the use of low [co2] to estimate diffusional and non-diffusional limitations of photosynthetic...

Plant, Cell and Environment

(2003)

26

, 585–594

© 2003 Blackwell Publishing Ltd

585

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2003? 2003

26?585594Original Article

Low CO

2

to estimate photosynthetic limitations in salt-stressed plantsM. Centritto

et al.

Correspondence: Mauro Centritto, Consiglio Nazionale delleRicerche (CNR), Istituto di Biologia Agroambientale e Forestale(IBAF), Via Salaria Km 29.300, 00016 Monterotondo Scalo(Roma), Italy. Fax:

+

39-06-9064492; e-mail: [email protected]

The use of low [CO

2

] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings

M. CENTRITTO

1

, F. LORETO

1

& K. CHARTZOULAKIS

2

1

CNR – Istituto di Biologia Agroambientale e Forestale, via Salaria km 29.300, 00016 Monterotondo Scalo (Roma), Italy and

2

NAGREF – Subtropical Plants and Olive Tree Institute, Agrokipio, 73100, Chania, Crete, Greece

ABSTRACT

In this study it has been shown that increased diffusionalresistances caused by salt stress may be fully overcome byexposing attached leaves to very low [CO

2

] (

~~~~

50

mmmm

molmol----

1

), and, thus a non-destructive-

in vivo

method to cor-rectly estimate photosynthetic capacity in stressed plants isreported. Diffusional (i.e. stomatal conductance,

g

s

, andmesophyll conductance to CO

2

,

g

m

) and biochemical limi-tations to photosynthesis (

A

) were measured in two 1-year-old Greek olive cultivars (Chalkidikis and Kerkiras) sub-jected to salt stress by adding 200 m

M

NaCl to the irrigationwater. Two sets of

A

–

C

i

curves were measured. A first setof standard

A

–

C

i

curves (i.e. without pre-conditioningplants at low [CO

2

]), were generated for salt-stressedplants. A second set of

A

–

C

i

curves were measured, on bothcontrol and salt-stressed plants, after pre-conditioningleaves at [CO

2

] of

~~~~

50

mmmm

mol mol----

1

for about 1.5 h to forcestomatal opening. This forced stomata to be wide open, and

g

s

increased to similar values in control and salt-stressedplants of both cultivars. After

g

s

had approached the max-imum value, the

A

–

C

i

response was again measured. Theanalysis of the photosynthetic capacity of the salt-stressedplants based on the standard

A

–

C

i

curves, showed low val-ues of the

J

max

(maximum rate of electron transport) to

V

cmax

(RuBP-saturated rate of Rubisco) ratio (1.06), thatwould implicate a reduced rate of RuBP regeneration, and,thus, a metabolic impairment. However, the analysis of the

A

–

C

i

curves made on pre-conditioned leaves, showed thatthe estimates of the photosynthetic capacity parameterswere much higher than in the standard

A

–

C

i

responses.Moreover, these values were similar in magnitude to theaverage values reported by Wullschleger (

Journal ofExperimental Botany

44, 907–920, 1993) in a survey of 109C

3

species. These findings clearly indicates that: (1) saltstress did affect

g

s

and

g

m

but not the biochemical capacityto assimilate CO

2

and therefore, in these conditions, thesum of the diffusional resistances set the limit to photosyn-

thesis rates; (2) there was a linear relationship (

r

2

====

0.68)between

g

m

and

g

s

, and, thus, changes of

g

m

can be as fastas those of

g

s

; (3) the estimates of photosynthetic capacitybased on

A

–

C

i

curves made without removing diffusionallimitations are artificially low and lead to incorrect inter-pretations of the actual limitations of photosynthesis; and(4) the analysis of the photosynthetic properties in terms ofstomatal and non-stomatal limitations should be replacedby the analysis of diffusional and non-diffusional limita-tions of photosynthesis. Finally, the C

3

photosynthesis modelparameterization using

in vitro

-measured and

in vivo

-measured kinetics parameters was compared. Applying the

in vivo

-measured Rubisco kinetics parameters resulted in abetter parameterization of the photosynthesis model.

