The influence of light quality on C4 photosynthesis under steady-state conditions in Zea mays and Miscanthus × giganteus: changes in rates of photosynthesis but not the efficiency of the CO2 concentrating mechanism
School of Biological Sciences, Molecular Plant Sciences, Washington State University, Pullman, WA 99164, USA
Differences in light quality penetration within a leaf and absorption by the photosystems alter rates of CO2 assimilation in C3 plants. It is also expected that light quality will have a profound impact on C4 photosynthesis due to disrupted coordination of the C4 and C3 cycles. To test this hypothesis, we measured leaf gas exchange, 13CO2 discrimination (Δ13C), photosynthetic metabolite pools and Rubisco activation state in Zea mays and Miscanthus × giganteus under steady-state red, green, blue and white light. Photosynthetic rates, quantum yield of CO2 assimilation, and maximum phosphoenolpyruvate carboxylase activity were significantly lower under blue light than white, red and green light in both species. However, similar leakiness under all light treatments suggests the C4 and C3 cycles were coordinated to maintain the photosynthetic efficiency. Measurements of photosynthetic metabolite pools also suggest coordination of C4 and C3 cycles across light treatments. The energy limitation under blue light affected both C4 and C3 cycles, as we observed a reduction in C4 pumping of CO2 into bundle-sheath cells and a limitation in the conversion of C3 metabolite phosphoglycerate to triose phosphate. Overall, light quality affects rates of CO2 assimilation, but not the efficiency of CO2 concentrating mechanism.
The reductive assimilation of inorganic carbon into organic plant material is mediated by nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP). These two high-energy molecules are required in specific reaction sequences at fixed stoichiometries, and their intracellular pools turn over rapidly (Kramer & Evans 2011). Imbalance in the production and consumption of NADPH and ATP associated with variation in environmental conditions, such as illuminating light quality, will alter the rate of photosynthetic CO2 assimilation. In C3 plants, the rates of CO2 assimilation were lower under blue compared with red and green light (Evans & Vogelmann 2003; Loreto, Tsonev & Centritto 2009). These differences in photosynthetic rates were partially attributed to variation in light quality absorptance between photosystems light harvesting antenna (Evans 1986) and subsequent production of ATP and NADPH. Additionally, unequal distribution of light energy within a leaf due to changes in light quality (Vogelmann & Evans 2002; Evans & Vogelmann 2003) may also contribute to the observed light quality differences in the rate of CO2 assimilation. The effects of light quality on the rate of CO2 assimilation in C4 species could be more profound given the anatomical and biochemical complexities of C4 photosynthesis; however, there are few publications addressing this issue.
Kranz-type C4 photosynthesis requires coordinated carbon metabolism in both the mesophyll cells and bundle-sheath cells (Hatch & Slack 1966). In the mesophyll cells, the C4 cycle is initiated by the fixation of bicarbonate (HCO3-) and phosphoenolpyruvate (PEP) by PEP carboxylase (PEPc) into 4-C acids. Ultimately, these 4-C acids are decarboxylated in the bundle-sheath cells, where the released CO2 is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) initiating the Calvin–Benson cycle. The unique compartmentalization of mesophyll C4 cycle and bundle-sheath C3 cycle creates a high CO2 environment around Rubisco (Furbank & Hatch 1987), reducing rates of ribulose-1,5-bisphosphate (RuBP) oxygenation to 2–3% of photosynthesis (Jenkins, Furbank & Hatch 1989). To achieve high photosynthetic efficiency, the activities of the C4 and C3 cycles must be effectively coordinated (Leegood & Walker 1999).
Variation in light environments, including both light quantity and quality, is expected to alter the coordination of C4 and C3 cycles because of the unequal distribution of excitation between the mesophyll and bundle-sheath cells (Evans, Vogelmann & von Caemmerer 2008; Tazoe et al. 2008; Kramer & Evans 2011). In fact, several studies have focused on the effect of light quantity in C4 photosynthesis (Henderson, von Caemmerer & Farquhar 1992; Cousins, Badger & von Caemmerer 2006; Tazoe et al. 2008; Kromdijk, Griffiths & Schepers 2010; Pengelly et al. 2010; Ubierna, Sun & Cousins 2011); however, little is known about the effects of light quality on the coordination of the C4 and C3 cycles during C4 photosynthesis. The differences in light quality penetration and absorption by the photosystems are expected to change the energy distribution between mesophyll and bundle-sheath cells. Indeed, Evans et al. (2008) observed that blue light reduced rates of net CO2 assimilation by 50% in the C4 plant Flaveria bidentis relative to white light at equal light intensities (350 µmol quanta m−2 s−1). This decrease in net CO2 assimilation under blue light was attributed to decreased penetration of blue light into the bundle-sheath cells and subsequent insufficient production of ATP for the C3 cycle to match the rate of mesophyll cell CO2 concentrating mechanism (Evans et al. 2008). This type of disruption of the C4 and C3 cycles would increase leakiness (ϕ), defined as the proportion of carbon fixed by PEPc which subsequently leaks out of the bundle-sheath cells (Farquhar 1983; Hatch, Agostino & Jenkins 1995). An increase in ϕ reduces the efficiency of the CO2 concentrating mechanism and potentially rates of C4 photosynthesis. However, ϕ was not tested in the earlier studies on the influence of light quality on C4 photosynthesis.
