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Reduced photorespiration and increased energy-use efficiency in young CO2-enriched sorghum leaves Blackwell Oxford, New NPH Trustees 0028-646X no 150 Original XXXXXXX 112 2001 Graphicraft 000 issue Phytologist UK no. of Article Science New Limited, Phytologist LtdHong Kong 2001
Asaph B. Cousins1, Neal R. Adam1,2, Gerard W. Wall2, Bruce A. Kimball2, Paul J. Pinter Jr2, Steven W. Leavitt3, Robert L. LaMorte2, Allan D. Matthias4, Michael J. Ottman5, Thomas L. Thompson4 and Andrew N. Webber1 1Department of Plant Biology and Center for the Study of Early Events in Photosynthesis, Arizona Sate University, PO Box 871601, Tempe, AZ, 85287–1601,
USA; 2USDA, Agricultural Research Service, US Water Conservation Laboratory, Phoenix, AZ, USA; 3Laboratory of Tree Ring Research, University of Arizona, Tucson, AZ 85721, USA; 4Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721, USA; 5Department of Plant Science, University of Arizona, Tucson, AZ 85721, USA
Summary Author for correspondence: Andrew N. Webber Tel: +1 480 965 8725 Fax: +1 480 965 6899 Email:
[email protected] Received: 8 August 2000 Accepted: 4 December 2000
• To determine the response of C4 plants to elevated CO2 it is necessary to establish whether young leaves have a fully developed C4 photosynthetic apparatus, and whether photosynthesis in these leaves is responsive to elevated CO2. • The effect of free-air CO2 enrichment (FACE) on the photosynthetic development of the C4 crop Sorghum bicolor was monitored. Simultaneous measurements of chlorophyll a fluorescence and carbon assimilation were made to determine energy utilization, quantum yields of carbon fixation (φCO2) and photosystem II (φPSII), as well as photorespiration. • Assimilation in the second leaf of FACE plants was 37% higher than in control plants and lower apparent rates of photorespiration at growth CO2 concentrations were exhibited. In these leaves, φPSII : φCO2 was high at low atmospheric CO2 concentration (Ca) due to overcycling of the C4 pump and increased leakiness. As Ca increased, φPSII : φCO2 decreased as a greater proportion of energy derived from linear electron transfer was used by the C3 cycle. • The stimulation of C4 photosynthesis at elevated Ca in young leaves was partially due to suppressed photorespiration. Additionally, elevated Ca enhanced energy-use efficiency in young leaves, possibly by decreasing CO2 leakage from bundle sheath cells, and by decreasing overcycling of the C4 pump. Key words: sorghum (Sorghum bicolor), C4 photosynthesis, free-air CO2 enrichment (FACE), elevated CO2, photorespiration, energy efficiency.
