Abstract

Mesophyll conductance, gm, was estimated from measurements of stomatal conductance to carbon dioxide transfer, gs, photosynthesis, A, and chlorophyll fluorescence for Year 0 (current-year) and Year 1 (1-year-old) fully sunlit leaves from short (2 m tall, 10-year-old) and tall (15 m tall, 120-year-old) Nothofagus solandrii var. cliffortiodes trees growing in adjacent stands. Rates of photosynthesis at saturating irradiance and ambient CO2 partial pressure, AsatQ, were 25% lower and maximum rates of carboxylation, Vcmax, were 44% lower in Year 1 leaves compared with Year 0 leaves across both tree sizes. Although gs and gm were not significantly different between Year 0 and Year 1 leaves and gs was not significantly different between tree heights, gm was significantly (19%) lower for leaves on tall trees compared with leaves on short trees. Overall, Vcmax was 60% higher when expressed on the basis of CO2 partial pressure at the chloroplasts, Cc, compared with Vcmax on the basis of intercellular CO2 partial pressure, Ci, but this varied with leaf age and tree size. To interpret the relative stomatal and mesophyll limitations to photosynthesis, we used a model of carbon isotopic composition for whole leaves incorporating gm effects to generate a surface of ‘operating values’ of A over the growing season for all leaf classes. Our analysis showed that A was slightly higher for leaves on short compared with tall trees, but lower gm apparently reduced actual A substantially compared with AsatQ. Our findings showed that lower rates of photosynthesis in Year 1 leaves compared with Year 0 leaves were attributable more to increased biochemical limitation to photosynthesis in Year 1 leaves than differences in gm. However, lower A in leaves on tall trees compared with those on short trees could be attributed in part to lower gm and higher stomatal, Ls, and mesophyll, Lm, limitations to photosynthesis, consistent with steeper hydraulic gradients in tall trees.

Introduction

The regulation of photosynthesis rate in leaves is closely related to leaf nitrogen concentration (Field and Mooney 1986, Evans 1989), with lower rates of photosynthesis in older leaves generally attributable to lower nitrogen concentrations and decreasing ratio of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) to chlorophyll content (Warren and Adams 2001). However, the biochemical limitation of photosynthesis does not account fully for the observed decreases in photosynthesis with leaf ageing (Warren 2006). The inclusion of stomatal limitation to photosynthesis, A, in models at leaf scales is widely accepted (Farquhar et al. 1980, Farquhar and Sharkey 1982), but there is increasing recognition that limitation to photosynthesis by mesophyll conductance is as important as stomatal limitation (Harley et al. 1992, Loreto et al. 1992, Flexas et al. 2008, Warren 2008). Inclusion of mesophyll conductance to CO2 transfer, gm, in models of photosynthesis reduces estimates of CO2 partial pressure at the chloroplasts, Cc, relative to the partial pressure in the intercellular air spaces, Ci. Using Cc rather than Ci corrects underestimation of the maximum rate of Rubisco carboxylation, Vcmax, derived from the analysis of A/Ci curves, which assumes that gm is infinitely large (Flexas et al. 2008, Warren 2008).

Recent reviews have summarized current understanding of the variability in gm between species and its dynamic regulation by environmental variables, but the mechanisms of the responses associated with anatomical, morphological and biochemical characteristics remain largely unknown (Flexas et al. 2008, Warren 2008). Early work (Evans and Loreto 2000) supported relationships between gm and structural properties of leaves, particularly mesophyll cell thickness and intercellular air space. However, measurements with a wide range of species show that while gm may be limited by leaf structure, it may vary widely within leaves of different species with a similar area to mass ratio (Flexas et al. 2008). It has also been suggested that gm may be related to the surface area of chloroplasts bordering sub-stomatal cavities (Evans 1999, Hanba et al. 2001). Parallel increases in gm and A have been observed during early leaf development in broadleaved trees (Miyazawa and Terashima 2001, Eichelmann et al. 2004), but gm tends to decrease with increasing leaf age following full expansion within weeks for herbaceous plants (Loreto et al. 1997) and deciduous trees (Grassi and Magnani 2005) and within years for evergreen broadleaved trees (Niinemets et al. 2005, 2006) and conifers (Ethier et al. 2006, Warren 2006).

In canopies, regulation of photosynthesis by leaf scale morphological and biochemical characteristics is further complicated by variability associated with light environment and nitrogen allocation to Rubisco activity (Niinemets et al. 2006). Further, hydraulic limitations may be responsible for age-related decline in photosynthesis and tree growth (Ryan and Yoder 1997, McDowell et al. 2002, 2005), although the mechanisms remain uncertain (Ryan et al. 2006). Photosynthesis is dependent on the diffusive pathways in leaves, and stomatal conductance to CO2 transfer, gs, and gm may be influenced at the tree scale by water potential gradients associated with differences in tree size during leaf development (Koch et al. 2004, Woodruff et al. 2008, 2009). Given that it is likely that hydraulic regulation of developmental changes in leaf properties and their associated effects on photosynthesis are attributable to path length effects on hydraulic conductance, it is more appropriate to interpret differences in gs and gm in relation to tree size rather than tree age (Mencuccini et al. 2005).

Warren et al. (2003) were the first to measure changes in gm with height through the crown of a 50-year-old, 30-m-tall coniferous Pseudotsuga menziesii tree and showed that gs and gm limited photosynthesis by 30 and 20%, respectively, but changes in gm with height were small. In contrast, Woodruff et al. (2009) measured reductions of 47 and 42% in gm and A, respectively, in P. menziesii trees ranging in height from 5 to 55 m and attributed these differences to increased needle thickness and mesophyll cell thickness with increased tree height associated with the effects of water potential gradients on needle development during expansion.

We used an existing successional sequence of Nothofagus solandrii var. cliffortiodes (mountain beech) trees growing at Craigieburn Forest, central South Island, New Zealand, to investigate the effects of leaf age (Year 0 and Year 1 leaves) and hydraulic constraints associated with tree size (2- and 15-m-tall trees) on stomatal and mesophyll limitations to photosynthesis. Our first hypothesis was that gs and gm would be lower in Year 1 leaves compared with Year 0 leaves because of increased path length for CO2 diffusion within older leaves. Our second hypothesis was that gs and gm would be lower for leaves of the same age on tall trees compared with short trees because of increased hydraulic constraints on leaf development in tall trees.

We estimated gm from measurements of photosynthesis and chlorophyll fluorescence using the ‘constant J’ method (see below; Loreto et al. 1992). We also used carbon isotopes to interpret the relationship between the long-term effects of leaf age and hydraulic path length on gs and gm. The stable carbon isotope composition of leaf tissue provides an integrated record of the balance of CO2 supply (stomatal conductance) and demand (photosynthesis) (Farquhar et al. 1982), and it is used commonly to interpret changes in Ci/Ca (Farquhar et al. 1989). McDowell et al. (2002) attributed linear relationships between isotopic composition and tree size to hydraulic constraints. Similarly, Woodruff et al. (2009) used measurements of carbon isotopic composition to derive relationships between A, gs and gm integrated across the lifetime of needles. In addition to interpretation of measurements of carbon isotopic composition, we extend the model of isotope discrimination to include a component attributable to gm following Barbour et al. (2010). Using this approach, we determined the potential combinations of gs, gm and A that give the measured isotopic values and we relate this to leaves of the same age from trees of different sizes.