Leakiness cannot be directly measured but it can be estimated by combining measurements of photosynthetic discrimination against 13C (Δ13C) (Evans et al. 1986) and the theoretical C4Δ13C model (Farquhar 1983). For example, this method has been used to verify ϕ changes when the coordination of the C4 and C3 cycles are disrupted in antisense Rubisco F. bidentis plants (von Caemmerer et al. 1997a,b). If differences in penetration of light qualities within a leaf result in an imbalance of energy for mesophyll and bundle-sheath cells carboxylation reactions, this would also result in changes in ϕ and consequently Δ13C. Additionally, preferential absorptance of blue light (Evans et al. 2008), relative to green and red light, by mesophyll cells has been suggested to increase in the C4 cycle disproportionately to the C3 cycle leading to an increase in ϕ (Evans et al. 2008).
Previous studies have shown a tight coordination of the C4 and C3 cycles under different light and CO2 availability (Leegood & von Caemmerer 1989). This coordination was achieved through the interconversion of key photosynthetic intermediates and sharing of reducing equivalents between the mesophyll and bundle-sheath cells (Leegood & von Caemmerer 1989; Leegood 1997). For example, the C4 and C3 cycles are connected through the interconversion of phosphoglycerate (PGA) and PEP by PGA-mutase and -enolase in the mesophyll cytosol (Huber & Edwards 1975; Furbank & Leegood 1984). Moreover, the reduction of PGA to triose phosphate relies, depending on C4 subtype, on NADPH generated in the mesophyll cells by linear electron flux (Hatch & Osmond 1976). Light quality-associated variation in the energy distribution between the C4 and C3 cycles is predicted to alter the generation of ATP and NADPH and the production of photosynthetic intermediates. However, it remains largely unknown to what extent C4 plants can coordinate the activities of C4 and C3 cycles in response to changes in light quality.
The objective of this study was to determine the effects of red, blue and green light on steady-state C4 photosynthesis and coordination of the C4 and C3 cycles. We measured leaf gas exchange, Δ13C, photosynthetic metabolite pools and Rubisco activation state under broad spectrum red (635 nm), green (522 nm), blue (460 nm) and white (equal parts red, green and blue) light in two economically and agriculturally important NADP-ME C4 plants Zea mays (FAO 2009) and Miscanthus × giganteus (Heaton, Dohleman & Long 2008; Dohleman & Long 2009). Additionally, under steady-state conditions leaf gas exchange and Δ13C measurements were used to estimate ϕ using the complete Δ13C model (Farquhar 1983). We hypothesized that lower rates of C4 photosynthesis under blue light compared with red and green would be caused by a disruption of the C4 and C3 cycles leading to an increase in ϕ.
MATERIALS AND METHODS
Zea mays seeds and Miscanthus × giganteus rhizomes were planted in 6 L pots. The plants were growing in a greenhouse with day temperature of 25–28 °C, night temperature of 20–25 °C and day length of 14 h. Illumination in the greenhouse was a combination of sunlight and supplementary light provided by 400 W high pressure sodium lamps. Plants were watered daily and fertilized weekly with Peters 20-20-20 (J.R. Peters, Inc., Allentown, PA, USA) and a slow release fertilizer (17-3-6 MPK). Four-week-old Z. mays and 3-month-old M. giganteus were used for the measurements.
Light response curve and quantum yield of CO2 assimilation
Light response curves were measured on the uppermost fully expanded leaves in four replicates of Z. mays and M. giganteus using the Li-6400xt gas exchange analyser with the opaque conifer chamber RGB light source (Li-Cor 6400-18; Li-Cor Inc., Lincoln, NE, USA). The light source generates a broad spectrum of red, green and blue light with peak wavelengths of 635, 522 and 460 nm and bandwidths of 16, 35 and 24 nm, respectively. White light was an equal mix of red, green and blue light. Light response curves were measured by decreasing light intensity from 2000, 1400, 900 and 1300, the highest light intensity achievable for white, red, green and blue light, respectively, to 0 µmol photon m−2 s−1. The CO2 mole fraction in the leaf chamber was maintained at 380 µmol mol−1, a leaf temperature of 25 °C and a relative humidity between 50 and 70% for all gas exchange measurements. The quantum yield of CO2 assimilation was estimated as the slope of the linear relationship between gross CO2 assimilation rates and incident photon flux density at low light intensity (≤200 µmol m−2 s−1) (Ehleringer & Björkman 1977).
