Carbon isotope discrimination as a diagnostic tool for C₄ photosynthesis in C₃-C₄ intermediate species

Highlight

Combined gas exchange and carbon isotope discrimination measurement and modeling is used to detect and estimate the contribution of the C₄ cycle in the C₃-C₄ intermediate species.

Abstract

The presence and activity of the C₄ cycle in C₃-C₄ intermediate species have proven difficult to analyze, especially when such activity is low. This study proposes a strategy to detect C₄ activity and estimate its contribution to overall photosynthesis in intermediate plants, by using tunable diode laser absorption spectroscopy (TDLAS) coupled to gas exchange systems to simultaneously measure the CO₂ responses of CO₂ assimilation (A) and carbon isotope discrimination (Δ) under low O₂ partial pressure. Mathematical models of C₃-C₄ photosynthesis and Δ are then fitted concurrently to both responses using the same set of constants. This strategy was applied to the intermediate species Flaveria floridana and F. brownii, and to F. pringlei and F. bidentis as C₃ and C₄ controls, respectively. Our results support the presence of a functional C₄ cycle in F. floridana, that can fix 12–21% of carbon. In F. brownii, 75–100% of carbon is fixed via the C₄ cycle, and the contribution of mesophyll Rubisco to overall carbon assimilation increases with CO₂ partial pressure in both intermediate plants. Combined gas exchange and Δ measurement and modeling is a powerful diagnostic tool for C₄ photosynthesis.

Introduction

C₄ photosynthesis is a highly efficient carbon fixation system characterized by the presence of a biochemical carbon pump with the capacity of increasing the CO₂ partial pressure (pCO₂) at the site of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) to concentrations higher than ambient air (Hatch et al., 1967; Hatch, 1987; Ehleringer et al., 1991). This increases photosynthetic rates and reduces photorespiration, potentially improving nitrogen and water use efficiency (Hibberd et al., 2008; Langdale, 2011). Most C₄ species show a common anatomical pattern, called Kranz anatomy, that leads to the separation of enzyme functions in two compartments, the mesophyll and the bundle sheath cell (Brown, 1975). CO₂ is first hydrated into bicarbonate in the mesophyll cell cytoplasm in a reversible reaction catalyzed by carbonic anhydrase (CA) (Badger and Price, 1994). Carbon is then fixed by phosphoenol pyruvate carboxylase (PEPC), localized exclusively in the mesophyll, into four-carbon acids that diffuse to the internally adjacent bundle sheath cell, where they are decarboxylated and the released CO₂ is refixed by Rubisco.

The most productive crops, such as maize, sorghum and sugar cane, are C₄ plants, exemplifying the higher efficiency of this system over the C₃ photosynthetic pathway present in most plant species, including major crops like wheat and rice. For this reason, there is currently a strong interest in implementing the advantages of C₄ photosynthesis in to C₃ crops with the aim of increasing yield, to keep pace with the food needs of a growing world population (von Caemmerer et al., 2012; Karki et al., 2013; Leegood, 2013). This kind of approach is boosting research on genetic, biochemical and physiological aspects of C₄ photosynthesis. However, the initial phases of these initiatives are not expected to produce fully functional C₄ plants, but plants showing incomplete C₄ phenotypes like those observed in C₃-C₄ intermediate species, which have been considered remnants of the evolution from C₃ ancestors to C₄ plants (Rawsthorne, 1992; Sage et al., 2011). They show Kranz or Kranz-like leaf anatomy, but the activity of C₄-related enzymes, such as PEPC, is lower compared to strict C₄ plants, and enzyme compartmentation is incomplete, with Rubisco and PEPC present in both the mesophyll and the bundle sheath cells (Cheng et al., 1988; Brown and Hattersley, 1989; Byrd et al., 1992). These factors reduce the efficiency of the carbon concentrating mechanism. In intermediate plants, a photorespiratory CO₂ pump, also known as the C₂ cycle or glycine shuttle, transports glycine formed during mesophyll photorespiration to the bundle sheath where it is decarboxylated and the CO₂ refixed, thus increasing overall CO₂ assimilation rate and reducing the effect of photorespiration (Monson et al., 1984; Sage et al., 2012; Schulze et al., 2013; Keerberg et al., 2014). The genus Flaveria has been the focus of numerous studies in the past because it comprises C₃, C₄ and C₃-C₄ intermediate species, the later showing different degrees of C₄ activity (Ku et al., 1983; McKown et al., 2005).

The C₄ cycle contribution to growth has been difficult to quantify in intermediate species. In these plants, a steeper initial slope in the CO₂ response of the CO₂ assimilation rate compared to a strict C₃ plantis expected. However, this trait is also affected by Rubisco content and its kinetic properties, so conclusions are not straightforward (von Caemmerer, 2000; von Caemmerer and Quick, 2000). Another important manifestation of C₄ activity in intermediate species is a reduction of the O₂ sensitivity of CO₂ assimilation and the compensation point (Γ) due to a proportion of Rubisco being contained in the bundle sheath (BS) and thus not in direct contact with air (Byrd and Brown, 1989; Dai et al., 1996). With the photorespiratory pump causing a similar effect, separating and quantifying the contribution of each biochemical pathway through this approach is not possible. The C₄ cycle activity relative to overall photosynthesis in intermediates has been estimated in the past by metabolite profiling, but recent reports indicate that metabolite accumulation is strongly dependent on the leaf zone sampled and its developmental stage (Monson et al., 1986; Leegood and von Caemmerer, 1994; Wang et al., 2014).

In order to develop a deeper understanding of the physiology of both natural and artificial C₃-C₄ intermediates, better tools are needed to evaluate the contribution of C₄ photosynthesis to overall assimilation. One signature of the activity of PEPC as the initial CO₂ fixation enzyme is a change in carbon isotopic discrimination (Δ) during photosynthesis. Whereas Rubisco has a strong preference for the lighter isotope, ¹²C, over the heavier isotope, ¹³C, PEPC is less discriminating, which causes an important difference in the biochemical fractionation between C₃ and C₄ plants (O’Leary, 1981; Farquhar, 1983). Incomplete C₄ photosynthesis in C₃-C₄ intermediates is also reflected in Δ, with both PEPC and mesophyll Rubisco acting as the initial CO₂ fixing enzymes and their relative activities determining the resulting Δ. Mathematical models describing CO₂ assimilation and isotopic discrimination in these plants have been previously developed (von Caemmerer and Hubick, 1989; von Caemmerer, 1992). However, attempts to characterize Flaveria intermediate species by studying carbon-isotope ratios in dry matter resulted in C₃-like profiles, and were interpreted as having little or no contribution of the C₄ system to plant growth, which was in contradiction to results from metabolite analysis (Monson et al., 1988; Byrd et al., 1992).