Key-words

:

Olea europea

;

A

–

C

i

curves; mesophyll conduc-tance; photosynthesis model; photosynthetic limitations;salinity stress; stomatal conductance.

INTRODUCTION

Carbon uptake is reduced by environmental stresses whichlower water activity, as expressed in the leaf water potential(Kramer & Boyer 1995). This is particularly so for waterstress (Lawlor 1995) and also for salinity stress (Munns1993), which firstly induces the so-called osmotic or water-deficit effect of salinity and, thus, reduces the ability ofplants to take up water. There have been many recent excit-ing advances in our understanding of the mechanisms bywhich photosynthesis responds to environmental factors.However, conflicts in the debate on the relative importanceof diffusive (Cornic 2000) and metabolic (Tezara

et al

. 1999)factors to the overall control of photosynthesis even undermild environmental stress still arise (Flexas & Medrano2002).

One of the earliest response to stress in general is adecrease in stomatal conductance (Jones 1973; Sharkey1990). Reducing stomatal conductance (

g

s

) is a major wayof decreasing water loss from the leaves. However, CO

2

diffusion into the leaves also decreases, leading to reducedinternal CO

2

partial pressure and consequently reducedrates of photosynthesis. An intriguing observation is thatwithin the range of leaf water status commonly occurring

586

M. Centritto

et al

.

© 2003 Blackwell Publishing Ltd,

Plant, Cell and Environment,

26,

585–594

in nature, the primary, if not exclusive, role on photosyn-thesis is played by

g

s

(Cornic 2000). In these conditions, thephotosynthetic capacity of the leaves is not impaired. How-ever, there are also suggestions that stomata are not themajor site of photosynthesis regulation, because environ-mental stress affects primarily the mesophyll metabolism(Lawlor 1995). A decrease in

g

s

is one of the earliestresponses to environmental stress, but if this situation con-tinues for long the mechanisms involved are more compli-cated than simply reduction of

g

s

and implicates non-stomatal limitations to photosynthesis, namely biochemicallimitations (Sharkey & Badger 1982; Sharkey & Seemann1989; Graan & Boyer 1990; Giménez, Mitchell & Lawlor1992; Quick

et al

. 1992; Tezara

et al

. 1999).The second step that can limit CO

2

diffusion toward thesites of fixation (the chloroplasts) is the conductance insidethe leaf mesophyll (

g

m

). Since indirect measurements ofmesophyll conductance have been made possible by usingdifferent techniques, an increasing body of evidence hasbeen accumulated that

g

m

is reduced under stress condi-tions, 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 allevi-ation by reopening and re-establishing gaseous exchangesbetween leaf and air. Photosynthesis may therefore recoverand even attain pre-stress rates if it is only dependent onCO

2

concentration in the leaf. The reduction of

g

m

, on thecontrary, has been long considered as irreversible, beingrelated to changes of mesophyll structure (Bongi & Loreto1989) or to a possible rearrangement of intercellular spaces(Delfine

et al

. 1998). However, Delfine

et al

. (1999) demon-strated that alleviation of a salinity stress prior to irrevers-ible biochemical damage also induced an increase of

g

m

,indicating that internal resistances to CO

2

diffusion do notincrease permanently under stress conditions. In our com-panion paper we have seen that the reduction of photosyn-thesis in moderately salt-stressed olive leaves is generallyattributable to the sum of stomatal and mesophyll resis-tances, with the reduction of

g

m

playing an important rolein reducing CO

2

internal concentration (Loreto, Centritto& Chartzoulakis 2003). We have also speculated that themechanism is similar in cultivars showing different sensitiv-ity of photosynthesis to the stress, and that cultivars whosephotosynthetic properties are less affected by salt stresshave inherently low

g

m

and chloroplast CO

2

concentration.With the experiments presented here we intend to answerto the questions whether both

g

s

and

g

m

may recover, andif photosynthesis may be concurrently re-established.

