Bundle Sheath Leakiness and Light Limitation during C₄ Leaf and Canopy CO₂ Uptake^,,

Abstract

Perennial species with the C₄ pathway hold promise for biomass-based energy sources. We have explored the extent that CO₂ uptake of such species may be limited by light in a temperate climate. One energetic cost of the C₄ pathway is the leakiness (φ) of bundle sheath tissues, whereby a variable proportion of the CO₂, concentrated in bundle sheath cells, retrodiffuses back to the mesophyll. In this study, we scale φ from leaf to canopy level of a Miscanthus crop (Miscanthus × giganteus hybrid) under field conditions and model the likely limitations to CO₂ fixation. At the leaf level, measurements of photosynthesis coupled to online carbon isotope discrimination showed that leaves within a 3.3-m canopy (leaf area index = 8.3) show a progressive increase in both carbon isotope discrimination and φ as light decreases. A similar increase was observed at the ecosystem scale when we used eddy covariance net ecosystem CO₂ fluxes, together with isotopic profiles, to partition photosynthetic and respiratory isotopic flux densities (isofluxes) and derive canopy carbon isotope discrimination as an integrated proxy for φ at the canopy level. Modeled values of canopy CO₂ fixation using leaf-level measurements of φ suggest that around 32% of potential photosynthetic carbon gain is lost due to light limitation, whereas using φ determined independently from isofluxes at the canopy level the reduction in canopy CO₂ uptake is estimated at 14%. Based on these results, we identify φ as an important limitation to CO₂ uptake of crops with the C₄ pathway.

Biomass production by perennial plant species can abate greenhouse gas emissions either by increased carbon sink activity and soil organic carbon sequestrations or by displacing fossil fuel emissions in the production of static energy (U.S. Department of Energy, 2006; Somerville, 2007). Perennial plant species with the C₄ photosynthetic pathway combine high productivity and resource use efficiency with low requirements for agronomic inputs; thus, they seem well equipped for biomass-based energy production (Lobell et al., 2008). For instance, Miscanthus (Miscanthus × giganteus hybrid) is a perennial C₄ grass with a recorded annual dry matter production up to 4 kg m⁻² (Heaton et al., 2004a, 2004b).

Thus, attributes of the C₄ pathway (potentially high productivity coupled with high nitrogen and water use efficiencies; Sage, 2004) can be coupled to the carbon sequestration potential of a rhizomatous perennial (Hansen et al., 2004; Clifton-Brown et al., 2007). Miscanthus has higher productivity than switchgrass (Panicum virgatum; Heaton et al., 2004a) and has sufficient cold tolerance (Beale et al., 1996; Naidu et al., 2003; Wang et al., 2008a, 2008b) for cultivation in temperate climatic conditions. However, we hypothesized that growth at low temperatures and low light in a temperate climate may reduce the conversion efficiencies of such a crop, and we set out to explore the specific limitations that may occur due to shading in a crop with a high leaf area index.

Our study focused on the leakiness (φ) of bundle sheath (BS) cells to CO₂ as a potential bottleneck in the photosynthetic performance of a Miscanthus canopy. The carbon-concentrating mechanism inherent to the C₄ pathway relies on spatial separation of carboxylases between mesophyll (M) cells (phosphoenolpyruvate carboxylase) and BS cells (Rubisco). Decarboxylation of organic acids within the BS generates high CO₂ concentrations adjacent to Rubisco, thereby suppressing oxygenase activity, but some of this CO₂ retrodiffuses and is refixed within the M cells. Such “leakage” in the system occurs through the BS cell walls, facilitated by the symplastic connections between M and BS, which are required for metabolite exchanges. Estimates for BS φ at the leaf level range between 10% and 40% (Osmond and Smith, 1976; Farquhar, 1983; Henderson et al., 1992; Hatch et al., 1995; Fravolini et al., 2002; Cousins et al., 2006, 2008; Tazoe et al., 2006, 2008; Kubasek et al., 2007). External factors associated with increasing φ are low temperature (Henderson et al., 1992; Kubasek et al., 2007), water stress (Farquhar, 1983; Bowman et al., 1989; Buchmann et al., 1996), low nitrogen nutrition (Meinzer and Zhu, 1998), and, most markedly, low light (Henderson et al., 1992; Buchmann et al., 1996; Cousins et al., 2006, 2008; Tazoe et al., 2006, 2008; Kubasek et al., 2007).

The loss of C₄ efficiency under low photon flux density (PFD) was recently quantified in a series of laboratory measurements and theoretical modeling studies (Von Caemmerer, 2000; Cousins et al., 2006, 2008; Tazoe et al., 2006, 2008) using real-time carbon isotope discrimination (Δ¹³C). However, it is not clear if the increase in φ results initially from light limitation of Rubisco activity, relative to phosphoenolpyruvate carboxylase activity, or increased Rubisco oxygenase and photorespiratory costs, or the ATP requirement for phosphoenolpyruvate regeneration (Von Caemmerer and Furbank, 2003), and whether this phenomenon occurs transiently or permanently following the transfer from high-light to low-light conditions.