Tunable diode laser (TDL) absorption spectroscopy allows relatively rapid measurements of Δ concurrently with gas exchange, and has been used to analyze and compare C₃ and C₄ species (Tazoe et al., 2011; von Caemmerer et al., 2014). The present work uses this technique, combined with mathematical modeling, as a tool to determine the presence and contribution of C₄ photosynthesis in C₃-C₄ intermediate plants. An updated mathematical model of carbon isotope discrimination for C₃-C₄ intermediate species is proposed, which considers the effect of mesophyll conductance and allows the calculation of the biochemical fractionation. The strategy was applied to the study of Flaveria bidentis (C₄), F. pringlei (C₃), F. floridana (C₃-C₄) and F. brownii (C₄-like). F. floridana has been described as a C₂ plant with elevated PEPC activity, but it was unclear if a C₄ cycle is actually contributing to total carbon assimilation in this species (Monson et al., 1986, 1988; Leegood and von Caemmerer, 1994; Dai et al., 1996). F. brownii, on the other hand, was initially considered a C₄ species, but later experiments proved incomplete enzyme compartmentation, with a small proportion of Rubisco activity present in the mesophyll cells, and it was then reclassified as a C₄-like intermediate species (Holaday et al., 1984; Monson et al., 1987; Moore et al., 1989). In the present study, concurrent Δ and gas exchange measurement and modeling allowed the detection and estimation of the C₄ cycle in the intermediate species, proving itself as a powerful diagnostic tool for C₄ photosynthesis.

Materials and methods

Plant material and growth conditions

Flaveria bidentis was propagated from seeds and F. pringlei, F. brownii and F. floridana were propagated from cuttings (Brown and Hattersley, 1989; Whitney et al., 2011). Plants were grown in 30 l pots in a garden soil mix fertilized with Osmocote (Scotts, Australia) in a glasshouse under natural light conditions, at 28/18°C day/night temperatures, respectively. Pots were watered daily.

Responses of CO₂ assimilation rate and CO₂ compensation point to O₂ partial pressure

Two Li-Cor 6400XTs (Li-Cor, USA) were used to measure CO₂ assimilation at a range of reference pCO₂ (388, 0, 24, 48, 73, 97, 145, 194, 291, 388, 485, 582 and 776 μbar). N₂ and O₂ were mixed in different ratios by mass flow controllers (Omega Engineering Inc., USA) to generate a range of O₂ partial pressures (pO₂; 20, 50, 100, 200 and 300mbar) supplied to the LI-6400s. Response curves of CO₂ assimilation rate (A) to intercellular pCO₂ (C_i), A/C _i curves, were repeated sequentially at each pO₂. The measurements were made at 25°C, a flow rate of 500 μmol s⁻¹ and 1500 μmol quanta m⁻² s⁻¹, inside a growth cabinet at 25°C. Four plants from each species were analyzed. The compensation point (Γ) was calculated from the A/C _i curves at each pO₂, as the intercellular CO₂ concentration where net CO₂ assimilation is zero.

To study the inhibitory effect of O₂ on assimilation rate, we compared the CO₂ assimilation rate at a reference pCO₂ of 380 μbar at each pO₂.

Concurrent gas exchange and Δ measurements and calculations of mesophyll conductance

Two Li-Cor 6400XTs (Li-Cor, USA) coupled to a tunable-diode laser absorption spectroscope (TDLAS, model TGA100A, Campbell Scientific, Inc., USA) as described in Tazoe et al. (2011) were used for concurrent measurements of gas exchange and carbon isotope discrimination (Bowling et al., 2003; Griffis et al., 2004; Pengelly et al., 2012; Evans and von Caemmerer, 2013). Plants were transferred from the glasshouse to a growth cabinet with fluorescence lights (TRIL1175, Thermoline Scientific Equipment, Australia) at 25°C and one young fully expanded leaf was placed in each of the 6cm² leaf chambers. Measurements were made at a leaf temperature of 25°C, a flow rate of 200 μmol s⁻¹, 1500 μmol quanta m⁻² s⁻¹ and 20mbar pO₂. The desired pO₂ was achieved as described above and supplied to the Li-Cors 6400. Reference pCO₂ was changed stepwise to 392, 980, 686, 490, 294, 196, 98, 49 and 392 μbar and measurements were made every 4min for at least 30min at each pCO₂. Dark respiration (R _d) was measured at the end of an A/C _i curve at 392 μbar pCO₂ and 20mbar pO₂ by switching off the Li-Cor lamp. Three or four plants from each species were analyzed. Δ was calculated as previously described (Evans et al., 1986; Evans and von Caemmerer, 2013).

Mesophyll conductance (g _m) was calculated for F. pringlei from concurrent gas exchange and Δ measurements at the above range of reference pCO₂ and 19mbar pO₂, applying the equations previously described and including the ternary effects of transpiration rate (Farquhar and Cernusak, 2012; Evans and von Caemmerer, 2013). This method is only valid for C₃ species. For intermediate and C₄ species, we assumed the same CO₂ response of g _m found in F. pringlei, and scaled the absolute value at ambient pCO₂ to obtain the best fit of the A and Δ models for the observed results (see Results section).

Mathematical models

The overall rate of net CO₂ assimilation (A) for C₃-C₄ intermediate plants was previously described (von Caemmerer, 1992, 2013):

$$A= A_{\text{s}}+ A_{\text{m}}$$ (1)

where A _m is the assimilation in the mesophyll and A _s is the assimilation in the bundle sheath, which are defined as:

$$A_{\text{s}}=V_{\text{p}}+\beta F_{\text{m}}-L$$ (2)

$$A_{\text{m}}=V_{\text{m}}-R_{\text{m}}-\left(1-\beta \right)F_{\text{m}}$$ (3)

so:

$$A=V_{\text{m}}-R_{\text{m}}-F_{\text{m}}+V_{\text{p}}-L$$ (4)

where V _p is PEPC carboxylation and β is the fraction of the CO₂ produced from photorespiration in the mesophyll (F _m) that is released in the bundle sheath. For simplification, bundle sheath respiration and photorespiration are not taken into account in eq. 4. The term L is the leak rate of CO₂ out of the bundle sheath, and can be expressed as:

$$L=\varphi (V_{\text{p}}+\beta F_{\text{m}})$$ (5)

and

$$A=V_{\text{m}}-R_{\text{m}}-F_{\text{m}}+V_{\text{p}}-\varphi (V_{\text{p}}+\beta F_{\text{m}})$$ (6)

where ϕ (leakiness) is the ratio of the leak rate of CO₂ out of the bundle sheath and the supply rate of CO₂ to the bundle sheath $(V_{\text{p}}+\beta F_{\text{m}})$ . When pO₂ is low, F _m can be considered 0.

