JXB Advance Access originally published online on August 13, 2004
Journal of Experimental Botany 2004 55(406):2313-2321; doi:10.1093/jxb/erh239
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RESEARCH PAPER |
The photosynthetic limitation posed by internal conductance to CO2 movement is increased by nutrient supply
School of Forest and Ecosystem Science, The University of Melbourne, Water Street, Creswick, VIC 3363, Australia
* Fax: +61 3 5321 4277. E-mail: crwarren{at}unimelb.edu.au
Received 9 March 2004; Accepted 30 June 2004
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Abstract |
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The internal conductance to CO2 supply from substomatal cavities to sites of carboxylation may pose a large limitation to photosynthesis, but little is known of how it is affected by nutrient supply. Knowing how internal conductance responds to nutrient supply is critical for interpreting the biochemical responses from A–Ci curves. The aim of this paper was to examine the response of gi and photosynthetic parameters to nutrient supply in glasshouse-grown seedlings of the evergreen perennial Eucalyptus globulus Labill. Seedlings were grown with five different nutrient treatments and gi was estimated from concurrent measurements of gas exchange and fluorescence. Internal conductance varied between 0.12 and 0.19 mol m–2 s–1 and the relative limitation of photosynthesis due to internal conductance was greater than the stomatal limitation. In most species these two limitations are rather similar, but in E. globulus stomatal limitations were abnormally low due to high stomatal conductance (0.31 to 0.39 mol m–2 s–1). The large positive response of photosynthesis to nutrient supply was not matched by changes in internal conductance, and thus the relative limitation of photosynthesis due to internal conductance increased with increasing nutrient supply. Failure to account for finite internal conductance led to estimates of Vcmax that were 60% of the true value, which, in turn, led to an underestimation of in vivo Rubisco specific activity (as Vcmax/Rubisco content). The specific activity of Rubisco in E. globulus (21 mol mol–1 s–1) was close to the maximum published estimates, and thus, despite these leaves containing a large fraction of N as Rubisco (38–44%) there was no evidence that Rubisco activity was down-regulated or that the enzyme was in excess.
Key words: Internal resistance, Jmax, mesophyll conductance, nitrogen, nutrient, photosynthesis, transfer conductance, Vcmax
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Introduction |
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For photosynthesis (A) to occur, CO2 must diffuse from the atmosphere to the sites of carboxylation. The concentration of CO2 at the sites of carboxylation (Cc) is less than atmospheric (Ca) owing to a series of gas-phase (air) and liquid-phase (mesophyll cell) resistances. In the gaseous phase, CO2 must diffuse across a boundary layer in the air above the foliage surface, through a stomatal opening, and across intercellular air spaces in the substomatal cavity. In the liquid phase there are resistances as CO2 enters the liquid phase at the surface of mesophyll cells, as CO2 diffuses within the cell to the chloroplast membrane, and from there to the sites of carboxylation (Aalto and Juurola, 2002
). It has been assumed that the concentration of CO2 in the substomatal cavities (Ci, for example, as measured by gas exchange) is uniform throughout the leaf and thus the same as Cc. There is mounting evidence that this assumption is invalid and Cc may be significantly less than Ci (Evans et al., 1986; Parkhurst and Mott, 1990; Lloyd et al., 1992; Epron et al., 1995; Ethier and Livingston, 2004). This drawdown in CO2 concentration between Ci and Cc is a function of rates of photosynthesis and the internal conductance 
Internal conductance affects the interpretation of the photosynthetic response to Ci and the biochemical photosynthesis model of Farquhar et al. (1980). The Farquhar model states that the rate of photosynthesis is limited by one of two (or in special cases three) processes, and that limitation shifts from one to another as CO2 concentration changes. At low CO2 concentrations, photosynthesis is limited by the maximum rate of Rubisco carboxylation (Vcmax), whereas at high CO2 concentrations photosynthesis is limited by the electron transport limited rate of RuBP regeneration (Jmax). Analysis of the 
response has become enormously popular and there are now data for a great many species growing under various conditions (Wullschleger, 1993; Wohlfart et al., 1999). The fashion in which the response is fitted assumes implicitly that there is no draw-down in CO2 concentration from Ci to Cc. It is now known that this assumption is incorrect (see above) and failure to account for finite gi results in erroneously low estimates of Vcmax (Epron et al., 1995; Bernacchi et al., 2002; Centritto et al., 2003; Warren et al., 2003a; Ethier and Livingston, 2004).