Materials and methods

Site and plant material

Measurements were made in an age sequence of N. solandrii var. cliffortiodes trees growing at Craigieburn Forest, central South Island, New Zealand (latitude 43.3°S, longitude 171.0°E, elevation ∼1100 m), consisting of plots of short trees in sapling (10 years) and tall trees in pole (120 years) stages of succession. Trees at the sapling stage had resulted from re-growth following disturbance from wind-throw of canopy trees and consisted of a dense canopy of trees with small diameter at 1.3 m above ground level (<30 mm) and a height of ∼2 m. The pole stand consisted of a large number of small-diameter (<200 mm) trees undergoing self-thinning, with heights of ∼15 m (Davis et al. 2003). The soils in both stands was a Katrine silt loam, predominantly high-country yellow-brown earths (Soil Survey Staff 1998), equivalent to Acidic Allophanic Brown Soil in the New Zealand classification (Hewitt 1998) and to Andic Dystrochrept in the USDA classification, derived from greywacke, loess and colluvium. Mean annual temperature and precipitation at the site are 8 °C and 1447 mm, respectively (McCracken 1980).

Sampling protocol and leaf characteristics

Gas exchange measurements were conducted in mid-summer (February) on Year 0 (current-year) and Year 1 (1-year-old) leaves collected from 12 replicate trees in the short and tall stands. Leaves of both ages were fully expanded and Year 0 and Year 1 leaves had emerged ∼3 and ∼15 months, respectively, before the measurements were made. Fully sunlit shoots were removed from trees using a pruning saw (short trees) and shotgun (tall trees), stems were re-cut under water immediately and samples were taken to the laboratory. The shoots were kept in water and exposed to ambient conditions until gas exchange measurements were made.

Following the measurements of photosynthesis, leaf samples were placed flat and photographed using a digital camera, and leaf area was estimated using digitizing software with the scanned images. Subsequently, the samples were dried at 70 °C and weighed to allow calculation of the ratio of leaf area to leaf dry mass, S. Dried samples were ground and measured for nitrogen concentration and carbon isotope composition with an isotope ratio mass spectrometer (Europa Scientific 20/20) interfaced to a Dumas elemental analyser (Europa Scientific ANCA-SL, Europa Scientific Ltd, Crewe, UK). Nitrogen concentrations are expressed on a mass, Nm, and area, Na, basis and isotope ratios are presented (as ‰) in the familiar delta notation as δ13C = (σsamplestandard) − 1, where σ is the isotope ratio (13C/12C) and the standard used is CO2 from Vee Dee Pee Belemnite (VDPB).

Measurements of photosynthesis

Measurements of photosynthesis were made using four portable photosynthesis systems (Model 6400; LiCor, Inc., Lincoln, NE, USA) equipped with CO2 control modules. Three photosynthesis systems were equipped with standard 20 × 30 mm chambers with light sources consisting of blue–red light-emitting diodes (Model 6400-02B) to provide irradiance (400–700 nm), Q, of varying intensity. One photosynthesis system was equipped with an integrated fluorescence detector (Model LI-6400-40 leaf chamber; LiCor, Inc.) for measurements of chlorophyll fluorescence (see below). The four photosynthesis systems were calibrated and matched for CO2 and water vapour concentrations before use. Measurements of gas exchange on excised shoots were started after stomatal conductance, gs, and photosynthesis, A, had reached maximum values and gs remained high during the entire measurement period. The length and width of the leaves were ∼5 mm, so several adjacent leaves were placed in the chamber. There was no contribution to gas exchange from leaf area covered by the gaskets around the edge of the chamber because all leaves were fully contained within the chamber and we assumed that the effects of stomatal heterogeneity on measurements of photosynthesis were insignificant. Values of stomatal conductance to CO2 transfer, gs, were calculated by dividing values for water vapour transfer by 1.6 (Jones 2002), accounting for differences in the rates of diffusion of water vapour and CO2 in air. All measurements were made at a constant leaf temperature of 20 °C, determined using an energy balance approach and maintained using thermoelectric coolers. Leaf-to-air vapour pressure deficit was generally between 0.5 and 1.1 kPa. All data are expressed on the basis of one-sided leaf surface area.

The response of photosynthesis, A, to varying intercellular CO2 partial pressure, Ci, was measured by varying the CO2 partial pressures in the leaf chamber, Ca, in 13 steps, decreasing from 150 to 0 Pa, using a flow rate of 500 μmol s−1 at a saturating irradiance, Q (400–700 nm), of 1000 μmol m−2 s−1. Measurements were recorded automatically at each Ca set point when photosynthesis had equilibrated, which was typically <2 min. We analysed the A/Ci curves using the Farquhar et al. (1980) model to estimate the maximum carboxylation rate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), Vcmax, the maximum rate of photosynthesis at saturating Q and ambient CO2 partial pressure (Ca = 38 Pa), AsatQ, the maximum rate of photosynthesis at saturating Q and saturating CO2 partial pressure (80 Pa), Amax, and gs at saturating Q and ambient CO2 partial pressure. Following Farquhar et al. (1980), calculation of these variables assumed that gm was infinitely large. We used the Michaelis–Menten constants of Rubisco described in Bernacchi et al. (2001), adjusted to 20 °C, such that Kc (rate constant for Rubisco carboxylation) = 23.12 Pa, Ko (rate constant for oxygenation) = 21.39 Pa and τ (specificity factor for Rubisco) = 2823 (Jordan and Ogren 1984). Subsequently, when fitting the response of A to the CO2 partial pressure at the chloroplasts, Cc, we used values of Kc = 15.34 Pa and Ko = 14.02 Pa adjusted to 20 °C following Bernacchi et al. (2002).

Measurements of chlorophyll fluorescence on light-adapted leaves were made simultaneously with measurements of photosynthesis at each value of Ci to determine the photochemical efficiency of photosystem II (ΦPSII) from (Fm′ − F)/Fm′, where F and Fm′ are the steady and maximal fluorescence, respectively (Schreiber et al. 1994). ΦPSII is related directly to the rate of electron transport, J (Genty et al. 1989), and is used to determine the portion of the A/Ci curve where J is constant.

We estimated values of the rate of mitochondrial respiration in the light, Rd, and the CO2 compensation point at the chloroplasts, Γ*, using the photo-compensation point method of Laisk and Oja (1998). Additional A/Ci curves were measured at three low levels of Q (300, 150 and 50 μmol m−2 s−1) at seven values of Ca (decreasing from 20 to 0 Pa) to generate relationships on the linear part of the A/Ci curve. Extrapolation of the common point of intersection of the three A/Ci curves to the A axis generated an estimate of Rd, while extrapolation to the Ci axis generated an estimate of the intercellular CO2 compensation point in the absence of day respiration, Ci*, and this was assumed to be equal to Γ* (DeLucia et al. 2003). Mean values of Rd and Γ* were calculated for each leaf class and used in the estimation of gm.