The estimation of photosynthetic limitations, both diffu-sional and metabolic, in stressed plants is complicated bythe possible patchy stomatal closure. To overcome the‘patchiness’ problem and separate the stomatal effects fromthe non-stomatal effects,

g

s

has been either manipulated bydecreasing leaf temperature in ambient air at a constantvapour pressure deficit (Cornic & Ghashghaie 1991), orremoved by measuring photosynthesis in very high [CO

2

](up to 0.017 MPa) using leaf disc oxygen electrode (Kaiser1987). In our study, we show that both the increased diffu-

sional resistances and the stomatal heterogeneities causedby salt stress may be fully overcome by exposing attachedleaves to very low [CO

2

] (

~

5 Pa). The rationale of ourapproach is that

g

s

is inversely correlated to [CO

2

] (Zeiger1983). Thus, the exposure of leaves to very low [CO

2

] for arelatively long time would force the stomata to be wideopen. To separate the different components affecting pho-tosynthetic capacity, we measured the

in vivo

photosyn-thetic biochemistry and diffusional limitations tophotosynthesis in one resistant (Kerkiras) and one suscep-tible (Chalkidikis) olive cultivar (Loreto

et al

. 2003).

MATERIALS AND METHODS

One-year-old plants of Greek olive cultivars (Chalkidikisand Kerkiras) were grown in 8.5 dm

3

pots in the glasshouseof the Subtropical Plants and Olive Tree Institute in Chania,Crete, Greece. There were eight plants per cultivar, four ofwhich were maintained in control conditions (control)whereas the other four were salt-stressed. The controlplants were irrigated twice daily through a closed recyclingsystem with a 50% strength Hoagland solution. The salttreatment started on 22 May 2000, when the plants haddeveloped shoots 15–20 cm long. The plants were salt-stressed by adding 200 m

M

NaCl to the irrigation water.Full details of the growth conditions are given in a compan-ion paper (Loreto

et al

. 2003)Measurements of photosynthetic photon flux density

(PPFD)-saturated CO

2

assimilation rate (

A

) in relation toleaf internal CO

2

concentration (

C

i

) were made between1000 and 1700 h over a range of CO

2

concentrationsbetween

~

40 and 1500

m

mol mol

-

1

. Measurements of the

A

–

C

i

response curves were made between 43 and 48 d afterbeginning the treatment, when no visible sign of toxicity(leaf chlorophyll chlorosis, burning of leaf edges or leafdropping) was observed. The

A

–

C

i

measurements weremade inside a laboratory on newly expanded leaves usinga portable gas exchange system (Licor 6400; Li-Cor Inc.,Lincoln NE, USA). The gas exchange cuvette window wasmodified to accommodate the fluorescence probe(MiniPAM; Walz, Effeltrich, Germany). The tip of the opticfibre of the MiniPAM was inserted in one of the windowextremities at an angle of 45

∞

. With this setting the opticfibre was placed at about 1 cm from the leaf without shad-ing it. To enable measurements of PPFD-saturated photo-synthetic rates, illumination of the leaf cuvette by naturalsunlight was supplemented with artificial light (provided bya white halogen lamp) to maintain PPFD over the leaf at~1200 mmol m-2 s-1. Air temperature in the laboratory wasmaintained between 25 and 26 ∞C, and the relative humid-ity in the leaf cuvette ranged between 47 and 50%.

A first set of standard A–Ci curves were generated on asalt-stressed plant of both Chalkidikis and Kerkiras, andalso on a salt-stressed plant of Throubolia (another varietyused in a parallel study, see the companion paper Loretoet al. 2003). These A–Ci curves were obtained with short-term measurements (~ 10 min for each data point), startingat [CO2] of 350 mmol mol-1 and progressively reducing the

Low CO2 to estimate photosynthetic limitations in salt-stressed plants 587

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 585–594

[CO2] to ~40 mmol mol-1; then, the [CO2] was progressivelyincreased up to ~1200 mmol mol-1. By the time the [CO2] of~1200 mmol mol-1 was reached, gs had decreased to valuesas low as 0.015 mol m2 s-1.