The author is aware of only one paper that has examined gi in plants grown with varying nutrient supply (Triticum; von Caemmerer and Evans, 1991), and it reported that gi was positively related to foliage N content per unit area. A subsequent paper suggested that the increase in gi was smaller than the increase in Rubisco carboxylation and thus the limitation posed by gi was greater in leaves with a high N content (Poorter and Evans, 1998). This type of experiment has not been replicated with other species and, in general, little is known of how gi responds to N supply. While not explicitly considering the effect of nutrient supply, several previous studies have reported gi at a range of N or Rubisco contents in leaves grown under different light intensities (Lloyd et al., 1992; Hanba et al., 1999; Piel et al., 2002; Warren et al., 2003a), in a developmental series (Hanba et al., 2001), in leaves of wild-type and anti-SSU Nicotiana (Evans et al., 1994), and among species (Lloyd et al., 1992; Hanba et al., 1999, 2001; Piel et al., 2002; Singsaas et al., 2004). It is not known if the relationship of gi with N or Rubisco varies among species or depends on the underlying cause of variation in N or Rubisco (e.g. light versus nutrient supply), and thus one aim of this paper was to determine if there are ‘universal’ N–gi and Rubisco–gi relationships based on a compilation of literature data.
The response of photosynthetic parameters to nutrient supply is widely reported (Walcroft et al., 1997; Warren et al., 2003b), but in none of these previous reports was gi determined. The underlying biochemical response (of photosynthesis) to nutrient supply may be confounded by the response of gi. If, for example, elevated nutrient supply increases Vcmax but gi is unaffected, the relative limitation due to gi will increase. A common observation in plants grown with a high N supply is a reduction in Vcmax/Rubisco content (i.e. specific activity), which is seen as evidence of over-investment in Rubisco (Warren and Adams, 2004). However, these studies were based on Vcmax determined from Ci, and thus the reduction in apparent Vcmax/Rubisco could simply indicate that the limitation due to gi is greater in high N plants, as suggested by Poorter and Evans (1998). The aim of this paper was to examine the response of gi and photosynthetic parameters to nutrient supply in glasshouse-grown seedlings of Eucalyptus globulus Labill. Seedlings were grown with five different nutrient treatments and gi was estimated from concurrent measurements of gas exchange and fluorescence (Harley et al., 1992).
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Materials and methods |
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Plant material and treatments
Seed of Eucalyptus globulus ssp. globulus (CSIRO ATSC seedlot 18725) was obtained from the Australian Tree Seed Centre (Kingston, ACT, Australia). On 25 August (late winter) seed was germinated in moist vermiculite in a glasshouse at the Forest Science Centre (Creswick, Victoria, Australia). Approximately one month later (26 September) germinants had one or two pairs of true leaves. At this stage germinants were carefully transferred to 6.4 l plastic pots (16 cm squarex25 cm high) filled with coarse sand. For the first month after transfer to pots, seedlings were irrigated to field capacity three times per week with balanced nutrient solution at one-quarter its normal concentration, while for the second month they received the same nutrient solution at half its normal concentration. The full-strength nutrient solution contained 8 mM N (as 4 mM NH4NO3), 0.67 mM P, 4 mM K, 2.9 mM Ca, 1.5 mM Mg, 1.5 mM S, 52 µM Mn, 49 µM Fe, 20 µM Cu, 15 µM Zn, 8 µM B, and 0.03 µM Mo. Previous studies have shown this nutrient solution produces optimal growth of E. globulus and other evergreen perennials (C Warren, unpublished data).
