Open Access

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

  • Carla E Brito1,
  • Horacio E Bown1Email author,
  • Juan-Pablo Fuentes1,
  • Nicolás Franck2 and
  • Jorge F Perez-Quezada3
Revista Chilena de Historia Natural201487:8

DOI: 10.1186/s40693-014-0008-0

Received: 14 February 2014

Accepted: 18 June 2014

Published: 16 August 2014

Abstract

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

Background

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-bis phosphate (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/Ci curves. 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 of gm 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 Ci rather 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.

Methods

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 CO2 concentration 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 gs became 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/Ci curve 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:
A = min A c , A q R d
(1)
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:
A c = V c max C i Γ * C i + K c 1 + O i / K o
(2)

where Vcmax is the maximum rate of Rubisco carboxylation under saturating RuBP and CO2, Ci and 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:
A q = J C i Γ * 4 C i + 2 Γ *
(3)

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:
A = αQ + A sat αQ + A sat 2 4 αθQ A sat 2 θ R dark
(4)

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 CO2 compensation 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 CO 2 concentration ( C i ) 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-gs where 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 = CiA/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 CO 2 concentration (A / C i 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 in Q. 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

Variable

Units

Schlerophyll species

ANOVA statistics

L. caustica

C. alba

Q. saponaria

A sat

(μmol m−2 s−1)

7.8 ± 0.7 a

9.5 ± 0.4 a

14.2 ± 0.8 b

***

α

(mol mol−1)

0.019 ± 0.002

0.024 ± 0.002

0.023 ± 0.002

ns

R dark

(μmol m−2 s−1)

0.7 ± 0.1

1.0 ± 0.2

0.6 ± 0.1

ns

Q sat

(μmol m−2 s−1)

987 ± 65 a

964 ± 56 a

1168 ± 57b

*

Q comp

(μmol m−2 s−1)

36 ± 8

51 ± 9

27 ± 4

ns

E

(μmol H2O m−2 s−1)

2133 ± 447

2913 ± 476

3492 ± 654

ns

1 / WUE

(mol H20 mol−1CO2 )

376 ± 93

347 ± 57

285 ± 60

ns

SLA

(m2 kg−1)

7 ± 0.4 a

10 ± 0.7 b

8.7 ± 0.5 ab

***

N a

(mmol m−2)

120.2 ± 8.9 a

195.5 ± 21 b

165.8 ± 17.6 ab

**

E n

(μmol mol−1 s−1)

69.8 ± 9.1 ab

56.4 ± 6.2 a

95.9 ± 8.8 b

**

A max

(μmol mol−1 s−1)

12.03 ± 0.86 a

17.01 ± 0.81 b

23.81 ± 1.18 c

***

dA/dCi

(mol mol−1)

0.038 ± 0.003 a

0.047 ± 0.002 a

0.070 ± 0.004 b

***

g m

(mol m−2 s−1 bar−1)

0.06 ± 0.005 a

0.065 ± 0.004 a

0.097 ± 0.007 b

***

g s

(mol m−2 s−1)

0.221 ± 0.03

0.221 ± 0.025

0.25 ± 0.018

ns

g m / g s

-

0.54 ± 0.18

0.47 ± 0.14

0.43 ± 0.06

ns

L s

-

0.07 ± 0.01

0.07 ± 0.02

0.08 ± 0.01

ns

L m

-

0.35 ± 0.03

0.42 ± 0.02

0.43 ± 0.02

ns

C a

(μmol mol−1)

368.3 ± 0.6

368.7 ± 1

368.7 ± 0.6

ns

C i

(μmol mol−1)

337.7 ± 7

336.5 ± 6.9

333.6 ± 3.7

ns

C c

(μmol mol−1)

220 ± 10.3

202.2 ± 8.2

192.8 ± 5.9

ns

Vcmax (Ci)

(μmol m−2 s−1)

26.8 ± 2.1 a

32.6 ± 1.1 a

49 ± 2.6 b

***

Jmax (Ci)

(μmol m−2 s−1)

56.3 ± 4.1 a

79.6 ± 3.8 a

111.4 ± 5.6 b

***

Jmax/ Vcmax (Ci)