Abbreviations A, measured rate of CO2 assimilation; A*, net CO2 fixation (A + RD); ANOVA, analysis of variance; Ca, atmospheric CO2 concentration; Cibs, CO2 concentration within the bundle sheath cell; DAP, days after planting; FACE, free-air CO2 enrichment; FITC, Fluorescein Isothiocyanate; Fm, maximal fluorescence (light); Fs, steady state fluorescence; PEPC, PEP, phosphoenol pyruvate carboxylase; PSII, photosystem II; rc, the resistance to CO2 diffusion from bundle sheath cells to the mesophyll cells intercellular space; rt total resistance to diffusion of CO2 from air to the chloroplast of bundle sheath cells; RD, dark respiration; Rubisco, Ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, Ribulose-1,5-bisphosphate; φCO2, quantum yield of CO2 fixation; φPSII, quantum yield of PSII. © New Phytologist (2001) 150: 275–284
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Introduction The onset of the industrial revolution and the exponential growth of the human population have caused changes to our environment at unprecedented rates. Continued anthropogenic activities are predicted to double (c. 700 µl l–1) the Earth’s atmospheric carbon dioxide concentration (Ca) by the end of this century (McElroy, 1994; Intergovernmental Panel on Climate Change, 1996). Rising Ca is likely to have a significant impact on plant development, metabolic regulation and net photosynthetic productivity ( Bowes, 1991; Stitt, 1991; Webber et al., 1994; Van Oosten & Besford, 1996). Increasing Ca has its most significant response on photosynthesis of terrestrial plants, in particular the assimilation of carbon by Ribulose bisphosphate carboxylase/oxygenase (Rubisco) (Wong, 1979; Sage et al., 1989). At current Ca, the carboxylation reaction in C3 plants is partially inhibited by the oxygenation of ribulose bisphosphate ( RuBP), which reduces the uptake of carbon (Sharkey, 1988; Sage, 1994). Elevated Ca suppresses the oxygenation of RuBP by Rubisco, resulting in increased rates of CO2 fixation ( Drake et al., 1997; Osborne et al., 1998). C4 plants are not expected to show an increased growth response in elevated Ca because they are able to concentrate CO2 at the site of Rubisco by 10–20 times that of atmospheric levels ( Furbank & Hatch, 1987; He & Edwards, 1996). However, several studies have shown that growth of C4 plants at elevated Ca stimulates an increase in biomass production, even under well watered conditions (Ghannoum et al., 1997; Ziska & Bunce, 1997; Lecain & Morgan, 1998; Maroco et al., 1999). One possible explanation for this increased biomass is that the immature C4 pathway in young leaves has C3-like characteristics; consequently, photosynthesis would respond to increasing Ca (Sionit & Patterson, 1984; Poorter et al., 1996). In many C4 plants, the CO2 pump requires compartmentalization of specific biochemical pathways within specialized and anatomically organized cell types (Hatch, 1988; Kanai & Edwards, 1999). In mesophyll cells of NADPH-type C4 plants CO2, in the form of HCO3–, is fixed by PEPC into oxaloacetate, which is then converted to malate and passively transported into the bundle sheath cells. Within bundle sheath cells, malate is decarboxylated to pyruvate, releasing CO2 that then enters the Calvin cycle. Two adenosine-triphosphate (ATP) molecules are required to regenerate phosphoenol pyruvate from the pyruvate produced by the NADP-malic enzyme catalysed malate decarboxylation reaction ( Kanai & Edwards, 1999). The minimum energy requirement of NADPH-type C4 plants is 5 ATP and 2 NADPH per CO2 fixed (Laisk & Edwards, 1998; Edwards & Baker, 1993). This translates to a minimum requirement of 12 photons per CO2 fixed by the Calvin cycle. Thus, the ratio of the quantum yield of PSII (φPSII) to the quantum yield of CO2 fixation (φCO2), φPSII : φCO2, is eight for NADPH-type C4 photosynthesis under optimal conditions (Furbank et al., 1990; Edwards & Baker, 1993). However, the quantum requirement for CO2 fixation, and the φPSII : φCO2