CO2 response curve and the maximum phosphoenolpyruvate carboxylase activity
CO2 response curves were measured on the uppermost fully expanded leaves in four individual plants of Z. mays and M. giganteus under white, red, green and blue light (900 µmol photons m−2 s−1) using the same gas exchange system as described above. For each CO2 response curve, the leaf chamber CO2 mole fraction was varied at a sequence of 380, 300, 250, 200, 150, 100, 80, 60, 40, 380, 450, 550, 650, 800, 1000, 1200 µmol mol−1. The initial linear part of the CO2 response curve was used to estimate the maximum phosphoenolpyruvate carboxylase (PEPc) activity (Vpmax). At low CO2, net CO2 assimilation rate (A) is given by (von Caemmerer 2000):
where Cm is mesophyll CO2 mole fraction, which was assumed to be equal to the intercellular air space (Ci); KP is the Michaelis–Menten constant of PEPc for CO2, which was assumed to be 80 µbar (von Caemmerer 2000); gbs is bundle-sheath conductance to CO2 (0.0046 and 0.01 mol m−2 s−1 for Z. mays and M. giganteus, respectively), which was estimated from photosynthetic discrimination measurements (described below); Rm is the mitochondrial respiration occurring in the mesophyll cells, which was assumed to be half of the dark respiration rate (Rd) (von Caemmerer 2000). Rd (obtained from light response curves) was 1.80 and 1.27 µmol m−2 s−1 for Z. mays and M. giganteus, respectively. Vpmax was then estimated from the least square difference between the measured and estimated rate of CO2 assimilation.
Transmittance, reflectance and absorptance of light by the leaf were measured with an integrating sphere (IS-040; Lab Sphere Inc., North Sutton, NH, USA) on similar leaves used for gas exchange in six replicates Z. mays and nine replicates M. giganteus under white, red, green and blue light. Illumination was provided by a custom-built light-emitting diode light source that can provide white light and red, green and blue light with peak wavelengths of 625, 527.5 and 457.5 nm and bandwidths of 10, 15 and 15 nm (Cree, Inc., Durham, NC, USA).
Steady-state gas exchange and online photosynthetic carbon isotope discrimination
Steady-state gas exchange and online photosynthetic carbon isotope discrimination against 13CO2 (Δ13C) were measured on the uppermost fully expanded Z. mays and M. giganteus leaves using the Li-6400xt gas exchange analyser coupled to a tunable diode laser absorption spectroscope (TDLAS, TGA 100A; Campbell Scientific, Logan, UT, USA). 12CO2 and 13CO2 mole fractions in the Li-Cor reference and sample cells were measured by the TDLAS concurrently with a CO2 free tank and two calibration tanks (Liquid Technology Corporation, Apopka, FL, USA) with known 12CO2 and 13CO2 mole fractions. The TDLAS sampled from each of the five sites at a flow rate of 150 µL min−1 and a frequency of 40 s per site. Only the last 10 s measurements at each site were used for the calculation of 12CO2 and 13CO2 mole fractions. The mole fractions of 12CO2 and 13CO2 in the reference and sample lines were calibrated using a gain and offset calculated from the two calibration tanks (Bowling et al. 2003; Ubierna, Sun & Cousins unpublished data). To achieve steady state, leaves were acclimated at 900 µmol m−2 s−1 with white, red, green or blue light for at least 1 h in the opaque conifer chamber at a CO2 mole fraction of 380 µmol mol−1. The irradiance of 900 µmol quanta m−2 s−1 was the maximum light intensity all light treatments could achieve with the Li-Cor 6400-18 RGB light source. Steady-state gas exchange and Δ13C were measured on four replicates Z. mays and M. giganteus, respectively.
where a (4.4‰) and s (1.8‰) are fractionations during CO2 diffusion in air (Craig 1953) and leakage of CO2 out of the bundle-sheath cells (Henderson, von Caemmerer & Farquhar 1992), respectively; Ca and Cs are CO2 mole fractions in the atmosphere, and the bundle-sheath cells, respectively. The terms b3 and b4 are defined as (Farquhar 1983):
where Rd is dark respiration rate (1.80 and 1.27 µmol m−2 s−1 for Z. mays and M. giganteus, respectively); Rm is mesophyll cell dark respiration rate, which is assumed to be half of the Rd (von Caemmerer 2000); Vc, Vo and Vp are Rubisco carboxylation rate, Rubisco oxygenation rate and PEP carboxylation rate, respectively [see Ubierna et al. (2011) for details on the modelling of Vc, Vo, Vp and Cs]; values for gbs were estimated as the value that minimized differences between predicted and observed photosynthetic discrimination under white light (Kromdijk et al. 2010; Ubierna et al. 2011), which resulted in gbs values of 0.0046 and 0.01 mol m−2 s−1 for Z. mays and M. giganteus respectively; is fractionation by Rubisco, 30‰ (Roeske & O'Leary 1984); represents the net effect of CO2 dissolution, hydration and PEPc activity, which at 25°C has a value of −5.7‰ (Farquhar 1983; Henderson et al. 1992); f is fractionation during photorespiration, 11.6‰ (Lanigan et al. 2008); e′ is fractionation associated with dark respiration, which is calculated as (Wingate et al. 2007):
where e is fractionation during decarboxylation, assumed to equal −6‰ (Ghashghaie, Duranceau & Badeck 2001; Sun, Resco & Williams 2010); δ13Cmeasurement (–31‰) is the carbon isotope signature of CO2 used for the online discrimination measurement, which was measured by the TDLAS; and δ13Cgrowth is the carbon isotope composition of CO2 in the greenhouse, which is assumed to be −8‰.