In this study, we set out to quantify the extent of φ during gas exchange at leaf and canopy scale for a 13-year-old Miscanthus stand at Teagasc Oak Park (Carlow, Ireland). Short-term field measurements of gas exchange and Δ¹³C at leaf level were used to compute φ and the corresponding loss of carbon assimilation under field conditions using the model for C₄ photosynthesis by Von Caemmerer and Furbank (1999) and Von Caemmerer (2000). In parallel, canopy isotopic flux densities, or “isofluxes” (CO₂ flux multiplied by isotopic signature), were constructed using eddy covariance data combined with [CO₂] and δ¹³C (carbon isotope composition relative to Vienna Pee Dee Belemnite standard) diurnal vertical profile measurements within the canopy and used to derive canopy Δ¹³C and φ values. We infer that while the effect of φ is higher at leaf level when measured in a well-coupled cuvette, potential carbon uptake is still significantly reduced by φ when assessed at the canopy scale. We conclude that leakage represents a considerable constraint on C₄ productivity, particularly in temperate climates, which is especially significant when the current and future importance of the C₄ pathway for food production (e.g. maize [Zea mays], sorghum [Sorghum bicolor], sugarcane [Saccharum officinarum]) and bioenergy production and CO₂ mitigation (Miscanthus, switchgrass) is taken into account.

RESULTS

Canopy Development

In Figure 1, we show the vertical distribution of leaf area through the canopy. The total leaf area index for the canopy was 8.3 ± 0.51 m² m⁻². A large proportion of this leaf area (2.8 ± 0.54 m² m⁻²) was located between 2 and 3 m height, with a smaller proportion (1.4 ± 0.49 m² m⁻²) located between 1 and 2 m. The section between 0 and 1 m also had a substantial fraction of the total leaf area (3.2 ± 0.80 m² m⁻²).

Figure 1.

Total leaf area index (m² m⁻²) and leaf area index separated into 1-m vertical sections. Error bars indicate 1 sd.

Potential Rate of Photosynthesis

In Figure 2, photosynthetic CO₂ uptake is depicted by electron transport capacity (J; determined using chlorophyll fluorescence; Fig. 2A) and CO₂ response (determined from gas exchange; Fig. 2B) as a function of height within the canopy. Due to the CO₂-concentrating mechanism of C₄ photosynthesis, light saturation of J (maximum electron transport capacity; J_max) typically occurred only at very high levels of PFD. At 3 m, J_max reached 142.4 ± 20.5 μequiv m⁻² s⁻¹ at PFD of 1,769 ± 42 μmol quanta m⁻² s⁻¹, whereas at 2.5 m, J_max was 104.1 ± 8.7 μequiv m⁻² s⁻¹ at PFD of 1,834 ± 41 μmol quanta m⁻² s⁻¹. At 2 m, J_max and saturating PFD were lower (54.8 ± 13.1 μequiv m⁻² s⁻¹ at 1,205 ± 21.5 μmol quanta m⁻² s⁻¹), and they were even lower at 1.5 m (26.6 ± 9.1 μequiv m⁻² s⁻¹ at 919 ± 41.6 μmol quanta m⁻² s⁻¹). At 1 m, J_max and saturating PFD were lowest (7.5 ± 1.8 μequiv m⁻² s⁻¹ at 553 ± 64.0 μmol quanta m⁻² s⁻¹), and there was very little response of J to light intensity. Mitochondrial (dark) respiration was also measured following 5 min in the dark, and averages yielded 0.89 ± 0.29 μmol m⁻² s⁻¹ for leaves at 3-m height, 0.71 ± 0.36 μmol m⁻² s⁻¹ for leaves at 2-m height, and 0.40 ± 0.25 μmol m⁻² s⁻¹ for leaves at 1-m height.

Figure 2.

A, Electron transport rate (J) at different levels of incident PFD for leaves at 1-, 1.5-, 2-, 2.5-, and 3-m height. Error bars indicate 1 sd. B, Light-saturated photosynthetic assimilation rates at different leaf intercellular CO₂ concentrations for leaves at 1-, 1.5-, 2-, 2.5-, and 3-m height. Measurements were taken at 1,600 μmol m⁻² s⁻¹ PFD and 22°C, and vapor pressure deficit was kept below 1 kPa.

While J_max at 2.5 and 2 m was reduced to 73% and 38% of values at 3 m, maximum photosynthetic capacity (A_max; Fig. 2B) values at 2.5 and 2 m were 113% and 74% of those at 3 m. Similarly, carboxylation efficiency (CE), calculated from the initial slopes of the CO₂ response curves (Fig. 2B), was 0.13 μmol CO₂ m⁻² s⁻¹ mol μmol⁻¹ at 3 m, 0.15 at 2.5 m, and 0.08 at 2 m. Below 2 m, CE decreased sharply to 0.04 at 1.5 m and 0.007 at 1 m, with a clear separation in CO₂ response between lower and higher leaf cohorts. Thus, whereas the light response of J had acclimated to a markedly lower capacity above 2 m, the transition in the decline of A_max and carboxylation efficiency occurred lower in the canopy.

Realized Rate of Photosynthesis

Vertical profiles of net CO₂ assimilation (A_n; Fig. 3, A–E), transpiration (E; Fig. 3, F–J), and stomatal conductance (g_s; Fig. 3, K–O), measured in situ on leaves throughout the canopy, are shown as a function of incident PFD. The observations reflected a steep light gradient within the canopy, with incident PFD below 2.5 m seldom higher than 50 μmol quanta m⁻² s⁻¹. High in the canopy at 3 m, A_n frequently was light saturated (10–12 μmol CO₂ m⁻² s⁻¹; Fig. 3A). At 2.5 m, most CO₂ fixation (3–8 μmol CO₂ m⁻² s⁻¹) occurred at PFD lower than 500 μmol quanta m⁻² s⁻¹ (Fig. 3B). Leaves at 2 m (Fig. 3C) were subject to very low light levels (0–75 μmol quanta m⁻² s⁻¹), but reasonable values of A_n were still sustained (0–7 μmol CO₂ m⁻² s⁻¹). At 1 and 1.5 m, the colimitation of light intensity and photosynthetic capacity clearly reduced A_n (0–2 μmol CO₂ m⁻² s⁻¹; Fig. 3, D and E).

Figure 3.

