SEARCH

SEARCH BY CITATION

Keywords:

  • Olea europea;
  • ACi curves;
  • mesophyll conductance;
  • photosynthesis model;
  • photosynthetic limitations;
  • salinity stress;
  • stomatal conductance

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In this study it has been shown that increased diffusional resistances caused by salt stress may be fully overcome by exposing attached leaves to very low [CO2] (∼ 50 µmol  mol−1), and, thus a non-destructive-in vivo method to correctly estimate photosynthetic capacity in stressed plants is reported. Diffusional (i.e. stomatal conductance, gs, and mesophyll conductance to CO2, gm) and biochemical limitations to photosynthesis (A) were measured in two 1-year-old Greek olive cultivars (Chalkidikis and Kerkiras) subjected to salt stress by adding 200 mm NaCl to the irrigation water. Two sets of ACi curves were measured. A first set of standard ACi curves (i.e. without pre-conditioning plants at low [CO2]), were generated for salt-stressed plants. A second set of ACi curves were measured, on both control and salt-stressed plants, after pre-conditioning leaves at [CO2] of ∼ 50 µmol mol−1 for about 1.5 h to force stomatal opening. This forced stomata to be wide open, and gs increased to similar values in control and salt-stressed plants of both cultivars. After gs had approached the maximum value, the ACi response was again measured. The analysis of the photosynthetic capacity of the salt-stressed plants based on the standard ACi curves, showed low values of the Jmax (maximum rate of electron transport) to Vcmax (RuBP-saturated rate of Rubisco) ratio (1.06), that would implicate a reduced rate of RuBP regeneration, and, thus, a metabolic impairment. However, the analysis of the ACi curves made on pre-conditioned leaves, showed that the estimates of the photosynthetic capacity parameters were much higher than in the standard ACi responses. Moreover, these values were similar in magnitude to the average values reported by Wullschleger (Journal of Experimental Botany 44, 907–920, 1993) in a survey of 109 C3 species. These findings clearly indicates that: (1) salt stress did affect gs and gm but not the biochemical capacity to assimilate CO2 and therefore, in these conditions, the sum of the diffusional resistances set the limit to photosynthesis rates; (2) there was a linear relationship (r2 = 0.68) between gm and gs, and, thus, changes of gm can be as fast as those of gs; (3) the estimates of photosynthetic capacity based on ACi curves made without removing diffusional limitations are artificially low and lead to incorrect interpretations of the actual limitations of photosynthesis; and (4) the analysis of the photosynthetic properties in terms of stomatal and non-stomatal limitations should be replaced by the analysis of diffusional and non-diffusional limitations of photosynthesis. Finally, the C3 photosynthesis model parameterization using in vitro-measured and in vivo-measured kinetics parameters was compared. Applying the in vivo-measured Rubisco kinetics parameters resulted in a better parameterization of the photosynthesis model.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Carbon uptake is reduced by environmental stresses which lower water activity, as expressed in the leaf water potential (Kramer & Boyer 1995). This is particularly so for water stress (Lawlor 1995) and also for salinity stress (Munns 1993), which firstly induces the so-called osmotic or water-deficit effect of salinity and, thus, reduces the ability of plants to take up water. There have been many recent exciting advances in our understanding of the mechanisms by which photosynthesis responds to environmental factors. However, conflicts in the debate on the relative importance of diffusive (Cornic 2000) and metabolic (Tezara et al. 1999) factors to the overall control of photosynthesis even under mild environmental stress still arise (Flexas & Medrano 2002).

One of the earliest response to stress in general is a decrease in stomatal conductance (Jones 1973; Sharkey 1990). Reducing stomatal conductance (gs) is a major way of decreasing water loss from the leaves. However, CO2 diffusion into the leaves also decreases, leading to reduced internal CO2 partial pressure and consequently reduced rates of photosynthesis. An intriguing observation is that within the range of leaf water status commonly occurring in nature, the primary, if not exclusive, role on photosynthesis is played by gs (Cornic 2000). In these conditions, the photosynthetic capacity of the leaves is not impaired. However, there are also suggestions that stomata are not the major site of photosynthesis regulation, because environmental stress affects primarily the mesophyll metabolism (Lawlor 1995). A decrease in gs is one of the earliest responses to environmental stress, but if this situation continues for long the mechanisms involved are more complicated than simply reduction of gs and implicates non-stomatal limitations to photosynthesis, namely biochemical limitations (Sharkey & Badger 1982; Sharkey & Seemann 1989; Graan & Boyer 1990; Giménez, Mitchell & Lawlor 1992; Quick et al. 1992; Tezara et al. 1999).

The second step that can limit CO2 diffusion toward the sites of fixation (the chloroplasts) is the conductance inside the leaf mesophyll (gm). Since indirect measurements of mesophyll conductance have been made possible by using different techniques, an increasing body of evidence has been accumulated that gm is reduced under stress conditions, and particularly in response to drought (Flexas et al. 2002) and to salinity (Bongi & Loreto 1989; Delfine et al. 1998; 1999). Stomata are known to respond to stress alleviation by reopening and re-establishing gaseous exchanges between leaf and air. Photosynthesis may therefore recover and even attain pre-stress rates if it is only dependent on CO2 concentration in the leaf. The reduction of gm, on the contrary, has been long considered as irreversible, being related to changes of mesophyll structure (Bongi & Loreto 1989) or to a possible rearrangement of intercellular spaces (Delfine et al. 1998). However, Delfine et al. (1999) demonstrated that alleviation of a salinity stress prior to irreversible biochemical damage also induced an increase of gm, indicating that internal resistances to CO2 diffusion do not increase permanently under stress conditions. In our companion paper we have seen that the reduction of photosynthesis in moderately salt-stressed olive leaves is generally attributable to the sum of stomatal and mesophyll resistances, with the reduction of gm playing an important role in reducing CO2 internal concentration (Loreto, Centritto & Chartzoulakis 2003). We have also speculated that the mechanism is similar in cultivars showing different sensitivity of photosynthesis to the stress, and that cultivars whose photosynthetic properties are less affected by salt stress have inherently low gm and chloroplast CO2 concentration. With the experiments presented here we intend to answer to the questions whether both gs and gm may recover, and if photosynthesis may be concurrently re-established.