ratio, may vary depending on the extent of CO2 leakage from the bundle sheath cells, over cycling of the C4 pump, and the contribution of the Q-cycle to the production of the proton motive force (Furbank et al., 1990; Edwards & Baker, 1993). The efficiency of the CO2 concentrating mechanism is dependent on the restriction of CO2 diffusion back from the bundle sheath cells into the mesophyll cells (Jenkins et al., 1989). The resistance to CO2 diffusion from bundle sheath cells to the intercellular space, rc (m2 s mol–1), and the total resistance to diffusion of CO2 from air to the bundle sheath cell chloroplast, rt (m2 s mol–1), have been calculated from measurements of photosynthesis, photorespiration, and isotope exchange (Brown & Byrd, 1993; He & Edwards, 1996). Lower rc values allow a higher rate of CO2 diffusion between the bundle sheath cells and mesophyll cells. When rc is low, a higher C4 cycle activity is required to maintain CO2 levels within the bundle sheath cells resulting in an increased φPSII : φCO2 ratio, and to a quantum requirement greater then the theoretical minimum (He & Edwards, 1996). Development of the C4 photosynthetic apparatus occurs early in maize leaf development. As young leaves emerge from the surrounding whorl formed by older leaves the expression and compartmentalization of the C4 photosynthetic apparatus is already complete (Langdale et al., 1988a; 1988b; Nelson & Langdale, 1989; Nelson & Dengler, 1992). Young maize leaves had lower rc values and a higher rate of photorespiration then older leaves (Dai et al., 1995), but it was not established if this observation correlated to a poorly developed C4 photosynthetic apparatus. By contrast, young Panicum leaves exhibited insignificant O2 inhibition of A indicating that photorespiration was completely suppressed in C4 plants (Ghannoum et al., 1998). These conflicting observations suggest that the efficiency of the CO2 concentrating mechanism was dependent on a variety of developmental and environmental signals and that it also may be species dependent. In order to understand how photosynthesis in C4 plants may respond to elevated CO2 it is necessary to determine if young leaves of field grown plants have a fully developed C4 photosynthetic apparatus, and whether or not photosynthesis in these leaves is more responsive to elevated Ca than older leaves. To address this issue we have monitored the effect of free-air carbon-dioxide enrichment (FACE) on the photosynthetic performance of the C4 cereal crop Sorghum bicolor. FACE conditions allow for a long-term study of the effects of elevated Ca on crops grown in field conditions with minimal disturbances to the microclimate (Hendrey et al., 1993). To follow the photosynthetic performance as Sorghum plants developed in FACE conditions, the uppermost fully expanded leaves were sampled at various days after planting (DAP). Simultaneous measurements of chlorophyll a fluorescence and carbon assimilation were made in order to determine energy utilization, quantum requirement of carbon fixation, photorespiration and enhancement of net photosynthesis (A*) of FACE-grown plants. The results demonstrate that leaves of young
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Sorghum plants exhibit more then twice the rate of photorespiration compared to leaves of older plants, and that in FACE conditions young plants exhibit a higher rate of A.
Materials and Methods An experiment was conducted to determine the effect of elevated CO2 on grain sorghum (Sorghum bicolor (L.) Moench) at the University of Arizona Maricopa Agricultural Center ( MAC), Maricopa, Arizona, USA. CO2 treatments The free-air CO2 enrichment ( FACE) technique was used to enrich the air in circular plots within a sorghum field similar to prior experiments ( Hendrey et al., 1993; Wechsung et al., 1995; Hunsaker et al., 1996; Kimball et al., 1999). Briefly, four replicate 25-m-diameter toroidal plenum rings constructed from 0.305-m-diameter pipe were placed in the field shortly after planting. Air enriched with CO2 was blown into the rings, and it exited through tri-directional jets in the vertical pipes at elevations near the top of the crop canopy. Wind direction, wind speed, and CO2 concentration were measured just outside of each FACE ring. A computer-control system used the wind speed and CO2 concentration information to adjust the CO2 flow rates to maintain the desired CO2 concentrations at the centres of the FACE rings. The system used the wind direction information to turn on only those stand pipes upwind of the plots, so that CO2-enriched air flowed across the plots no matter which way the wind blew. The CO2 flow rates were updated every second, and the choice of which vertical pipes to release from was updated every 4 s. The 1-min-average CO2 concentrations were within 10% of the set point 87% of the time ( Hendrey et al., 1993; Nagy et al., 1994). Air blowers were installed in the nonCO2-enriched ambient control plots to provide air movement similar to that of the FACE plots. Like the 1996 and 1997 experiments ( Kimball et al., 1999) the FACE plots were enriched to a target 200 µl l–1 above control. The FACE treatment was applied continuously from emergence to harvest. The mean daytime values were 566 µl l–1 and 373 µl l–1 and the mean nighttime values were 607 µl l–1 and 433 µl l–1 for FACE and control, respectively.