On 20 November, differential nutrient treatments were imposed. 25 seedlings were randomly assigned to one of five nutrient treatments. The five nutrient treatments were obtained by dilution of full-strength nutrient solution, and thus all nutrients (not just N) varied. However, for the sake of convenience nutrient treatments are referred to based on their elemental N concentrations: 0.5, 1.0, 2.0, 4.0, and 8.0 mM N. Seedlings were irrigated to field capacity with nutrient solution four times per week, and with water on the remaining days. At no time were soil water deficits allowed to develop, and thus larger seedlings were irrigated with water or nutrient solution more frequently than smaller seedlings. From October until the end of the experiment, temperatures inside the glasshouse varied between 17 °C and 23 °C, while leaf-to-air vapour pressure deficit was typically less than 1 kPa. Seedlings were exposed to full (glasshouse) sunlight at all times, which was approximately 70% of external sunlight, with weekly average PPFD varying between 33 and 41 mol m–2 d–1.
Photosynthesis measurements were made on five seedlings per treatment between 29 December and 2 January (6 weeks after nutrient treatments commenced). All measurements were made on the youngest fully expanded leaves.
Specific leaf area and leaf nitrogen concentration
The specific leaf area (SLA, m2 leaf area kg–1 dry mass) was determined for a leaf opposite the one used for photosynthesis measurements. Leaves were excised, placed in damp plastic bags and transported to the laboratory where projected area was determined with a planimeter (LI-3000A/LI-3050A, Li-Cor). Dry mass was determined after 72 h at 70 °C. Leaves were subsequently ground to a fine powder in a mixer mill (MM301, Retsch, Haan, Germany) and analysed for total N by Dumas combustion.
Leaf Rubisco content
Four leaf discs (0.56 cm2 each) were punched from the leaves used for photosynthesis measurements, placed in a 2 ml Eppendorf microfuge tube (Safe-Lock tube 2.0 ml, Eppendorf AG, Hamburg, Germany), frozen in liquid N and stored at –80 °C until analysis. Rubisco was quantified by capillary electrophoresis using a method modified from Warren et al. (2000). Samples were removed from the freezer and kept frozen while adding two 3 mm diameter stainless steel ball bearings and approximately 0.2 ml of polyvinylpolypyrrolidone. Frozen samples were rapidly ground in a mixer mill (MM301, Retsch, Haan, Germany). 1.0 ml of cold (0–4 °C) extraction buffer (50 mmol l–1 TRIS-HCl, 0.1 mol l–1 2-mercaptoethanol, 1% w/v SDS, and 15% v/v glycerol) was added to each tube and samples were extracted by shaking for 90 s with the mixer mill. The extract was centrifuged for 2 min in a microfuge, the supernatant removed, and the pellet was re-extracted with an additional 1.0 ml of extraction buffer. The supernatants were pooled, mixed and then recentrifuged at room temperature. A 150 µl aliquot was precipitated with the methanol/chloroform/water procedure (Wessel and Flügge, 1984), taken up in 400 µl of extraction buffer and denatured by heating at 95–100 °C for 10 min in a water bath. Samples were stored at 0–4 °C for no greater than 6 h prior to analysis. Capillary electrophoresis was performed with a Beckman P/ACE MDQ system (Beckman-Coulter, Fullerton, CA) fitted with a photo-diode array detector and controlled by a computer running System Gold software (Beckman-Coulter). The separation of proteins was performed in SDS 14–200 gel buffer (Beckman-Coulter) in a SDS-coated fused-silica capillary (100 µm i.d.x31.2 cm long, eCap SDS 14–200 capillary, Beckman-Coulter). To reduce analysis time, samples were injected from the outlet side, resulting in an effective length from injection to detection window of 11.2 cm. Electrophoresis was conducted at 20 °C, a constant voltage of 9 kV, with 0.5 psi of pressure applied at both ends of the capillary. Samples were injected at 0.5 psi for 15 s, and protein–SDS complexes were detected at 220 nm. The capillary was rinsed sequentially between successive electrophoretic runs with 1 M HCl for 60 s, followed by SDS 14-200 gel buffer for 120 s. Standard curves for purified Rubisco were highly linear (r2=0.99) over the range of 0.05 to 1 mg ml–1. Standard curves constructed by serial dilution of a leaf extract were also highly linear (r2=0.99), and this, combined with 95% recovery of Rubisco in a spike and recovery test, suggests quantification was unaffected by the complex matrix of E. globulus leaves (Warren et al., 2000). Rubisco concentrations were initially calculated on a leaf area basis, but were subsequently converted to a dry mass basis using measured SLA. The fraction of total foliar nitrogen present in Rubisco was calculated using the assumption that Rubisco contains 16.7% N.