The estimation of photosynthetic limitations, both diffusional and metabolic, in stressed plants is complicated by the possible patchy stomatal closure. To overcome the ‘patchiness’ problem and separate the stomatal effects from the non-stomatal effects, gs has been either manipulated by decreasing leaf temperature in ambient air at a constant vapour pressure deficit (Cornic & Ghashghaie 1991), or removed by measuring photosynthesis in very high [CO2] (up to 0.017 MPa) using leaf disc oxygen electrode (Kaiser 1987). In our study, we show that both the increased diffusional resistances and the stomatal heterogeneities caused by salt stress may be fully overcome by exposing attached leaves to very low [CO2] (∼ 5 Pa). The rationale of our approach is that gs is inversely correlated to [CO2] (Zeiger 1983). Thus, the exposure of leaves to very low [CO2] for a relatively long time would force the stomata to be wide open. To separate the different components affecting photosynthetic capacity, we measured the in vivo photosynthetic biochemistry and diffusional limitations to photosynthesis in one resistant (Kerkiras) and one susceptible (Chalkidikis) olive cultivar (Loreto et al. 2003).

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

One-year-old plants of Greek olive cultivars (Chalkidikis and Kerkiras) were grown in 8.5 dm3 pots in the glasshouse of the Subtropical Plants and Olive Tree Institute in Chania, Crete, Greece. There were eight plants per cultivar, four of which were maintained in control conditions (control) whereas the other four were salt-stressed. The control plants were irrigated twice daily through a closed recycling system with a 50% strength Hoagland solution. The salt treatment started on 22 May 2000, when the plants had developed shoots 15–20 cm long. The plants were salt-stressed by adding 200 mm NaCl to the irrigation water. Full details of the growth conditions are given in a companion paper (Loreto et al. 2003)

Measurements of photosynthetic photon flux density (PPFD)-saturated CO2 assimilation rate (A) in relation to leaf internal CO2 concentration (Ci) were made between 1000 and 1700 h over a range of CO2 concentrations between ∼ 40 and 1500 µmol mol−1. Measurements of the ACi response curves were made between 43 and 48 d after beginning the treatment, when no visible sign of toxicity (leaf chlorophyll chlorosis, burning of leaf edges or leaf dropping) was observed. The ACi measurements were made inside a laboratory on newly expanded leaves using a portable gas exchange system (Licor 6400; Li-Cor Inc., Lincoln NE, USA). The gas exchange cuvette window was modified to accommodate the fluorescence probe (MiniPAM; Walz, Effeltrich, Germany). The tip of the optic fibre of the MiniPAM was inserted in one of the window extremities at an angle of 45°. With this setting the optic fibre was placed at about 1 cm from the leaf without shading it. To enable measurements of PPFD-saturated photosynthetic rates, illumination of the leaf cuvette by natural sunlight was supplemented with artificial light (provided by a white halogen lamp) to maintain PPFD over the leaf at ∼ 1200 µmol m−2 s−1. Air temperature in the laboratory was maintained between 25 and 26 °C, and the relative humidity in the leaf cuvette ranged between 47 and 50%.

A first set of standard ACi curves were generated on a salt-stressed plant of both Chalkidikis and Kerkiras, and also on a salt-stressed plant of Throubolia (another variety used in a parallel study, see the companion paper Loreto et al. 2003). These A–Ci curves were obtained with short-term measurements (∼ 10 min for each data point), starting at [CO2] of 350 µmol mol−1 and progressively reducing the [CO2] to ∼ 40 µmol mol−1; then, the [CO2] was progressively increased up to ∼ 1200 µmol mol−1. By the time the [CO2] of ∼ 1200 µmol mol−1 was reached, gs had decreased to values as low as 0.015 mol m2 s−1.

To remove the effect of stomatal limitation on A caused by salt stress (and allow an estimate of the photosynthetic capacity at high gs), another set of A–Ci curves was generated, on three to four of both the control and the stressed plants of Chalkidikis and Kerkiras, using a different methodology. After measuring A at [CO2] of 350 µmol mol−1, the [CO2] was brought down to ∼ 50 µmol mol−1 and leaves were exposed at this [CO2] for about 1.5 h to force stomatal opening. When gs approached the maximum value (Fig. 1), [CO2] was progressively increased up to 350 µmol mol−1. After the measurement made at 350 µmol mol−1, the [CO2] was immediately increased up to 1500 µmol mol−1, and then progressively decreased back to 350 µmol mol−1. The measurements of A at increasing Ci after exposing leaves at ∼ 50 µmol mol−1, were relatively fast (each measurement lasted 3–4 min). During these A–Ci measurements, A was measured three times per plant at [CO2] of 350 µmol  mol−1 (before and after exposing the leaves at [CO2] of ∼ 50 µmol mol−1, and after progressively decreasing [CO2] from 1500 µmol mol−1). On these occasions, photosynthesis, stomatal conductance and chlorophyll fluorescence yield were simultaneously measured. The fluorescence yield (i.e. the quantum yield of PSII in the light, ΔF/Fm) was measured using a saturating pulse (10000 µmol m−2 s−1) of white light. The mesophyll (or internal) conductance to CO2 between the intercellular spaces and the chloroplasts was calculated by using the fluorescence-gas exchange method as explained by Harley et al. (1992). The measurements of electron transport under low (2%) O2, to calibrate the system under non-photorespiratory conditions, were made at the end of the A–Ci curves. As predicted by the theory (Harley et al. 1992), the electron transport under low O2 was the same when measured by fluorescence and gas-exchange.

image

Figure 1. Time course of exposure to changes in ambient [CO2] (Ca; triangles) and in its corresponding calculated intercellular [CO2] (Ci; circles) (a, c), and the effect of these changes on stomatal conductance (gs) (b, d) of a typical Chalkidikis (a, b) and Kerkiras (c, d) plant during A–Ci measurements. The measurements were made between 43 and 48 d after beginning the salt treatment. Filled symbols, control plants; open symbols, salt-stressed plants.