Estimation of gm using the constant J method

Mesophyll conductance, gm, was estimated using the ‘constant J’ method (Loreto et al. 1992), which assumes that when the rate of photosynthetic electron transport, J, becomes constant, further increases in A with increasing Ci are due to suppression of photorespiration because the rate of carboxylation progressively substitutes the rate of oxygenation (Harley et al. 1992, Long and Bernacchi 2003, Singsaas et al. 2003). In these conditions, photosynthesis is related to the CO2 partial pressure at the site of fixation in the chloroplasts, Cc, and the relative specificity of Rubisco to CO2 and O2, which is normally described by the chloroplastic CO2 compensation point, Γ*. Estimates of gm were obtained from values of Γ* and Rd and data from the linear part of the A/Ci response following Harley et al. (1992). Values of gm were obtained from three or more measurements of photosynthesis from the A/Ci response curves at high Ci partial pressure when the rate of electron transport, J, was constant (Singsaas et al. 2003, Warren 2006). The value for gm was resolved iteratively as the value that provided the lowest variance of J associated with A.

Data analysis

All analyses were undertaken at the leaf level using SPSS software (SPSS for Windows, version 11.0.1, 2001; SPSS, Inc., Chicago, IL, USA). Variables were tested for normality and homogeneity of variance, and logarithmic transformations were made as necessary to meet the underlying statistical assumptions of the models. The main and interactive effects of leaf age and tree size were tested using analysis of variance.

Stomatal and mesophyll limitations to photosynthesis

The responses of photosynthesis to ambient, Ca, intercellular, Ci, and chloroplastic, Cc, CO2 partial pressures were derived from estimates of average values of gs and gm for each of the four leaf classes using the relationships (Farquhar and Sharkey 1982)  

(1)
formula

This assumes that boundary layer effects were negligible (Lanigan et al. 2008), which is reasonable, considering the small size of Nothofagus leaves and the good mixing within the leaf chamber. The limitations to photosynthesis imposed by stomatal, Ls, and mesophyll, Lm, conductance were estimated from calculations of A using measured values of gs and gm or assuming that they were infinite, following the procedure adopted by Warren et al. (2003). Rates of photosynthesis, An, at saturating irradiance (1000 μmol m−2 s−1) and ambient CO2 partial pressure (38 Pa) were estimated using average measurements of stomatal, gs, and mesophyll gm, conductance. Rates of photosynthesis at saturating irradiance when Cc = Ci were estimated using measured gs and assuming gm was infinite, Am, and rates of photosynthesis at saturating irradiance when Ci = Ca were estimated using measured gm and assuming gs was infinite, As. Stomatal and mesophyll limitations to photosynthesis were then calculated from  

(2)
formula

Estimation of the effects of gm on operating values of A and gs

Fractionation of carbon isotope composition during photosynthesis, Δ, is described by Farquhar et al. (1989) as  

(3)
formula
where a (4.4‰) and b (assumed to be 27‰) are diffusional and biochemical isotope fractionation factors, respectively. However, it is the chloroplastic CO2 partial pressure, Cc, that is important for discrimination, as is evident when the full derivation of Eq. (3) is considered (Brugnoli and Farquhar 2000, Barbour et al. 2010):  
(4)
formula
where ab (2.9‰, noting that we have assumed very high boundary layer conductance for Nothofagus leaves given their small size) and a1 (0.7‰) are fractionations associated with diffusion through the boundary layer and leaf water, respectively; bs is fractionation as CO2 moves into solution (1.1‰ at 25 °C), Cs is the CO2 partial pressure at the leaf surface, and e (−3‰, Bickford et al. 2009) and f (11.6‰, Lanigan et al. 2008) are fractionations associated with mitochondrial respiration in the light and photorespiration, respectively. Γ* is the CO2 compensation point in the absence of mitochondrial respiration, and Rd is measured as described above. Note that the value of b in Eq. (4) is ∼30‰, the true discrimination by Rubisco and other carboxylating enzymes, rather than the approximate value to account for other fractionations within the mesophyll as in Eq. (3) above.

Measured values of leaf tissue isotopic composition, δ13Cp, were converted to discrimination, Δ, using  

(5)
formula
where δ13Ca is the isotopic composition of CO2 in air and was assumed to be −8‰. Equations (3) and (4) may be solved for A at a range of values for stomatal conductance to CO2, gs, incorporating Eq. (1) using values for the parameters listed above. The model was implemented using the Solver function in Excel (version 2007) where a value of gs was input and Eqs (1), (3) and (4) were solved iteratively for A until Δ matched the measured value for each leaf class. We assumed that Eqs (3) and (4) relate directly to bulk leaf material, i.e., that post-photosynthetic fractionation within the leaves is negligible, although we recognize that this is not strictly true. For example, lignin is known to be ∼4‰ more depleted in δ13C than sucrose (Schmidt and Gleixner 1998) and lignin content likely varies with leaf age (Miyazawa et al. 2003) and covaries with the ratio of leaf area to dry mass in trees of different heights (Niinemets et al. 1999). For this reason, we restrict our interpretation of fitted values to a comparison between the simple model (Eq. 3) that assumes infinite gm and the more comprehensive model (Eq. 4) that includes gm for each leaf class.

Results

Leaf properties and rates of photosynthesis

Leaf area to mass ratio, S, was significantly lower for Year 1 leaves compared with Year 0 leaves in stands of both ages (average difference 5%), and S was also significantly lower for both ages in tall trees (120-year-old) compared with short trees (10-year-old, average difference was 9%, Table 1). This was associated with a significantly lower value for nitrogen concentration per unit mass, Nm, in Year 1 leaves compared with Year 0 leaves (average difference 18%) but, on a leaf area basis, nitrogen concentration, Na, was not significantly different. Values of Na were significantly higher for tall trees compared with short trees (average difference 10%). Rates of photosynthesis at saturating irradiance and saturating CO2 partial pressure, Amax, and at saturating irradiance and ambient CO2 partial pressure, AsatQ, were significantly lower for Year 1 leaves (both by 25%) compared with values for Year 0 leaves in the stands of both tree sizes.

Table 1.

 Leaf characteristics and rates of photosynthesis for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees.

Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
S (m2 kg−15.58 ± 0.21 5.25 ± 0.15 5.01 ± 0.12 4.80 ± 0.15 L 0.026 
     T 0.014 
     L × T ns 
Nm (mmol g−10.87 ± 0.02 0.70 ± 0.01 0.84 ± 0.02 0.71 ± 0.02 L  < 0.0001 
     T ns 
     L × T 0.028 
Na (mmol m−2158.0 ± 6.2 133.5 ± 4.5 168.9 ± 6.0 149.5 ± 6.0 L 0.078 
     T 0.007 
     L × T ns 
Amax (μmol m−2 s−128.2 ± 1.4 20.3 ± 0.8 25.0 ± 0.8 19.6 ± 1.0 L  < 0.0001 
     T ns 
     L × T 0.046 
AsatQ (μmol m−2  s−116.1 ± 1.1 12.1 ± 0.5 14.7 ± 0.7 10.9 ± 0.7 L  < 0.0001 
     T ns 
     L × T ns 
Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
S (m2 kg−15.58 ± 0.21 5.25 ± 0.15 5.01 ± 0.12 4.80 ± 0.15 L 0.026 
     T 0.014 
     L × T ns 
Nm (mmol g−10.87 ± 0.02 0.70 ± 0.01 0.84 ± 0.02 0.71 ± 0.02 L  < 0.0001 
     T ns 
     L × T 0.028 
Na (mmol m−2158.0 ± 6.2 133.5 ± 4.5 168.9 ± 6.0 149.5 ± 6.0 L 0.078 
     T 0.007 
     L × T ns 
Amax (μmol m−2 s−128.2 ± 1.4 20.3 ± 0.8 25.0 ± 0.8 19.6 ± 1.0 L  < 0.0001 
     T ns 
     L × T 0.046 
AsatQ (μmol m−2  s−116.1 ± 1.1 12.1 ± 0.5 14.7 ± 0.7 10.9 ± 0.7 L  < 0.0001 
     T ns 
     L × T ns 

Data shown are mean ± standard error values for the leaf area to mass ratio, S, nitrogen concentration on a mass, Nm, and area, Na, basis, rates of photosynthesis at saturating irradiance and saturating CO2 partial pressure, Amax, and at saturating irradiance and ambient CO2 partial pressure, AsatQ. Significance of the main effects of leaf age, L, tree size, T, and their interaction, L × T, are shown with the P values or as not significant, ns, when P > 0.05.