Simultaneous fluorescence and gas exchange measurements
Simultaneous gas exchange and chlorophyll fluorescence measurements were made with an open gas exchange system (LI-6400, Li-Cor, Lincoln, NE, USA) and integrated fluorescence chamber head (LI-6400–40). The photochemical efficiency of photosystem II (
PSII) was determined by measuring steady-state fluorescence (F') and maximal fluorescence (
) during a light-saturating pulse (Genty et al., 1989):
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The rate of linear electron transport (Jf) is related to PSII:
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is the total leaf absorptance, the factor 0.5 describes the distribution of light between the two photosystems and is assumed to be 0.5. Jf is not strictly related to linear electron transport because fluorescence primarily measures upper cell layers and is thus not representative of the whole leaf. Furthermore, the distribution of light between photosystems is hard to determine. Owing to these uncertainties, no a priori assumptions were made regarding relationships between fluorescence and linear electron transport, and instead chose to determine an empirical relationship. This was done by measuring a light-response curve under non-photorespiratory conditions and rests on the assumption that under non-photorespiratory conditions linear electron transport should be wholly associated with gross photosynthesis. Such a ‘calibration’ procedure obviates the need for measuring leaf absorptance and assumptions regarding the distribution of light between photosystems and representativeness of fluorescence to whole-leaf processes. In E. globulus, this relationship was highly linear (R2=0.96) with a non-significant intercept for all leaves (Fig. 1), suggesting there were no significant alternative electron sinks.
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To construct a ‘calibration’ curve, reference gas was supplied from a cylinder containing a mixture of 450 µmol mol–1 CO2 in 1% O2 (Linde Gas, Australia). Leaf temperature was controlled at 25 °C, leaf-to-air vapour pressure deficit was maintained at 0.8–1.3 kPa using a dew-point generator (LI-610, Li-Cor). Leaves were acclimated to a PPFD of 2000 µmol m–2 s–1 for at least 30 min, or until stomatal conductance, net photosynthesis, and fluorescence were steady. Thereafter, PPFD was decreased, in ten steps, from 2000 µmol m–2 s–1 to 0 µmol m–2 s–1. At each PPFD, leaves were acclimated for at least 5 min until stomatal conductance, net photosynthesis, and fluorescence reached a steady-state, and then gas exchange and fluorescence parameters were recorded.
After the last point was measured (i.e. at 0 µmol m–2 s–1 PPFD), the gas supply was changed to 450 µmol mol–1 CO2 in 21% O2, and PPFD was increased to 2000 µmol m–2 s–1. These measurements were subsequently used to estimate gi. Leaves were allowed to acclimate to these non-photorespiratory conditions for at least 30 min, or until gas exchange and fluorescence were steady. Thereafter, a light response curve was generated in the same fashion as for non-photorespiratory conditions.
The CO2 and H2O sensitivity of the LI-6400 is affected by O2 concentration of the analysis gas. The effect of O2 on CO2 sensitivity is small enough to be ignored, whereas the change in H2O sensitivity of approximately 3% (between 1% and 21% O2) has a measurable effect on the estimation of stomatal conductance and Ci. Therefore, the measured H2O concentration was corrected using an empirical correction reported by Ghannoum et al. (1998).