Download figure to PowerPoint

Values for the photosynthetic parameters Vcmax (RuBP-saturated rate of Rubisco), Jmax (maximum rate of electron transport), Amax(the net CO2 assimilation rate under conditions of PPFD and CO2 saturation) and Rd (mitochondrial respiration in the light per unit leaf area) were obtained by fitting the mechanistic model of CO2 assimilation proposed by Farquhar, von Caemmerer & Berry (1980) to individual A–Ci response data using the method developed by de Pury & Farquhar (1997), in which A is given as

  • A = vc − 0.5vo − Rd(1)

where vc and vo are the carboxylation rate and the oxygenation rate of Rubisco, respectively, and 0.5 is the stoichiometry between O2 uptake by RubP (ribulose bisphosphate) oxygenase and photorespiratory CO2 evolution (Jordan & Ogren 1984); vc can not be larger than the minimum rates of Rubisco-limited carboxylation (Ac) and electron transport-limited RuBP regeneration (Aj). Thus:

  • vc = min{AcAj}(2)

where

  • Ac = Vcmax(Ci − Γ*)/[Ci + Kc(1 + O/Ko)](3)

and

  • Aj = J(Ci − Γ*)/4(Ci + 2Γ*)(4)

where Γ* is the CO2 photocompensation point (compensation point for photosynthesis in the absence of dark respiration, i.e. the value of Ci at which vc = 0.5 vo) (Laisk 1977), Ko and Kc are the Michaelis–Menten constants for O2 and CO2, respectively, and O is the O2 partial pressure in the intercellular space, taken to be 0.21 mol mol−1. The value of Vcmax was determined from the slope of the A–Ci curve at [CO2] of 40–200 µmol mol−1, assuming that the resistance to CO2 diffusion inside the leaf mesophyll is taken as zero (i.e. gm = ∞). Amax and Jmax were determined from the saturating portion of the curve at high [CO2] (i.e. in non-photorespiratory conditions). Γ*, Ko, Kc, Jmax, Vcmax, and Rd have the following temperature dependencies:

  • Γ* = P[Pv25 + 1.68(T − 25) + 0.012(T − 25)2](5)
  • Ko = Pv25 · exp[Ea(T − 25)/(298R(T + 273))](6)
  • Kc = Pv25 · exp[Ea(T − 25)/(298R(T + 273))](7)
  • Jmax = Jmax25 · exp[Ea(T − 25)/(298RT)]{[1 +  exp((298S − H)/(298R))]/[1 + exp((ST − H)/(RT))]}(8)
  • Vcmax = Vcmax25 · exp[Ea(T − 25)/(298R(T + 273))](9)
  • Rd = Rd25 · exp[Ea(T − 25)/(298R(T + 273))](10)

where P is the atmospheric pressure in bars, Pv25 is the photosynthetic parameter value at 25 °C (Table 1), T is the leaf temperature (°C), R is the universal gas constant (8.314 J mol−1 K−1), Ea is the activation energy (Table 1), S is the electron-transport temperature response parameter (0.71 kJ K−1), and H is the curvature parameter of Jm(T) (220 kJ mol−1). The fitting model developed by de Pury & Farquhar (1997) was run with either in vitro-measured Pv25 and Ea values (Badger & Collatz 1977; Jordan & Ogren 1984; Brooks & Farquhar 1985; von Caemmerer et al. 1994) (Table 1a) or with the in vivo Rubisco kinetics parameters measured by Bernacchi et al. (2001), with the exception of the Ea of Jmax which was measured in vitro (Table 1b). Fitting the model involved an optimization procedure in which the parameter values were optimized by adjusting them so as to minimize the sums of residuals between observed and modelled assimilation values over a range of Ci.

Table 1.  The (a) in vitro-measured and (b) in vivo-measured (with the exception of the activation energy of Jmax) photosynthesis kinetics parameters (Pv25) at 25 °C and their activation energies (Ea, kJ mol−1, describing the parameter temperature dependence responses) used in the method developed by de Pury & Farquhar (1997) to fit the mechanistic model of CO2 assimilation proposed by Farquhar et al. (1980) to individual A–Ci response data
ParameterPv25Ea
  1. The Rubisco parameters Kc, Ko and Γ* are appropriate to an infinite gm. Kc, Michaelis–Menten constant for CO2; Kc, Michaelis–Menten constant for O2; Vcmax (photosynthetic Rubisco capacity per unit leaf area), Jmax (potential rate of electron transport rate per unit leaf area), Rd (mitochondrial respiration in the light per unit leaf area); Γ*, CO2 photocompensation point. avon Caemmerer et al. (1994); bBadger & Collatz (1977); cFarquhar et al. (1980); dJordan & Ogren (1984); eBrooks & Farquhar (1985); fBernacchi et al. (2001).

(a) in vitro values
Kc40.4a59.40b
Ko24.8 × 103a36.00b
Vcmax64.80b
Jmax37.00c
Rd66.40c
Γ*42.70e29.00d
(b) in vivo values
Kc40.49f79.43f
Ko27.84 × 103f36.38f
Vcmax65.33f
Jmax37.00c
Rd46.39f
Γ*42.75f37.83f

RESULTS AND DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The relationship between PPFD-saturated CO2 assimilation rate and leaf internal CO2 concentration are widely used to measure the in vivo multi-enzyme kinetic properties of photosynthesis and, thus, to ascertain the in vivo biochemical limitation to photosynthesis (Laisk & Oja 1998). The parameters describing plant photosynthetic capacity (Vcmax, Amax, and Jmax) are increasingly used in mechanistic models predicting the effects of global change on growth processes (Centritto & Jarvis 1999; Beerling & Woodward 2001; Centritto 2002) and, thus, it is of paramount importance to estimate them correctly. To estimate the biochemical limitations to photosynthesis, Wullschleger (1993) made a retrospective analysis of A–Ci curves and calculated Vcmax and Jmax for 109 C3 species. He applied the Farquhar et al. (1980) C3 model of photosynthesis using in vitro-measured Rubisco kinetics parameters. In our study we first compare the Wullschleger's (1993) results to the photosynthetic parameters of salt-stressed plants of Chalkidikis, Kerkiras, and Throubolia (Table 2a) and of control and salt-stressed plants of Chalkidikis and Kerkiras pre-conditioned at low [CO2] (Table 3a), obtained by fitting the Farquhar et al. (1980) model of leaf photosynthesis to individual A–Ci curves using in vitro-measured Rubisco kinetics parameters. Secondly, we compare the model parameterization by using in vitro-measured (Table 1a) and in vivo-measured (Table 1b) photosynthesis kinetics parameters. Finally, we analyse the diffusional limitations to photosynthesis.