Stomatal and mesophyll conductance

Overall, the values of stomatal conductance to CO2 transfer, gs, and mesophyll conductance, gm, were not significantly different between leaf ages. However, gs and gm were 24 and 19% lower, respectively, for leaves on tall trees compared with short trees (Table 2). The mean ratio of gm/gs for leaves within each treatment was similar, with an overall average (±SE) value of 0.93 ± 0.06 mol mol−1.

Table 2.

Estimates of stomatal and mesophyll characteristics for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees.

Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
gs (mmol m−2 s−189.9 ± 13.2 76.1 ± 9.3 67.4 ± 7.1 58.7 ± 6.3 L ns 
     T ns 
     L × T ns 
gm (mmol m−2 s−173.7 ± 3.5 84.9 ± 6.4 56.3 ± 2.6 72.7 ± 7.1 L ns 
     T 0.001 
     L × T ns 
gm/gs (mol mol−10.94 ± 0.15 0.92 ± 0.11 0.83 ± 0.11 1.00 ± 0.14 L ns 
     T ns 
     L × T ns 
δ13C (‰) −28.21 ± 0.18 −28.78 ± 0.17 −27.04 ± 0.26 −27.50 ± 0.23 L ns 
     T  < 0.0001 
     L × T ns 
Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
gs (mmol m−2 s−189.9 ± 13.2 76.1 ± 9.3 67.4 ± 7.1 58.7 ± 6.3 L ns 
     T ns 
     L × T ns 
gm (mmol m−2 s−173.7 ± 3.5 84.9 ± 6.4 56.3 ± 2.6 72.7 ± 7.1 L ns 
     T 0.001 
     L × T ns 
gm/gs (mol mol−10.94 ± 0.15 0.92 ± 0.11 0.83 ± 0.11 1.00 ± 0.14 L ns 
     T ns 
     L × T ns 
δ13C (‰) −28.21 ± 0.18 −28.78 ± 0.17 −27.04 ± 0.26 −27.50 ± 0.23 L ns 
     T  < 0.0001 
     L × T ns 

Data shown are mean ± standard error values for stomatal conductance to CO2 transfer, gs, mesophyll conductance, gm, and the δ13C isotopic composition of leaves. Significance of the main effects of leaf age, L, tree size, T, and their interaction, L × T, are shown with the P values or as not significant, ns, when P > 0.05. Values for gs needed to be logarithmically transformed before the analysis of variance was undertaken.

Analysis of the responses of photosynthesis, A, to CO2 partial pressure in ambient air, Ca, in intercellular spaces, Ci, and at the chloroplasts, Cc, highlighted the higher rates of photosynthesis in Year 0 leaves compared with Year 1 leaves. Further, values of Cc were higher for Year 0 leaves compared with Year 1 leaves (Figure 1). From these responses and Eqs (1) and (2), the limitation to photosynthesis by stomatal conductance, Ls, was similar for leaves of both ages from short and tall trees except for Year 1 leaves on short trees where Ls was lower (Table 3). Limitations to photosynthesis by mesophyll conductance, Lm, were higher than Ls for all leaves and values were higher for Year 0 leaves than for Year 1 leaves on trees of both sizes.

Figure 1.

Graphical analysis of the response of photosynthesis, A, to CO2 partial pressure in ambient air, Ca, in intercellular spaces, Ci, and at the chloroplasts, Cc, for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees. Rates of photosynthesis, An, at saturating irradiance (1000 μmol m−2 s−1) and ambient CO2 partial pressure (38 Pa) were estimated using the mean measurements of stomatal conductance, gs, and mesophyll conductance, gm, given in Table 2 with Eq. (1). Rates of photosynthesis at saturating irradiance when Cc = Ci were estimated using measured gs and assuming gm is infinite, Am, and rates of photosynthesis at saturating irradiance when Ci = Ca were estimated using measured gm and assuming gs is infinite, As. Stomatal, Ls, and mesophyll, Lm, limitations estimated from Eq. (2) and the data in the figure are given in Table 3.

Figure 1.

Graphical analysis of the response of photosynthesis, A, to CO2 partial pressure in ambient air, Ca, in intercellular spaces, Ci, and at the chloroplasts, Cc, for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees. Rates of photosynthesis, An, at saturating irradiance (1000 μmol m−2 s−1) and ambient CO2 partial pressure (38 Pa) were estimated using the mean measurements of stomatal conductance, gs, and mesophyll conductance, gm, given in Table 2 with Eq. (1). Rates of photosynthesis at saturating irradiance when Cc = Ci were estimated using measured gs and assuming gm is infinite, Am, and rates of photosynthesis at saturating irradiance when Ci = Ca were estimated using measured gm and assuming gs is infinite, As. Stomatal, Ls, and mesophyll, Lm, limitations estimated from Eq. (2) and the data in the figure are given in Table 3.

Table 3.

Estimates of stomatal, Ls, and mesophyll, Lm, limitations to photosynthesis for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees calculated from analysis of the average response of photosynthesis, A, to CO2 partial pressure in ambient air, Ca, intercellular spaces, Ci, and at the chloroplasts, Cc, shown in Figure 1 and Eqs (1) and (2) for each leaf class.

Tree age (years) 10 10 120 120 

 
Leaf age (years) 
Ls 0.31 0.18 0.35 0.33 
Lm 0.52 0.22 0.55 0.39 
Ls/Lm 0.60 0.82 0.64 0.85 
Tree age (years) 10 10 120 120 

 
Leaf age (years) 
Ls 0.31 0.18 0.35 0.33 
Lm 0.52 0.22 0.55 0.39 
Ls/Lm 0.60 0.82 0.64 0.85 

Parameters describing photosynthesis

The mitochondrial CO2 compensation point at the chloroplasts, Γ* (mean ± SE = 3.1 ± 0.23 Pa), and the rate of mitochondrial respiration in the light, Rd (mean ± SE = 1.6 ± 0.17 μmol m−2 s−1), were very similar for leaves in both leaf age and tree size classes. The maximum rate of Rubisco carboxylation, Vcmax, calculated on the basis of Ci was 44% higher for Year 0 leaves compared with Year 1 leaves and significantly higher within both tree size classes (Table 4), consistent with differences in Amax and Asat (Table 1). There were no significant differences in Vcmax with tree size for Year 0 or Year 1 leaves.

Table 4.

Estimates of the maximum rate of Rubisco carboxylation, Vcmax, for Year 0 (current-year) and Year 1 (1-year-old) N. solandrii var. cliffortiodes leaves on short (10-year-old) and tall (120-year-old) trees.

Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
Vcmax, Ci basis (μmol m−2 s−143.5 ± 2.4 24.0 ± 1.2 37.0 ± 1.9 21.3 ± 2.0 L  < 0.0001 
     T ns 
     L × T 0.048 
Vcmax, Cc basis (μmol m−2 s−169.0 ± 6.2 29.0 ± 2.0 58.7 ± 3.8 42.3 ± 6.4 L  < 0.0001 
     T ns 
     L × T 0.016 
Tree age (years) 10 10 120 120 ANOVA statistics 

 
Leaf age (years)  
Vcmax, Ci basis (μmol m−2 s−143.5 ± 2.4 24.0 ± 1.2 37.0 ± 1.9 21.3 ± 2.0 L  < 0.0001 
     T ns 
     L × T 0.048 
Vcmax, Cc basis (μmol m−2 s−169.0 ± 6.2 29.0 ± 2.0 58.7 ± 3.8 42.3 ± 6.4 L  < 0.0001 
     T ns 
     L × T 0.016 

Data shown are mean ± standard error values for Vcmax calculated on the basis of intercellular CO2 concentration, Ci, and CO2 concentration at the chloroplasts, Cc. Significance of the main effects of leaf age, L, tree size, T, and their interaction, L × T, are shown with the P values or as not significant, ns, when P > 0.05.

Values of Vcmax expressed on the basis of Cc were higher than those expressed on the basis of Ci, and differences between leaf age, but not tree size, were highly significant (Table 4). The mean (±SE) increase in Vcmax calculated on the basis of Cc rather than Ci across all leaves was 60 ± 3%.

Effects of gm on operating values of A and gs

The δ13C value of leaves from taller trees was significantly less depleted than that of leaves from shorter trees (Table 2), consistent with lower values of Cc in the tall trees. In contrast, differences in δ13C composition between leaf ages were not significant.

Results from fitting Eqs (1), (3), (4) and (5) to the data allowed us to construct ‘operating surfaces’ of the potential combinations of gs and A that give the measured Δ13C values for leaves in each age class (Figure 2). This demonstrates that the leaves were operating at photosynthetic rates well below measured AsatQ when gm was taken into consideration. Using measured values of gs (Table 2), A was fitted as being between 4.1 and 4.8 μmol m−2 s−1 when gm was included. Ignoring gm (using Eq. (3) rather than Eq. (4)) resulted in values of A nearly two times higher than those including gm for all leaf classes.

Figure 2.

Possible combinations of A and gs from measured Δ13C of Year 0 (current-year; a and c) and Year 1 (1-year-old; b and d) leaves from short (10-year-old, a and b) and tall (120-year-old, c and d) trees when gm is excluded (Eq. (3), solid line, open symbols) and included (Eq. (4), dashed line, closed symbols). The points indicate fitted values of A when gs was assumed to equal values given in Table 2. See text for further explanation.

Figure 2.

Possible combinations of A and gs from measured Δ13C of Year 0 (current-year; a and c) and Year 1 (1-year-old; b and d) leaves from short (10-year-old, a and b) and tall (120-year-old, c and d) trees when gm is excluded (Eq. (3), solid line, open symbols) and included (Eq. (4), dashed line, closed symbols). The points indicate fitted values of A when gs was assumed to equal values given in Table 2. See text for further explanation.

Discussion

We have demonstrated that S, Nm and Amax were lower in Year 1 leaves compared with Year 0 leaves but we did not detect significant differences in gs or gm. We could not detect significant differences in gs for leaves of the same age from tall trees compared with those on short trees, but values of gm were lower in leaves from tall trees. Values of foliar δ13C were less depleted for leaves of the same age on tall trees compared with short trees, consistent with more pronounced limitation to photosynthesis from diffusive conductance, possibly attributable to lower gm associated with thicker leaves. However, lower values of Vcmax in Year 1 leaves compared with Year 0 leaves suggest more pronounced biochemical limitation of photosynthesis, possibly associated with lower nitrogen concentrations in Year 1 leaves. Our results provide further support for the notion that there is more pronounced hydraulic limitation to photosynthesis with increasing tree height.

Our values of gm were lower than maximum values reported for a wide range of species in earlier reviews (Manter and Kerrigan 2004, Flexas et al. 2008, Warren 2008), although Flexas et al. (2008) emphasize the dynamic nature of gm and the high variability associated with different species, growing conditions and environmental variables. Warren (2008) pointed out that it is more useful to compare the drawdown in CO2 partial pressure between Ca and Ci and between Ci and Cc among species than absolute values of gm. For the leaves in our study (data not shown), the mean values of Ca − Ci and Ci − Cc were 15.6 and 9.8 Pa, respectively, and these are very similar to the mean values of 13.6 and 8.8 Pa reported for woody deciduous trees (Warren 2008). Further, the ratio of gm/gs was close to unity for all leaves, similar to the value of 0.82 mol mol−1 for deciduous trees (Warren 2008).

Variability in gm with leaf age is likely attributable to both anatomical and biochemical factors (Warren 2008). Niinemets et al. (2005) attributed lower rates of photosynthesis in older leaves of shrub species to lower Nm and reduced amounts of photosynthetic proteins, but lower values of gm were attributable to higher cell wall fractions in older leaves. The increased cell wall fraction in older Quercus ilex leaves led to increased limitations to diffusion resulting from structural acclimation to the light environment and accumulation of structural compounds (Niinemets et al. 2006). In contrast to these findings, despite differences in gs and gm in needles ranging in age from current year to 3 years in Pinus pinaster, Warren (2006) found no differences in stomatal or mesophyll limitations to photosynthesis with increasing needle age. Lower rates of photosynthesis with increasing needle age were attributed to lower Rubisco activity. However, lower values of gm are generally associated with thicker leaves (Warren and Adams 2006). Although mechanistic explanations to account for differences in gm in relation to leaf structural properties are not known, data from a wide range of species suggest that leaf structure sets the upper limit for gm in leaves with high values of S (younger leaves) but actual gm varies in response to other environmental variables (Flexas et al. 2008).

Despite significantly higher values of S in Year 0 leaves compared with Year 1 leaves at our site, the lack of significant differences in gs and gm, but marked differences in Nm and Vcmax between leaf ages support the conclusion that differences in the rate of photosynthesis are attributable to biochemical limitation of photosynthesis, possibly associated with low nitrogen availability. The values of Nm for Nothofagus were low when compared with other New Zealand native broadleaved, nitrogen-fixing species (Dungan et al. 2003, Whitehead et al. 2005), non-nitrogen-fixing species (Whitehead and Walcroft 2005) and mature Nothofagus fusca (Hollinger 1996). In an earlier study at our site, Clinton et al. (2002) suggested that low Nm in both the 10- and 120-year-old stands could be attributable to reduced soil nitrogen availability, possibly resulting from disturbance, compared with an adjacent mature (150+ years old) stand, leading to lower rates of productivity. We also found that Na for the two leaf ages was not significantly different, suggesting that lower Nm in Year 1 leaves might be due to dilution of nitrogen with increased mass per unit area (lower S), as reported in the Mediterranean shrub species Q. ilex by Niinemets et al. (2006). Further support for biochemical limitation to photosynthesis is provided by reconstruction of the response curves of photosynthesis to ambient, intercellular and chloroplastic CO2 partial pressure (Figure 1) and estimates of stomatal, Ls, and mesophyll, Lm, limitations to photosynthesis (Table 3). The rates of photosynthesis in Year 1 leaves were much lower than those in Year 0 leaves but Lm was lower in Year 1 leaves, suggesting that biochemical limitation to photosynthesis was higher for Year 1 leaves compared with Year 0 leaves.