-

2.15 ± 0.1

2.44 ± 0.07

2.29 ± 0.07

ns

Vcmax (Cc)

(μmol m−2 s−1)

45.5 ± 5.1 a

64.4 ± 3.8 a

98.3 ± 5.7 b

***

Jmax (Cc)

(μmol m−2 s−1)

60.4 ± 4.4 a

89.9 ± 4.2 b

126.4 ± 6.1 c

***

Jmax/ Vcmax (Cc)

-

1.41 ± 0.07

1.43 ± 0.06

1.31 ± 0.05

ns

R d *

(μmol m−2 s−1)

1.0 ± 0.1 a

1.7 ± 0.2 b

1.5 ± 0.1 b

***

Ci*

(μmol mol−1)

33 ± 4.2 ab

23.2 ± 2.3 a

34 ± 2.4 b

*

Γ*

(μmol mol−1)

55.5 ± 5.4

56.8 ± 4.1

59.2 ± 5.9

ns

Values are presented as means (± 1 SE, n=15) for the light-saturated rate of photosynthesis (Asat), apparent maximum quantum efficiency (α), 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) and L. 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. alba and 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) and C. 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 gm values (~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 gm values 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 low gm/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. caustica exhibits 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 + 100.49 gm, 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 g m . 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 Vcmax and 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 Jmax were 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. alba and 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 Ci basis for Q. saponaria, C. alba and L. caustica, respectively.

Although there were significant differences in values of Jmax and Vcmax among species, the Jmax/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, J max , and the maximum rate of Rubisco carboxylation, V cmax , calculated on the basis of chloroplastic CO 2 concentration, C c (solid line) and intercellular CO 2 concentration, C i (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 for C. alba, a = 101.296 for Q. saponaria, r2 = 0.62, P < 0.001). It is worth noting that L.caustica exhibited the lowest intercept followed by C. alba and Q. saponaria consistently for both the Vcmax/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.

Conclusions

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.

Appendix

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

Abbreviations

Symbol

Description

Units

A sat

light-saturated rate of photosynthesis at ambient Ca

μmol m−2 s−1

R dark

respiration rate at zero irradiance

μmol m−2 s−1

Q sat

Light saturation point determined as the Q value for which assimilation is 90% of Asat

μmol m−2 s−1

Q comp

Light compensation point determined as the Q value for which the rate of photosynthesis exactly matches the rate of respiration

μmol m−2 s−1

α

apparent maximum quantum efficiency

mol mol−1

C a

CO2 concentration in the air outside the leaf

μmol mol−1

C i

CO2 concentration in intercellular air spaces

μmol mol−1

C c

CO2 concentration in the chloroplast

μmol mol−1

Γ*

chloroplastic CO2 compensation point

μmol mol−1

C i *

intercellular CO2 compensation point in the absence of day respiration

μmol mol−1

R d *

rate of mitochondrial respiration in the light

μmol m−2 s−1

E N

instantaneous photosynthetic nitrogen use efficiency

μmol mol−1 s−1

L s

relative stomatal (plus boundary layer) limitation to photosynthesis

%

L m

relative mesophyll limitations to photosynthesis

%

g s

stomatal (plus boundary layer) conductance to CO2 diffusion

mol m−2 s−1

g m

Mesophyll transfer conductance to CO2 diffusion

mol m−2 s−1 bar−1

V cmax

in vivo maximum rate of ribulose-1,5-bis phosphate (RuBP) carboxylase-oxygenase (Rubisco) carboxylation

μmol CO2 m−2 s−1

J max

electron transport driving regeneration of RuBP

μmol electrons m−2 s−1

A max

the rate of photosynthesis near saturating Ci (800 μmol mol−1)

μmol CO2 m−2 s−1

N a

foliage nitrogen concentration on an area basis

mmol m−2

M

Leaf area to mass ratio

m2 kg−1

VPD

Leaf-to-air vapour pressure deficit

kPa

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Facultad de Ciencias Forestales y de la Conservación de la Naturaleza, Universidad de Chile
(2)
Centro de Estudios de Zonas Áridas, Universidad de Chile
(3)
Facultad de Ciencias Agronómicas, Universidad de Chile

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