gi was estimated with the ‘variable J method’ (Harley et al., 1992):
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*=38.7 (von Caemmerer et al., 1994; Bernacchi et al., 2002)
Measurement of the CO2 response of photosynthesis
The CO2 response of photosynthesis was determined 1 d after fluorescence measurements. Leaves were enclosed in a 2x3 cm broadleaf chamber with integrated light source (LI-6400–02B, Li-Cor). Using the broadleaf chamber, as opposed to the fluorescence chamber, increased the enclosed leaf area and the signal-to-noise ratio by a factor of 3, a critical factor when making photosynthesis measurements at low Ca and PPFD. Measurements were made at 25±1 °C and a leaf-to-air vapour pressure deficit of 0.8–1.3 kPa. The molar flow rate through the chamber was varied between 300 and 750 µmol s–1 depending on the rates of photosynthesis and transpiration.
For each leaf an curve was generated at 2000 µmol m–2 s–1 PPFD. Leaves were exposed to 400 µmol mol–1 CO2 in air and a PPFD of 2000 µmol m–2 s–1 until rates of photosynthesis and transpiration were steady. After this, Ca was increased to 1500 µmol mol–1 and an curve was generated by decreasing, stepwise, Ca to 50 µmol mol–1. Over the Ca range from 150 to 50 µmol mol–1, measurements were made at 25 µmol mol–1 intervals so as to ensure adequate characterization of the initial portion of the curve. At each Ca photosynthesis was allowed to stabilize for at least 3 min, and three successive measurements were made at 1 min intervals to ensure stability.
Data were not corrected for diffusion of CO2 into and out of the leaf chamber because this was insignificant with this experimental set-up. Preliminary experiments established that with a molar flow rate of 300 µmol s–1 (the lowest flow rate used in these experiments) there was never more than 1.5 µmol mol–1 diffusion of CO2 into an empty chamber maintained at 50 µmol m–1 CO2.
Vcmax and Jmax were determined from CO2 response data fitted to the photosynthesis model of Farquhar et al. (1980), essentially as described previously (Warren et al., 2003b). For 
a value of 736 µmol mol–1 was used (von Caemmerer et al., 1994), while 0.24 was assumed for the apparent quantum yield of electron transport.
Estimation of Cc and relative limitation on photosynthesis imposed by gi and gs
Using estimated gi and measured A and Ci, Cc was calculated as:
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; Warren et al., 2003a). Estimates of A were based on CO2-response curves and measured gi and gs. Rates of net photosynthesis were estimated assuming gi and gs were as measured (An, the light-saturated rate of photosynthesis at Ca=360 µmol mol–1), assuming gi was infinite and gs as measured (Ail, the light-saturated rate of photosynthesis at 
), or assuming gi as measured and gs was infinite (Asl, the light-saturated rate of photosynthesis at Ci=360 µmol mol–1). The relative limitations due to internal resistances (Li) and stomatal resistances (Ls) were estimated as:
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Results |
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Specific leaf area and leaf content of nitrogen and Rubisco
Specific leaf area (SLA) was not significantly affected by differential nutrient treatments, despite a trend for SLA to increase with increasing N supply (Table 1). Nitrogen content per unit area varied significantly among nutrient treatments, increasing from 0.7 g m–2 to 1.3 g m–2 with an increase in N supply from 0.5 mM to 8.0 mM. This difference was proportionally greater on a mass basis (12–27 mg g–1; data not shown). The content per unit area of Rubisco increased from 1.8 g m–2 to 3.0 g m–2 with increasing N supply, but this was only marginally significant (P=0.08) owing to large variation within treatments. On a mass basis, Rubisco increased from 32 to 61 mg g–1, and this difference was clearly significant (P=0.01; data not shown). There was no effect of nutrient supply on the proportion of total N allocated to Rubisco, which varied between 38% and 44% among treatments.