Table 2.  Values of the photosynthetic parameters in salt-stressed plants of Chalkidikis, Kerkiras, and Throubolia. These values were obtained by fitting the Farquhar et al. (1980) model of leaf photosynthesis to the individual A–Ci response curves shown in Fig. 2 using either (a) in vitro-measured Pv25 and Ea values or (b) in vivo-measured Pv25 and Ea values (with the exception of the activation energy of Jmax)
 JmaxVcmaxAmaxJmax : VcmaxRdNe
  1. Jmax, potential rate of electron transport rate per unit leaf area (µmol m−2 s−1); Vcmax, photosynthetic Rubisco capacity per unit leaf area (µmol m−2 s−1); Amax, maximum photosynthetic rate at saturating PPFD (µmol m−2 s−1), Rd, mitochondrial respiration in the light per unit leaf area (µmol m−2 s−1); Ne, number of electrons consumed per CO2 fixed in non-photorespiratory conditions.

(a) in vitro values
Throubolia72.073.310.50.98−0.727.37
Chalkidikis81.080.411.11.01−2.028.91
Kerkiras85.671.211.71.20−2.249.05
Mean79.5 ± 3.275.0 ± 2.311.1 ± 0.31.06 ± 0.06−1.66 ± 0.398.44 ± 0.44
(b) in vivo values
Throubolia64.3065.5410.500.98−0.656.53
Chalkidikis76.3772.7311.101.05−1.157.67
Kerkiras76.7362.9311.701.22−1.127.25
Mean72.47 ± 3.3467.07 ± 2.3911.10 ± 0.281.08 ± 0.06−0.97 ± 0.137.15 ± 0.27
Table 3.  Values of the photosynthetic parameters in control and salt-stressed plants Chalkidikis and Kerkiras. These values were obtained by fitting the Farquhar et al. (1980) model of leaf photosynthesis to the individual A–Ci response curves shown in Fig. 3 (i.e. after exposing plants at [CO2] of ∼ 50 µmol mol−1 for about 1.5 h to force stomatal opening) using either (a) in vitro-measured Pv25 and Ea values or (b) in vivo-measured Pv25 and Ea values (with the exception of the activation energy of Jmax)
 ChalkidikisKerkirasMean
ControlSaltControlSalt
  1. Jmax, potential rate of electron transport rate per unit leaf area (µmol m−2 s−1); Vcmax, photosynthetic Rubisco capacity per unit leaf area (µmol m−2 s−1); Amax, maximum photosynthetic rate at saturating PPFD (µmol m−2 s−1), Rd, mitochondrial respiration in the light per unit leaf area (µmol m−2 s−1); Ne, number of electrons consumed per CO2 fixed in non-photorespiratory conditions. All figures ± one standard error, n = 3–4; letters (a, b, c) indicate significant differences at P < 0.05 in the same line.

(a) in vitro values
Jmax113.29 ± 1.55a118.21 ± 2.20a124.87 ± 3.89a121.19 ± 3.27a119.39 ± 2.12
Vcmax 71.84 ± 0.95a 74.62 ± 0.58ab 77.72 ± 1.65bc 79.16 ± 0.78c 75.84 ± 1.41
Amax 20.13 ± 0.47a 21.90 ± 0.76ab 24.10 ± 1.12b 23.38 ± 0.79b 22.38 ± 0.76
Jmax : Vcmax  1.58 ± 0.04a  1.58 ± 0.03a  1.61 ± 0.02a  1.53 ± 0.03a  1.57 ± 0.01
Rd −1.34 ± 0.02a −1.29 ± 0.27a −0.80 ± 0.12a −1.25 ± 0.14a −1.17 ± 0.11
Ne  6.03 ± 0.073a  5.77 ± 0.185a  5.45 ± 0.093a  5.49 ± 0.091a  5.69 ± 0.118
(b) in vivo values
Jmax106.59 ± 3.24a108.17 ± 4.02a111.30 ± 4.99a113.63 ± 5.92a109.92 ± 1.37
Vcmax 66.95 ± 2.51a 66.70 ± 3.28a 68.23 ± 2.56a 70.31 ± 3.21a 68.05 ± 0.71
Amax 20.13 ± 0.47a 21.90 ± 0.76ab 24.10 ± 1.12b 23.38 ± 0.79b 22.38 ± 0.76
Jmax : Vcmax  1.60 ± 0.05a  1.63 ± 0.04a  1.63 ± 0.03a  1.62 ± 0.02a  1.62 ± 0.01
Rd −0.65 ± 0.03a −0.61 ± 0.02a −0.60 ± 0.07a −0.66 ± 0.04a −0.63 ± 0.01
Ne  5.48 ± 0.087a  5.08 ± 0.043a  4.74 ± 0.059a  5.00 ± 0.024a  5.07 ± 0.131

Comparing the biochemical limitations

Based on the in vivo and in vitro analysis of photosynthetic capacity, it has been suggested that environmental stresses which lower water activity, lead to metabolic impairment (Sharkey & Badger 1982; Lawlor 1995). Particularly, it was shown that mild water stress led to limited RuBP regeneration in field-grown grapevines (Escalona, Flexas & Medrano 1999). An apparently similar response was found in our study, when standard A–Ci responses (i.e. measurements made without pre-conditioning plants at low [CO2]) were measured on three olive cultivars (Chalkidikis, Kerkiras, and Throubolia) subjected to salt stress (Fig. 2a). These A–Ci response curves had steep initial slopes but approached saturation at low Ci (about 300 µmol mol−1) with mean Amax, averaged across the three cultivars, of 11.1 µmol m−2 s−1 (Table 2). Mean Vcmax, averaged across the three cultivars, was 75 µmol m−2 s−1 (Table 2a). This value is very close to the average Vcmax of 64 µmol m−2 s−1 reported in the survey by Wullschleger (1993) in his retrospective analysis of the A–Ci curves from a large number of C3 species made in PPFD conditions. However, the three olive cultivars had a mean Jmax value of 79.5 µmol m−2 s−1 (Table 2a) which is, in contrast, much lower than that of 134 µmol m−2 s−1 averaged by Wullschleger (1993) across 109 C3 species. Consequently, the average Jmax : Vcmax ratio was much lower than that reported in the survey by Wullschleger (1993), namely 1.06 and 1.64, respectively. This low value of Jmax and, consequently of Amax, would indicate a reduced rate of RuBP regeneration according to the Farquhar et al. (1980) model. Hence, these findings would support the hypothesis that environmental stress affects primarily the mesophyll carbon metabolism, at least when CO2 is not a limiting factor. Under these conditions, the strong reduction of the photochemical efficiency observed in these salt-stressed leaves (Loreto et al. 2003) may effectively indicate the onset of these carbon metabolism limitations.

image

Figure 2. The relationship between (a) net CO2 assimilation rate (A) and intercellular [CO2] (Ci), and (b) stomatal conductance (gs) and Ci in saturating PPFD (∼ 1200 µmol m−2 s−1) in salt-stressed plants of Chalkidikis (○), Kerkiras (▿) and Throubolia (□).