Our data demonstrate that gs and gm were generally lower in the tall trees compared with the short trees for both leaf ages, but only the difference in gm was significant. Evidence from several studies suggests that gm declines with increasing water stress over periods from several days to weeks (Loreto et al. 1997, Flexas et al. 2008), but there have been few studies of changes in gm with increasing height within tree canopies. Warren et al. (2003) showed an increase in gm from 160 to 200 mmol m−2 s−1 between the lower and upper canopy in a 34-m-tall P. menziesii tree, but this difference was likely associated with higher levels of irradiance or higher leaf nitrogen concentration in the upper canopy. In contrast, Woodruff et al. (2009) found a decrease in gm of 1.1 mmol m−2 s−1 per metre increase in height when measured in P. menziesii trees ranging in height from 5 to 55 m. They concluded that decreasing gm with increasing tree height was associated with an increasing water potential gradient and resulting effects of turgor on leaf expansion in taller trees.

Enrichment of foliage δ13C with increasing tree height (McDowell et al. 2002, 2005) is consistent with a height-related increase in water use efficiency resulting from increased hydraulic limitation. Although this may be more pronounced in upper canopies where foliage is fully exposed to high irradiance, the effect may be obscured in the lower canopy because of shading effects on stomatal conductance and photosynthesis (Waring and Silvester 1994). In our study, lack of significant differences in gs, Amax and Asat between leaves of the same age on both short and tall trees suggests the absence of shading effects in trees of different heights. However, the highly significant enrichment in foliage δ13C, the decrease in gm, and higher Ls and Lm for leaves of the same age on tall trees compared with those on short trees provide evidence to support the hydraulic limitation hypothesis (McDowell et al. 2002, 2005) and its effect on gm. This effect is independent of a biochemical limitation to photosynthesis as shown by the lack of differences in Vcmax with tree height.

The range in our data was not sufficient to allow us to reveal relationships between A and gm but other studies have shown that these variables scale proportionally (Loreto et al. 1992, DeLucia et al. 2003, Warren et al. 2003, Bown et al. 2009), although the relationship varies with the magnitude of gm (Ethier and Livingston 2004, Warren and Adams 2006, Warren 2008). Variability in gm is often not explained well by differences in Amax, partly because of errors in estimating gm (Warren 2008) and because of differences in the relationship between gm and Amax among species (Warren 2004). This results in differences in the magnitude of the gradient between Ci and Cc and thus differences in the interpretation of Vcmax and Jmax from A/Ci rather than A/Cc curves. Most values of Vcmax reported in the literature are based on calculations from A/Ci curves (Farquhar et al. 1980), which assume that gm is infinitely large and therefore there is no reduction in CO2 partial pressure from Ci to Cc, leading to underestimation of Vcmax (Ethier and Livingston 2004). In our study, calculations of Vcmax using A/Cc curves were 60% higher than those using A/Ci curves, which is exactly the same increase as the mean value for a range of species compiled by Warren (2008), noting the need to use appropriate kinetic constants (Bernacchi et al. 2002, Ethier and Livingston 2004).

We are aware that our estimates of gm using the constant J method are insensitive to changes in Rd but very sensitive to errors in Γ* (Harley et al. 1992). We minimized these errors using independent estimates of Γ* (Pons et al. 2009), avoided sensitivity of Γ* to temperature (Warren 2008) by making all measurements at the same temperature and maximized the number of measurements in the curvature region of the A/Ci curve (Ethier and Livingston 2004). Further, we minimized errors in Rd from leakage of CO2 across the gasket in the leaf chamber (Pons and Welschen 2002) because leaves were fully contained within the chamber except for petioles passing through the gasket. Further, the flow rate was set to ensure a reasonable difference in CO2 partial pressure between inside and outside the chamber (Pons et al. 2009).

Estimates of instantaneous water use efficiency obtained from measurements of A/gs′, where gs′ is stomatal conductance to water vapour, would not have been appropriate in our study because we were working with detached branches. However, estimates of water use efficiency from measurements of foliar δ13C can be used as an integrated measure for the life of the leaf, as described by Woodruff et al. (2009). Our measurements showing that δ13C values were less depleted for leaves of the same age on tall trees compared with short trees provide support for a height-related increase in water use efficiency, which is consistent with the observation of increasing hydraulic limitation of photosynthesis with increasing height (Ryan and Yoder 1997, McDowell et al. 2002, 2005, Woodruff et al. 2009). Fitting the model of carbon isotope discrimination using long-term integrated measurements of foliage δ13C and Eq. (4) to show the potential operating combination of A and gs highlighted the importance of gm in regulating photosynthesis. The surfaces showed that, when using measured values of gs, A was overestimated substantially when gm was excluded from the model. The model allows interpretation of the response of A to a wide range in gs and is not constrained to the values measured on the sampled shoots.

Independent estimates of gm combined with our model of carbon isotope fractionation provide a powerful approach to estimating actual, integrated operating values of A within set limits of gs. Incorporation of fractionation attributable to gm in the model highlighted the significance of limitation to photosynthesis by gm and could be used to analyse responses of gm to other environmental variables.

In conclusion, our data and model of carbon isotope fractionation that included discrimination attributable to gm provide further evidence for strong limitation to photosynthesis from mesophyll conductance that is more pronounced in tall trees compared with short trees. However, differences in photosynthesis between leaves of different ages on trees of the same height were more strongly attributable to differences in biochemical limitation than gm. Our findings also confirm the underestimation of Vcmax if the effects of gm are ignored, highlighting the need to incorporate responses of gm to environmental variables in models of canopy photosynthesis.

Funding

Funding for this work was provided by the Foundation for Research, Science and Technology, New Zealand.

Acknowledgements

We are grateful to Gilbert Ethier for early discussion on data interpretation and to Markus Löw for undertaking statistical analysis. We thank Rob Allen and Murray Davis for permission to use the field plots and John Hunt for collecting the leaves using a shotgun.