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Response of gi and photosynthetic parameters to nutrient supply
The rate of net photosynthesis was significantly affected by nutrient supply, increasing from 10 µmol m–2 s–1 to 16 µmol m–2 s–1 as nutrient supply increased from 0.5 mM N to 8.0 mM N (Table 2). Stomatal conductance was unaffected by nutrient supply, but there were significant differences in Ci among treatments with Ci generally lower in those plants receiving a greater nutrient supply. There were significant differences in internal conductance (gi) among treatments, with gi being noticeably lower in seedlings receiving 0.5 mM N compared with those receiving a more concentrated nutrient supply. However, gi did not vary in a monotonic fashion with nutrient supply, and thus there was a (minor) reduction in gi at the highest nutrient supply. Cc was, on average, 81 µmol mol–1 lower than Ci, and there was a general trend for Cc to decrease with increasing nutrient supply. The relative limitation posed by stomatal conductance was in all cases less than that due to internal conductance. There was a general trend for both limitations to increase with increasing nutrient supply (see also Fig. 3).
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r2=0.38; 
r2=0.32; 
r2=0.57; 
r2=0.28.Vcmax,Ci was, on average, 60% of Vcmax,Cc, while Jmax,Ci was 96% of Jmax,Cc. These differences were both significantly different (paired t-test, P<0.001). Vcmax and Jmax (on Ci and Cc bases) increased with increasing nutrient supply from 0.5 mM to 4.0 mM N, while from 4.0 mM to 8.0 mM N there was no further increase in either parameter (Table 3). Specific activity of Rubisco (estimated as Vcmax/Rubisco) did not vary with nutrient supply; however, estimates were significantly greater on a Cc basis (mean=21 mol mol–1 s–1) than on a Ci basis (mean=14 mol mol–1 s–1).
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Relationship of gi to N and Rubisco
A compilation of literature data with the author's showed that there was no common or ‘universal’ relationship of gi with either N or Rubisco (Fig. 2). Instead, relationships varied in strength and form among functional groups. E. globulus is a perennial evergreen angiosperm, but it generally had higher gi per unit N or Rubisco than other perennial angiosperms. Relationships with N content per unit area were significant only for annual angiosperms (Triticum and Nicotiana) and E. globulus. Notably, the slope of this relationship (‘gi per unit N’) was steeper in annual angiosperms than E. globulus, for which the relationship was significant but weak (r2=0.18). Relationships with Rubisco were not significant in annual angiosperms, primarily because of four Phaseolus with low gi. With the exception of these four points, annual angiosperms had higher gi than other groups at common N or Rubisco. The slope of gi–Rubisco relationships was similar in E. globulus and the group of perennial angiosperms, but gi was, on average, 0.065 mol m–2 s–1 higher in E. globulus than the other perennial angiosperms.
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r2=0.18, n=25; annual angiosperms, 
r2=0.66, n=12; relationships were not significant for perennial angiosperms (n=37) or conifers (n=5). gi was significantly related to Rubisco: E. globulus gi=0.0152 Rubisco+0.132, r2=0.18, n=25; perennial angiosperms, gi=0.020 Rubisco+0.065, r2=0.20, n=25; relationships were not significant for annual angiosperms (n=10); for conifers there were only two concordant gi–Rubisco measurements.The internal and stomatal limitations to photosynthesis were both positively correlated with contents per unit area of N and Rubisco (Fig. 3). When compared at common N or Rubisco, internal limitations were greater than stomatal limitations.
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Discussion |
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Internal conductance in seedlings of the evergreen perennial E. globulus varied between 0.12 and 0.19 mol m–2 s–1 (Table 2), which is similar to previous reports for this species (Loreto et al., 1992
) and is consistent with results from other species with similar rates of photosynthesis (Evans and Loreto, 2000; Warren et al., 2003a). Furthermore, the mean draw-down from Ci to Cc of 81 µmol mol–1 was very similar to the mean value of 78 µmol mol–1 derived from a literature review by Evans and Loreto (2000).