Download figure to PowerPoint

The short-term response of stomatal conductance to stepwise changes of Ci showed that gs reached values as low as 0.015 mol m−2 s−1 at Ci of ∼ 450 µmol mol−1 in salt-stressed plants (Fig. 2b). To understand whether the photosynthetic capacity parameters derived from the A–Ci response curves shown in Fig. 2a had been affected by such low values of gs, both control and salt-stressed plants of Chalkidikis and Kerkiras were exposed to low [CO2] to force stomatal opening and remove the effect of stomatal limitation, before measuring a new set of A–Ci curves (Fig. 1). Stomatal conductance of both control and salt-stressed plants responded positively to low [CO2]. Maximum stomatal opening was reached in about 1.5 h, and gs was increased to similar values in control and salt-stressed plants of both cultivars: gs of control and salt-stressed plants was increased to an average of 0.22 and 0.20 mol m−2 s−1, respectively, in Chalkidikis (Fig. 1b), and to an average of 0.19 and 0.18 mol m−2 s−1, respectively, in Kerkiras (Fig. 1b). After gs had approached the maximum value, the A–Ci response was again measured by progressively increasing [CO2] up to 350 µmol mol−1, then [CO2] was suddenly increased up to 1500 µmol mol−1, and in subsequent steps progressively decreased back to 350 µmol mol−1.

These new A–Ci curves, made on pre-conditioned plants, showed that photosynthesis reached a maximum in all leaves at Ci values between 800 and 900 µmol mol−1 (Fig. 3). In general, Kerkiras plants (Fig. 3b) had higher photosynthetic capacity than Chalkidikis plants (Fig. 3a). Likewise, salt-stressed plants had a slightly higher photosynthetic capacity than control plants. However, analysis of the data using the Farquhar et al. model (Farquhar et al. 1980; de Pury & Farquhar 1997) fitted to individual A–Ci shows that the best-fit of Vcmax, Jmax, Amax and Rd were not statistically different in control and salt-stressed plants of both cultivars (Table 3). This indicates that the biochemical capacity of photosynthesis was similar between cultivars and salt treatment.

image

Figure 3. The A–Ci relationship measured after exposing Chalkidikis (a) and Kerkiras (b) plants at [CO2] of ∼ 50 µmol  mol−1 for about 1.5 h to force stomatal opening (see Fig. 1). The measurements were made in saturating PPFD (∼ 1200 µmol m−2  s−1) on three to four plants on both control (□) and salt-stressed (•) plants.

Download figure to PowerPoint

Interestingly, by comparing the A–Ci curves shown in Figs 2 and 3, it is evident that whereas mean Vcmax was almost identical in the two sets of A–Ci curves, the estimates of the best-fit Jmax and Amax were much higher in the A–Ci curves of plants pre-conditioned at low [CO2] (Table 3) than in standard A–Ci responses (Table 2). The Jmax of pre-conditioned plants had a mean value, averaged across salt-stressed and control saplings of both Chalkidikis and Kerkiras cultivars, of 119.39 µmol m−2 s−1 (Table 3a) which was similar in magnitude to the average value reported in the survey by Wullschleger (1993).

Moreover, in the pre-conditioned plants, the value of 1.57 of the mean Jmax : Vcmax ratio (Table 3a) was similar to that of 1.64 averaged by Wullschleger (1993) across 109 C3 species. In the pre-conditioned plants a positive linear correlation was observed between the best-fit estimates of Jmax and Vcmax (Fig. 4), which indicates a tight co-ordination between the activities of thylakoid proteins (photochemistry) and soluble proteins (Calvin cycle) to match each other. This relationship was not observed in salt-stressed plants before pre-conditioning. The ability to maintain a constant ratio between the carboxylation and light-harvesting activities across a wide range of environmental conditions, originates from a functional balance between RuBP consumption and RuBP regeneration. Thus, the A–Ci curves on salt-stressed leaves after pre-conditioning at low [CO2] clearly indicate that (1) salt stress did not affect the biochemical capacity to assimilate CO2, and (2) the estimates of photosynthetic capacity without removing stomatal limitation are artificially low and may lead to a wrong interpretation of the actual limitation of photosynthesis. Moreover, because gs was strongly increased also in control plants pre-conditioned at low [CO2] (Fig. 1), it is reasonable to speculate that at least in sclerophytic leaves the A–Ci curves should be assessed after pre-conditioning plants in low [CO2]. Finally, the path-dependent reduction in Vcmax and gs, put forward by Prioul, Cornic & Jones (1984) and then revised by Jones (1985), Assmann (1988), and more recently by Wilson, Baldocchi & Hanson (2000), has not been found in our study, because Vcmax was almost identical in the two sets of A–Ci curves (Figs 2a & 3) despite the remarkable differences in gs between non-pre-conditioned (Fig. 2b) and pre-conditioned (Fig. 1) plants.

image

Figure 4. Relationships between the maximum rate of electron transport (Jmax) and the maximum rate of carboxylation (Vcmax), derived from best-fit estimates for individual A–Ci curves in salt-stressed plants of Chalkidikis, Kerkiras, and Throubolia (open symbols) and of salt-stressed and control plants of Chalkidikis and Kerkiras (closed symbols) pre-conditioned at [CO2] of ∼ 50 µmol mol−1 for about 1.5 h to force stomatal opening (see Fig. 1). The linear relationship (r2 = 0.78) between these two components of photosynthetic capacity of the pre-conditioned plants had a slope of 1.57 (i.e. Jmax/Vcmax).