Steady-state levels for photosynthetic assimilation rate (A–E), transpiration rate (F–J), and stomatal conductance (K–O) at different levels of incident PFD. Measurements were taken under field conditions for leaves at (bottom to top) 1-m (n = 53), 1.5-m (n = 59), 2-m (n = 91), 2.5-m (n = 64), and 3-m (n = 84) height.

At 2.5 and 3 m, g_s ranged between 0.05 and 0.15 mol m⁻² s⁻¹ (Fig. 3, K and L), with generally higher conductances at 3 m than at 2.5 m for comparable light levels, which correspondingly led to higher rates of water loss in leaves at 3 m (Fig. 3, F and G). At 1 and 1.5 m, g_s remained below 0.03 mol m⁻² s⁻¹ (Fig. 3, N and O), and it increased to just above 0.06 mol m⁻² s⁻¹ at 2 m (Fig. 3M), but only for light levels above 50 μmol quanta m⁻² s⁻¹. Corresponding transpiration rates were less than 0.4 mmol water m⁻² s⁻¹ for leaves at 1 and 1.5 m (Fig. 3, I and J) and increased with light intensity to 1.5 mmol water m⁻² s⁻¹ at 2 m (Fig. 3H).

In conclusion, leaves at 2.5 and 3 m receive higher incident PFD than leaves at lower locations and consequently exhibit much higher in situ rates of CO₂ fixation and water loss than leaves at 1 and 1.5 m, while leaves at 2 m frequently achieve comparable assimilation and transpiration rates to leaves higher in the canopy.

Leaf Δ¹³C and φ

In Figure 4, we show the results for instantaneous Δ¹³C and corresponding ratios of intercellular CO₂ and ambient CO₂ partial pressures (p_i/p_a) from gas exchange. Also, corresponding derived values of φ are shown (derived from Eq. 3 below). Δ¹³C observations for individual leaves covered a wide range of values. Δ¹³C above 350 μmol quanta m⁻² s⁻¹ PFD showed some variation (Fig. 4A), ranging between 2.6‰ and 6.9‰. At lower PFD, Δ¹³C measurements were much more variable, ranging from 0.5‰ (1.5 m) to 16.5‰ (1 m). There was also a wide range of p_i/p_a values derived from gas exchange during the isotopic determinations, ranging from 0.25 (high PFD, high in the canopy) to 0.8 (shaded leaves, low in the canopy). BS φ to CO₂, derived from p_i/p_a and Δ¹³C using Equation 3 below, showed a similar trend (Fig. 4C), with relatively lower values of φ above 350 μmol quanta m⁻² s⁻¹ PFD higher in the canopy and a wide range of values below this threshold light intensity lower in the canopy. At higher light intensity, φ was mostly between 0.2 and 0.5 for leaves at 3 m. Below 350 μmol quanta m⁻² s⁻¹ PFD, the range for φ increased with values up to 0.8 (specifically found at 1 m and 48 μmol quanta m⁻² s⁻¹).

Figure 4.

A, Short-term Δ¹³C measured concurrently with gas exchange at natural light intensity for leaves at 1-m (n = 3), 1.5-m (n = 1), 2-m (n = 2), 2.5-m (n = 8), and 3-m (n = 9) height. B, Ratio between internal and ambient CO₂ mole fractions. The line plot represents nonlinear regression [p_i/p_a = (a × b)/(b + PFD), where a = 0.8738, b = 353.2301, r² = 0.55] used in Figure 6 to calculate canopy φ. C, φ calculated with Equation 1 using measurements of Δ¹³C (A) and the ratio between internal and ambient CO₂ mole fractions (B). The line plot shows the best fit of φ [(a × b)/(b + PFD), where a = 0.6409, b = 763.0974, r² = 0.16] used in Figure 6 to calculate A_n.

Canopy CO₂ Uptake, Δ¹³C, and φ

In Figure 5, we present eddy covariance flux measurements of ecosystem CO₂ exchange with corresponding levels of incident PFD as well as canopy Δ¹³C (derived with the eddy covariance-flask method, as described by Bowling et al. [2003]). Except for measurements in the early morning and late afternoon, the canopy was thoroughly mixed and storage flux below the eddy covariance measurements was insignificant (determined by vertical profile measurements of CO₂ concentrations within the canopy), apart from measurements between 6:00 and 7:00 am and between 6:00 and 7:00 pm, which were not used for canopy Δ¹³C determinations.

Figure 5.

Accumulated data for August 28 to September 1, 2007. A, Incident PFD. B, Eddy covariance measurements of net ecosystem CO₂ exchange. Direction of net flux is indicated by plus/minus sign, and negative (positive) flux indicates net CO₂ uptake (net respiration). C, Canopy Δ¹³C calculated by assigning isotopic signatures (from regressions in Table I) to gross CO₂ uptake derived from daytime net CO₂ fluxes (B) and ecosystem respiration based on nighttime regression with soil temperature (y = 0.5505e^0.0748T; r² = 0.887). D, Canopy Δ¹³C (C) as a function of incident PFD.

Ecosystem net CO₂ exchange (Fig. 5B) showed a clear diurnal pattern, with nocturnal respiration mainly correlated with temperature (1°C bin-averaged r² = 0.887) and net uptake mostly controlled by incident PFD (Fig. 5A). The δ¹³C of ecosystem respiration was determined by taking the y intercept of a Keeling plot (linear geometric mean regression of 1/[CO₂] versus δ¹³C) assuming a two-source mixing model (Keeling, 1958, 1961). For our 13-year-old stand (Table I), δ¹³C of respiration was still very much influenced by a C₃ carbon isotope signal from the previous cropping system, which is consistent with the low turnover of soil organic carbon in Miscanthus ecosystems (Hansen et al., 2004; Clifton-Brown et al., 2007).

