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Revista chilena de historia natural

versión impresa ISSN 0716-078X

Rev. chil. hist. nat. vol.87  Santiago  2014 



Mesophyll conductance constrains photosynthesis in three common sclerophyllous species in Central Chile


Carla E BritoI; Horacio E BownI, *; Juan-Pablo FuentesI; Nicolás FranckII; Jorge F Perez-QuezadaIII

IFacultad de Ciencias Forestales y de la Conservación de la Naturaleza, Universidad de Chile, Casilla 9206, Santiago, Chile
IICentro de Estudios de Zonas Áridas, Universidad de Chile, Casilla 129, Coquimbo, Chile
IIIFacultad de Ciencias Agronómicas, Universidad de Chile, Casilla 1004, Santiago, Chile



BACKGROUND: Quillaja saponaria Mol., Cryptocarya alba Mol. Looser, and Lithraea caustica Molina Hook et Arn., are common sclerophyllous species in Mediterranean Central Chile. Mesophyll conductance, gm, may strongly limit photosynthesis in these semiarid environments.
RESULTS: Simultaneous measurements of gas exchange and chlorophyll fluorescence were carried out in 45 nursery plants from these species to determine diffusional and biochemical limitations to photosynthesis. Values of stomatal conductance, gs, were greater than those of mesophyll conductance, gm, while their ratio (gm/gs) was not influenced by species being on average 0.47. Relative limitations posed by mesophyll conductance to photosynthesis, Lm, (0.40 ± 0.02) were high compared to those imposed by stomata, Ls (0.07 ± 0.01). The average CO2 concentration in the intercellular air spaces (Ci) was 32
µmol mol-1 lower than in the atmosphere (Ca), while the average CO2 concentration in the chloroplasts (Cc) was 131 µmol mol-1 lower than Ci independent of species. Maximal rates of Rubisco carboxylation, Vcmax, and maximal electron transport rates driving regeneration of RuBP, Jmax, ranged from 13 to 66 µmol CO2 m-2 s-1 and from 33 to 148 µmol electrons m-2 s-1, respectively, and compare well to averages for C3 plants.
CONCLUSIONS: Photosynthetic performance was in the series: Q. saponaria > C. alba
 ≥ L. caustica, which can be attributed first to mesophyll conductance limitations, probably mediated by leaf anatomical traits and then to species specific foliage N partitioning strategies.

Keywords: Chloroplastic CO2 concentration; Electron transport; Mesophyll conductance; Rubisco carboxylation; Sclerophyllous species



The biochemical model of leaf photosynthesis originally proposed by Farquhar et al. (1980) and later improved (von Caemmerer and Farquhar, 1981; Sharkey, 1985; Harley and Sharkey,1991 is widely used in ecophysiological research for describing CO2 exchange processes and to scale carbon exchange from leaves to canopies (Baldocchi and Harley 1995; McMurtrie et al.1992; Whitehead et al. 2004). In this model, photosynthesis is considered to be limited by the maximal rate of ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) carboxylation, Vcmax, and by the maximal electron transport rate driving regeneration of RuBP,Jmax, and by triose phosphate utilization (TPU) (Farquhar et al. 1980; von Caemmerer 2000; von Caemmerer and Farquhar 1981). These parameters are fitted to the response of photosynthesis, A, to the CO2 concentration in the intercellular air spaces, Ci, known as A/Cicurves. Values of Ci, are estimated from stomatal conductance to CO2 transfer, gs, and the ambient CO2 concentration external to the leaf, Ca. This model has been used to explain the mechanisms of photosynthetic acclimation to elevated atmospheric CO2 (Griffin et al. 2000; Hogan et al. 1996; Kellomaki and Wang 1996; Murray et al. 2000; Turnbull et al. 1998), the influence of global warming on plant carbon budgets (Turnbull et al. 2002), and for identifying factors limiting photosynthesis under water stressed conditions in Mediterranean plants (Grassi and Magnani 2005; Gulias et al. 2002; Niinemets et al. 2009b), among others.

In most studies, the mesophyll conductance of CO2 from the intercellular air spaces to the sites of Rubisco carboxylation in the chloroplasts, gm, has been considered to be sufficiently large to be negligible (Farquhar et al. 1980). However, there is a growing awareness that the significance ofgm in limiting photosynthesis can be similar to the limitation imposed by stomata (Flexas et al.2008; Harley et al. 1992; Loreto et al. 1992; von Caemmerer 2000; Warren and Adams 2006; Warren et al. 2003). As a result, Ci is greater than the CO2 concentration in the chloroplasts, Cc, and values of Vcmax and Jmax are underestimated when fitted to estimates of Cirather than Cc.

Arid and semiarid lands account for 41% of Chile's continental territory, covering about 31 million ha (Benites et al. 1994). Within this area, semiarid sclerophyllous (i.e. a woody plant with small coriaceous evergreen leaves dominant of the Mediterranean region) shrublands and forests extends from 32-36° S latitude (~345,000 ha) in Central Chile (Armesto et al. 2007; CONAF 1999); exhibiting high levels of endemism, and are therefore considered a priority for biodiversity conservation (Arroyo et al. 2004). These ecosystems are subjected to high radiative and water stresses that limit their development and reproduction (Cabrera 2002); and, additionally, have a long history of degradation by human activity, which may represent an important adaptive pressure (Galmes et al. 2007). Therefore, understanding how environmental limitations are imposed on the carbon budget is relevant to accurately estimate carbon uptake and water use by sclerophyllous species.