Download figure to PowerPoint

Comparing the model parameterization

The parameterization of the model developed by de Pury & Farquhar (1997) showed that Jmax, Vcmax, Rd, and Ne(the number of electrons consumed per CO2 fixed in non-photorespiratory conditions, that is, Ne = Jmax/(A + Rd)) were lower using Rubisco kinetics parameters measured in vivo (Tables 2b & 3b) than those obtained using in vitro-measured parameters (Tables 2a & 3a). In contrast, the Jmax : Vcmax ratio was increased by parameterizing the model with the in vivo kinetics parameters estimated by Bernacchi et al. (2001). However, these differences were not significant at the 5% level in salt-stressed plants of Chalkidikis, Kerkiras, and Throubolia (Table 2). Whereas in control and salt-stressed plants of Chalkidikis and Kerkiras pre-conditioned at low [CO2], these differences were significant. In fact, applying the Bernacchi et al. (2001) in vivo kinetics parameters caused a significant decrease at P < 0.05 in Jmax, Rd, and Ne, and at P < 0.01 in Vcmax, and a significant increase at P < 0.001 in the Jmax : Vcmax ratio (Table 3b) with respect to the values estimated by using Rubisco kinetics parameters measured in vitro (Table 3a). The mean number of electrons consumed per CO2 fixed in non-photorespiratory conditions was reduced from 5.69 (Table 3a) to 5.07 (Table 3b) by applying the Bernacchi et al. (2001) in vivo parameters. This latter value is not far from the theoretical values of four electrons per CO2 fixed in non-photorespiratory conditions [i.e. Jmax= 4(A + Rd)] (Farquhar et al. 1980), assuming that light were equally distributed between the photosystems I and II, and that alternative electron sinks were absent (i.e. electrons were not used for processes other than photosynthesis, such as pseudocyclic electron transport and nitrogen assimilation), and is similar to values obtained in other experiments both in C3 and C4 plants (Genty & Harbison 1996). The reduced value in Ne obtained with the Bernacchi et al. (2001) in vivo parameters, along with the increase in the Jmax : Vcmax ratio and, above all, the reduction in Rd, indicates a better parameterization of the photosynthetic model.

Diffusional limitations

We have suggested that the sum of stomatal and mesophyll resistances set the photosynthesis rates of salt-stressed leaves in ambient [CO2] (Loreto et al. 2003). With these experiments we have proved that both these additive resistances can be removed by pre-conditioning salt-stressed leaves to low [CO2] (Table 4). Short-term changes in [CO2] not only affected gs, but also affected gm. In fact, gm measured at [CO2] of 350 µmol mol−1 was not constant, but was linearly related (r2 = 0.68) to gs (Fig. 5). The factors setting mesophyll conductance were believed to be constitutive and essentially dependent on leaf anatomy (Evans et al. 1994; Syvertsen et al. 1995). However, a recent study has demonstrated that the water-deficit effect of salinity did not affect photochemistry, but induced a reversible decrease of gm which, in turn, was responsible of transient reduction of photosynthesis in an herbaceous species (Delfine et al. 1999). Our current experiments also suggest that changes of gm can be as fast as those of gs (Fig. 5); and also that the sum of the diffusional resistances set the limit to photosynthesis rates. This is the reason why there was no relationship between A measured at CO2 concentration of ∼ 350 µmol mol−1 and the CO2 drawdown caused by stomatal resistance, whereas a clear relationship was found between A and CO2 drawdown when this was caused by the sum of stomatal and mesophyll resistances (Fig. 6, and Loreto et al. 2003).

Table 4.  Assimilation rate (A, µmol m−2 s−1), stomatal conductance (gs, mol m−2 s−1), and mesophyll conductance to CO2 (gm, mol m−2 s−1) measured at [CO2] of 350 µmol mol−1 in control and salt-stressed plants of Kerkiras and Chalkidikis during leaf exposure to the changes in [CO2] shown in Fig. 1
 ControlSalt-stressed
AgsgmAgsgm
  1. These parameters were measured three times per plant at [CO2] of 350 µmol mol−1: before (pre) and after (post) exposing the leaves at [CO2] of ∼ 50 µmol mol−1, and after progressively decreasing [CO2] from 1500 µmol mol−1 (end). Data are means of three to four plants per treatment ± 1 SEM.

Kerkiras:
Pre 7.34 ± 0.570.08 ± 0.0110.085 ± 0.008 2.65 ± 0.480.02 ± 0.0050.048 ± 0.007
Post12.87 ± 1.890.17 ± 0.0040.120 ± 0.01212.92 ± 1.450.15 ± 0.0230.162 ± 0.018
End 8.04 ± 0.970.08 ± 0.0110.079 ± 0.010 7.70 ± 1.450.07 ± 0.0130.034 ± 0.011
Chalkidikis:
Pre 6.12 ± 1.200.10 ± 0.0280.062 ± 0.011 2.92 ± 0.420.02 ± 0.0030.028 ± 0.009
Post11.83 ± 0.550.21 ± 0.0060.107 ± 0.01612.90 ± 0.670.18 ± 0.0160.141 ± 0.011
End 6.37 ± 0.700.11 ± 0.0090.088 ± 0.010 7.93 ± 0.630.07 ± 0.0070.064 ± 0.006
image

Figure 5. Linear relationship between the mean data of mesophyll conductance to CO2 (gm) and stomatal conductance (gs) shown in Table 4 (when [CO2] = 350 µmol mol−1 for pre, post and end). Data combined from Chalkidikis and Kerkiras plants; r2 = 0.68; gm = 0.56 gs + 0.026. Filled symbols, control plants; open symbols, salt-stressed plants.

Download figure to PowerPoint

image

Figure 6. Relationship between photosynthesis and CO2 drawdown between ambient and intercellular [CO2] (open symbols) or ambient and chloroplast [CO2] (filled symbols) (data combined from control and salt-stressed plants of Chalkidikis and Kerkiras). These measurements were made three times per plant at [CO2] of 350 µmol mol−1: before and after exposing the leaves at [CO2] of ∼ 50 µmol mol−1, and after progressively decreasing [CO2] from 1500 µmol mol−1 (see Fig. 1).