Leaves similar in age and position to those used for the steady-state gas exchange and Δ13C measurements were rapidly freeze-clamped in situ using a custom-built rapid kill system attached to a Li-6400 gas exchange analyser (Badger, Sharkey & von Caemmerer 1984; Hendrickson et al. 2008). CO2 mole fraction of the custom-built leaf chamber attached to the rapid kill system was controlled by a Li-Cor 6400 gas exchange analyser. A custom-built light-emitting diode array provided white light and red, green and blue light with peak wavelengths of 625, 527.5 and 457.5 nm and bandwidths of 10, 15 and 15 nm (Cree, Inc.), and leaf temperature was controlled by a water bath. Leaves were acclimated at 900 µmol quanta m−2 s−1, a sample CO2 mole fraction of 380 µmol mol−1, and a leaf temperature of 25 °C under each light condition for at least 1 h before being rapidly freeze-clamped between two liquid nitrogen cooled copper rods. The frozen leaf disc was stored in an −80 °C deep freezer prior to extraction for photosynthetic metabolites and Rubisco activation state assays.
Photosynthetic metabolite measurements
Leaf discs (approximately 4.5 cm2) were ground to a fine powder in liquid nitrogen in a pre-cooled pestle and mortar and extracted with 1 m HCLO4 (Leegood & von Caemmerer 1988). Pyruvate and PEP were measured consecutively in the same assay by monitoring changes in absorptance at 340 nm (Leegood & Furbank 1984). Triose phosphate (TP), PGA and RuBP were measured consecutively in the same coupled enzyme assay as described previously (He et al. 1997), using Rubisco purified from tobacco. Assays were tested by known concentration standards purchased from Sigma-Aldrich (St. Louis, MO, USA).
Rubisco activation state
Rubisco activity was determined by the incorporation of 14CO2 into acid-stable products at 25 °C (Salvucci & Anderson 1987). The initial activity was determined by adding 20 µL of the leaf extract to the reaction mixture [100 mm Tricine-NaOH pH 8.0, 10 mm MgCl2, 10 mm NaH14CO3 (0.2 µCi µmol−1) and 0.4 mm RuBP] and quenching the reaction after 30 s with 100 µL acid (1 N HCl/4 N formic acid). Total activity was measured after incubating 20 µL of the same leaf extract in the reaction mixture for 3 min at 25 °C minus RuBP, to carbamylate all Rubisco catalytic sites. Subsequently, the reaction was initiated with RuBP and quenched after 30 s. Rubisco activation state was determined by the ratio of initial to total Rubisco activity.
Linear regression analysis was conducted to estimate quantum yield of CO2 assimilation and correlations between steady-state net assimilation rate and Vpmax or photosynthetic metabolites. The effects of light quality on net assimilation rate in light and CO2 response curves were assessed by one-way repeated measures analysis of variance (anova). One-way repeated measures anova was conducted to assess light quality effects on leaf light absorptance, steady-state net assimilation rate, stomatal conductance, the ratio of leaf intercellular air space to ambient CO2 partial pressure (pi/pa), Δ13C and leakiness. One-way anova was conducted to assess light quality effects on quantum yield of CO2 assimilation, Vpmax, photosynthetic metabolites and Rubisco activation state. Tukey's post hoc test was performed to test within species between light differences in quantum yield of CO2 assimilation, Vpmax, steady-state net assimilation rate, pi/pa, Rubisco activation state and metabolite pools. All statistical analyses were carried out using SAS version 9.0 (SAS Institute Inc., Cary, NC, USA). Average values are reported as the arithmetic mean ± 1 standard error.
Light and CO2 response curves
Net CO2 assimilation rate (A) increased with increasing light intensity under white, red, green and blue light in both Z. mays and M. giganteus (Fig. 1a,c). For irradiances > 300 µmol quanta m−2 s−1, A was significantly lower under blue than white, red and green light. Significant light quality differences in quantum yield of CO2 assimilation (ΦCO2) were detected in Z. mays and M. giganteus (P < 0.01, Table 1). Compared with white light [Z. mays (0.033 ± 0.001 µmol CO2µmol−1 quanta) and M. giganteus (0.035 ± 0.001 µmol CO2µmol−1 quanta)], ΦCO2 values increased under red light [Z. mays (0.041 ± 0.003 µmol CO2µmol−1 quanta) and M. giganteus (0.043 ± 0.001 µmol CO2µmol−1 quanta)], but decreased under blue light (0.025 ± 0.001 µmol CO2µmol−1 quanta for both Z. mays and M. giganteus). There were no apparent differences in ΦCO2 values between white and green light for either Z. mays or M. giganteus (Table 1).