V _m and R _m are Rubisco carboxylation and day respiration in the mesophyll, respectively. V _p and V _m are calculated as described in von Caemmerer (2000):

$$V_{\text{m}}=\frac{C_{\text{m}}·V_{\text{m},\text{max}}}{C_{\text{m}}+K_{\text{c}}(1+\frac{\text{O}}{K_{\text{o}}})}$$ (7)

$$V_{\text{p}}=\frac{C_{\text{m}}·V_{\text{p},\text{max}}}{C_{\text{m}}+K_{\text{p}}}$$ (8)

and

$$C_{\text{m}}=C_{\text{i}}-\frac{A}{g_{\text{m}}}$$ (9)

where C _m and C _i are mesophyll and intercellular pCO₂, respectively. K _c and K _o are the Michaelis-Menten constants for CO₂ and O₂ respectively, expressed as a partial pressure. Although the pCO₂ in the cytosol (site of PEPC carboxylation) and the chloroplast (site of Rubisco carboxylation) of the mesophyll cell are presumably different due to diffusional limitations, the same value (C _m) was assumed in both compartments (von Caemmerer, 2000, 2013; Tholen and Zhu, 2011).

When the rate of PEP regeneration is limiting, V _p=V _pr, where V _pr is a constant. V _m,max is the maximum Rubisco carboxylation in the mesophyll, and V _p,max is the maximum PEPC carboxylation (Table 1). When RuBP becomes limiting, V _m in eq. 6 can be given by an electron transport limited rate (W _j), as previously described (von Caemmerer, 2000, 2013).

Table 1.

Values assigned to variables for model fitting purposes

When fitting F. brownii as a strict C₄ and F. floridana as a strict C₃ species, values were assigned to obtain the best fitting without considering measured enzyme activities.

Variable	Definition	F. pringlei	F. bidentis	F. brownii	F. brownii (strict C 4)	F. floridana	F. floridana (strict C 3)	Origin of the value
a	Fractionation during diffusion in air (‰)	4.4	4.4	4.4	4.4	4.4	4.4	Farquhar (1983)
ab	Fractionation during diffusion through the boundary layer (‰)	2.9	2.9	2.9	2.9	2.9	2.9	Griffiths et al. (2007)
abs	Leaf absorptance	0.8	0.8	0.8	0.8	0.8	0.8	von Caemmerer (2000)
al	Fractionation during diffusion in water (‰	0.7	0.7	0.7	0.7	0.7	0.7	Griffiths et al. (2007)
β	Fraction of the photorespired CO2 released in the bundle sheath	1	1	1	1	1	1	Assigned
b3	Fractionation during carboxylation by Rubisco (‰)	29	29	29	29	29	29	Roeske and O’Leary (1984)
b4	Combined fractionation by the C4 cycle (‰)	na	−5.7	−5.7	−5.7	−5.7	na	O’Leary (1981)
bs	Fractionation during CO2 dissolution in water (‰	1.1	1.1	1.1	1.1	1.1	1.1	von Caemmerer (1992)
c	gm scaling constant	0.666a	0.8b	0.666b	0.666b	0.78b	0.78b	a, measured in this work; b, assigned from model fitting
e	fractionation during mitochondrial respiration	2.91	3.54	3.51	3.51	3.72	3.72	Calculated as e=δ13Ccylinder-δ13Catmosphere
F	Correction coefficient for spectral quality	0.15	0.15	0.15	0.15	0.15	0.15	von Caemmerer (2000)
Jt	Total electron transport rate (μmol electrons m−2 s−1)	120	400	440	700	250	0	Assigned [von Caemmerer (2000), eq. 5.17]
Jm	Electron transport rate allocated to mesophyll C3 cycle	120	0	40	0	200	240	Assigned [von Caemmerer (2000), eq. 5.17]
KC	Rubisco Michaelis–Menten constant for CO2 (μbar)	359	605	383	383	395	395	Kubien et al. (2008)
KO	Rubisco Michaelis–Menten constant for O2 (μbar)	528 000	507 000	300 000	300 000	544 000	544 000	Kubien et al. (2008)
KP	PEPC Michaelis–Menten constant for PEP (μbar)	n.a.	80	80	n.a.	80	n.a.	Bauwe (1986)
Rd	Mitochondrial respiration (μmol m−2 s−1)	0.6	0.4	1.3	1.3	1.7	1.7	Measured in the dark in this work
s	Fractionation during leakage (‰)	n.a.	1.8	1.8	1.8	1.8	n.a.	von Caemmerer (1992)
Vm, max	Maximum Rubisco carboxylation rate in the mesophyll (μmol m−2 s−1)	60a	0b	15b	0b	90a	130b	a, measured in this work; b, assigned
VP, max	Maximum PEP carboxylation rate (μmol m−2 s−1)	0a	90a	80a	80b	15a	0b	a, measured in this work; b, assigned
VPr	PEP regeneration rate (μmol m−2 s−1)	0	36	32	50	8	0	Assigned
φ	Leakiness	n.a.	0.28	0.21	0.3	0.40	n.a.	Assigned from model fitting
θ	Empirical curvature factor	0.1	0.1	0.1	0.1	0.1	0.1	Ubierna et al. (2011)

n.a., not applicable.

Theory developed by Farquhar et al. (1982) and Farquhar (1983) showed that photosynthetic carbon isotope discrimination can be described by equations having diffusion and biochemistry dependent terms. The equation of Δ presented by (Griffiths et al., 2007), which takes into account the effect of g _m, was modified to incorporate the ternary effects of transpiration rate as suggested by Farquhar and Cernusak (2012):

$$\begin{array}{c}\Delta =\frac{1}{1-t}{a}^{\prime }+\frac{1+t}{1-t}(a_{\text{l}}+b_{\text{s}}-{\Delta }_{\text{bio}})\frac{A}{g_{\text{m}}\cdot C_{\text{a}}}\\ +\frac{1}{1-t}[\left(1+t\right){\Delta }_{bio}-a^{\prime }]\frac{C_{\text{i}}}{C_{\text{a}}}\end{array}$$ (10)

where a _l is the fractionations during diffusion in water and b _s is the fractionation as CO₂ enters solution. The term $t=\frac{\left(1+a^{\text{'}}\right)E}{2g_{ac}^t}$ , where E denotes the transpiration rate and $g_{ac}^t$ the total conductance to CO₂ diffusion including boundary layer and stomatal conductance. The symbol aʹ denotes the combined fractionation during diffusion in the boundary layer and in air, and is calculated as:

$$a^{\prime }=\frac{a_{\text{b}}\left(C_{\text{a}}-C_{\text{l}}\right)+a(C_{\text{l}}-C_{\text{i}})}{(C_{\text{a}}-C_{\text{i}})}$$ (11)

where a is the fractionation during diffusion in air, a _b is the fractionation during diffusion in the boundary layer, and C _a, C _l, C _i are the pCO₂ in the air, leaf surface and intercellular space respectively. The biochemical fractionation, Δ _bio, is the integrated net biochemical discrimination, and depends on the biochemistry of net CO₂ uptake (Griffiths et al., 2007).