Table I.

Regressions to derive ecosystem gross fluxes (values of parameters ± se)

Regression	Intercept	Slope	r2	No.
Nocturnal Keeling plot	−25.885 ± 0.801	6,334 ± 318	0.942	25
Daytime [CO2] versus δ13C	13.519 ± 2.441	−0.06031 ± 0.00673	0.776	20

Canopy Δ¹³C followed a diurnal trend, with lower values around midday and slightly increased values in morning and afternoon (Fig. 5C). Figure 5D shows the canopy Δ¹³C determinations as a function of incident PFD, revealing a similar pattern as the measurements of leaf Δ¹³C (Fig. 4A). Except for two outliers, canopy Δ¹³C remained between 1.3‰ and 4.3‰ above 350 μmol m⁻² s⁻¹ PFD. Below this light intensity, the range of canopy Δ¹³C values steadily expanded, with values between 1.5‰ and 6.3‰.

Losses of Potential CO₂ Fixation Due to φ

Canopy A_n was modeled (according to the model by Von Caemmerer and Furbank [1999] and Von Caemmerer [2000], with some adjustments; see Supplemental Appendix S1) for 3 d to show how φ would reduce net carbon gain on the basis of either the leaf or canopy data presented above (Figs. 4 and 5, respectively; hereafter referred to as “leaf level” and “canopy level”). Measured vertical canopy profiles of temperature, PFD, and photosynthetic capacity were used to compute a vertical profile of A_n, which was then multiplied by leaf area at each height and summed to obtain canopy A_n. Expression of individual leaves in canopy A_n, therefore, was weighted by their assimilation rate. In order to estimate the impact of φ, we calculated theoretical values for canopy A_n without leakage (φ = 0) and compared these values with canopy A_n obtained with our estimates for φ at leaf or canopy level (Fig. 6A).

Figure 6.

Results for August 29 to August 31, 2007 (Julian days 242–244). A, Modeled canopy A_n for two scenarios: φ based on leaf level measurements of Δ¹³C and on canopy Δ¹³C (rates are depicted as percentages of φ = 0). φ is taken from regression φ = a × b/(b + PFD), where a = 0.6409 and b = 763.0974 for leaf level (Figure 4C) and a = 0.4014 and b = 470.342 for canopy level (based on nonlinear fit of the canopy model in Figure 5C, using Eq. 3). B, Cumulative modeled canopy A_n for φ = 0, based on leaf level measurements of Δ¹³C and on canopy Δ¹³C. Scenarios are compared with total cumulative canopy A_n with φ = 0.

In Figure 6A, the 100% datum represents canopy A_n with φ set to zero. Both leaf- and canopy-level modeled values of canopy A_n were reduced because of φ. When canopy A_n was calculated with the use of φ based on canopy-level determinations, the reduction due to leakage was lower than when canopy A_n was derived from leaf-level measurements. The decrease in canopy A_n would be most severe when light was limiting early in the morning or in the evening, when canopy A_n was predicted to be only 43% (leaf level) or 62% (canopy level) of canopy A_n without leakage of CO₂. Canopy A_n at midday was predicted to range from 85% (leaf level) to 94% (canopy level) of canopy A_n without leakage.

In order to assess the cumulative effect of φ, A_n was also summed over 3 d. Cumulative canopy A_n was 68% (leaf level) to 86% (canopy level) of canopy A_n without CO₂ leakage (Fig. 6B), indicating that cumulative losses of canopy CO₂ fixation due to φ over 3 d were between 14% and 32%.

DISCUSSION

This study makes a major contribution to understanding the constraints to CO₂ uptake within Miscanthus, an important biomass crop. We have evaluated the impact of φ, an intriguing physiological correlate of the C₄ pathway, which has previously been investigated largely under laboratory conditions. The data have demonstrated that φ provides a significant constraint to carbon assimilation at leaf and canopy level under field conditions when estimated using real-time Δ¹³C techniques. We now evaluate the implications of our findings for integrating leaf-level and canopy-level gas-exchange processes and scaling likely physiological constraints to Miscanthus productivity in a temperate climate.

Gas Exchange and Fluorescence

Measurements of assimilation rate under controlled conditions comply well with previous findings. The high-light saturation points in Figure 2A are consistent with reported values for C₄ photosynthesis (Usuda, 1987; Beale et al., 1996, 1997). Carboxylation efficiency under saturating light (initial slope; Fig. 2B) as well as gas exchange under field conditions (Fig. 3) at 3 and 2.5 m agreed with findings by Beale et al. (1996, 1997) for the third leaf of Miscanthus (top down) during the second half of the growing season. Below 2 m, A_n decreased rapidly, which can be explained by leaf age and associated lower enzymatic activity (Usuda, 1984) as well as by early stages of leaf senescence at 1-m height. At canopy level, half-hourly averages of net ecosystem exchange were consistent with long-term measurements above Miscanthus (F. Carroll and M.B. Jones, unpublished data).

In the absence of simultaneous measurements of A_n and J under field conditions, we are not able to make any direct comparison of how A_n responded to changes in light use efficiency caused by φ. Also, measurements of the quantum yield of PSII are known to be influenced by quantitative and qualitative light history of the leaf as well as leaf temperature (Edwards and Baker, 1993). Figure 2B was determined under steady-state laboratory conditions, and so interpolating the ratio of J versus A_n, which should increase at low PFD based on our φ determinations (assuming that cyclic electron flow occurs at a constant fraction of J and absence of other significant electron sinks), could only be inferred on a qualitative basis. However, we note that for a fast-growing crop, leaves that were previously fully exposed were subsequently shaded by the developing canopy. For our observations of Miscanthus, it was evident that J_max was down-regulated higher in the canopy (2.5 m), while photosynthetic capacity (CE, A_max) was sustained lower in the canopy (2 m). The implications for the effect of φ on the ratio between J and A_n, therefore, need to be addressed in a more detailed study.