Download figure to PowerPoint

Recently Wilson et al. (2000) applied the path-dependent method (Prioul et al. 1984; Jones 1985; Assmann 1988) to quantify stomatal versus non-stomatal limitations to PPFD-saturated A of chestnut oak and sugar maple in response to drought. They estimated that stomatal limitations to photosynthesis accounted for about 75%, whereas non-stomatal limitations did not exceed 25%. However, they did not measure gm and did not pre-conditioned plants at [CO2]. Consequently, they could not separate diffusional and non-diffusional limitations of photosynthesis. Therefore, if the linear relationship between gm and gs found in our study in control and salt-stressed olive plants holds also in drought conditions, and also the A–Ci curves of water-stressed plants respond positively to pre-conditioning leaves at [CO2], the size of the stomatal limitations to photosynthesis estimated by Wilson et al. (2000) would have to be consistently reduced. It is well known that stomata open in response to low [CO2]. The reasons why mesophyll conductance also recover after pre-conditioning leaves at low [CO2] is however, unknown and deserves further investigation.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The assessment of reduced photosynthetic efficiency under stress conditions in terms of stomatal and non-stomatal limitations has been a standard practice over the last 30 years (Jones 1973; Bradford & Hsiao 1982; Cornic, Prioul & Louason 1983; Jones 1985; Assmann 1988; Bethke & Drew 1992; Wilson et al. 2000; Noormets et al. 2001; Flexas & Medrano 2002). However, these two terms have been often regarded as quite separate levels of control as if there were no reciprocal influences. To complicate matters, although it is clear that stomatal limitations to photosynthesis describe the diffusional resistances in the gas phase of the CO2 transport pathway between the ambient air and the carboxylation sites, the interpretations of non-stomatal limitations to photosynthesis are somehow confusing. Non-stomatal limitations include both physical limitations, namely internal (or mesophyll) resistances to CO2 diffusion in the gas and liquid phase, and biochemical limitations, namely carboxylation rate and efficiency, to photosynthesis. There is much analysis in the literature in which the different role played by these two components of the mesophyll properties in determining the non-stomatal limitations to photosynthesis have been ignored or confused. This can give misleading indications on the response of photosynthesis to environmental stresses, because it can lead to the conclusion that photosynthesis capacity is directly reduced by environmental stresses, even under circumstances in which there may be no real damage to the photosynthetic apparatus. However, during the last decade, three methods have been developed to estimate in vivo the internal conductance to CO2 diffusion between the intercellular spaces and the chloroplasts (Loreto et al. 1992; Evans & Loreto 2000). This allows the separation of the physical and biochemical components of non-stomatal limitations to photosynthesis. Based on this methodology, we have presented a non-destructive-in vivo method to estimate the photosynthetic capacity in stressed plants by separating the diffusional limitations from the non-diffusional limitations. Our findings clearly indicates that: (1) salt stress did affect gs and gm but not the biochemical capacity to assimilate CO2. Therefore, in these conditions the sum of the diffusional resistances set the limit to photosynthesis rates; (2) there was a linear relationship between gm and gs, and, thus, changes of gm can be as fast as those of gs; (3) the estimates of photosynthetic capacity based on A–Ci curves made without removing diffusional limitations are artificially low and lead to incorrect interpretations of the actual limitations of photosynthesis; and (4) the analysis of the photosynthetic properties in terms of stomatal and non-stomatal limitations should be replaced by the analysis of diffusional and non-diffusional limitations of photosynthesis. Finally, applying in vivo-measured Rubisco kinetics parameters instead of the in vitro-measured parameters results in a better parameterization of the Farquhar et al. (1980) C3 photosynthesis model.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This work has been supported by the Italian and Greek Ministry of Foreign Affairs within the frame of the Italian – Greek Bilateral Cooperation for Research and Development (Contract no. GSRT-18345). The assistance of Ms. Maria Moutsopoulou during gas-exchange measurements is gratefully acknowledged.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • Assmann S.M. (1988) Stomatal and non-stomatal limitations to carbon assimilation: an evaluation of the path-dependent method. Plant, Cell and Environment 11, 577582.
  • Badger M.R. & Collatz G.J. (1977) Studies on the kinetic mechanism of ribulose-1,5-bisphosphate carboxylase and oxygenase reactions, with particular reference to the effect of temperature on kinetic parameters. Carnegie Institute of Washington Yearbook 76, 355361.
  • Beerling D.J. & Woodward F.I. (2001) Vegetation and the Terrestrial Carbon Cycle: Modelling the First 400 Million Years. Cambridge University Press, Cambridge, UK.
  • Bernacchi C.J., Singsaas E.L., Pimentel C., Portis A.R. Jr & Long S.P. (2001) Improved temperature response functions for models of Rubisco-limited photosynthesis. Plant, Cell and Environment 24, 253259.
  • Bethke P.C. & Drew M.C. (1992) Stomatal and non-stomatal components to inhibition of photosynthesis in leaves of Capsicum annuum during progressive exposure to NaCl salinity. Plant Physiology 99, 219226.
  • Bongi G. & Loreto F. (1989) Gas-exchange properties of salt-stressed olive (Olea europea L.) leaves. Plant Physiology 90, 14081416.
  • Bradford K.J. & Hsiao T.C. (1982) Physiological response to moderate water stress. In Physiological Plant Ecology II: Water Relations and Carbon Assimilation (eds O.L.Lange, P.S.Nobel, C.B.Osmond & H.Ziegler), pp. 263324. Springer-Verlag, Berlin, Germany.
  • Brooks A. & Farquhar G.D. (1985) Effects of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light. Planta 165, 397406.
  • Von Caemmerer S., Evans J.R., Hudson G.S. & Andrews T.J. (1994) The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco. Planta 195, 8897.
  • Centritto M. (2002) Interactive effects of elevated [CO2] and drought on peach seedlings. Plant Biosystems 5, 177188.
  • Centritto M. & Jarvis P.G. (1999) Long-term effect of elevated carbon dioxide concentration and provenance on four clones of Sitka spruce (Picea sitchensis (Bong.) Carr) II. Photosynthetic capacity and nitrogen use efficiency. Tree Physiology 19, 807817.