When Δ and g _m are known, Δ _bio can be solved from equation 10, resulting in:

$${\Delta }_{\text{bio}}=\frac{\Delta -\frac{1}{1-t}a\text{'}\frac{C_a-C_i}{C_a}-\frac{1+t}{1-t}{a}_i\frac{A}{g_{\text{m}}\cdot C_{\text{a}}}}{\frac{1+t}{1-t}(\frac{C_{\text{i}}}{C_{\text{a}}}-\frac{A}{g_{\text{m}}\cdot C_{\text{a}}})}$$ (12)

Because g _m was obtained from combined measurement of Δ and gas exchange in the C₃ species F. pringlei, Δ and g _m are not independent and we could not estimate Δ _bio from eq. 12. For the intermediate and C₄ species, g _m was calculated independently of the Δ measurements as described in the Materials and Methods section, so Δ _bio could be estimated from eq. 12 for F. floridana, F. brownii and F. bidentis.

For modeling purposes, or when Δ is unknown, Δ _bio can be derived from von Caemmerer’s (1992) equation A17:

$$\begin{array}{l}\frac{R_{\text{i}}}{R_{\text{p}}}=1+(b_3-\frac{f{F}_{\text{m}}+e{R}_{\text{m}}}{A})+\frac{A_{\text{s}}}{A}[\left(b_3-s\right)\varphi \\ +\frac{\left(b_4-b_3\right)V_{\text{p}}-f\beta F_{\text{m}}}{V_{\text{p}}+\beta F_{\text{m}}}]-\frac{f{F}_{\text{s}}+e{R}_{\text{s}}}{A}\varphi \end{array}$$

where R _i and R _p are the molar abundance ratios of ¹³C/¹²C in the intercellular space and the photosynthetic product, respectively.

$${\Delta }_{\text{bio}}=\frac{R_{\text{i}}}{R_{\text{p}}}-1$$

Thus:

$$\begin{array}{l}{\Delta }_{\text{bio}}=(b_3-\frac{f{F}_{\text{m}}+e{R}_{\text{m}}}{A})+\frac{A_{\text{s}}}{A}[\left(b_3-s\right)\varphi \\ +\frac{\left(b_4-b_3\right)V_{\text{p}}-f\beta F_{\text{m}}}{V_{\text{p}}+\beta F_{\text{m}}}]-\frac{f{F}_{\text{s}}+e{R}_{\text{s}}}{A}\varphi \end{array}$$ (13)

The factor b ₃ is the Rubisco fractionation, and b ₄ is the combined fractionation of PEP carboxylation and the preceding isotope equilibrium during dissolution of CO₂ and conversion to bicarbonate; s is the fractionation during leakage of CO₂ out of the bundle sheath; e is the fractionation during mitochondrial respiration; f is the fractionation during photorespiration; R _m and R _s are the mitochondrial respiration rates in the mesophyll and the bundle sheath in the light, respectively. It was assumed that R _d=R _m+R _s, and R _m=R _s=0.5R _d. The factors F _m and F _s are the photorespiration rates derived from Rubisco oxygenation in the mesophyll and the bundle sheath, respectively. When pO₂ is low, F _m and F _s are close to 0, so equation 13 simplifies to:

$${\Delta }_{\text{bio}}=(b_3-\frac{e{R}_{\text{m}}}{A})+\frac{A_{\text{s}}}{A}[\left(b_3-s\right)\varphi +\left(b_4-b_3\right)]-\frac{e{R}_{\text{s}}}{A}\varphi$$ (14)

The parameter e needs to account for differences between the isotopic composition of CO₂ during plant growth and during the measurements, because the substrates used during respiration are most likely carbohydrates assimilated before the experiment (Wingate et al., 2007). No fractionation during mitochondrial respiration was assumed in this work, so e was calculated as the difference between δ¹³C in the CO₂ cylinder used during the experiments and δ¹³C in the atmosphere during growth conditions (e=δ¹³C_cylinder-δ¹³C_atmosphere) (Tazoe et al., 2009; Pengelly et al., 2010). In this work, δ¹³C_cylinder was between −4.12‰ and −5.14‰, and δ¹³C_atmosphere was assumed to be −8‰ (Table 1).

In vitro enzyme activity assays

Leaf discs (0.5cm²) were collected from the leaves used for gas exchange experiments and frozen in liquid nitrogen immediately after the experiment. Soluble protein was extracted by grinding one frozen leaf disc in a cold Tenbroeck homogenizer with 0.5ml extraction buffer [50mM HEPES, 1mM EDTA, 0.1% (v/v) Triton X-100, 10mM DTT, 1% (w/v) PVPP, 1% (v/v) protease inhibitor cocktail (Sigma), pH 7.8]. Extracts were centrifuged at 13000rpm for 30s. Spectrophotometric assays were performed to determine Rubisco and PEPC activities as described in Pengelly et al. (2010).

CA activity was measured in the same extract used for PEPC and Rubisco activity measurements, using a membrane inlet mass spectrometer to measure the rates of ¹⁸O exchange from labeled ¹³C¹⁸O₂ to H₂ ¹⁶O at 25°C with a subsaturating total carbon concentration of 1mM (Badger and Price, 1989; von Caemmerer et al., 2004; Cousins et al., 2008). The hydration rates were calculated from the enhancement in the rate of ¹⁸O loss over the uncatalyzed rate, and the nonenzymatic first-order rate constant was applied at pH 7.4 (k _c=6.22x10⁻¹¹/[H⁺]+3.8x10⁻²=0.0396), appropriate for the mesophyll cytosol, at a CO₂ concentration of 8μM, which is approximately the CO₂ concentration in the mesophyll of F. bidentis (Jenkins et al., 1989; von Caemmerer et al., 2004). When CA is in the chloroplast, which is tipically the case in C₃ plants like F. pringlei, our calculations underestimate its in planta activity by ~10% due to the effect of the higher chloroplastic pH on k _c (k _c=0.0442 at pH 8).