To remove the effect of stomatal limitation on A causedby salt stress (and allow an estimate of the photosyntheticcapacity at high gs), another set of A–Ci curves was gener-ated, on three to four of both the control and the stressedplants of Chalkidikis and Kerkiras, using a different meth-odology. After measuring A at [CO2] of 350 mmol mol-1, the[CO2] was brought down to ~50 mmol mol-1 and leaves wereexposed at this [CO2] for about 1.5 h to force stomatalopening. When gs approached the maximum value (Fig. 1),[CO2] was progressively increased up to 350 mmol mol-1.After the measurement made at 350 mmol mol-1, the [CO2]was immediately increased up to 1500 mmol mol-1, and thenprogressively decreased back to 350 mmol mol-1. The mea-surements of A at increasing Ci after exposing leaves at~50 mmol mol-1, were relatively fast (each measurementlasted 3–4 min). During these A–Ci measurements, A wasmeasured three times per plant at [CO2] of 350 mmolmol-1 (before and after exposing the leaves at [CO2] of~50 mmol mol-1, and after progressively decreasing [CO2]from 1500 mmol mol-1). On these occasions, photosynthesis,stomatal conductance and chlorophyll fluorescence yieldwere simultaneously measured. The fluorescence yield (i.e.the quantum yield of PSII in the light, DF/F¢m) was measuredusing a saturating pulse (10000 mmol m-2 s-1) of white light.The mesophyll (or internal) conductance to CO2 betweenthe intercellular spaces and the chloroplasts was calculatedby using the fluorescence-gas exchange method as explainedby Harley et al. (1992). The measurements of electron trans-port under low (2%) O2, to calibrate the system under non-photorespiratory conditions, were made at the end of theA–Ci curves. As predicted by the theory (Harley et al. 1992),the electron transport under low O2 was the same whenmeasured by fluorescence and gas-exchange.

Values for the photosynthetic parameters Vcmax (RuBP-

saturated rate of Rubisco), Jmax (maximum rate of electrontransport), Amax (the net CO2 assimilation rate under con-ditions of PPFD and CO2 saturation) and Rd (mitochondrialrespiration in the light per unit leaf area) were obtained byfitting the mechanistic model of CO2 assimilation proposedby Farquhar, von Caemmerer & Berry (1980) to individualA–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 oxygen-ation rate of Rubisco, respectively, and 0.5 is the stoichiom-etry between O2 uptake by RubP (ribulose bisphosphate)oxygenase and photorespiratory CO2 evolution (Jordan &Ogren 1984); vc can not be larger than the minimum ratesof Rubisco-limited carboxylation (Ac) and electron trans-port-limited RuBP regeneration (Aj). Thus:

vc = min{Ac, Aj} (2)

where

Ac = Vcmax(Ci - G*)/[Ci + Kc(1 + O/Ko)] (3)

and

Aj = J(Ci - G*)/4(Ci + 2G*) (4)

where G* is the CO2 photocompensation point (compensa-tion point for photosynthesis in the absence of dark respi-ration, i.e. the value of Ci at which vc = 0.5 vo) (Laisk 1977),Ko and Kc are the Michaelis–Menten constants for O2 andCO2, respectively, and O is the O2 partial pressure in theintercellular space, taken to be 0.21 mol mol-1. The value ofVcmax was determined from the slope of the A–Ci curve at[CO2] of 40–200 mmol mol-1, assuming that the resistance toCO2 diffusion inside the leaf mesophyll is taken as zero (i.e.gm = •). Amax and Jmax were determined from the saturatingportion of the curve at high [CO2] (i.e. in non-photorespiratory conditions). G*, Ko, Kc, Jmax, Vcmax, and Rd

have the following temperature dependencies:

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 mea-surements were made between 43 and 48 d after beginning the salt treatment. Filled sym-bols, control plants; open symbols, salt-stressed plants.