Table 1. Leaf parameters under different light qualities
Quantum yield of CO2 assimilation (ΦCO2; µmol CO2µmol quantum−1), the maximum rate of phosphoenolpyruvate carboxylase (Vpmax; µmol m−2 s−1), leaf absorptance (%), steady-state photosynthetic discrimination (Δ13C; ‰), leakiness (ϕ) and Rubisco activation state (%) under white, red, green and blue light (at an irradiance of 900 µmol quanta m−2 s−1) in Zea mays and Miscanthus × giganteus. Data are reported as the arithmetic mean ± 1 standard error (n = 4–9). F-values, numerator degrees of freedom (ndf), denominator degrees of freedom (ddf) and P-values from analysis of variance are reported. Different letters within each species indicate significant differences (P < 0.05) between light treatments.
0.033 ± 0.001a
0.041 ± 0.003b
0.032 ± 0.002a
0.025 ± 0.001c
78.7 ± 10.4a
80.9 ± 11.8a
75.2 ± 9.0a
60.5 ± 3.8b
84 ± 1
85 ± 1
81 ± 2
85 ± 1
2.6 ± 0.3
2.6 ± 0.1
2.5 ± 0.3
2.7 ± 0.4
0.14 ± 0.01
0.14 ± 0.008
0.14 ± 0.003
0.15 ± 0.01
Rubisco activation state
95.0 ± 4.2
98.7 ± 2.8
93.7 ± 2.7
94.5 ± 2.6
0.035 ± 0.001a
0.043 ± 0.001b
0.036 ± 0.001a
0.025 ± 0.001c
52.2 ± 4.0a
53.8 ± 5.7a
50.4 ± 5.0a
40.0 ± 2.1b
78 ± 2
83 ± 2
78 ± 3
85 ± 3
3.3 ± 0.3
3.6 ± 0.2
3.1 ± 0.5
3.1 ± 0.3
0.19 ± 0.01
0.21 ± 0.008
0.18 ± 0.02
0.17 ± 0.01
Rubisco activation state
96.1 ± 4.1
97.9 ± 2.9
95.8 ± 3.3
95.7 ± 2.8
For all light treatments, A increased rapidly with Ci, and approached maximum values when Ci was above 100 and 150 µbar for Z. mays and M. giganteus, respectively (Fig. 1b, d). The maximum phosphoenolpyruvate carboxylase activity (Vpmax) was affected by light quality in both Z. mays (P = 0.03) and M. giganteus (P < 0.01, Table 1); with the Vpmax values under blue light [Z. mays (60.5 ± 3.8 µmol m−2 s−1) and M. giganteus (40.0 ± 2.1 µmol m−2 s−1)] significantly lower than under white [Z. mays (78.7 ± 10.4 µmol m−2 s−1) and M. giganteus (52.2 ± 4 µmol m−2 s−1)], red [Z. mays (80.9 ± 11.8 µmol m−2 s−1) and M. giganteus (53.8 ± 5.7 µmol m−2 s−1)] and green light [Z. mays (85.2 ± 9.0 µmol m−2 s−1) and M. giganteus (50.4 ± 5.0 µmol m−2 s−1)].
Steady-state gas exchange, photosynthetic 13C discrimination and leakiness
At an irradiance of 900 µmol quanta m−2 s−1, A was significantly lower under blue light [Z. mays (20.8 ± 0.8 µmol m−2 s−1) and M. giganteus (16.6 ± 0.8 µmol m−2 s−1)] than under white light [Z. mays (28.7 ± 1.5 µmol m−2 s−1) and M. giganteus (23.4 ± 1.5 µmol m−2 s−1)]. However, there were no statistically significant differences in steady-state A between white and red, or green light (Fig. 2a). Absorption of light was similar across all light treatments for both Z. mays (P = 0.17) or M. giganteus (P = 0.13, Table 1). Despite the strong effects of light quality on A, stomatal conductance was independent of light quality in both species (P > 0.05, Fig. 2b). The ratio pi/pa was similar across light treatments in Z mays (P = 0.65), but significantly different in M. giganteus (P = 0.01), with the lowest and highest pi/pa values in red and blue light, respectively.
In both species, photosynthetic discrimination against 13CO2 (Δ13C) and leakiness (ϕ) were independent of light quality (Table 1). However, ϕ was different across species (P < 0.01); with ϕ in Z. mays (white, 14.1 ± 1.2%; red, 13.8 ± 0.8%; green, 14.4 ± 0.3%; blue, 15.1 ± 1.3%) lower than those in M. giganteus (white, 19.1 ± 1.2%; red, 21.4 ± 0.8%; green, 18.0 ± 1.8%; blue, 16.6 ± 1.3%) (Fig. 3; Table 1).