Results

O₂ response of CO₂ assimilation rate and compensation point

The effect of pO₂ on CO₂ assimilation rate and the compensation point (Γ) was measured at 380 μbar reference CO₂, an irradiance of 1500 μmol quanta m⁻² s⁻¹ and 25 °C (Fig. 1).

Fig. 1.

The responses of (a) CO₂ assimilation rate, A and (b) compensation point (Γ) in F. pringlei, F. floridana, F. brownii and F. bidentis to changes in atmospheric pO₂. Assimilation rate is expressed as a percentage of the assimilation rate at 19mbar O₂ (average of 28.7±1.13 μmol m⁻² s⁻¹ for F. pringlei, 24.2±1.52 for F. floridana, 20.6±1.2 for F. brownii and 21.7±0.49 for F. bidentis). Measurements were made at 25°C and 385 μbar CO₂ (R), and an irradiance of 1500 μmol m⁻² s⁻¹. Values represent averages and standard error of four replicates.

In F. pringlei, increasing pO₂ caused a decrease in CO₂ assimilation rate, a response typical of a C₃ plant. Consistent with this, the Γ increased with increasing pO₂, ranging from 5.6 ubar at 19mbar O₂ to 53 µbar at 285mbar O₂.

In the C₄ species F. bidentis, the effect of oxygen was very small, with only a 5% decrease in CO₂ assimilation rate at the highest tested pO₂. Γ in these plants barely changed with pO₂, and ranged from 0.2 to 1.2 μbar.

The effect of O₂ on Γ in F. brownii was also very small and similar to the C₄ species F. bidentis, ranging from 1.3 to 3.1 μbar (Fig. 1b). However, the inhibitory effect of O₂ on CO₂ assimilation rate was more pronounced, and resulted in an intermediate response of CO₂ assimilation rate to increasing pO₂ (Fig. 1a).

The O₂ response of Γ in F. floridana was intermediate between C₃ and C₄ species (2.3–18μbar; Fig. 1b), as has been previously shown (Ku et al., 1991). However, in our experiments the inhibitory effect of O₂ on photosynthesis was smaller than that previously reported by these authors and strikingly similar to that in F. brownii when pO₂ was 200mbar or lower, despite the important differences in the enzyme compartmentation between these two species (Fig. 1a). Only at 290mbar O₂ the inhibition of photosynthesis was higher for F. floridana, with a reduction of a 22%, compared to that in F. brownii (15% inhibition).

Stomatal conductance and C _i increased slightly with pO₂, with the exception of F. bidentis, which remained stable, and were considerably higher in the C₃ species F. pringlei at any pO₂ (Supplementary Fig. S1 at JXB online).

Rubisco, PEPC and CA activity

In vitro Rubisco, PEPC and CA activities were analyzed in extracts from the same leaves on which the concurrent gas exchange and Δ measurements were made (Fig. 2). Rubisco activity was higher in F. floridana (average of 74.9 μmol m⁻² s⁻¹), followed by F. pringlei (60.5 μmol m⁻² s⁻¹), F. brownii (49.2 μmol m⁻² s⁻¹) and F. bidentis (39.7 μmol m⁻² s⁻¹). PEPC activity was lowest in F. pringlei (2.9 μmol m⁻² s⁻¹) and, notably, four times higher in F. floridana (13.8 μmol m⁻² s⁻¹). F. brownii showed a PEPC activity closer to that of F. bidentis (79.3 and 91.8 μmol m⁻² s⁻¹ respectively). CA activity was similar and high in F. bidentis and F. brownii (1278.7 and 1464.5 μmol m⁻² s⁻¹ respectively), and lower in F. pringlei and F. floridana (614.9 and 623.6 μmol m⁻² s⁻¹ respectively).

Fig. 2.

(a) In vitro Rubisco, PEPC and CA activities in F. pringlei, F. floridana, F. brownii and F. bidentis, measured from samples of the same leaves used for gas exchange and expressed on a leaf area basis. (b) PEPC to Rubisco activity ratio in these experiments. Values represent mean and standard error of four experimental replicates.

The relative activity of PEPC to Rubisco was lowest in F. pringlei and highest in F. bidentis (Fig. 2b). F. floridana showed a PEPC:Rubisco ratio 3.4 times greater than the C₃ species, and F. brownii was closer to the C₄ species.

CO₂ assimilation rate and carbon isotope discrimination

Measurements of carbon isotope discrimination concurrently with gas exchange were performed under a range of CO₂ concentrations at 19mbar O₂ on 3–4 plants from each species (Fig. 3). At this low pO₂, photorespiration is greatly reduced and the effect of the C₂ cycle is negligible. Thus, small differences in the level of C₄ activity or mesophyll Rubisco activity are easier to detect.

Fig. 3.

Concurrent measurements of (a, d) CO₂ assimilation rate, A, (b, e) carbon isotope discrimination, Δ, and (c, f) the ratio of intercellular to ambient CO₂, C _i/C _a, as a function of intercellular CO₂ (C _i) in F. pringlei, F. floridana, F. brownii and F. bidentis. Values represent averages and standard error of 4 replicates. Measurements were made at 19 mbar O₂, a leaf temperature of 25°C and an irradiance of 1500 μmol m⁻²s⁻¹.

F. pringlei and F. bidentis showed the typical C₃ and C₄ response of CO₂ assimilation rate to increasing C _i, respectively (Fig. 3a). The initial slope of the A/C _i curve in F. floridana was closer to that in the C₃ species, F. pringlei, whereas that of F. brownii was more similar to that of the C₄ species, F. bidentis, although in both intermediate species the sharp saturation typical of the C₄ species was missing. The maximum apparent assimilation rates in both intermediates were higher than those of the C₃ and C₄ species.

Carbon isotope discrimination measured over the defined range of pCO₂ provided clear differences between the four species (Fig. 3b). Δ was greatest in F. pringlei at any C _i, ranging from 16‰ to 24.4‰. Discrimination in F. floridana followed a similar trend than that in the C₃ species, with Δ generally increasing with C _i, but Δ was lower than in F. pringlei across the whole experimental range, ranging from 12.2‰ to 18.6‰. The response of C _i/C _a to CO₂ concentration was parallel to that of Δ in F. pringlei and F. floridana, reflecting the strong dependence of Δ on the ratio C _i/C _a in C₃ species and also in F. floridana (Fig. 3c). The initial decrease of Δ in F. pringlei is also caused by a drop in C _i/C _a, which is in turn driven by a reduction of stomatal conductance with increasing C _i when C _i is lower than 200μbar.