Photosynthetic measurements Measurements were made on the uppermost fully expanded leaf obtained from an undisturbed portion of the canopy in 1999 between DAP 6 and 60. At DAP 6 leaf 2 was the uppermost; DAP 9 and 19 were leaf 3 and 5, respectively. Material was sampled before 07:30 hours to avoid any effects of photoinhibition or drought as previously described by Osborne et al. (1998). Plants were removed with soil still surrounding the roots and stored in sealed plastic bags at 10°C in darkness until measurements were made. The uppermost fully expanded leaf was excised below the ligule under water. The base of the leaf was kept in water and the mid-portion was placed into a 6400–06 PAM 2000 adapter cuvette (Li-Cor, Inc. Lincoln, NE, USA) which fits the fibreoptic probe of the pulse-modulated fluorometer (PAM 2000, Walz, Effeltrich, Germany) above the leaf at a 60 degree angle. The entire width of the leaf was measured until leaves became large enough to use just one side of the mid-vein. Leaves were dark adapted for a minimum of 1 h, after which simultaneous measurements of chlorophyll a fluorescence and gas exchange were made to determine the dark respiration rate (R D ). Subsequently, the cuvette was illuminated with c. 800 µmol photon m–2 s–1 by a 400 W Agrosun Halide lamp (Hydroform Inc., Petaluma, CA, USA). Leaf temperature was maintained at 30 ± 1°C. Leaf samples were acclimated for c. 1 h until steady state photosynthesis and chlorophyll a fluorescence was reached under ambient gas concentrations. Gas exchange and chlorophyll a measurements were made at CO2 concentrations of 75, 200, 370, 570 and 700 µl l–1. The carbon dioxide was supplied from CO2 canisters and the Li-Cor 6400 computer software (Li-Cor Inc.) was used to adjust concentrations. At each CO2 concentration, A and fluorescence were measured in air containing 21% O2 and then 2% O2. Premixed gasses were supplied from pressurized tanks containing 21% and 2% oxygen with a nitrogen balance (Air America Liquide, Pheonix, USA) which fed directly to the intake pumps of the Li-Cor 6400 (Li-Cor Inc.). The quantum yield of PSII was calculated as φPSII = (Fm′–Fs)/Fm′ (Genty et al., 1989). The quantum yield of CO2 fixation (φCO2 = A*/absorbed photosynthetic photon flux density (PPFD) ) was calculated as the ratio of net CO2 fixation to PPFD absorbed (Oberhuber & Edwards, 1993, Oberhuber et al., 1993). Leaf absorbance was determined using an integrating sphere (P. Pinter, Pers. Comm.).
Crop culture Certified grain sorghum seed ( Dekalb DK54), which had been treated with fungicide (Captan, Chloropyrifos-methyl, Fluxofenium and Metalaxyl), was planted into relatively dry soil in north–south rows spaced 0.76 m (30 inches) apart at a rate of 318 000 seeds ha–1. Immediately after planting, erection of the FACE and control apparatus commenced and was completed when the first irrigation was applied to all plots.