There is extensive work on comparisons of gm between species and plant functional groups (Loreto et al. 1992; De Lucia et al. 2003; Hassiotou et al. 2009; Niinemets et al. 2009b). However, little is known about the extent to which gm regulates the rate of photosynthesis in sclerophyllous species of central Chile. Consequently we chose to work with three native sclerophyllous species:Quillaja saponaria Mol., Cryptocarya alba Mol. Looser and Lithraea caustica Molina Hook et Arn., to determine stomatal, mesophyll and biochemical limitations to photosynthesis. Specifically, we assessed whether mesophyll conductance to CO2 induces similar constrains to photosynthesis across different sclerophyllous species.


Plant material

Plant material consisted of 15 randomly selected plants from each of the following species: Q. saponaria, C. alba and L. caustica, from a nursery of the Faculty of Forestry and Nature Conservation at University of Chile, Santiago, Chile. Seeds were sown in the winter 2008, and transferred during spring 2008 and spring 2009 to 12 × 15 cm (0.25 L) and 20 × 30 cm (2 L) black polyethylene bags filled with an even mixture of composted plant residues, soil and sand. No fertilizers were applied. Plants developed under 46% shading (in order to mimic the light environment experienced by saplings in their natural habitat) and received weekly irrigation during winter, and daily irrigation to field capacity in other seasons. At the time of measurements, we selected fifteen homogenous plants of Q. saponaria, C. alba and L. caustica which exhibited an average (±1 SD) plant height of 82.9 ± 14.4 cm, 63.4 ± 18.9 cm, and 47.6 ± 15.3 cm; and a collar diameter of 7.5 ± 0.8 mm, 9.1 ± 1.4 mm, and 11.0 ± 2.6 mm, respectively. All gas exchange and chlorophyll fluorescence measurements were taken in a laboratory without thermal regulation, with open windows to allow circulation of fresh air. Measurements took 45 days; with only one plant measured per day, spread in the summer period from December 20, 2010 to March 15, 2011; six months after plants were transferred from small to large containers. Gas exchange measurements for each individual plant typically took all day between 10 am and 6 pm; and followed the exact same sequence of measurements for all plants (see below). We alternated species each day in order to avoid differences brought about by day to day meteorological changes.

Gas exchange measurements

Simultaneous measurements of gas exchange and chlorophyll a fluorescence were carried out on the 45 selected plants, using a portable photosynthesis system (CIRAS-2, PP Systems, MA, USA) equipped with an integrated chlorophyll fluorescence and gas exchange chamber (PLC6-U Auto Leaf Cuvette, PP System, MA, US). Plants were shifted from the nursery to the laboratory the day before measurements were undertaken.

For each plant, a fully-developed leaf in the upper third of the plant was chosen and placed inside a circular 2.5 cm2 (18 mm in diameter) cuvette. Temperature in the cuvette (block) was maintained at 25ºC while leaf-to-air vapour pressure deficit (VPD) was maintained below 1 kPa. Each leaf was left to equilibrate inside the cuvette for 10 min at about 368 ± 3 µmol mol-1 CO2concentration and saturating irradiance (2,000 µmol photons m-2 s-1), before measuring the response of net assimilation (A) to intercellular CO2 concentration (Ci). External CO2 concentration (Ca) was supplied with a CO2 mixer across the sequence 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200 and 1500 µmol mol-1, with saturating irradiance, Q (400 - 700 nm), maintained at 2,000 µmol m-2 s-1. Measurements were recorded after values of A, Ci and gsbecame stable but with a minimum waiting time of 3 min at each step within the sequence. At each value of Ci, measurements of fluorescence for the light-adapted leaf were made simultaneously to the gas exchange measurements. Values of F and Fm' (the steady and maximal fluorescence respectively), were used to calculate photochemical efficiency of photosystem II, ΦPSII. The response of net assimilation to irradiance (A/Q curves) was measured immediately after each A/Cicurve ended, across the Q sequence: 0, 50, 100, 150, 200, 300, 400, 500, 800, 1000, 1200, 1400, 1600, 1800 and 2000 µmol m-2 s-1, with ambient CO2 concentration maintained at 368 ± 3 µmol mol-1 using a CO2 mixer. The A/Ci and A/Q curves were measured on the same foliage sample.

Models fitted

The biochemical model of leaf photosynthesis by Farquhar et al. (1980) describes the rate of photosynthesis (A) as:

where Ac and Aq are the photosynthetic rates limited by Rubisco carboxylation and by electron transport rate respectively, and min {} indicates the minimum of these two rates. Rd is the rate of daytime respiration resulting from processes other than photorespiration. The photosynthetic rate limited by Rubisco carboxylation (Ac) is given by:

where Vcmax is the maximum rate of Rubisco carboxylation under saturating RuBP and CO2, Ciand Oi are the intercellular CO2 and O2 concentrations, Γ* is the CO2 compensation concentration in the absence of day respiration, and Kc and Ko are the Michaelis constants for CO2 and O2, respectively.