Most often the relative limitation due to internal conductance is slightly less than that due to stomatal conductance (Epron et al., 1995; Warren et al., 2003a), but in E. globulus internal limitations (0.19 to 0.38) were consistently greater than stomatal limitations (0.05 to 0.23). This difference is not due to higher than normal internal limitations (see above), but was instead due to lower than usual stomatal limitations due to the high gs of E. globulus seedlings (0.31 to 0.39 mol m–2 s–1). These high stomatal conductances are not unusual for E. globulus seedlings, with similar or higher gs at this vapour pressure deficit (0.8 to 1.3 kPa) being reported by Sasse and Sands (1996). Similarly high gs and low stomatal limitations were also noted in seedlings of Eucalyptus grandis (Grassi et al., 2002), so this may be a more general trait of Eucalyptus seedlings. Stomatal conductance of E. globulus (and most species) is reduced by high vapour pressure deficits and soil water deficits (Sasse and Sands, 1996), and thus one could argue that the predominance of internal limitations in the present study is a consequence of low vapour pressure deficits and saturated soils. However, this cannot explain interspecific variation in internal versus stomatal limitations because most previous examinations of gi and gs have also used saturated soils and similar vapour pressure deficits (Warren et al., 2003a). In any case, conditions that reduce gs are likely to cause (more or less) proportional reductions in gi (Roupsard et al., 1996; Delfine et al., 1999; Warren et al., 2004). If this is the case, internal limitations may always be greater than stomatal limitations in E. globulus.
The relative limitation of photosynthesis due to internal conductance increased with increasing nutrient supply and was positively correlated with leaf contents of N and Rubisco (Fig. 3). This is because there was a large positive response of photosynthesis to nutrient supply, whereas nutrient supply had a rather small and inconsistent effect on gi (Table 2) and the correlation of gi with leaf contents of nitrogen or Rubisco was positive but rather weak (Fig. 2). A similar response to nutrient supply was found in Triticum where the increase in gi was smaller than the increases in biochemical capacity (von Caemmerer and Evans, 1991). To date, there are only data from two species, but this effect of gi in dampening the biochemical response to nutrient supply may well be ubiquitous given that it occurred in such disparate species. Stomatal conductance, like gi, did not vary consistently with nutrient supply and thus also curtailed the biochemical response to nutrients.
Failure to account for finite gi affects the calculation of Vcmax, which in turn affects the determination of Rubisco specific activity (as Vcmax/Rubisco content). Neglecting the draw-down from Ci to Cc resulted in estimates of Vcmax that were 60% of the ‘true’ Vcmax (Table 3; Epron et al., 1995; Warren et al., 2003a; Ethier and Livingston, 2004) and had a proportional effect on Vcmax/Rubisco content. In vitro activity assays are unreliable in many sclerophyllous species owing to high concentrations of secondary metabolites that interfere with analysis, and thus it may be preferable to determine in vivo specific activity as Vcmax/Rubisco content (Warren et al., 2003b). However, the present study highlights that this method is very sensitive to gi. If Ci-based Vcmax had been used it would have appeared that E. globulus had a low Rubisco specific activity (14 mol mol–1 s–1) compared with published estimates of 24 mol mol–1 s–1 (von Caemmerer et al., 1994), and this would have been interpreted as evidence that E. globulus over-invests in Rubisco. When estimates were based on Cc, this discrepancy in specific activities between E. globulus (21 mol mol–1 s–1) and published estimates largely disappears, and one is left to conclude that E. globulus contains no more Rubisco than is necessary for photosynthesis.
Evergreen perennials are commonly thought to allocate a smaller fraction of N to the photosynthetic machinery than herbaceous annuals (Field and Mooney, 1986), but this is not the case in E. globulus. Seedlings contained from 38–44% of N as Rubisco, values that are higher than normally reported for perennial evergreens (Warren et al., 2000) and at the upper end of values reported for many herbaceous annuals (Evans, 1989). Previous examples of perennial evergreens allocating a large fraction of N to Rubisco have mostly been associated with over-investment in Rubisco and its putative use as a storage protein with luxury N supply (Warren and Adams, 2004). However, this was not the case in E. globulus seedlings because specific activity did not decrease with increasing nutrient supply, but was instead close to published maximum values in all cases (see above).