  • Cornic G. (2000) Drought stress inhibits photosynthesis by decreasing stomatal aperture – not by affecting ATP synthesis. Trends in Plant Science 5, 187188.
  • Cornic G. & Ghashghaie J. (1991) Effects of temperature on net CO2 assimilation and photosystem II quantum yield of electron transfer of French bean leaves (Phaseolus vulgaris L.) during drought. Planta 185, 255260.
  • Cornic G., Prioul J.-L. & Louason G. (1983) Stomatal and non-stomatal contribution in the decline in leaf net CO2 uptake during rapid water stress. Physiologia Plantarum 58, 295301.
  • Delfine S., Alvino A., Villani M.C. & Loreto F. (1999) Restrictions to CO2 conductance and photosynthesis in spinach leaves recovering from salt stress. Plant Physiology 119, 11011106.
  • Delfine S., Alvino A., Zacchini M. & Loreto F. (1998) Consequences of salt stress on conductance to CO2 diffusion, Rubisco characteristics and anatomy of spinach leaves. Australian Journal of Plant Physiology 25, 395402.
  • Escalona J.M., Flexas J. & Medrano H. (1999) Stomatal and non-stomatal limitations of photosynthesis under water stress in field-grown grapevines. Australian Journal of Plant Physiology 26, 421433.
  • Evans J.R. & Loreto F. (2000) Acquisition and diffusion of CO2 in higher plant leaves. In Photosynthesis: Physiology and Metabolism (eds R.C.Leegood, T.D.Sharkey & S.Von Caemmerer), pp. 321351. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  • Evans J.R., Von Caemmerer S., Setchell B.A. & Hudson G.S. (1994) The relationship between CO2 transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of Rubisco. Australian Journal of Plant Physiology 21, 475495.
  • Farquhar G.D., Von Caemmerer S. & Berry J.A. (1980) A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149, 7890.
  • Flexas J. & Medrano H. (2002) Drought-inhibition of photosynthesis in C3 plants: stomatal and non-stomatal limitations revised. Annals of Botany 89, 183189.
  • Flexas J., Bota J., Escalona J.M., Sampol B. & Medrano H. (2002) Effects of drought on photosynthesis in grapevines under field conditions: an evaluation of stomatal and mesophyll limitations. Functional Plant Biology 29, 461471.
  • Genty B. & Harbison J. (1996) Regulation of light utilization for photosynthetic electron transport. In Photosynthesis and the Environment (ed. N.R.Baker), pp. 6799. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  • Giménez C., Mitchell V.J. & Lawlor D.W. (1992) Regulation of photosynthetic rate of two sunflower hybrids under water stress. Plant Physiology 98, 516524.
  • Graan T. & Boyer J.S. (1990) Very high CO2 partially restores photosynthesis in sunflower at low water potential. Planta 181, 378384.
  • 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 Physiology 98, 14291436.
  • Jones H.G. (1973) Limiting factors in photosynthesis. New Phytologist 72, 10951106.
  • Jones H.G. (1985) Partitioning stomatal and non-stomatal limitations to photosynthesis. Plant, Cell and Environment 8, 95104.
  • Jordan D.B. & Ogren W.L. (1984) The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase. Planta 161, 308313.
  • Kaiser W.M. (1987) Effect of water deficit on photosynthetic capacity. Physiologia Plantarum 71, 142149.
  • Kramer P.J. & Boyer J.S. (1995) Water Relations of Plants and Soils. Academic Press, San Diego, CA, USA.
  • Laisk A. (1977) Kinetics of Photosynthesis and Photorespiration in C3-plants. Nauka, Moscow, Russia (in Russian).
  • Laisk A. & Oja V. (1998) Dynamics of Leaf Photosynthesis: Rapid Response Measurements and Their Interpretations. CSIRO Publishing, Collingwood, Australia.
  • Lawlor D.W. (1995) The effects of water deficit on photosynthesis. In Environmental and Plant Metabolism: Flexibility and Acclimation (ed. N.Smirnoff), pp. 129160. BIOS Scientific Publishers, Oxford, UK.
  • Loreto F., Centritto M. & Chartzoulakis K. (2003) Photosynthetic limitations in olive cultivars with different sensitivity to salt stress. Plant, Cell and Environment, 26, 595601.
  • Loreto F., Harley P.C., Di Marco G. & Sharkey T.D. (1992) Estimation of mesophyll conductance to CO2 flux by three different methods. Plant Physiology 98, 14371443.
  • Munns R. (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell and Environment 16, 1524.
  • Noormets A., Sôber A., Pell E.J., Dickson R.E., Podila G.K., Sôber J., Isebrands J.G. & Karnosky D.F. (2001) Stomatal and non-stomatal limitation to photosynthesis in two trembling aspen (Populus tremuloides Michx.) clones exposed to elevated CO2 and/or O3. Plant, Cell and Environment 24, 327336.
  • Prioul J.L., Cornic G. & Jones H.G. (1984) Discussion of stomatal and non-stomatal limitation in leaf photosynthesis decline under stress conditions. In Advances in Photosynthesis Research (ed. C.Sybesma) Vol. IV, pp. 375378. Martinus-Nijhoff/Dr W. Junk Publishers, The Hague, The Netherlands.
  • De Pury D.G.G. & Farquhar G.D. (1997) Simple scaling of photosynthesis from leaves to canopies without the errors of big-leaf models. Plant, Cell and Environment 20, 537557.
  • Quick W.P., Chaves M.M., Wendler R., David M., Rodrigues M.L., Passaharinho J.A., Pereira J.S., Adcock M.D., Leegood R.C. & Stitt M. (1992) The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions. Plant, Cell and Environment 15, 2535.
  • Sharkey T.D. (1990) Water stress effects on photosynthesis. Photosynthetica 24, 651.
  • Sharkey T.D. & Badger M.R. (1982) Effects of water stress on photosynthetic electron transport, photophosphorylation and metabolite levels of Xanthium strumarium cells. Planta 156, 199206.
  • Sharkey T.D. & Seemann J.R. (1989) Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves. Plant Physiology 89, 10601065.
  • Syvertsen J.P., Lloyd J., McConchie C., Kriedemann P.E. & Farquhar G.D. (1995) On the site of biophysical constraints to CO2 diffusion through the mesophyll of hypostomatous leaves. Plant, Cell and Environment 18, 149157.
  • Tezara W., Mitchell V.J., Driscoll S.D. & Lawlor D.W. (1999) Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature 401, 914917.
  • Wilson K.B., Baldocchi D.D. & Hanson P.J. (2000) Quantifying stomatal and non-stomatal limitations to carbon assimilation resulting from leaf aging and drought in mature deciduous tree species. Tree Physiology 20, 787797.
  • Wullschleger S.D. (1993) Biochemical limitations to carbon assimilation in C3 plants – a retrospective analysis of the A/Ci curves from 109 species. Journal of Experimental Botany 44, 907920.
  • Zeiger E. (1983) The biology of stomatal guard cells. Annual Review of Plant Physiology 34, 441475.

Received 8 October 2002; received in revised form 14 October 2002; accepted for publication 14 October 2002