In F. bidentis, as expected from a C₄ plant, discrimination was low (2–4‰) and decreased slightly with increasing C _i. Δ in F. brownii was similar to F. bidentis at C _i under 95 μbar (3.5–2.6‰), but above that the value of Δ increased with increasing C _i, to a maximum of 6.1‰.

Measured Δ is shown with respect to C _i/C _a in Fig. 4. The theoretical lines assume infinite mesophyll conductance, which explains why both F. pringlei and F. floridana fell below the theoretical response for C₃ plants, with Δ and C _i/C _a generally lower in F. floridana. In F. bidentis, the result was as predicted by a theoretical CO₂ response of Δ for a C₄ plant when $\varphi =0.25,$ whereas F. brownii only fitted the expected response at low C _i/C _a, with Δ higher than predicted at high C _i/C _a.

Fig. 4.

Observed carbon isotope discrimination, Δ expressed as a function of the ratio of intercellular to ambient CO₂, C _i/C _a, in F. pringlei, F. floridana, F. brownii and F. bidentis. Values are the same as plotted in Fig. 3. Solid line represents the theoretical response of Δ to C _i/C _a in C₃ plants ($\Delta =4.4+\frac{C_i}{C_a}(29-4.4)$ ; (Roeske and O’Leary, 1984; Evans et al., 1994). Dashed line represents the theoretical response of Δ to C _i/C _a in C₄ plants, $\Delta =4.4+\frac{C_i}{C_a}[-5.7-4.4+\varphi \left(29-1.8\right)]$ (Henderson et al., 1992) when φ=0.25.

Modeling CO₂ assimilation rate and carbon isotope discrimination in C₃-C₄ intermediate species

In order to evaluate the contribution of the C₄ cycle to overall photosynthesis in the intermediate species F. floridana and F. brownii, the mathematical models proposed here for A and Δ responses to C _i (eqs 6 and 10, respectively) were fitted concurrently to the observed results (Fig. 5). By simultaneously fitting both models using the same set of parameters, the accuracy of the predictions increases because some combinations of assigned constants that may result in a good fit for one of the models are unacceptable for the other. For comparison, the same strategy was also applied to the C₃ and C₄ species (see Supplementary Fig. S2).

Fig. 5.

Comparison between modeled and measured responses of CO₂ assimilation rate, A, and carbon isotope discrimination, Δ, to variation in intercellular pCO₂, C _i, in the C₃-C₄ intermediate species F. floridana and F. brownii. Measured A (a) and Δ (b) as a function of C _i in F. floridana (empty triangles), compared with the modeled responses predicted by C₃-C₄ photosynthetic model assuming an active C₄ cycle (solid lines) or no C₄ cycle activity (dashed lines). Measured A (c) and Δ (d) as a function of C _i in F. brownii (white squares), compared with the modelled responses using the C₃-C₄ models assuming Rubisco activity in the mesophyll cells (solid lines) or a strict compartmentalization of Rubisco in the bundle sheath cells (dashed lines). Parameters used for model simulations are presented in Table 1.

Table 1 shows the values assigned for fitting purposes and their source. Rubisco K _C and K _O (Michaelis–Menten constants for CO₂ and O₂, respectively) in the four Flaveria species analyzed here have been previously reported (Kubien et al., 2008), and V _c,max and V _p,max are from our own in vitro experiments. We assigned reasonable values for maximum electron transport (J _max). Leakiness$(\varphi )$ was assigned so that the sum of the squares of the variances between the measured and modeled A, and between the measured and modeled Δ, was minimum. The distribution of Rubisco between the mesophyll and the bundle sheath in the intermediate species can be adjusted in the models by the assigned V _m,max (maximum rate of Rubisco carboxylation in the mesophyll) value. When V _m,max equals the V _c,max observed in vitro, all Rubisco is in the mesophyll. A lower asigned V _m,max indicates that part of the Rubisco activity is contained in the bundle sheath cells.

Mesophyll conductance (g _m) for F. pringlei was calculated from concurrent gas exchange and carbon isotope discrimination measurements at 19mbar O₂ and a range of reference pCO₂ as previously described (Tazoe et al., 2011; Farquhar and Cernusak, 2012; Evans and von Caemmerer, 2013). Results show that g _m decreases from 0.62±0.1 to 0.33±0.03mol m^–2 s^–1 bar^–1 with increasing C _i when atmospheric pCO₂ is lower than ambient, and then remains stable at higher pCO₂ (Fig. 6). The CO₂ dependence of g _m in F. pringlei is described by the polinomial function g _m=10^–6·C _i ²−0.0013·C _i+c, where c=0.666. In C₄ and C₃-C₄ intermediate species, g _m cannot be obtained from concurrent gas exchange and Δ ¹³C measurements, so the same CO₂ dependence of g _m was assumed for F. bidentis, F. brownii and F. floridana, and the constant c was calculated from model fitting so that the sum of variances between the measured and modeled A, and between the measured and modeled Δ, was minimum (Table 1). The resulting g _m are shown in Fig. 6. Methods for obtaining g _m in C₄ and C₃-C₄ intermediate species, based on ¹⁸O discrimination measurements, are currently being developed (S. von Caemmerer, unpublished results).

Fig. 6.

Response of mesophyll conductance (g _m) to changes in atmospheric pCO₂. In F. pringlei, g _m was calculated from concurrent gas exchange and Δ measurements made at 19mbar pO₂. The values for F. floridana, F. brownii and F. bidentis were assigned assuming the same response of g _m to C _i as observed in F. pringlei, scaled from model fitting.

The A and Δ responses to increasing C _i predicted with this strategy were reasonably close to the measured values for F. pringlei and F. bidentis (Supplementary Fig. S2).

In an exercise to prove the predictive value of these models for the presence of low levels of activity of the C₄ component, we attempted to fit the models for F. floridana under two different premises. In one case, we assumed a certain level of effective C₄ cycle contribution to overall carbon assimilation (Fig. 5a, b, solid lines). In the second case, we considered no C₄ activity and values were assigned to obtain the best possible fitting ignoring the measured enzyme activities (Fig. 5a, b, dashed lines). The models could only be fitted to the measured values of Δ and A if some C₄ activity, specified by a V _p,max close to our in vitro measurements, was assumed.

A similar approach was used with F. brownii. In one case, the models were fitted assuming the presence of Rubisco in the mesophyll, and in the other case the model was fitted as if it were a strict C₄ plant (Fig. 5c, d). The predicted responses approached the measured values only if ~30% of Rubisco activity was located in the mesophyll (V _m,max =15 μmol m⁻² s⁻¹; observed in vitro V _c,max =50 μmol m⁻² s⁻¹).