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Immunolocalization Leaf sections from the same leaves used for the photosynthesis measurements were fixed in a FAA fixative (2% formaldehyde, 50% ethanol and 5% glacial acetic acid) over night at room temperature (Robertson & Leech, 1995). Sections were then dehydrated in an ethanol series and a tert-butal alcohol series before being embedded into paraplast-plus (Oxford
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Labware, St. Louis, MO, USA). Transverse sections (10 µm) were cut using a Spencer No. 820 rotary microtome (American Optical Company, Buffalo, NY, USA) and adhered to a poly lysine coated slide. Sections were de-waxed in xylenes and re-hydrated in an ethanol series. Subsequently, the slides were placed in a phosphate buffer PBS (0.16 M NaCl, 8.0 mM Na2HPO4, 2.7 mM KCl and 1.5 mMKH2PO4 ) for 15 min then incubated at 4°C over night with the polyclonal primary antibodies in 0.5% BSA in PBS. Sections were then washed and incubated with Fluorescein Isothiocyanate (FITC) conjugated secondary IGg antibodies for 1 h ( Jackson-immuno, West Grove, PA, USA). Protein compartmentalization was then visualized on a Leica DM RBE microscope equipped with a Leica TCS NT confocal scanning head equipped with manufactures filters set up for FITC dies (Leica, Heidelberg, Germany). Images were composed and analysed using Adobe Photoshop 5.0 (Adobe systems Inc., San Jose, CA, USA). Statistical analysis All gas exchange and fluorescence measurements were replicated three times at each DAP for control and FACE grown plants. Photosynthetic data determined under growth CO2 concentrations were analysed using PROC MIXED (SAS Inc., Cary, NC, USA) for a two-way ANOVA where sources of variation tested were CO2 treatment, DAP and their interaction. The effects of O2 concentration, DAP and their interaction on the photosynthetic rates were tested by a two-way ANOVA separately for both control and FACE plants. The objective of testing the photosynthetic rates at two levels of O2 was to determine the response of each leaf on a DAP under growth CO2 concentrations. Therefore, differences between the means of the two O2 treatments on each leaf were analysed by a one-tailed Student–t-test. The effects of CO2 treatment and DAP on the slope and intercept of the relationship between φPSII and φCO2 was also examined by a two-way ANOVA.
Results Photosynthesis at growth CO2 concentrations To determine the response of net photosynthesis (A*) of Sorghum leaves to growth in control and FACE Ca conditions, gross carbon assimilation (A, µmol m– 2 s–1) and dark respiration rates (R D, µmol m–2 s–1), not shown, were measured on the uppermost fully expanded leaves between DAP 6 and DAP 60, Table 1. The percent stimulation of A in FACE grown plants is shown in Table 1. Photosynthesis of the second leaves of FACE-grown plants (DAP 6) was 37% (P < 0.001) greater than that of control-grown plants. A less pronounced increase in assimilation was observed in leaves of older FACEgrown plants measured later in the growth season. One explanation for the higher rates of photosynthesis in the FACE-grown plants is that elevated Ca suppressed the rate of photorespiration. The extent of photorespiration occurring under the different growth conditions was therefore assessed by measuring photosynthesis at growth Ca in air containing either 21% or 2% O2 (Fig. 1). Reducing the partial pressure of O2 will increase the efficiency of the carboxylation reaction and stimulate the rate of carbon assimilation, if photorespiration is occurring at a significant rate (Bowes, 1996). By suppressing photorespiration, A* was stimulated in leaves of both control-grown (P < 0.05) and FACE-grown (P < 0.05) plants (Fig. 1a,b) although no DAP by O2 interactions were significant for either CO2 treatment (F5,22 = 0.2 and F5,22 = 0.3, respectively). However, when comparing the O2 response from each leaf individually, A* in the second leaves was stimulated by 16% (P = 0.07) in leaves of control-grown plants, and by 8.8% (P = 0.24) in leaves of FACE-grown plants (Fig. 1c,d). A smaller (c. 5%) stimulation was observed in older leaves later in the season. We were also interested to determine if the extent of photorespiration would increase at lower Ca values, as is typically observed in C3 plants. In mature maize leaves, decreasing Ca
Gross photosynthesis (A)a µmol CO2 m–2 s–1 DAP
Control
FACE
6 9 19 23 38 60
23.33 (± 2.05) 27.86 (± 2.73) 29.40 (± 2.15) 22.23 (± 1.51) 26.46 (± 1.31) 25.91 (± 0.29)
31.90 (± 2.61) 28.00 (± 1.66) 26.33 (± 1.29) 25.80 (± 1.35) 29.91 (± 1.54) 29.85 (± 0.93)
% difference P