Rubisco activation state and photosynthetic metabolites
At a light intensity of 900 µmol m−2 s−1, Rubisco activation state was not affected by different light quality for either Z. mays or M. giganteus (Table 1) averaging 95 ± 1% for Z. mays and 96 ± 1% for M. giganteus across all light treatments. However, leaf content of pyruvate and TP, but not in PEP and PGA differed under the different light treatments (Table 2). Under blue light there was an increase in pyruvate, but a reduction in the TP content compared with white, red and green light for both Z. mays and M. giganteus (Table 2). There was a significant effect of light quality on RuBP, content in M. giganteus (P < 0.01), but not in Z. mays (P = 0.24).
Table 2. Leaf metabolite concentrations
Pools of pyruvate (µmol m−2), phosphoenolpyruvate (PEP; µmol m−2), triose phosphate (TP, µmol m−2), phosphoglycerate (PGA, µmol m−2) and ribulose-1,5-bisphosphate (RuBP, µmol m−2) in Zea mays and Miscanthus × giganteus under 900 µmol quanta m−2 s−1 of white, red, green and blue light. Data are reported as the arithmetic mean ± 1 standard error (n = 5–8). F-values, numerator degrees of freedom (ndf), denominator degrees of freedom (ddf) and P-values from analysis of variance are reported. Different letters within each species indicate significant differences (P < 0.05) between light treatments.
146 ± 7a
142 ± 9a
149 ± 14ab
192 ± 12b
40.5 ± 3.2
42.5 ± 3.5
40.7 ± 2.9
39.6 ± 2.9
182 ± 11a
197 ± 12a
175 ± 9a
123 ± 5b
187 ± 7
189 ± 10
198 ± 12
188 ± 14
35.0 ± 3.1
39.6 ± 3.6
40.3 ± 3.4
44.4 ± 2.9
104 ± 4a
82 ± 4a
101 ± 4a
145 ± 9b
54.0 ± 4.7
59.6 ± 5.2
61.5 ± 3.0
58.4 ± 4.9
146 ± 7a
155 ± 7a
142 ± 6a
77 ± 7b
209 ± 8
194 ± 14
205 ± 9
189 ± 10
22.0 ± 1.3a
26.3 ± 2.2ab
25.4 ± 3.5a
36.3 ± 2.5b
In both species, the ratio of TP/PGA was the lowest (P < 0.01) under blue light (Fig. 4a), whereas RuBP/PGA was larger under blue light relative to white, red and green light, although significant only in M. giganteus (P < 0.01, Fig. 4b). No light quality effects were detected in the PGA/PEP (Fig. 4c).
Correlations between steady-state A, Vpmax and photosynthetic metabolites
There was a strong positive correlation (Fig. 5a) between steady-state A and Vpmax across white, red, green and blue light in Z. mays (r2 = 0.99, P < 0.01) and M. giganteus (r2 = 0.95, P = 0.02). For both species, across light treatments, steady-state A was negatively correlated (r2 = 0.95, P < 0.02) with pyruvate contents (Fig. 5b) but positively correlated (r2 = 0.94, P < 0.03) with TP contents (Fig. 5c).
Effects of light quality on carbon assimilation
Rates of net CO2 assimilation were lower under blue light compared with the other light treatments in both NADP-ME C4 plants Z. mays and M. giganteus (Figs 1 & 2a). Similar reductions in photosynthetic rates under blue light have been observed in the C3 species Spinacia oleracea L. (Evans & Vogelmann 2003; Evans et al. 2008), Nicotiana tabacum (Loreto et al. 2009) and Platanus orientalis (Loreto et al. 2009), as well as in the C4 species F. bidentis (Evans et al. 2008). Additionally, the quantum yield of CO2 assimilation has also been found to be wavelength dependent, with the maximum quantum yield occurring near 600 nm, declining rapidly at wavelengths shorter than 400 nm and greater than 680 nm (McCree 1972; Evans 1987). Accordingly, we found that the quantum yield of CO2 assimilation was lower under blue light (460 nm) than under white, red (635 nm) or green (522 nm) light. The reduced photosynthetic rate and quantum yield under blue light were not a result of light quality-associated differences in Rubisco activation state or leaf absorption (Table 1), rather they may have resulted from: (1) light quality-induced changes in stomatal conductance; (2) blue light-induced chloroplast movement; (3) changes in light distribution throughout the leaf; and (4) differences in leaf photosystem I and photosystem II absorptance.
Blue light induces stomatal opening (Sharkey & Raschke 1981) with zeaxanthin (Zeiger & Zhu 1998) or phototropin (Kinoshita et al. 2001) acting as photoreceptors, modifying guard cells turgor and the aperture of stomata. However, there were no blue light effects on stomatal conductance in N. tabacum and P. orientalis at an irradiance of 300 µmol m−2 s−1 as the percentage of blue light was increased from 0 to 80% (Loreto et al. 2009). In our study, steady-state stomatal conductance (gs) was not significantly different across light treatments (Fig. 2b). Therefore, light quality effects on rate of CO2 assimilation (Fig. 2a) did not result from light quality-associated differences in gs (Fig. 2b) and CO2 supply for photosynthesis (Fig. 2c).