The photosynthetic rate limited by RuBP regeneration driven by electron transport (Aq) is given by:

where J is the rate of electron transport at a given irradiance Q.

Prioul and Chartier (1977) described the response of A to Q by a non-rectangular hyperbola as:

where θ is the convexity of the non-rectangular hyperbola, α is the initial slope of the A/Q curve (often referred as 'apparent maximum quantum efficiency'), Asat is the light-saturated photosynthetic capacity and Rdark the respiration rate at zero irradiance. Individual A/Q response curves were fitted using Eq. 4 in order to estimate α, Asat and Rdark. The light saturation point,Qsat, was calculated as the irradiance for which 90% of Asat was achieved.

Values of the rate of mitochondrial respiration in the light, Rd*, and the chloroplastic CO2compensation point, Γ*, were estimated for each sample using the Laisk method (von Caemmerer 2000). Briefly, the A/Ci response was measured at three levels of low irradiance (Q = 50, 200 and 400 µmol m-2 s-1) for six increasing values of Ca from 25 to 400 µmol CO2 mol-1. Linear relationships between A and Ci were fitted and the point of intersection of the three lines was taken as Rd* (y-axis) and Γ* (x-axis) (Figure 1).

Figure 1 The response of net photosynthesis (A) to intercellular CO2 concentration (Ci) at three different irradiances (Q =400, 200 and 50 µmol m-2 s-1, 400 - 700 nm) for a representative foliage sample. Linear relationships between A and Ci were fitted for each Q level and their intersections averaged to yield a point which when projected to the A axis was taken as the rate of day respiration (Rd*) and when projected to the Ci axis was taken as the intercellular CO2 compensation concentration in the absence of day respiration (Ci*). The mitochondrial CO2 compensation concentration (Γ*) was calculated as Γ* = Ci* + Rd* / gm (von Caemmerer 2000), where gm is mesophyll conductance to CO2 transfer. Method originally proposed by Laisk, A. (von Caemmerer 2000) to determine Ci* and Rd. This figure is equivalent to the one presented by De Lucia et al. (2003) but with data drawn from this study.


The photosynthesis model described by Farquhar et al. (1980) (Eq. 1) was fitted to the A/Ci and A/Cc curves by non-linear least squares regression (SigmaPlot, Version 12.1, SPSS, Chicago, IL). Values of Vcmax and Rd were estimated from the lower part of the A/Ci or A/Cc curve (Ci or Cc < 220 µmol mol-1) (Eq. 1 and 2), and these values were then used to estimate Jmax over the entire range of measured Ci or Cc (Eq. 1,2 and 3). Michaelis-Menten constants of Rubisco for CO2 and O2, Kc and Ko, used in the fitting (25 °C) were 404.9 µmol mol-1 and 278.4 mmol mol-1, respectively as described by Bernacchi et al. (2001).

Calculations of mesophyll conductance

Mesophyll conductance, gm, was estimated using the "constant J" method (Harley et al. 1992; Loreto et al. (1992) where J is the rate of electron transport (Figure 2b). This method uses data in the RuBP-regeneration limited portion of the A/Ci curve, where rates of electron transport are constant. Within this region, further increases in photosynthesis with increasing Ci are due to suppression of photorespiration as the rate of carboxylation progressively substitutes the rate of oxygenation. Thus, photosynthesis is related to Cc and the relative CO2/O2 specificity of Rubisco, normally described by the chloroplastic CO2 compensation point, Γ*. The constant J method is sensitive to errors in both the rate of mitochondrial respiration in the light, Rd*, and Γ* and the approach assumes that both J and gm are constant across the range of Ca concentrations used for the measurements (Harley et al. 1992; Pons et al. (2009). The relationship of gm with Γ*, Rd*and intercellular CO2 compensation point in the absence of day respiration, Ci* is given by Γ* = Ci* + Rd*/gm (Peisker and Apel 2001; von Caemmerer 2000).

Figure 2 Graphic description of the constant J method to determine transfer conductance. (a) The rate of net photosynthesis (A; open circles) and photochemical efficiency of photosystem II (ΦPSII; solid triangles) as a function of the intercellular CO2 concentration (Ci) for a representative foliage sample. Solid lines represent a least-squares fit to the A/Ci and ΦPSII/Ci response. Open squares are observed values used to estimate mesophyll conductance (gm). These values are within the portion of the A/Ci response where ΦPSII indicated that electron transport rate was constant. (b) The variance of estimated electron transport rates, J, for different values of gm. Values of J were estimated for each of the four A values indicated as open squares in Figure 2a using the equation given by Harley et al. (1992). The gm that minimized the variance of J estimates for this foliage sample was 0.054 mol m-2 s-1 bar-1. This figure is equivalent to the one presented by Harley et al. (1992) and De Lucia et al. (2003) but with data drawn from this study.