A comparison of Δ and Δ _bio highlights the fact that CO₂ diffusion processes have a large influence on Δ (Figs 3, 7). Δ _bio was calculated from eq. 12 using gas exchange and Δ measured values. Calculation of Δ _bio factors out the contribution from CO₂ diffusion and shows that the biochemical fractionations are different in the species analyzed. In F. floridana, Δ _bio was high and increasing with C _i. In F. brownii, Δ _bio also increased with increasing C _i, whereas in the C₄ species F. bidentis Δ _bio generally decreases with C _i.

Fig. 7.

Biochemical fractionation (Δ _bio), as a function of intercellular CO₂ (C _i) in F. floridana, F. brownii and F. bidentis. Δ _bio was calculated from eq. 12 using the combined gas exchange and Δ measurements shown in Fig. 3. Δ _bio could not be calculated for F. pringlei because g _m is obtained from Δ measurements in this species, so both factors are not independent. Values represent averages and standard error of four replicates.

The A and Δ responses to C _i could be modeled assuming a constant g _m without important differences (data not shown). However, the calculation of biochemical fractionation (Δ _bio) from eq. 12 is dependent on g _m, and thus the dependence of g _m on C _i must have an effect on Δ _bio. To show the magnitude of this effect, the C _i response of Δ _bio was calculated from eq. 12 and the gas exchange and Δ measurements, assuming either variable g _m, assigned as previously explained in this section, or constant g _m, calculated as the average of the variable g _m values obtained for each species (see Supplementary Fig. S3). As a reference, the C _i response of Δ _bio was calculated from eq. 14 (modelled Δ _bio) after fitting the models for the C _i responses of A and Δ using variable g _m.

Estimation of the C₄ (bundle sheath) photosynthesis contribution to total photosynthesis

The relative contribution of the bundle sheath to total photosynthesis in the intermediate species was estimated from A _s in eq. 2, after fitting the models to our observed results (Fig. 8). Because the experiments were performed under low O₂, photorespiration is greatly reduced and it can be assumed that all the CO₂ assimilated in the bundle sheath is transported by the C₄ cycle. The contribution of the bundle sheath to total photosynthesis in both F. floridana and F. brownii decreased with increasing C _i. In F. brownii, almost all carbon was fixed by Rubisco in the bundle sheath at very low C _i, but up to 25% of fixation occurred via Rubisco in the mesophyll at high C _i. In F. floridana, the maximum estimated contribution of the bundle sheath photosynthesis via the C₄ cycle was 21% at very low C _i and it dropped to 12% at the highest C _i analyzed.

Fig. 8.

CO₂ response of the estimated contribution of the C₄ cycle and the mesophyll C₃ cycle in the intermediate species F. floridana and F. brownii, expressed as a percent of total CO₂ assimilation rate, under low pO₂.

Discussion

Effect of O₂ on carbon assimilation and compensation point

The oxygen responses of CO₂ assimilation and the compensation point have been used in the past as a tool to identify and characterize C₃-C₄ intermediate species (Sayre and Kennedy, 1977; Monson et al., 1984; Dai et al., 1996; Vogan et al., 2007). As only mesophyll Rubisco is exposed to air oxygen, its effect on CO₂ assimilation and Γ decreases with increasing proportions of the enzyme allocated to the bundle sheath. However, it is difficult to separate and quantify the effects of the C₂ and C₄ cycles from studies on the O₂ response of CO₂ assimilation, as both cycles contribute to reduce the negative effect of photorespiration in carbon assimilation and the compensation point. Moreover, the efficiency of the C₂ cycle varies between different intermediate species, as does the contribution of the C₄ cycle (Cheng et al., 1988; Keerberg et al., 2014).

In this work, the O₂ response of carbon assimilation, and especially Γ, in F. brownii was very close to that of the C₄ species F. bidentis. A highly efficient C₂ cycle would have a greater impact on the O₂ sensitivity of Γ than on carbon assimilation and that, combined with high in vitro PEPC and CA activities at the same level as the C₄ species F. bidentis, eliminates the effect of pO₂ on Γ almost completely (Cheng et al., 1988; Ku et al., 1991). Previous studies initially classified F. brownii as a C₄ species, but it was later demonstrated that the enzyme compartmentation is incomplete in this plant (Monson et al., 1987; Ku et al., 1991). The small proportion of Rubisco present in the mesophyll is reflected in the sensitivity of assimilation rate to pO₂.

CA activity in F. floridana is similar to F. pringlei but PEPC activity is four times higher (13.8 μmol m⁻² s⁻¹), consistent with Ku et al. (1991) and supporting the hypothesis of an active C₄ cycle. However, PEPC activity is still low when compared with F. bidentis (91.8 μmol m⁻² s⁻¹), indicating that the activity of the C₄ cycle in this plant is small. In our experiments, the O₂ sensitivity of Γ in F. floridana is intermediate, and the O₂ response of CO₂ assimilation rate is remarkably close to that of F. brownii.

Previous studies have reported a C₃-like O₂ response in F. floridana (Dai et al., 1996; Monson et al., 1986), which differs from our observations. Although the reason for this discrepancy is not known, it must be noted that O₂ sensitivity measurements are affected by variation of parameters like temperature or stomatal conductance between measurements at different pO₂. These interactions increase the difficulty of estimating the activity of the C₄ cycle from O₂ response experiments.

Signature of C₄ photosynthesis in the CO₂ response of Δ in intermediate species

The different CO₂ responses of Δ in the intermediate C₃-C₄ species, relative to the C₃ or C₄ species, can be attributed to the different ratios of PEPC/Rubisco activity in the mesophyll. The lower Δ observed in F. floridana, relative to F. pringlei, is partially attributable to a lower C _i/C _a, but their different Δ _bio indicates an influence of the PEPC to Rubisco ratio, especially at low C _i.

Interestingly, F. brownii and F. bidentis show similar Δ at low C _i, but it increases in F. brownii with increasing pCO₂ instead of decreasing as in the C₄ plant. This particular response can be attributed to the activity of the small fraction of Rubisco in the mesophyll that would have a stronger influence at high pCO₂. In F. floridana, Rubisco is abundant in the mesophyll but PEPC activity is low, and as a consequence the greatest effect of the C₄ cycle activity is observed at very low pCO₂, with a greater reduction of Δ compared to the C₃ species. Both results indicate that the contribution of mesophyll Rubisco to overall assimilation is more important under high pCO₂, and of the C₄ cycle at low pCO₂. The fact that environmental conditions affect the contribution of C₄ photosynthesis may explain ambiguous results on previous analyses of dry matter δ¹³C in F. floridana and other intermediates, which showed C₃-like ratios (Monson et al., 1988; Byrd et al., 1992). δ¹³C is a result of carbon discrimination during the leaf growth, thus it integrates the effect of variable environmental conditions. In the online experiments presented here, instant discrimination is measured under controlled conditions, highlighting their influence. By performing the analyses under low pO₂, the effect of photorespiration and subsequent refixation through the C₂ cycle is greatly reduced, emphasizing the differences in biochemical fractionation caused by the presence of C₄ activity.