Alternatively, lower photosynthetic rates under blue light could be due to chloroplast movement. It has long been observed that blue light induces chloroplast movement (Senn 1908) to prevent photodamage under strong light. This phenomenon has been observed in both C3 and C4 plants (Inoue & Shibata 1974; Kagawa et al. 2001; Yamada et al. 2009; Luesse, DeBlasio & Hangarter 2010; Maai et al. 2011). However, in C4 species, chloroplast movement under blue light was only observed in mesophyll cell chloroplasts, but not in bundle-sheath chloroplast (Yamada et al. 2009). Recent studies indicate that phototropins and specific protein kinases are activated by blue light and act as photoreceptors controlling chloroplast movement (Kagawa et al. 2001; DeBlasio et al. 2003; Wada, Kagawa & Sato 2003). If mesophyll chloroplast movement was the primary contributor for the observed low rate of CO2 assimilation under blue light, then we should have observed differences in CO2 assimilation among white, red and green light, because white light contained 1/3 of blue light (≈ 333 µmol m−2 s−1), which is enough to induce mesophyll chloroplast movement (Maai et al. 2011).
A third mechanism that could account for the observed reduction in C-assimilation under blue light is an imbalance in the energy available for the C4 and C3 cycles, originated by the low penetration of blue light within the leaf. Blue light is strongly absorbed by the surface mesophyll cells, whereas green and red light penetrate deeper into leaves (Vogelmann & Han 2000; Vogelmann & Evans 2002). In the C3 plant S. oleracea, the profile of light absorptance was tightly associated with the profile of carbon fixation (Sun, Nishio & Vogelmann 1998; Evans & Vogelmann 2003). The effect of light penetration is likely to also impact C4 species, as C4 photosynthesis requires coordination of mesophyll C4 cycle and bundle-sheath C3 cycle. Despite sharing of reducing equivalents between the C4 and C3 cycles, bundle-sheath cells need to generate approximately 50% of the total required ATP to fulfil the energy requirement of the Calvin–Benson cycle (Kanai & Edwards 1999). Indeed, Evans et al. (2008) observed that at a light intensity of 350 µmol m−2 s−1, blue light caused a 25% reduction, relative to white light, in the rate of CO2 assimilation of the C3 species S. oleracea, but a 50% reduction in the C4 species F. bidentis. Preferential absorptance of blue light by the surface mesophyll cells would lead to enhancement in C4 cycle and reduced C3 cycle activities (Evans et al. 2008). Under these conditions, one could expect an increase in leakiness (ϕ). However, our results show that both steady-state Δ13C and ϕ were similar under all light treatments (Fig. 3; Table 1). This suggests that C4 photosynthesis is able to coordinate the activities of the C4 and C3 cycles under different light environments to optimize C gain. Possible regulatory mechanisms are discussed in detail in subsequent sections.
For calculations of ϕ, we followed Ubierna et al. (2011) recommendations and used a more complete formulation to describe Δ13C (Eqn 4) that avoids overestimation of ϕ. Values for bundle-sheath conductance (gbs) were estimated as the value that minimized differences between predicted and observed photosynthetic discrimination under white light (Kromdijk et al. 2010; Ubierna et al. 2011). This resulted in gbs values of 0.0046 and 0.01 mol m−2 s−1 for Z. mays and M. giganteus, respectively, which are within the range of values previously suggested for this parameter of 0.005–0.02 mol m−2 s−1 (He & Edwards 1996; von Caemmerer & Furbank 2003).
Finally, low photochemical efficiency of blue light could be explained by differences in the antenna absorption spectra between the photosystems I and II (Evans 1986). Under blue light, more quanta are absorbed by photosystem II than photosystem I (Evans 1986); this imbalance is likely to reduce the overall efficiency of electron flux and the production of ATP and NADPH. Accordingly, an insufficient supply of ATP to the C4 cycle under blue light would result in reduced CO2 assimilation. Supporting this idea, we observed significantly lower values for Vpmax under blue light than under white, red and green light (Table 1), suggesting that blue light was not efficiently driving mesophyll cell electron flux. The strong positive correlations between steady-state net assimilation rate and Vpmax across light species in both Z. mays and M. giganteus (Fig. 5a) further confirm that low assimilation rates and quantum yields under blue light resulted from insufficient CO2 pumping capacity of the C4 cycle. Although Vpmax is estimated in part from an assumed Michaelis–Menten constant of PEPc for CO2 (KP), it is unlikely that KP changes with light quality. Therefore, differences in Vpmax between Z. mays and M. giganteus may not be absolute; however, the response to changes in light quality within a species will be accurate. This decrease in Vpmax is expected to decrease bundle-sheath CO2 concentrations (Cs). We used the light-limited C4 photosynthesis model (von Caemmerer 2000) to estimate Cs (µmol mol−1) which was lower under blue light compared with white, red and green light in both Z. mays (white 1241 ± 17; red 1280 ± 13; green 1221 ± 18; blue 1078 ± 11) and M. giganteus (white 738 ± 18; red 763 ± 17; green 740 ± 18; blue 667 ± 9). Very low Cs values could potentially result in incomplete suppression of photorespiration and increased ϕ (Kromdijk et al. 2010; Ubierna et al. 2011); however, for an irradiance of 900 µmol quanta m−2 s−1, we found no light quality effect on ϕ, suggesting that Cs was large enough to efficiently suppress photorespiration. Further studies are needed to evaluate coordination of C4 and C3 cycles at low light intensities for red, green and blue light.