The photochemical efficiency of photosystem II (ΦPSII) was estimated from chlorophyll fluorescence measurements as (Fm' - F) / Fm', where F and Fm' are the steady and maximal fluorescence in the light-adapted sample respectively (Schreiber et al. (1994). The values of ΦPSII are directly proportional to the rate of electron transport through photosystem II, and therefore can be used to determine the portion of the A/Ci curve where the rate of electron transport is constant (Genty et al. (1989) (Figure 2a). Optimal values for gm were resolved iteratively from three or more measurements of photosynthesis at high values of Ci that correspond with constant rates of electron transport (Singsaas et al. 2004; Warren (2006). This was done using the Generalized Reduced Gradient Nonlinear Solving Method for nonlinear optimization included in the Solver Tools of Microsoft Excel with measurements of A, Ci, Γ*, Rd* to resolve the value of gm that best explained changes in photosynthesis with changes in Ci indicated by minimum variance in J (Harley et al. 1992; Singsaas et al. 2004; Warren 2006) (Figure 2b).

Stomatal and mesophyll limitations to photosynthesis

A/Ci response and values for gs and gm, were used to partition stomatal, mesophyll and biochemical limitations to photosynthesis. Values of stomatal conductance to CO2 transfer were calculated by gs = A/(Ca-Ci) dividing values by 1.64 (Jones 1992). Relative stomatal limitations were calculated using the method of Farquhar and Sharkey (1982) as Ls = 1 - Aa-gs / Ai-gswhere Aa-gs and Ai-gs are the actual value of A and the value estimated when gs is infinite, respectively. Similarly, following Bernacchi et al. (2002), relative limitation to photosynthesis imposed by gm was calculated as: Lm = 1 - Aa-gm / Ai-gm where Aa-gm and Ai-gm are the actual value of A and the value estimated when gm is infinite, respectively. The CO2 concentration in the chloroplasts, Cc, was calculated from Cc = Ci - A/gm.

Foliage surface area and nutrient concentrations

Following the completion of A/Ci and A/Q curves, the leaf was carefully removed from the cuvette, scanned to determine leaf area by optical methods, dried at 70°C to constant mass and then both measurements used to calculate the foliage area to mass ratio, M. Foliage samples were finely ground, Dumas combusted and N concentrations determined by thermo conductivity (LECO, TruSpec CN, US) at the Laboratory of Soil and Foliar Analysis at Universidad Católica de Chile, Santiago, Chile. Nitrogen foliage concentrations are expressed on a hemi-surface area basis (Na) and photosynthetic nitrogen-use efficiency, EN, defined as Asat/Na.

Data analysis

All statistical analyses were undertaken at the plant level using The R System (R Core Team 2013). Variables were tested for normality and homogeneity of variance and transformations were made as necessary to meet the underlying statistical assumptions of the models used. All values are presented as means ± 1 standard error (n = 15) unless stated otherwise. A one way analysis of variance was used to compare diffusional and biochemical limitations to photosynthesis between sclerophyllous species. Tukey's least significant difference test was used to distinguish among individual means where applicable with a confidence level of P < 0.05. Differences in slopes and intercepts in the linear relationships between photosynthetic parameters and foliar nitrogen among sclerophyllous species were tested for significance by analysis of covariance.

Results and discussion

Overall photosynthetic performance

The Farquhar et al. (1980) model was fitted to the A/Ci curves while the Prioul and Chartier (1977) model to the A/Q curves measured for each leaf sample (n = 45). There was excellent correspondence between modelled and observed data independent of the sclerophyllous species (Figure 3).

Figure 3 The response of net assimilation to either (a) photosynthetically active radiation (A/Q curve) or (b) internal CO2 concentration (A/Ci curve) for Q. saponaria, C. alba and L. caustica. The Prioul and Chartier (1977) model was fitted to the A/Q curve while the Farquhar et al. (1980) model to the A/Ci curve.


Light response curves (Figure 3a) were very similar among species up to an irradiance level of about 500 µmol photons m-2 s-1, changing drastically thereafter with clearly distinct maximums of photosynthetic rates in the series: Q. saponaria > C. alba > L. caustica which reflects that the three species exhibited a similar initial pseudo-linear slope of the A/Q curve (the apparent maximum quantum efficiency: α) averaging 0.033 ± 0.007 mol CO2 mol-1 quanta. The rate of photosynthesis at saturating irradiance (2000 µmol m-2 s-1) and ambient CO2 concentration (Asat), on the other hand, was significantly greater in Q. saponaria (14.2 ± 0.8 µmol CO2 m-2 s-1) compared to C. alba (9.5 ± 0.4 µmol CO2 m-2 s-1) and L. caustica (7.8 ± 0.7 µmol m-2 s-1) (Figure 3a; Table 1). The light saturation point, Qsat, which followed a similar pattern as Asat, was consistently higher inQ. saponaria (1168 ± 57 µmol photons m-2 s-1) compared to C. alba (964 ± 56 µmol photons m-2 s-1) and L. caustica (987 ± 65 µmol photons m-2 s-1). The rate of respiration in the absence of light at ambient CO2 concentration (Rdark) was 0.77 ± 0.10 µmol CO2 m-2 s-1 independent of the species. The light compensation point, or the irradiance value at which the rate of photosynthesis equals zero, did not differ across species, being 38 ± 4 µmol photons m-2 s-1. The rate of transpiration at saturating irradiance (2000 µmol m-2 s-1) and ambient CO2 concentration (E) was 2831 ± 309 µmol H2O m-2 s-1 while the instantaneous water use efficiency (E/ Asat) was 337 ± 41 mol H2O mol-1 CO2, unaffected by species (Table 1).