Δ¹³C

At high PFD (above 350 μmol quanta m⁻² s⁻¹), Δ¹³C for leaves at different heights matched well with values generally assigned to C₄ photosynthesis (for reviews, see Farquhar, 1983; O'Leary, 1988). Δ¹³C at lower PFD tended to be much more variable, ranging from 0.5‰ (1.5 m) to 16.5‰ (1 m), which is consistent with likely changes in the coupling of C₄ and C₃ cycles, variations in photosynthetic capacity, local microenvironment, and associated enzyme activity between leaves. Also, Δ¹³C for individual leaves, when calculated using Equation 4 below, becomes increasingly less precise when CO₂ depletion is low relative to the CO₂ concentration in air. The observation that higher values of Δ¹³C seemed to occur more often at low PFD deep in the canopy is consistent with previously reported values under low PFD for Amaranthus cruentus (Tazoe et al., 2006, 2008) and Flaveria bidentis (Cousins et al., 2006).

Remarkably, the increase in Δ¹³C at low PFD was also observed when derived independently at canopy level. Canopy Δ¹³C mostly reflected Δ¹³C processes by leaves high in the canopy. Since Δ¹³C at leaf level is weighted in canopy Δ¹³C according to the rate of CO₂ uptake relative to other leaves, predictably leaves high in the canopy with higher assimilation rates influence canopy A_n and Δ¹³C more than leaves at lower locations. The method used to determine canopy Δ¹³C (Bowling et al., 2003) depended on measurements of ecosystem CO₂ exchange together with isotopic compositions of ecosystem respiration and daytime canopy air. A single isotopic respiratory signal had to be assumed for the complete ecosystem, which is likely to be an oversimplification, given the difference between current (C₄) and previous (C₃) vegetation on soil respiration (Buchmann and Ehleringer, 1998) and light effects on respiration of aboveground biomass (Atkin et al., 1998).

The ecosystem respiratory flux was defined by an exponential regression between soil temperature and nocturnal eddy covariance measurements of CO₂ efflux (Lloyd and Taylor, 1994), which could only be firmly established by bin averaging the ecosystem respiration measurements in 1°C increments. A similar problem was found by Bowling et al. (2003) and appears to be quite common in previous reports of net ecosystem partitioning into gross assimilation and respiration fluxes (Reichstein et al., 2005; Knohl et al., 2008). Bowling et al. (2003) explained part of the variability by spatial variation of soil moisture content, and Knohl et al. (2008) implied nonhomogeneity of the soil profile. Both factors may have contributed in our study, especially since diurnal measurements of soil respiration as well as soil temperature and moisture content were also spatially variable (data not shown).

Increased Δ¹³C at Low Light Intensity

Our results support previous observations of an increase in Δ¹³C at low light intensity. The underlying mechanisms have been analyzed extensively. Based on studies of photosynthetic behavior in C₄ plants, Leegood et al. (1989) argued that the energy supply for the assimilatory pathway (production of ATP and NADPH) declines drastically following a transition from high to low PFD. Stimulation of cyclic electron flow and Q-cycle activity (Furbank et al., 1990) might make up for part of the ATP shortage (by cyclic partitioning of electrons around the PSI complex and altering the ATP-NADPH production ratio). However, we can find no evidence in the literature for increased activity of these alternative electron cycling processes as a result of lower PFD; so ultimately, photosynthesis remains limited by the energy supply.

Energy limitation could increase measured Δ¹³C in several ways. First, an increase in the relative contribution from mitochondrial respiration under low light could vary Δ¹³C (Duranceau et al., 2001), particularly if respiratory CO₂ is enriched in ¹³C (Ghashghaie et al., 2003). Alternatively, Henderson et al. (1992) stated that with reduced phosphoenolpyruvate regeneration slowing C₄ cycle activity under low light, a decrease in the BS CO₂ concentration or CO₂-O₂ ratio would allow the relative contribution of photorespiration to increase. The extra energy needed for the photorespiratory cycle would limit C₃ cycle activity compared with C₄ cycle activity, which would lead to an increase in leakage of CO₂ (Von Caemmerer et al., 1997a, 1997b) from BS cells, thereby allowing more of the isotopic fractionation by Rubisco (30‰) to be expressed. Recently, Tazoe et al. (2008) may have provided evidence for an increase in φ as a result of energy limitation under low PFD, since the increase in Δ¹³C and, hence, φ, following a transfer to low PFD, was lower in Amaranthus cruentus plants grown under low light intensity compared with high-light-grown plants. This may have been caused by a slightly altered relative composition (within the limits of C₄ pathway plasticity) of the photosynthetic apparatus in the plants grown under low PFD, resulting in an improved ability to sustain carboxylation efficiency in BS cells at low light. The relative changes in light use and carboxylation efficiency within the canopy (Fig. 2; see discussion above) certainly suggest that differing degrees of acclimation occur at reduced light intensity, once fully expanded leaves are overtopped by the newly growing canopy.