Coordination of C4 and C3 cycles
Despite a 29% reduction in steady-state photosynthetic rates under blue light relative to white light (Fig. 2a), ϕ was independent of light quality, suggesting that under steady-state conditions both Z. mays and M. giganteus successfully coordinated the activities of the C4 and C3 cycles. Measurements of photosynthetic metabolite pools confirmed that the activities of C4 and C3 cycles were proportionally reduced under blue light (Table 2). Previous studies on photosynthetic metabolite pools have demonstrated a regulation of the fluxes through the C4 and C3 cycles for a wide range of light intensities, probably through interconversion of photosynthetic intermediates and sharing of reducing equivalent between the mesophyll and bundle-sheath cells (Leegood & von Caemmerer 1988, 1989; Furbank 2011).
For example, because of the lack of photosystem II in bundle-sheath chloroplasts of most NADP-ME C4 plants (Chapman & Hatch 1981), reduction of PGA to triose phosphate typically relies on NADPH produced through linear electron flux in the mesophyll cells. Therefore, C3 cycle activity is coordinated to some extent with C4 cycle activity. We observed an accumulation of pyruvate under blue light (Table 2) and pyruvate content was negatively correlated with the rate of CO2 assimilation (Fig. 5b), which may reflect insufficient ATP supply for the phosphorylation of pyruvate. An increase in pyruvate pool size with decreasing light intensity has also been observed in Z. mays (Usuda 1987; Leegood & von Caemmerer 1989). The ratio RuBP/PGA was also greater under blue light than under white, red and green light (Fig. 4b), suggesting that the C3 cycle was not limited by RuBP supply under these conditions. However, we observed strong positive correlations between the TP pool and rate of CO2 assimilation (Fig. 5c) and a decrease in the TP/PGA (Fig. 4a), similar to previous studies (Leegood & von Caemmerer 1988, 1989). The change in TP/PGA was caused by differences in TP as PGA content was similar across all light treatments (Table 2). A lack of PGA response to changes in photosynthetic rates above 500 µmol quanta m−2 s−1 was also observed in a previous study on Z. mays, suggesting no correlation between PGA content and rate of CO2 assimilation under high light conditions (Leegood & von Caemmerer 1989).
The interconversion of PEP and PGA has been proposed as a mechanism for co-regulating the C4 and C3 cycles in C4 plants (Leegood & von Caemmerer 1989; Furbank, Hatch & Jenkins 2000). For example, a correlation has been demonstrated between PGA and PEP pool sizes under different environmental conditions (Furbank & Leegood 1984; Leegood & von Caemmerer 1989), with the ratio PGA/PEP ≈ 4 (Leegood & von Caemmerer 1989). We observed that PGA/PEP remained fairly constant across light species for both Z. mays and M. giganteus (Fig. 4c), and averaged 4.8 and 3.6 for Z. mays and M. giganteus, respectively. Constancy in the ratio PGA/PEP suggests a tight coordination of the activities of C4 and C3 cycles under different light treatments. In addition to the existing evidence that C4 and C3 cycles are highly coordinated under changing light intensity, CO2 availability (Leegood & von Caemmerer 1989) and Rubisco activase content (Hendrickson et al. 2008), we demonstrate here that the activities of C4 and C3 cycles are also regulated under different steady-state illuminating light qualities.
Low rates of CO2 assimilation and quantum yield of CO2 assimilation under blue light relative to white, red and green light in Z. mays and M. giganteus may have resulted from a decrease in blue light penetration and imbalance in antenna absorptance between photosystems I and II, rather than from blue light-induced stomatal openness and chloroplast avoidance movement. The energy limitation under blue light affected both C4 and C3 cycles, as we observed a reduction in C4 pumping of CO2 into bundle-sheath cells (lower Vpmax under blue light) and a limitation in the conversion of C3 metabolite PGA to triose phosphate (strong positive correlation between TP and A). Accordingly, steady-state CO2 leakiness, derived from comparing measured Δ13C and the complete Δ13C model, was not significantly affected by light quality suggesting that Z. mays and M. giganteus managed to coordinate the activities of C4 and C3 cycles to maintain photosynthetic efficiency under these steady-state conditions.
This research was supported by the Office of Basic Energy Science, US Department of Energy DE-FG02_09ER16062 and instrumentation obtained through an NSF Major Research Instrumentation grant (#0923562). We thank C. Cody for plant growth management and Dr. Steve Long for Miscanthus plant material.