Table 1 Comparison of leaf and photosynthetic parameters for three common sclerophyll species in Central Chile

Values are presented as means (± 1 SE, n=15) for the light-saturated rate of photosynthesis (Asat), apparent maximum quantum efficiency (á), the rate of mitochondrial respiration at zero irradiance (Rdark), light saturation (Qsat) and compensation point (Qcomp), evapotranspiration (E), the inverse of water use efficiency (1/WUE), specific leaf area (SLA), the nitrogen content on area basis (Na), photosynthetic nitrogen-use efficiency (EN), Ci-saturated rate of photosynthesis (Amax) and inicial slope of the A/Ci curve (dA/dCi), mesophyll (gm) and stomatal (gs) conductance to CO2 diffusion, and their ratio (gm/gs), relative stomatal, Ls, and mesophyll, Lm, limitations to photosynthesis, the external (Ca), intercellular (Ci) and chloroplastic (Cc) CO2 concentration, maximal rate of Rubisco carboxylation (Vcmax), maximal rate of electron transport driving regeneration of RuBP (Jmax), the ratio Jmax / Vcmax on Ci and Cc basis, the rate of mitochondrial respiration in the light (Rd * ), the intercellular (Ci *), and chloroplastic (Γ*) CO2 compensation concentration in the absence of mitochondrial respiration. Separation of means was determined by a Tukey test where applicable. Different letters between species indicate that means were significantly different at P<0.05. Significance of species on photosynthetic parameters are shown as: ns, non significant; *, significant at P<0.05; **, significant at P<0.01; ***, significant at P<0.001.


The response of photosynthetic rates (A) to internal CO2 concentration (Ci) (Figure 3b), changed drastically among species for the whole range of Ci values in the series: Q. saponaria > C. alba > L. caustica. Consequently the initial slope of the A/Ci curves (dA/dCi; often referred to as 'carboxylation efficiency') was greater in Q. saponaria (0.070) compared with C. alba (0.047) andL. caustica (0.038). A similar trend was observed for the rate of photosynthesis near saturating Ci(800 µmol mol-1), Amax, being 12.03, 17.01 and 23.81 µmol CO2 m-2 s-1 for L. caustica, C. albaand Q. saponaria, respectively (Table 1).

Mesophyll conductance

Mesophyll conductance (gm) was similar between L. caustica (0.060 mol CO2 m-2 s-1 bar-1) andC. alba (0.065 mol CO2 m-2 s-1 bar-1), but collectively significantly lower than Q. saponaria(0.097 mol CO2 m-2 s-1 bar-1). Although unsignificantly, gs tended to be greater for Q. saponaria (0.250 mol CO2 m-2 s-1) compared to C. alba (0.227 mol CO2 m-2 s-1) and L. caustica (0.221 mol CO2 m-2 s-1). The ratio gm/gs was on average 0.47 ± 0.08, independent of species (Table 1). Relative limitations posed by mesophyll conductance to photosynthesis, Lm, (0.40 ± 0.02) were high compared to those imposed by stomata, Ls (0.07 ± 0.01) and not significantly influenced by species. The average CO2 concentration in the intercellular air spaces (Ci) was 31.7 µmol mol-1 lower than in the atmosphere (Ca), while the average CO2 concentration in the chloroplasts (Cc) was 130.6 µmol mol-1 lower than in Ci , independent of the species (Table 1).

The gm values found in this study were generally lower than those reported for other plant functional groups. Flexas et al. (2008) argues that gm is associated with leaf forms and plant functional groups, rather than evolutive trends: herbaceous plants exhibit generally the largest gmvalues (~0.4 mol CO2 m-2 s-1 bar-1), perennial herbs and woody deciduous angiosperms display intermediate values (~0.2 mol CO2 m-2 s-1 bar-1), while woody evergreen plants exhibit gmvalues slightly above and below 0.1 mol CO2 m-2 s-1 bar-1 in angiosperms and gymnosperms, respectively. The mean value across all angiosperm sclerophyllous species studied was 0.073 mol CO2 m-2 s-1 bar-1, similar to those found by Niinemets et al. (2009b) for Australian sclerophyllous species (0.087 mol CO2 m-2 s-1 bar-1).

There seems to be a consistent pattern for sclerophyllous species to exhibit both low gm and lowgm/gs values (Gulias et al. 2002; Niinemets et al. 2009b; Tomas et al. 2013; Warren 2004). Consequently, differences between Ca and Ci are relatively small compared to the difference between Ci and Cc. This drawdown of CO2 implies that relative limitations imposed by mesophyll conductance to photosynthesis are much greater than those posed by stomata. Warren (2004) found that mesophyll limitations (Lm ~ 0.19 to 0.38) were greater than stomatal limitations (Ls ~ 0.05 to 0.23) for Eucalyptus globulus which are similar to our study. Equivalent mesophyll conductance limitations were observed for other sclerophyllous species (Lm ~ 0.20-0.50) by Niinemets et al. (2009b). This seems to be explained by leaf anatomical traits such as thickness, density and the leaf mass to area ratio (Flexas et al. 2012; Niinemets et al. 2009b; Tomas et al. 2013) and also by tissue and cell anatomical traits, such as chloroplast surface area exposed to the intercellular air spaces, cell wall thickness and palisade tissue path length, among others (Tomas et al. 2013; Flexas et al. 2012; Tosens et al. 2012; Terashima et al. 2011; Hassiotou et al. 2009).