BS φ

At high light intensity, φ was similar to reported values in NADP-malic enzyme C₄ photosynthesis of 0.2 to 0.3 in maize (Henderson et al., 1992) and 0.3 to 0.4 in sugarcane (Meinzer et al., 1994; Meinzer and Saliendra, 1997). The maximum values for φ at low light intensity (below a threshold PFD of 350 μmol quanta m⁻² s⁻¹) were comparable to reported values of 0.65 at 150 μmol quanta m⁻² s⁻¹ for F. bidentis by Cousins et al. (2006) and of 0.76 at 40 μmol quanta m⁻² s⁻¹ for A. cruentus by Tazoe et al. (2008). Other contributions to the measured leaf-level variability of Δ¹³C and φ could be related to the input parameters (p_i/p_a, b₄, b₃, s, and a) and simplifying assumptions for Equation 3 below. The assigned parameter value of −6‰ (at 22°C) to b₄ is based on the assumption that carbonic anhydrase (CA) is abundant enough to maintain isotopic equilibrium between CO₂ and HCO₃⁻. However, if CA activity is too low to maintain equilibrium, b₄ becomes less negative and Δ¹³C increases (leading to an overestimation of φ with Eq. 3), which was shown by Cousins et al. (2006) with F. bidentis RNA antisense CA plants. Von Caemmerer et al. (2004) argued that CA activity was too high to maintain steady-state assimilation rates in F. bidentis; however, Gillon and Yakir (2000, 2001) have reported that CA activity in C₄ monocots could limit photosynthetic rates. Equation 3 requires high CO₂ concentration in BS cells relative to that in M cells. If this requirement was not met, φ might have been overestimated by 10% or more for very low BS CO₂ concentrations (Tazoe et al., 2008). Also, the gradient between intercellular and M CO₂ concentrations was assumed to be negligible in Equation 3. Results of a sensitivity analysis with the extended model by Farquhar (1983) showed that a gradient of 30 μmol mol⁻¹ would cause a mean absolute difference in calculated φ of 0.026.

Furthermore, there might be additional fractionations associated with photorespiration (Gillon and Griffiths, 1997; Lanigan et al., 2008), variable mitochondrial respiration between old and young leaves (Villar et al., 1995), or different carbon sources for respiration (Ghashghaie et al., 2003). A sensitivity analysis (Table II) was undertaken, using the extended model by Farquhar (1983), with various combinations of values for mitochondrial respiration, the fractionation factor assigned to respiration (e), and carboxylation rate in BS cells (V_c). The results were relatively insensitive to most of the modeled combinations, and only when V_c was low (3 μmol m⁻² s⁻¹) and mitochondrial respiration was high (1.2 μmol m⁻² s⁻¹) did mean absolute differences in φ range from −0.06 to 0.08 (depending on the value of e; Table II). The observed dark respiration rate in leaves at 1, 2, and 3 m in our study (0.40, 0.71, and 0.89 μmol m⁻² s⁻¹, respectively) suggest that such errors would not occur, due to the relative reduction in both assimilation and respiration rates low in the canopy.

Table II.

Sensitivity of leaf level calculations of φ to different values of mitochondrial respiration rate (R_d), fractionation factor associated with mitochondrial respiration (e), and carboxylation rate in BS cells (V_c)

The extended model by Farquhar (1983) was used to calculate the mean absolute difference of φ between the extended model and the simplified version without respiratory fractionation (Eq. 3). Phosphoenolpyruvate carboxylation and photorespiration rates were set at 1.5 and 0.03 times V_c, respectively. A value of 11.5‰ was used for the fractionation factor associated with photorespiration, based on recent estimates by Lanigan et al. (2008).

Rd	e	Vc	Mean Difference in φ
μmol m−2 s−1	‰	μmol m−2 s−1
0.4	6	30	0.0080
0.4	−6	30	0.0032
0.4	6	3	0.0306
0.4	−6	3	−0.018
1.2	6	30	0.0129
1.2	−6	30	−0.0016
1.2	6	3	0.0852
1.2	−6	3	−0.0614

Implications of Results for Canopy CO₂ Fixation

Assuming that φ is the major correlate of variations in leaf and canopy Δ¹³C, we suggest that φ significantly reduces canopy net assimilation rate. Having calculated φ from leaf-level Δ¹³C and simultaneous gas exchange, and also independently from canopy Δ¹³C and PFD and temperature measurements within the canopy, there were relative reductions in cumulative canopy A_n ranging from 14% to 32%. There are implications for losses of carbon-fixing capacity in densely planted C₄ crops. Screening for varieties with low Δ¹³C under a range of limiting light conditions might be a means to increase the productivity of Miscanthus (or other C₄ crops), especially in the temperate climate conditions of northwestern Europe. Additionally, the observed variations in the canopy profile of J_max and A_max may provide a mechanistic understanding of the energetic factors controlling φ.

In conclusion, our results have established that retrodiffusion or leakage of CO₂ from BS cells is a major limitation to carbon gain at leaf and canopy scales under field conditions. While additional laboratory studies are required to evaluate the exact nature of this energetic inefficiency at low light (perhaps through the analysis of transient responses of φ to light in sun- and shade-acclimated plants), there was an agreeable consistency in response to incident PFD measured independently for individual leaves and their cumulative light limitation within the upper part of a full C₄ crop canopy. Second, we have been able to demonstrate how φ, as a function of leaf area index and incident PFD within the canopy, can be translated into canopy-scale carbon partitioning and potential productivity. Additional work is required to confirm the consistency of these responses during canopy development and to allow more precise partitioning between respiratory components and canopy net assimilation. However, carbon isotopes have provided a powerful means to analyze instantaneous photosynthetic limitation at leaf and canopy scale and allowed us to derive estimates of the likely reduction due to φ in CO₂ uptake for a C₄ crop limited by light and temperature in a northern temperate environment. The good agreement between previous measurements of φ versus PFD from controlled environment studies (Henderson et al., 1992; Meinzer et al., 1994; Meinzer and Saliendra, 1997; Cousins et al., 2006; Tazoe et al., 2008) and our observations under field conditions might also suggest that within-canopy measurements of PFD can be used as a rough proxy for φ losses due to light limitation arising from self shading.