References

Barbour
M.M.
Warren
C.R.
Farquhar
G.D.
Forrester
G.
Brown
H.
2010
.
Variability in mesophyll conductance between barley genotypes, and effects on transpiration efficiency and carbon isotope discrimination
.
Plant, Cell Environ
 .
33
:
1176
1185
.
Bernacchi
C.J.
Singsaas
E.L.
Pimentel
C.
Portis
A.R.
Long
S.P.
2001
.
Improved temperature response functions for models of Rubisco-limited photosynthesis
.
Plant, Cell Environ
 .
24
:
253
259
.
Bernacchi
C.J.
Portis
A.R.
Nakano
H.
von Caemmerer
S.
Long
S.P.
2002
.
Temperature response of mesophyll conductance. Implications for the determination of rubisco enzyme kinetics and for limitations to photosynthesis in vivo
.
Plant Physiol
 .
130
:
1992
1998
.
Bickford
C.P.
McDowell
N.G.
Erhardt
E.B.
Hanson
D.T.
2009
.
High-frequency field measurements of diurnal carbon isotope discrimination and internal conductance in a semi-arid species, Juniperus monosperma
.
Plant, Cell Environ
 .
32
:
796
810
.
Bown
H.E.
Watt
M.S.
Mason
E.G.
Clinton
P.W.
Whitehead
D.
2009
.
The influence of nitrogen and phosphorus supply and genotype on mesophyll conductance limitations to photosynthesis in Pinus radiata
.
Tree Physiol
 .
29
:
1143
1151
.
Brugnoli
E.
Farquhar
G.D.
2000
.
Photosynthetic fractionation of carbon isotopes. In
Photosynthesis
 :
Physiology and Metabolism. Eds
.,
Leegood
R.C.
Sharkey
T.D.
von Caemmerer
S.
Kluwer Academic Publishers
,
The Netherlands
, pp
399
434
.
Clinton
P.W.
Allen
R.B.
Davis
M.R.
2002
.
Nitrogen storage and availability during stand development in a New Zealand Nothofagus forest
.
Can. J. For. Res
 .
32
:
1
9
.
Davis
M.R.
Allen
R.B.
Clinton
P.W.
2003
.
Carbon storage along a stand development sequence in a New Zealand Nothofagus forest
.
For. Ecol. Manag
 .
177
:
313
321
.
DeLucia
E.H.
Whitehead
D.
Clearwater
M.J.
2003
.
The relative limitation of photosynthesis by mesophyll conductance in co-occurring species in a temperate rainforest dominated by the conifer Dacrydium cupressinum
.
Funct. Plant Biol
 .
30
:
1197
1204
.
Dungan
R.J.
Whitehead
D.
Duncan
R.
2003
.
Seasonal and temperature dependence of photosynthesis and respiration for two co-occurring broad-leaved tree species with contrasting leaf phenology
.
Tree Physiol
 .
23
:
561
568
.
Eichelmann
H.
Oja
V.
Rasulov
B.
Padu
U.
Bichele
I.
Pettai
H.
Niinemets
Ü.
Laisk
A.
2004
.
Development of leaf photosynthetic parameters in Betula pendula Roth leaves: correlations with photosystem I density
.
Plant Biol
 .
6
:
307
318
.
Ethier
G.J.
Livingston
N.J.
2004
.
On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar–von Caemmerer–Berry leaf photosynthesis model
.
Plant, Cell Environ
 .
27
:
137
153
.
Ethier
G.J.
Livingston
N.J.
Harrison
D.L.
Black
T.A.
Moran
J.A.
2006
.
Low stomatal and internal conductance to CO2 versus Rubisco deactivation as determinants of the photosynthetic decline of ageing evergreen leaves
.
Plant, Cell Environ
 .
29
:
2168
2184
.
Evans
J.R.
1989
.
Photosynthesis and nitrogen relationships in leaves of C3 plants
.
Oecologia
 
78
:
9
19
.
Evans
J.R.
1999
.
Leaf anatomy enambes more equal access to light and CO2 between chloroplasts
.
New Phytol
 .
143
:
93
104
.
Evans
J.R.
Loreto
F.
2000
.
Acquisition and diffusion of CO2 in higher plant leaves. In
Photosynthesis: Physiology and Metabolism
 .
Leegood
R.C.
Sharkey
T.D.
von Caemmerer
S.
Kluwer Academic Publishers
,
The Netherlands
, pp
321
351
.
Farquhar
G.D.
Sharkey
T.D.
1982
.
Stomatal conductance and photosynthesis
.
Annu. Rev. Plant Physiol
 .
33
:
317
345
.
Farquhar
G.D.
von Caemmerer
S.
Berry
J.A.
1980
.
A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species
.
Planta
 
149
:
78
90
.
Farquhar
G.D.
O'Leary
M.H.
Berry
J.A.
1982
.
On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves
.
Aust. J. Plant Physiol
 .
9
:
121
137
.
Farquhar
G.D.
Ehleringer
J.R.
Hubick
K.T.
1989
.
Carbon isotope discrimination and photosynthesis
.
Annu. Rev. Plant Physiol. Plant Mol. Biol
 .
40
:
503
537
.
Field
C.
Mooney
H.A.
1986
.
The photosynthesis–nitrogen relationship in wild plants. In
On the Economy of Plant Form and Function. Ed
 .
Givnish
T.
Cambridge University Press
,
Cambridge
, pp
25
55
.
Flexas
J.
Ribas-Carbó
M.
Diaz-Espejo
A.
Galmés
J.
Medrano
H.
2008
.
Mesophyll conductance to CO2: current knowledge and future prospects
.
Plant, Cell Environ
 .
31
:
602
621
.
Genty
B.
Briantais
J.M.
Baker
N.R.
1989
.
The relationship between quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence
.
Biochim. Biophys. Acta
 
990
:
87
92
.
Grassi
G.
Magnani
F.
2005
.
Stomatal, mesophyll conductance and biochemical limitations to photosynthesis as affected by drought and leaf ontogeny in ash and oak trees
.
Plant, Cell Environ
 .
28
:
834
849
.
Hanba
Y.T.
Miyazawa
S.I.
Kogami
H.
Tershima
I.
2001
.
Effects of leaf age on internal CO2 transfer conductance and photosynthesis in tree species having different types of shoot phenology
.
Aust. J. Plant Physiol
 .
28
:
1075
1084
.
Harley
P.C.
Loreto
F.
Di Marco
G.
Sharkey
T.D.
1992
.
Theoretical considerations when estimating the mesophyll conductance to CO2 flux by analysis of the response of photosynthesis to CO2
.
Plant Physiol
 .
98
:
1429
1443
.
Hewitt
A.D.
1998
.
New Zealand soil classification. Landcare Research Science Series 14, No. 1
.
Manaaki Whenua Press, Lincoln
,
New Zealand
.
Hollinger
D.Y.
1996
.
Optimality and nitrogen allocation in a tree canopy
.
Tree Physiol
 .
16
:
627
634
.
Jones
H.G.
2002
.
Plants and microclimate
,
2nd edn
.
Cambridge University Press
,
Cambridge
,
428 p
.
Jordan
D.B.
Ogren
W.L.
1984
.
The CO2/O2 specificity of ribulose, 1, 5-bisphosphate carboxylase/oxygenase. Dependence on ribulose bisphosphate concentration, pH and temperature
.
Planta
 
161
:
308
313
.
Koch
G.W.
Sillet
S.C.
Jennings
G.M.
Davis
S.D.
2004
.
The limits to tree height
.
Nature
 