C.albaThere are only a few studies on leaf anatomical traits for the sclerophyllous species considered in this study. Q. saponaria generally exhibits lower density palisade parenchyma and spongy mesophyll compared to C. alba and L. caustica (Gotor 2008); and additionally L. causticaexhibits substantially thicker cell walls compared to the other sclerophyllous species (Montenegro 1984). We also found L.caustica leaves to be thicker and denser, as shown by its lower foliage area to mass ratio (M) (7.0 m2 kg-1), compared to Q. saponaria (8.7 m2 kg-1) and C. alba(10 m2 kg-1). Tomas et al. (2013) found that for sclerophyllous species gm was strongly constrained by cell wall thickness (e.g. L. caustica). Such differences in leaf, tissue and cell anatomical traits may at least partially explain why values of gm and photosynthetic rates are in the series: Q. saponaria > C. alba ≥ L. caustica.

In our study, values of Asat were strongly and positively correlated with gm (Asat = 1.92 + 100.49gm, r2 = 0.61, P < 0.001). Intercepts (F2,38 = 8.07, P < 0.01) but not slopes (F2,38 = 1.6, P = 0.21 ) of this linear relationship were influenced by species (Figure 4). Values of gs and gm were uncorrelated (r2 = 0.03, P = 0.29). An increase in the rate of photosynthesis with increasing gm is consistent with previous findings from a wide range of species (Flexas et al. 2004; Grassi and Magnani 2005; Loreto et al. 1992; Niinemets et al. 2009b; Singsaas et al. 2004; Tomas et al. 2013; von Caemmerer and Evans 1991; Warren et al. 2003). Using the variable J method and carbon isotopes to estimate gm for 15 angiosperm species, Loreto et al. (1992) estimated that the slope of the relationship between gm (mol m-2 s-1 bar-1) and the rate of photosynthesis at saturating irradiance, Asat (µmol m-2 s-1) was 0.025 (i.e. Asat = 40 gm). However, the magnitude of this slope in our study was 3.45 times greater. Grassi and Magnani (2005) estimated that the slope of the relationship between gm and Asat was 0.0132 (i.e. Asat = 75.7 gm) for oak trees, being our slope 1.8 times greater. This confirms that mesophyll conductance strongly limited the photosynthetic rates in the sclerophyllous species included in our study, compared to other plant groups; although they compare well with other sclerophyllous species.

Figure 4 The relationship between Asat and gm. Asat stands for the rate of photosynthesis at saturating irradiance and ambient CO2 concentration; and, gm for mesophyll conductance. The Asat/gm linear relationship was highly significant (Asat = 2.4 + 109 gm, r2 = 0.6, P < 0.001; without intercept: Asat = 138 gm). Intercepts (F2,38 = 8.07, P < 0.01) but not slopes (F2,38 = 1.6, P = 0.21) of the Asat/gm linear relationships differed between species. Dashed-lines are Asat = 40 gm, r2 = 0.89, determined for 15 angiosperms species by Loreto et al. (1992).


The 'constant J method' used to estimate gm is sensitive to errors in both the rate of mitochondrial respiration in the light, Rd*, and the chloroplastic CO2 compensation concentration in the absence of mitochondrial respiration, Γ*. Values of Γ* in our study were very similar across sclerophyllous species (F2,41 = 0.26, P = 0.78), with a mean value of 50.7 µmol mol-1 (Table 1). In contrast, Rd*, was significantly lower (F2,41 = 9.87, P < 0.001) in L. caustica (1.0 µmol CO2 m-2 s-1) compared to C. alba (1.7 µmol CO2 m-2 s-1) and Q. saponaria (1.5 µmol CO2 m-2 s-1) (Table 1).

A recent paper by Gu and Sun (2014) suggests that the so-called Laisk method to estimate Γ*and Rd* seems to be invalid, and hence an alternative estimate for gm in our study would reduce the uncertainty related to our results. We tested whether our results for gm withhold when using values found in the literature for Γ* and Rd*. We used values for tobacco from Brooks and Farquhar (1985) (Set 1: Rd = 0.8 µmol m-2 s-1, Γ* = 36.9 µmol mol-1) and for spinach from Bernacchi et al. (2001) (Set 2: Rd = 1 µmol m-2 s-1, Γ* = 42.8 µmol mol-1), which have been commonly used to calculate gm for evergreen species (Gallé et al. 2011; Hassiotou et al. 2009; Niinemets et al. 2009a; Niinemets et al. 2009b). We did not find significant differences within each species when comparing the three estimates of gm i.e. using our estimates using the Laisk method, Set 1 and Set 2 (Q. saponaria: P = 0.48, C. alba: P = 0.38, L. caustica: P = 0.12). Tomas et al. (2013a, b) performed a similar analysis for deciduous, semideciduous and evergreen trees and herbs plants, obtaining similar values of gm independent of the values chosen for Γ*y Rd*. This shows that our results hold independent of having used the Laisk method to estimate Γ*y Rd*.