Although the CO₂ response of A is also influenced by different relative activities of mesophyll Rubisco and PEPC, the effect of each enzyme in this case is difficult to separate. The greater initial slope of the A/C _i curve in F. floridana, compared with F. pringlei, reflects the slightly greater PEPC activity detected in our in vitro assays, but could also be attributed to higher Rubisco activity. In the same sense, the initial slope of the A/C _i curve in F. brownii and F. bidentis are similar and typically C₄, whereas their Δ are different.

Concurrent model fitting reveals C₄ activity in F. floridana

The strategy to evaluate the contribution of the C₄ cycle to total carbon assimilation in intermediate species presented in this work is based on concurrently measuring and model-fitting the CO₂ responses of carbon assimilation and discrimination.

Mathematical modeling has proved to be a powerful tool to get a deeper insight into the biochemical and physiological basis of the observed responses of carbon assimilation and discrimination, and it has been used to estimate parameters such as the maximum carboxylase activity of Rubisco in vivo (V _C,max) and g _m in C₃ species, or V _P,max and leakiness in C₄ systems (Tazoe et al., 2011; Ubierna et al., 2011; Walker et al., 2013; Sharwood and Whitney, 2014). However, in most cases there is more than one unknown variable in the equations that represent those responses. This is especially problematic in intermediate species, where the number of factors affecting those responses is greater than in C₃ or C₄ plants. By concurrently fitting the CO₂ responses of A and Δ in each experiment with the same set of constants, the range of values that can be assigned to these variables to obtain a satisfactory fitting is reduced. In this work, the activities of photosynthetic enzymes were analyzed in vitro to further reduce the number of unknowns, providing more accurate predictions. This method confirmed the presence of Rubisco activity in the mesophyll of F. brownii, which was already known (Cheng et al., 1988), but more interestingly indicated that F. floridana harbors an active C₄ cycle. This C₄ activity causes a change in the biochemical fractionation, compared to F. pringlei, which is evident at any C _i analyzed. This is consistent with the increased activity of PEPC and previous observations based on ¹⁴CO₂ pulse-chase experiments (Monson et al., 1986; von Caemmerer and Hubick, 1989). It is important to note that other studies based on δ¹³C analyses, metabolite dynamics and O₂ response of carbon assimilation and Γ were unable to conclusively prove a contribution of the C₄ cycle to overall photosynthesis in F. floridana, and the presence of a futile C₄ cycle was proposed where most or all the CO₂ released in the bundle sheath is not fixed and leaks back to the mesophyll (Monson et al., 1988; Leegood and von Caemmerer, 1994; Dai et al., 1996). However, other authors have already indicated that in F. floridana the C₄ cycle may contribute up to 50% of the total CO₂ fixation (Ku et al., 1991). In this work, the contribution of the mesophyll and the bundle sheath Rubisco to overall carbon assimilation was calculated for F. brownii and F. floridana. In both intermediate species, the contribution of the C₄ cycle, or bundle sheath Rubisco, is highest at very low pCO₂, and decreases with increasing pCO₂. This reflects the lower apparent K _c of PEPC compared to that of Rubisco (Bauwe, 1986; Kubien et al., 2008).

An improved equation describing CO₂ response of Δ in intermediate species

An equation describing photosynthetic carbon isotope discrimination (Δ) that is applicable for C₃, C₄ and C₃-C₄ photosynthesis is provided and applied in this study. It allows the calculation of the biochemical fractionation occurring for the different photosynthetic pathways as a function of C _i and takes into account g _m and the ternary effects of transpiration rate. The biological relevance of g _m, and its influence on Δ, has been reported extensively and incorporated in mathematical models for C₃ species (Evans et al., 1986; von Caemmerer and Evans, 1991; Tazoe et al., 2011). When mesophyll conductance is considered in C₃ species, C _c (pCO₂ at the site of Rubisco) can be estimated and is lower than C _i, and this affects the estimates of Rubisco carboxylations. The same applies in intermediate species, where assimilation and discrimination by mesophyll Rubisco is dependent on the concentration of CO₂ diffusing from the intercellular space. For model fitting purposes, the calculated C _c was used as the available CO₂ for both PEPC and mesophyll Rubisco in the case of the intermediate species. The models presented in this work assume that pCO₂ is the same in the cytosol and the chloroplast.

The effect of pCO₂ on g _m has been studied by other authors, with results depending on the species analyzed. Whereas previous results showed that g _m is not affected by pCO₂ in wheat (Tazoe et al., 2009), other authors reported an inverse correlation in several C₃ species (Flexas et al., 2007; Tazoe et al., 2011). We observed that g _m is dependent on pCO₂ in the C₃ F. pringlei, and assumed that the same is true for the C₄ and intermediate species analyzed. Although the effect of using either constant or variable g _m on the models of the CO₂ responses of carbon assimilation and discrimination has only a minor effect at low C _i, it is important for the calculation of Δ _bio and thus the contribution of the C₄ and C₃ cycles to overall carbon assimilation, especially at low C _i. The fact that Δ _bio is similar when calculated using either constant or variable g _m in F. brownii and F. bidentis reflects the lower relevance of g _m when the CO₂ concentrating mechanism is expressed at high levels.

Conclusion

Concurrent Δ and gas exchange measurements and modeling provide a powerful diagnostic tool for C₄ photosynthesis. Performing the measurements under controlled environmental conditions, especially low pO₂, allows the detection and estimation of the C₄ cycle activity in C₃-C₄ intermediate species even when it is low. This approach confirmed the presence of active Rubisco in the mesophyll of F. brownii, and revealed a contribution of the C₄ cycle to total carbon assimilation in F. floridana. However, the carbon isotope signal is complex and not all its components are well understood, so some caution is required. We show for example that a CO₂ dependence of g_m affects the calculation of the biochemical fractionation, and thus the contribution of the C₄ cycle to overall CO₂ assimilation.

Supplementary data

Supplementary data are available from JXB online.

Figure S1. Responses of C _i and stomatal conductance to changes in atmospheric pO₂.

Figure S2. Models of CO₂ response of assimilation rate and carbon isotope discrimination in the C₃ and C₄ species.

Figure S3. Effect of assuming constant or variable g _m in the calculation of the biochemical fractionation.