428
:
851
854
.
Laisk
A.
Oja
V.
1998
.
Dynamics of leaf photosynthesis: rapid response measurements and their interpretations
.
CSIRO Publishing, Melbourne
,
Australia
.
Lanigan
G.J.
Betson
N.
Griffiths
H.
Seibt
U.
2008
.
Carbon isotope fractionation during photorespiration and carboxylation in Senecio
.
Plant Physiol
 .
148
:
2013
2020
.
Long
S.P.
Bernacchi
C.J.
2003
.
Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error
.
J. Exp. Bot
 .
54
:
2393
2401
.
Loreto
F.
Harley
P.C.
DiMarco
G.
Sharkey
T.D.
1992
.
Estimation of mesophyll conductance to CO2 flux by three different methods
.
Plant Physiol
 .
98
:
1437
1443
.
Loreto
F.
Delfine
S.
Alvino
A.
1997
.
On the contribution of mesophyll resistance to CO2 diffusion to photosynthesis limitation during water and salt stress
.
Acta Hortic
 .
449
:
417
422
.
Manter
D.K.
Kerrigan
J.
2004
.
A/Ci curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance
.
J. Exp. Bot
 .
55
:
2581
2588
.
McCracken
I.J
.
1980
.
Mountain climate in the Craigieburn Range, New Zealand. In
Mountain Environments and Subalpine Growth. Eds
 .
Benecke
U.
Davies
M.R.
New Zealand Forest Service, Wellington
,
New Zealand
, ppp
41
60
.
McDowell
N.G.
Phillips
N.
Lunch
C.K.
Bond
B.J.
Ryan
M.G.
2002
.
An investigation of hydraulic limitation and compensation in large, old Douglas-fir trees
.
Tree Physiol
 .
22
:
763
774
.
McDowell
N.G.
Licata
J.
Bond
B.J.
2005
.
Environmental sensitivity of gas exchange in different-sized trees
.
Oecologia
 
145
:
9
20
.
Mencuccini
M.
Martinez-Vilalta
J.
Vanderklein
D.
Hamid
H.A.
Korakaki
E.
Lee
S.
Michiels
B.
2005
.
Size-mediated ageing reduces vigour in trees
.
Ecol. Lett
 .
8
:
1183
1190
.
Miyazawa
S.I.
Terashima
I.
2001
.
Slow development of leaf photosynthesis in an evergreen broad-leaved tree, Castanopsis sieboldii: relationships between leaf anatomical characteristics and photosynthetic rate
.
Plant, Cell Environ
 .
24
:
279
291
.
Miyazawa
S.I.
Makino
A.
Terashima
I.
2003
.
Changes in mesophyll anatomy and sink–source relationships during leaf development in Quercus glauca, an evergreen tree showing delayed leaf greening
.
Plant, Cell Environ
 .
26
:
745
755
.
Niinemets
Ü.
Kull
O.
Tenhunen
J.D.
1999
.
Variability in leaf morphology and chemical composition as a function of canopy light environment in coexisting deciduous trees
.
Int. J. Plant Sci
 .
160
:
837
848
.
Niinemets
Ü.
Cescatti
A.
Rodeghiero
M.
Tosens
T.
2005
.
Leaf internal diffusion conductance limits photosynthesis more strongly in older leaves of Mediterranean evergreen broad-leaved species
.
Plant, Cell Environ
 .
28
:
1552
1566
.
Niinemets
Ü.
Cescatti
A.
Rodeghiero
M.
Tosens
T.
2006
.
Complex adjustments of photosynthetic potentials and internal diffusion conductance to current and previous light availabilities and leaf age in Mediterranean evergreen species Quercus ilex
.
Plant, Cell Environ
 .
29
:
1159
1178
.
Pons
T.L.
Welschen
R.A.M.
2002
.
Overestimation of respiration rates in commercially available clamp-on leaf chambers. Complications with measurement of net photosynthesis
.
Plant, Cell Environ
 .
25
:
1367
1372
.
Pons
T.L.
Flexas
J.
von Caemmerer
S.
Evans
J.R.
Genty
B.
Ribas-Carbo
M.
Brugnoli
E.
2009
.
Estimating mesophyll conductance to CO2: methodology, potential errors, and recommendations
.
J. Exp. Bot
 .
60
:
2217
2234
.
Ryan
M.R.
Yoder
B.J.
1997
.
Hydraulic limits to tree height and tree growth
.
Bioscience
 
47
:
232
242
.
Ryan
M.R.
Phillips
N.
Bond
B.J.
2006
.
The hydraulic limitation hypothesis revisited
.
Plant, Cell Environ
 .
29
:
367
381
.
Schmidt
H.-L.
Gleixner
G.
1998
.
Carbon isotope effects on key reactions in plant metabolism and 13C-patterns in natural compounds. In
Stable Isotopes: The Integration of Biological, Ecological and Geochemical Processes. Ed
 .
Griffiths
H.
Bios Scientific Publishers
,
Oxford, UK
, ppp
13
25
.
Schreiber
U.
Bilger
W.
Neubauer
C.
1994
.
Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis
. In
Ecophysiology of Photosynthesis
 .
Schulze
E.D.
Caldwell
M.M.
Springer
,
Berlin
, ppp
49
70
.
Singsaas
E.L.
Ort
D.R.
DeLucia
E.H.
2003
.
Elevated CO2 effects on mesophyll conductance and its consequences for interpreting photosynthetic physiology
.
Plant, Cell Environ
 .
27
:
41
50
.
Soil Survey Staff.
1998
.
Keys to soil taxonomy
,
8th edn
.
United States Department of Agriculture, Natural Resources Conservation Service
,
Washington, DC, USA
,
326 p
.
Waring
R.H.
Silvester
W.B.
1994
.
Variation in foliar 13C values within crowns of Pinus radiata trees
.
Tree Physiol
 .
20
:
637
643
.
Warren
C.R.
2004
.
The photosynthetic limitation posed by internal conductance to CO2 movement is increased by nutrient supply
.
J. Exp. Bot
 .
55
:
2313
2321
.
Warren
C.R.
2006
.
Why does photosynthesis decrease with needle age in Pinus pinaster?
Trees Struct. Funct
 .
20
:
157
164
.
Warren
C.R.
2008
.
Stand aside stomata, another actor deserves centre stage: the forgotten role of internal conductance to CO2 transfer
.
J. Exp. Bot
 .
59
:
1475
1487
.
Warren
C.R.
Adams
M.A.
2001
.
Distribution of N, Rubisco and photosynthesis in Pinus pinaster and acclimation to light
.
Plant, Cell Environ
 .
24
:
597
609
.
Warren
C.R.
Adams
M.A.
2006
.
Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use in photosynthesis
.
Plant, Cell Environ
 .
29
:
192
201
.
Warren
C.R.
Ethier
G.J.
Livingston
N.J.
Grant
N.J.
Turpin
D.H.
Harrison
D.L.
Black
T.A.
2003
.
Transfer conductance in second growth Douglas-fir (Pseudotsuga menzisii (Mirb.) Franco) canopies
.
Plant, Cell Environ
 .
26
:
1215
1227
.
Whitehead
D.
Walcroft
A.S.
2005
.
Forest and shrubland canopy carbon uptake in relation to foliage nitrogen concentration and leaf area index: a modelling analysis
.
Ann. For. Sci
 .
62
:
525
535
.
Whitehead
D.
Boelman
N.T.
Turnbull
M.H.
Griffin
K.G.
Tissue
D.T.
Barbour
M.M.
Hunt
J.E.
Richardson
S.
Peltzer
D.A.
2005
.
Photosynthesis and reflectance indices for rainforest species in ecosystems undergoing progression and retrogression along a soil fertility chronosequence in New Zealand
.
Oecologia
 
144
:
233
244
.
Woodruff
D.R.
Meinzer
F.C.
Lachenbruch
B.
2008
.
Height related trends in leaf xylem anatomy and hydraulic characteristics in a tall conifer: safety versus efficiency in foliar water transport
.
New Phytol
 .
180
:
90
99
.
Woodruff
D.R.
Meinzer
F.C.
Lachenbruch
B.
Johnson
D.M.
2009
.
Coordination of leaf structure and gas exchange along a height gradient in a tall conifer
.
Tree Physiol
 .
29
:
261
272
.