Biochemical limitations to photosynthesis

Values of Vcmax (range 13 - 66, mean 36 µmol m-2 s-1) and Jmax (range 33 - 148, mean 82 µmol m-2 s-1) in our study were within the range compiled for 109 C3 plant species by Wullschleger (1993) (Vcmax: range 6 - 194, mean 64 µmol m-2 s-1; Jmax: range 17 - 372, mean 134 µmol m-2 s-1). Wullschleger (1993) also provided specific values of Vcmax (range 11 - 119, mean 47 µmol m-2 s-1) and Jmax (range 29 - 237, mean 104 µmol m-2 s-1) for temperate hardwoods and sclerophyllous species (Vcmax: range 35 - 71, mean 53 µmol m-2 s-1; Jmax: range 94 - 167, mean 122 µmol m-2 s-1); which are similar to the ones found in our study. Pena-Rojas et al. (2004) observed values of 29 µmol CO2 m-2 s-1 and 59 µmol electrons m-2 s-1 for Vcmaxand Jmax, respectively for Quercus ilex. Niinemets et al. (2009b) observed values of 37 µmol CO2 m-2 s-1 and 89 µmol electrons m-2 s-1 for Vcmax and Jmax, respectively for different Australian sclerophyllous species. This emphasizes the fact that photosynthetic capacity in our study was more limited by gm than by biochemical limitations, as our values of Vcmax and Jmaxwere greater than those reported by Pena-Rojas et al. (2004) and Niinemets et al. (2009b) and comparable to averages for C3 hardwoods.

The maximal rate of Rubisco carboxylation (Vcmax) calculated on a Ci basis, were significantly greater in Q. saponaria (49.0 µmol CO2 m-2 s-1) compared to C. alba (32.6 µmol CO2 m-2 s-1) and L. caustica (26.8 µmol CO2 m-2 s-1) (Table 1). Corresponding values of Vcmax on a Cc basis were 98.3, 64.4 and 45.5 µmol CO2 m-2 s-1, respectively. Therefore, values of Vcmax calculated on a Cc basis were 101%, 97% and 70% greater than those on a Ci basis for Q. saponaria, C. albaand L. caustica, respectively.

Similar to Vcmax, the maximal rate of electron transport driving regeneration of RuBP (Jmax) calculated on a Ci basis, were significantly greater in Q. saponaria (111.4 µmol electrons m-2 s-1) compared to C. alba (79.6 µmol electrons m-2 s-1) and L. caustica (56.3 µmol electrons m-2 s-1) (Table 1). Corresponding values of Jmax on a Cc basis were 126.4, 89.9 and 60.4 µmol electrons m-2 s-1, respectively. Therefore, values of Jmax were 13%, 13% and 7% greater on a Cc than a Cibasis for Q. saponaria, C. alba and L. caustica, respectively.

Although there were significant differences in values of Jmax and Vcmax among species, theJmax/Vcmax ratio was constant both on a Ci (2.29 ± 0.05) and Cc (1.38 ± 0.04) basis across species (Table 1). Similarly, the relationship between Jmax and Vcmax was highly significant both on a Ci(Jmax = 2.267 Vcmax, r2 = 0.82, P < 0.001) and Cc (Jmax = 1.288 Vcmax, r2 = 0.81, P < 0.001) basis (Figure 5).

Figure 5 Relationship between the maximum rate of electron transport driving regeneration of RuBP,Jmax, and the maximum rate of Rubisco carboxylation, Vcmax, calculated on the basis of chloroplastic CO2 concentration,Cc (solid line) and intercellular CO2 concentration, Ci (dotted line). On a Cc basis: Jmax = 1.288 Vcmax,r2 = 0.81, P < 0.001; on a Ci basis: Jmax = 2.267 Vcmax, r2 = 0.82, P < 0.001


Most values of Vcmax and Jmax reported in the literature are calculated from A/Ci response curves, rather than A/Cc curves, with the implicit assumption that mesophyll conductance is infinitely large. When this assumption is invalid, values of Vcmax and Jmax are underestimated (Ethier and Livingston 2004; Ethier et al. 2006; Grassi and Magnani 2005; Harley et al. 1992; Long and Bernacchi 2003; Loreto et al. 1992; Manter and Kerrigan 2004; von Caemmerer 2000; Niinemets et al. 2009a). To prove this assumption Grassi and Magnani (2005) showed that the relationship between Vcmax,ci and Vcmax,cc values result in a slope of 1.62 (r2 = 0.94), showing that the Ci calculation underestimated the real photosynthetic capacity of the leaf. This relation is similar to the one found in our study, which resulted in a slope of 1.28 (r2 = 0.76).