MATERIALS AND METHODS

Site Description

Measurements were carried out at Teagasc Agricultural Research Centre (Oak Park, Carlow, Ireland) on a 13-year-old stand of Miscanthus (Miscanthus × giganteus) in the summer of 2007. The plot was rainfed and unfertilized except for one application of 50 kg ha⁻¹ nitrogen fertilizer in 2006. Harvest of previous-year stems took place in early April 2007.

Potential Rate of Photosynthesis

Five leaves were randomly selected from 1-, 1.5-, 2-, 2.5-, and 3-m height within the canopy to assess electron transport rate (J; μmol m⁻² s⁻¹) as a function of incident PFD. All measurements were done just above the end of the visible mid rib. First, the quantum yield of PSII (Φ_PSII) was measured using pulse amplitude-modulated (PAM) chlorophyll fluorescence at a range of light intensities with a mini-PAM photosynthetic yield analyzer (Heinz Walz) according to Genty et al. (1989):

(1)

where F_m′ and F_t refer to maximum fluorescence at saturating light pulse and steady-state fluorescence immediately prior to the pulse. J could then be calculated at each light level with the use of Equation 2:

(2)

where PFD represents the absorbed PFD, and 0.5 is a factor that accounts for the partitioning of energy between PSI and PSII. Leaf absorptance was set at 0.84, which has been shown to be valid for a wide range of growth conditions in Miscanthus (Farage et al., 2006). As a control, we also measured the chlorophyll content of different leaves (n = 5) from each height using the protocol by Lichtenthaler (1987), but no significant differences were found.

CO₂ responses of net assimilation rate were analyzed on three leaves that were cut at the ligule, with the cut end placed in water and recut under water, 3 cm from the base (Beale et al., 1996). The leaves were positioned in the leaf cuvette (area = 6 cm²) of a LI6400 portable, open gas-exchange system (Li-Cor Biosciences) illuminated by a Walz Fiber Illuminator FL440 with controllable intensity, with a model 400-F fiberglass connection and projector to filter heat, and left for 30 min to acclimatize to saturating light (1,600 μmol quanta m⁻² s⁻¹) at ambient CO₂. Leaf temperature was set at 22°C, while the vapor pressure deficit was kept below 1 kPa. After reaching steady state, the photosynthetic assimilation rate as a function of internal CO₂ concentration was determined from high to low external CO₂ concentrations (380, 150, 100, 50, and 40 μmol mol⁻¹).

Mitochondrial respiration not associated with photorespiration (R_d) was measured on different leaves from 1, 2, and 3 m (n = 5) by gas exchange with the LI6400 (leaf temperature set at 22°C), with readings taken after 5 min in the darkened cuvette.

Realized Rate of Photosynthesis

Leaves were randomly selected at 1-, 1.5-, 2-, 2.5-, and 3-m height within the canopy, and (still attached) photosynthetic assimilation rate, transpiration rate, and stomatal conductance were measured under natural light and temperature by placing them in the leaf cuvette of the LI6400 portable, open gas-exchange system, while being careful not to change the angle and orientation of the leaf. A reading was taken after steady state was reached (approximately 5 min).

Use of Δ¹³C to Calculate BS φ

Hattersley (1976) first suggested that variation in δ¹³C of C₄ species may reflect variations in the amount of leakage. An increase in BS φ results in higher Δ¹³C, because fractionation by Rubisco becomes more expressed. This relationship is used in Equation 3, developed by Farquhar (1983) and Henderson et al. (1992), which allows calculation of BS φ if Δ¹³C and p_i/p_a are known.

(3)

where a is the fractionation during diffusion of CO₂ in air (4.4‰), b₄ is the combined fractionation of phosphoenolpyruvate carboxylation (2.2‰) and the isotopic equilibrium during dissolution of CO₂ and conversion to bicarbonate, yielding a net value of −6‰ (at 22°C; Mook et al., 1974), b₃ is the fractionation by Rubisco (30‰), and s (1.8‰) is the fractionation during leakage.

Δ¹³C Measured Concurrently with Gas Exchange

To measure Δ¹³C during gas exchange, the exhaust tube of a LI6400 portable photosynthesis system was connected to a cryogenic water and CO₂ trapping-purification line as described by Griffiths et al. (1990). CO₂ was trapped and purified for 15 min. The obtained CO₂ samples were stored in sealed glass vials and analyzed afterward with a VG SIRA dual-inlet isotope ratio mass spectrometer (modified and maintained by Pro-Vac Services), and values were corrected for the presence of N₂O and ¹⁷O. The δ¹³C of CO₂ measured before and after photosynthetic depletion yields the net discrimination using the following equation by Evans et al. (1986):

(4)

where

(5)

where δ_e and δ_o represent the isotopic compositions of CO₂ relative to the PDB standard, and c_e and c_o represent the CO₂ mole fractions in air entering and leaving the cuvette, respectively. The Δ¹³C values obtained in this way thus reflect average values for 15 min of continuous photosynthetic discrimination.

Canopy Microclimate Record

A 3.5-m-tall pole with cross bars at 1-, 2-, and 3-m height was erected within the canopy with the cross bars facing north/south. PFD quantum sensors (SKP-215; Skye Instruments) were mounted on the cross bars facing south and thermocouples (Cu-Co) were mounted facing north. Also, a thermocouple and a soil moisture probe (SM200; Delta-T Devices) were inserted 0.02 m below the soil surface. A data-logger (CR21X; Campbell Scientific) was used to record and store average values every 10 min.