The allocation of a large fraction of N to Rubisco with a high specific activity (Vcmax/Rubisco), and small stomatal limitations, resulted in rates of photosynthesis per unit N that were at the upper end of observations for evergreen perennials and amongst some of the highest observed in herbaceous species (Poorter and Evans, 1998). These photosynthetic characteristics are most often associated with rapidly growing early successional and/or herbaceous species (‘weeds’), but their occurrence in E. globulus seedlings is almost certainly related to the fact that this species germinates in spring when soils are wet and thus, for at least the first few months, have ample water and nutrients.
Strong N–gi and Rubisco–gi relationships have been reported in individual studies (von Caemmerer and Evans, 1991), but when all the studies were collated it is clear that N–gi or Rubisco–gi relationships vary among functional groups and are generally weak (Fig. 2). In many respects, these trends in N–gi or Rubisco–gi relationships are analogous to those in An–N relationships (Field and Mooney, 1986). However, unlike An–N relationships for which there is a strong mechanistic underpinning, it is not clear why there should be a causal relationship of N or Rubisco with gi. Coincidental relationships of gi with N or Rubisco may arise for one of several reasons. (i) Several authors have argued that gi is largely constitutive and determined by leaf structural traits such as cell wall thickness and the surface area of chloroplasts exposed to intercellular air spaces (Evans et al., 1994; Syvertsen et al., 1995). Hence, correlations of gi with N and Rubisco may reflect an underlying correlation with anatomical and morphological traits such as the surface area of chloroplasts exposed to intercellular air spaces (Singsaas et al., 2004). (ii) Leaf structural traits fail to explain the rapid responses of gi to environmental conditions (Bernacchi et al., 2002; Centritto et al., 2003), which are instead better explained by the actions of carbonic anhydrase and/or aquaporins (Gillon and Yakir, 2000; Terashima and Ono, 2002). It seems unlikely that nutrient supply provides a mechanistic link of N or Rubisco with carbonic anhydrase or aquaporins. This is because carbonic anhydrase and aquaporins have a very low N cost, and thus there is no reason to expect a correlation between nutrient supply and leaf contents of carbonic anhydrase and/or aquaporins. As argued by Singsaas et al. (2004) any correlation of Rubisco with gi may result from a correlation between Rubisco and carbonic anhydrase rather than any direct mechanism. The absence of a mechanistic link is supported by the results of Evans et al. (1994) who found that the relationship between Rubisco and gi was broken in antisense-Rubisco-transformed plants.
Leaf structural traits and enzymatic processes are both correlated with gi, but at this stage one cannot ascribe trends in gi among functional groups to any particular suite of characters and the nature of N–gi and Rubisco–gi relationships remains obscure.
Fluorescence estimates of gi are sensitive to * and Rd (Harley et al., 1992), but use of different estimates of * and Rd has no tangible effect on the results for E. globulus. It was reasoned, as have others (Harley et al., 1992), that * is an intrinsic property of Rubisco and thus varies little among species or with growing conditions. It was chosen to ignore estimates of the intercellular * (
see Warren et al., 2003a), which left several estimates of ‘true’ *. The chosen estimate of * (38.7 µmol mol–1; von Caemmerer et al., 1994) is very similar to the 37.4 µmol mol–1 reported by Bernacchi et al. (2002), but less than the widely used 45 µmol mol–1 of Jordan and Ogren (1984). When data were recalculated assuming *=45 µmol mol–1, treatment differences were unaffected and estimates of gi were, on average, 0.07 mol m–2 s–1 higher. The relative limitation due to internal conductance was reduced by around 0.07, but treatment differences were unaffected and in all cases the internal limitations were greater than stomatal limitations. Another uncertainty in these calculations is the assumption that mitochondrial respiration measured in the dark (‘dark respiration’) is a valid estimate of mitochondrial respiration in the light (Rd). Inhibition of mitochondrial respiration in the light has been reported in many species (Atkin et al., 2000), but the recalculation of data assuming that respiration was inhibited by 50% led to estimates of gi that were only 0.01 mol m–2 s–1 lower. Hence, these data are only marginally affected by assumptions regarding * and Rd, and treatment differences (or the lack thereof) are not affected.
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Funding from the Australian Research Council supported this work. Frank Jones is thanked for expert technical assistance.
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