There is mounting evidence that gm changes with chloroplastic CO2 concentration and irradiance, although some of that variation can be an artifact due to the mathematical methods employed (Gu and Sun 2014). Also Tholen et al. (2012) suggests there are limitations in the precise estimate of gm when taking several A values from varying CO2 concentrations and rates of photorespiration to estimate gm. Taking aside artefactual responses, it seems that gm initially increases, then peaks to decline thereafter with increasing Cc; while gm seems to increase with irradiance for the same level of Cc (Flexas et al. 2007). We are aware that converting A/Ci into A/Cc curves assuming a constant gm can be invalid; but still useful for comparing our results with previous studies. For instance, the parameters and results found in our study were similar for the photosynthesis model stated by Farquhar et al. (1980) with model parameters in normal scenarios of Vcmax ~ 50 µmol CO2 m-2 s-1, Jmax ~ 125 µmol electrons m-2 s-1, and dark respiration rates ~ 1.25 µmol CO2 m-2 s-1. These values were approximate to an 'average' C3 leaf based on Cc-derived estimates and in the range of the results for the three sclerophyllous species of our study; therefore emphasizing that our species were more limited by gm that biochemical parameters. We are also aware that using 'standard' Rubisco kinetics from tobacco to calculate gm, Vcmax and Jmax may lead to biases (Walker et al. 2013) to parameterize photosynthesis in the sclerophyllous species considered in this study. This emphasizes the need for future work to estimate Rubisco kinetics for sclerophyllous species in central Chile.

Foliar nitrogen and photosynthetic parameters

Photosynthetic rates are known to be closely related to foliar nitrogen concentrations (Field and Mooney 1986; Walcroft et al. 1997; Grassi et al. 2002; Ripullone et al. 2003). This is explained by the high proportion of total nitrogen partitioned to the carboxylating enzyme Rubisco (Sage and Pearcy 1987; Evans 1989; Warren and Adams 2002; Takashima et al. 2004) and also by the strong coupling effect among capacities Vcmax and Jmax (von Caemmerer and Farquhar 1981; Wullschleger 1993; Sharkey 1985).

At the plant level, observed Na ranged almost sixfold from 54 to 339 mmol N m-2 being significantly greater in C. alba (195.5 mmol m-2) compared to Q. saponaria (165.8 mmol m-2) and L. caustica (120.2 mmol m-2) (Table 1). Values of gm were uncorrelated to Na (r2 = 0.001, P = 0.40); but as expected values of Vcmax and Jmax on a Ci basis significantly increased with Na(F1,38 > 5.95, P < 0.019). Intercepts (F2,38 > 31.3, P < 0.001) but not slopes (F2,38 < 1.42, P > 0.25) of the Vcmax/Na and Jmax/Na linear relationships were significantly different between species (Vcmax = a + 0.075 Na, with a = 17.728 for L. caustica, a = 32.489 for C. alba, a = 39.204 for Q. saponaria, r2 = 0.61, P < 0.001; Jmax = a + 0.149 Na, with a = 38.349 for L. caustica, a = 85.976 forC. alba, a = 101.296 for Q. saponaria, r2 = 0.62, P < 0.001). It is worth noting that L.causticaexhibited the lowest intercept followed by C. alba and Q. saponaria consistently for both theVcmax/Na and the Jmax/Na relationships which can be attributed to reasons other than foliage N (i.e. foliage N was accounted for in the slope of these relationships). We then may speculate that intercept differences among species can be attributed to distinct leaf N investment strategies. Consistently we found significant differences in Nitrogen use efficiency (En), being greatest in Q. saponaria (95.9 µmol CO2 mol N s-1), compared to L. caustica (69.8 µmol CO2 mol N s-1) and C. alba (56.4 µmol CO2 mol N s-1) (Table 1). Such differences in En, may suggest that Q. saponaria, that exhibits a greater photosynthetic capacity, may invest proportionally more N to Rubisco compared to L. caustica and C. alba that exhibit poorer photosynthetic performance.


In conclusion, values of stomatal conductance, gs, were greater than those of mesophyll conductance, gm, while their ratio (gm/gs) was not influenced by species, being on average 0.47. Therefore, the relative limitations imposed by gm were high (Lm ~ 0.40, Ci-Cc ~ 131 µmol mol-1) compared to those imposed by gs (Ls ~ 0.07, Ca-Ci ~ 32 µmol mol-1). Consequently photosynthetic rates in our study were mainly limited by gm as biochemical limitations Vcmax and Jmax compare well to averages for C3 plants. Photosynthetic performance was in the series: Q. saponaria > C. alba ≥ L. caustica which can be attributed first to mesophyll conductance limitations, probably mediated by leaf anatomical traits and then to species specific foliage N partitioning strategies.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CB was involved in the original idea and carried out all measurements of gas exchange. HB participated in the design of the study, performed the statistical analysis and wrote together with CB the first draft. JPQ and JPF contributed significantly in the presentation of results and discussion. NF substantially contributed in the discussion. All authors read and approved the final manuscript.


During this work the corresponding author was supported by the National Science and Technology Commission (CONICYT) through the project FONDECYT 1090259 "Disturbance mediated water and nutrient stresses regulate carbon assimilation and allocation in sclerophyll ecosystems in Central Chile: A process-based approach". We thank Corporación Nacional Forestal for their support to carry out FONDECYT 1090259, and for providing accommodation and technical advice within the National Reserve "Roblería de los Cobres de Loncha". We also thank Mrs. Cristina Sáez N. (Universidad de Chile), Mr. Roberto Cerda R. (CONAF) and Mr. Eric Campos (Universidad de Chile) for their kind advice and valuable technical skills. The experiments and measurements undertaken for this paper comply with the current laws of Chile.



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Received: 14 February 2014
Accepted: 18 June 2014
Published: 16 August 2014


* Corresponding author:



Abbreviations used throughout the text can be found in Table 2.

Table 2 Abbreviations