severe soil water deficit by elevated growth [CO2] have been also reported for other crop plant species [25, 37]. There is abundant evidence that Rubisco can be modulated by growth at elevated [CO2 ]. However, claims of down-regulation of the enzyme activity need careful evaluation, as the basis on which Rubisco activity is expressed may alter or nullify the observation [6, 7]. In this study, elevated growth [CO2] reduced the extractable activities of Rubisco, expressed on a leaf fresh weight basis, in soybean, peanut and rice. Also, it should be noted that the Rubisco activities reported here (Figs. 1-6 and Table 3) were the Vma. values, which would reflect more truly and ideally the potential activities of the enzyme than the activities as normally obtained at rate-saturating HCO3 concentrations [16]. In addition to a down-regulation of Rubisco activity by long-term [CO2] enrichment, there was also a concomitant reduction in activity of the enzyme for soybean and peanut at high growth temperatures, and for rice under severe depletion in soil water availability. Such reductions were more prominent for the Rubisco initial than total activity. For rice, although both elevated [CO2] and drought reduced the activity of Rubisco, the reduction, however, was less for the elevated-0O2 stressed plants than for the ambient-0O 2 stressed plants. The Km(CO 2) values of HCO31Mg2-activated Rubisco for soybean, peanut and rice of this study were in the range of those reported for other C3 plant species [14, 15]. Preincubation of the enzyme extract with Mg2 and HCO3 before assay for its

Photosynthesis and Rubisco at rising CO 2 and climate changes total activity raised the Km(CO 2) values by up to threefold, and the values by up to twofold (Table 3). As Rubisco released from intact chloroplasts during extraction is only a partially activated enzyme, it will be further activated in the presence of Mg2 and HCO3 in the incubation medium. In higher plants, the eight active sites of the Rubisco holoenzyme appear to function independently of one another, and the activation state of Rubisco represents the portion of the sites that is catalytically competent [19]. The-activation state of Rubisco has been shown to change in response to variations in light, CO 2, air temperature and soil water status conditions during growth of test plants [8, 21, 33, 39-411. For a variety of crop plant species, there is reduction of the Rubisco activation state under elevated [CO 2], high temperature and soil water deficit growth conditions [8, 21 2 42]. For soybean, peanut and rice of this study, Rubisco activation state was reduced by 7% by elevated growth [CO2 ], but 8-30% by high temperatures and 34% by severe drought. There were no clear-cut alterations in Km(CO2) for the initial Rubisco of soybean, peanut and rice, and for the Mg +2 /HCO3 - -activated (total) Rubisco of peanut and rice, grown from seed sowing to plant maturity under double-ambient [CO2 ]. For the activated enzyme of soybean, there was about 30%. increase in Km(CO 2) at double-ambient growth [CO2]. In rice, the K,.,,, of activated Rubisco is unaffected by either [CO 2] or temperature used for growth, whereas in peanut and soybean the K0, of activated Rubisco increased by 5-10% at double-ambient [CO2 ] and 10-20% at high growth temperatures [7, 8, 361. The either moderate increases or hardly noticed changes in K,(CO2) and Ka, for soybean, peanut and rice, grown only for one season at double-ambient [CO 2], do not necessarily imply that the C 3-type Rubisco will stay unaltered as atmospheric [CO 2] continues to increase in the future. As it took over thousands to millions of years for the evolutionary rise of the C4 photosynthesis syndrome to develop as well as for the diversification of C4 photosynthesis to progress, multiple successive generations of growth under elevated CO2 might be required for the C 3 Rubisco to evolve towards a more effective type enzyme.

39

The plant Arabidopsis, with its many available mutants and favorable features of short growth cycle and small size, could be additionally used, in parallel with other C3 crops, as a valuable model experimental plant for addressing this still unknown question in CO2 enrichment research. Substantial reductions in Rubisco protein concentration and levels of both Rubisco large and small subunit transcripts, in addition to modifications in a variety of physiological, biochemical and ultrastructural parameters, have been reported for Arabidopsis grown at elevated [CO2] [12, 43]. ACKNOWLEDGEMENTS This work was partially supported by the Florida Agricultural Experiment Station and Agronomy Department with the University of Florida in Gainesville, Florida. We thank Ms. Joan Anderson for her skillful laboratory assistance, and Mr. Wayne Wynn for his engineering support. REFERENCES Schneider, S. H. 2001, Nature, 411, 17. Hoffert, M. 1., Caldeira, K., Benford, G., Criswell, D. R., Green, C., Herzog, H., Jam, A. K., Kheshgi, H. S., Lackner, K. S., Lewis, J. S., Lightfoot, H. D., Manheimer, W., Mankins, J. C., Mauel, M. E., Perkins, L. J., Schlesinger, M. E., Volk, T., and Wigley, T. M. L. 2002, Science, 298, 981. 3.

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