Another pole with air inlets on the cross bars was connected to the air inlet of a LI6400 portable, open gas-exchange system via 4-mm-diameter Teflon tubing and a switching manifold to measure and record within-canopy CO₂ concentrations at 1-, 2-, 3-, and 3.5-m height. During the recordings, the exhaust tube of the LI6400 leaf cuvette was connected to the trapping-purification line, and CO₂ was trapped and purified for 5 min and stored in sealed glass vials. The isotopic ratio of purified CO₂ was determined with a VG SIRA dual-inlet isotope ratio mass spectrometer and corrected for the presence of ¹⁷O and N₂O.

Canopy Characterization

Leaf area index was measured with a Sunscan System SS-1 (Delta-T Devices). Measurements were made with the sunprobe at 0-, 1-, 2-, and 3-m height to analyze the specific leaf area index of vertical sections within the canopy as well as the total leaf area of the stand (0-m measurements).

Ecosystem CO₂ Exchange

Net ecosystem exchange (NEE) of CO₂ was measured using an eddy covariance system consisting of an open-path infrared gas analyzer (Li-Cor LI-7500) coupled to a three-dimensional sonic anemometer (Soluent R3; Gill Instruments). The sonic anemometer and air intake were positioned 1 m above the canopy. CO₂ concentrations were measured with the open-path LI-7500 infrared gas analyzer. Measurements were recorded at 20 Hz on a CR23X datalogger (Campbell Scientific), and 30-min average fluxes were processed using the EdiRe software version 1.4.3.987 (Edinburgh University). Data quality and stability were tested using the methods of Foken and Wichura (1995), and data were discarded based on a lower u* threshold of 0.1 (m s⁻¹), high sd in measured CO₂ concentration, and CO₂ concentration and flux values outside of approved bandwidths. The footprint model of Kormann and Meixner (2001) was used to determine the percentage of measured flux originating from within the measurement plot, when less than 70% of the data were discarded.

Canopy CO₂ Uptake and Isotope Discrimination

Data from diurnal measurements (August 28–29 and August 31–September 1, 2007) as well as eddy covariance flux measurements (August 28–September 1, 2007) were used to reconstruct canopy CO₂ uptake and short-term Δ¹³C using the method described in detail by Bowling et al. (2003).

A regression (exponential, ordinary least squares) was made of nocturnal eddy covariance measurements of ecosystem respiratory fluxes against soil temperature (bin averaged per 1°C). This regression was extrapolated over daytime to partition daytime gross ecosystem respiratory flux. The daytime gross ecosystem respiratory flux was then subtracted from the daytime total net flux, measured with eddy covariance, which yielded the canopy CO₂ uptake gross flux.

We assumed that daytime net ecosystem CO₂ fluxes were composed of gross fluxes of ecosystem respiration (F_R) and canopy uptake (F_A; Eq. 6), and net ecosystem uptake fluxes were assigned a negative sign by micrometeorological convention.

(6)

Analogously, net isofluxes were also assumed to consist of gross respiration and photosynthetic isofluxes (Eq. 7).

(7)

The isoflux of ecosystem respiration [(δ¹³C_R)F_R] was determined by multiplying the daytime gross respiratory flux with the intercept of the nocturnal Keeling plot (geometric mean regression of 1/[CO₂] versus δ¹³C; Keeling, 1958, 1961). Net daytime isofluxes (δ¹³CNEE) were calculated by multiplying the daytime net ecosystem exchange fluxes with corresponding isotopic signatures using daytime geometric mean regression of CO₂ concentrations versus δ¹³C. Since (δ¹³C_R)F_R, δ¹³CNEE, ambient isotopic signature of CO₂ (δ¹³C_a), and gross fluxes of canopy CO₂ uptake (F_A) were identified, based on mass balance and negligible storage flux in NEE measurements (checked with diurnal vertical CO₂ concentration profiles), the isotopic contribution of canopy CO₂ uptake (δ_A) became the only unknown variable and could be solved.

Losses of Potential CO₂ Fixation Due to φ

We used the following approach to calculate the implications of φ at leaf level on canopy CO₂ uptake (A_n). Based on the leaf-level determinations, a function for hyperbolic decay [φ = (a × b)/(b + PFD)] was fitted to define φ as a function of incident PFD (Fig. 4C). Measured incident PFD and temperature at 1, 2, and 3 m from August 29 to August 31 were used to represent the range of conditions defining CO₂ fixation. A vertical profile of A_n was calculated using the model for C₄ photosynthesis by Von Caemmerer and Furbank (1999) and Von Caemmerer (2000) with some adaptations (see Supplemental Appendix S1), which was multiplied by a profile of leaf area index and summed to yield canopy A_n. We calculated potential canopy A_n without CO₂ leakage (φ = 0) and compared it with canopy A_n computed with φ determined by the fitted relationship between φ and PFD in Figure 4C.

A similar approach was used in combination with Equation 3 to translate measurements of canopy Δ¹³C into φ for each height. First, leaf-level data were used to derive a regression between incident PFD and p_i/p_a (Fig. 4B). Based on previous results (Cousins et al., 2006; Tazoe et al., 2006, 2008) and the model by Von Caemmerer and Furbank (1999) and Von Caemmerer (2000), we used a descriptive function for hyperbolic decay [φ = (a × b)/(b + PFD)] to describe φ as a function of incident PFD. The measured incident PFD within the canopy, together with Equation 3, then allowed the calculation of A_n and Δ¹³C for each layer. Δ¹³C weighted by A_n was used to calculate canopy Δ¹³C, which was compared with measured canopy Δ¹³C. This comparison allowed fitting parameters a and b by means of nonlinear regression, and the best fit was used to produce results for canopy A_n based on canopy-level determinations of φ (as described in the previous paragraph).

The Ventana Simulation Environment Vensim DSS for Windows, version 5.6a Double Precision (Ventana Systems), was used for all calculations and nonlinear regression.

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Appendix S1. Modeling A_n for C₄ photosynthesis.