Kerkiras

Time (min)

0 20 40 60 80 100 120 140

Ca,

Ci( m

mol

mol

-1)

0

300

600

900

1200

1500

Time (min)

0 20 40 60 80 100 120 140

g s(m

olm

-2s-1

)

0.00

0.05

0.10

0.15

0.20

0.25(c) (d)

Chalkidikis

Ca,

Ci( m

mol

mol

-1)

0

300

600

900

1200

1500

g s(m

olm

-2s-1

)

0.00

0.05

0.10

0.15

0.20

0.25(a) (b)

588 M. Centritto et al.


G* = 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 thephotosynthetic parameter value at 25 ∞C (Table 1), T is theleaf temperature (∞C), R is the universal gas constant(8.314 J mol-1 K-1), Ea is the activation energy (Table 1), Sis 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 & Ogren1984; Brooks & Farquhar 1985; von Caemmerer et al. 1994)(Table 1a) or with the in vivo Rubisco kinetics parametersmeasured by Bernacchi et al. (2001), with the exception ofthe Ea of Jmax which was measured in vitro (Table 1b). Fit-ting the model involved an optimization procedure in whichthe parameter values were optimized by adjusting them so

as to minimize the sums of residuals between observed andmodelled assimilation values over a range of Ci.

RESULTS AND DISCUSSION

The relationship between PPFD-saturated CO2 assimila-tion rate and leaf internal CO2 concentration are widelyused to measure the in vivo multi-enzyme kinetic proper-ties of photosynthesis and, thus, to ascertain the in vivobiochemical limitation to photosynthesis (Laisk & Oja1998). The parameters describing plant photosyntheticcapacity (Vcmax, Amax, and Jmax) are increasingly used inmechanistic models predicting the effects of global changeon growth processes (Centritto & Jarvis 1999; Beerling &Woodward 2001; Centritto 2002) and, thus, it is of para-mount importance to estimate them correctly. To estimatethe biochemical limitations to photosynthesis, Wullschleger(1993) made a retrospective analysis of A–Ci curves andcalculated Vcmax and Jmax for 109 C3 species. He applied theFarquhar et al. (1980) C3 model of photosynthesis using invitro-measured Rubisco kinetics parameters. In our studywe first compare the Wullschleger’s (1993) results to thephotosynthetic parameters of salt-stressed plants ofChalkidikis, Kerkiras, and Throubolia (Table 2a) and ofcontrol and salt-stressed plants of Chalkidikis and Kerkiraspre-conditioned at low [CO2] (Table 3a), obtained by fittingthe Farquhar et al. (1980) model of leaf photosynthesis toindividual A–Ci curves using in vitro-measured Rubiscokinetics parameters. Secondly, we compare the modelparameterization by using in vitro-measured (Table 1a) andin vivo-measured (Table 1b) photosynthesis kineticsparameters. Finally, we analyse the diffusional limitationsto photosynthesis.

Comparing the biochemical limitations

Based on the in vivo and in vitro analysis of photosyntheticcapacity, it has been suggested that environmental stresseswhich lower water activity, lead to metabolic impairment(Sharkey & Badger 1982; Lawlor 1995). Particularly, it wasshown that mild water stress led to limited RuBP regener-ation in field-grown grapevines (Escalona, Flexas &Medrano 1999). An apparently similar response was foundin our study, when standard A–Ci responses (i.e. measure-ments 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 butapproached saturation at low Ci (about 300 mmol mol-1)with mean Amax, averaged across the three cultivars, of11.1 mmol m-2 s-1 (Table 2). Mean Vcmax, averaged across thethree cultivars, was 75 mmol m-2 s-1 (Table 2a). This value isvery close to the average Vcmax of 64 mmol m-2 s-1 reportedin the survey by Wullschleger (1993) in his retrospectiveanalysis of the A–Ci curves from a large number of C3

species made in PPFD conditions. However, the three olivecultivars had a mean Jmax value of 79.5 mmol m-2 s-1

(Table 2a) which is, in contrast, much lower than that of

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

Parameter Pv25 Ea

(a) in vitro valuesKc 40.4a 59.40b

Ko 24.8 ¥ 103a 36.00b

Vcmax ~ 64.80b

Jmax ~ 37.00c

Rd ~ 66.40c

G* 42.70e 29.00d

(b) in vivo valuesKc 40.49f 79.43f

Ko 27.84 ¥ 103f 36.38f

Vcmax ~ 65.33f

Jmax ~ 37.00c

Rd ~ 46.39f

G* 42.75f 37.83f

The Rubisco parameters Kc, Ko and G* are appropriate to aninfinite gm. Kc, Michaelis–Menten constant for CO2; Kc, Michaelis–Menten constant for O2; Vcmax (photosynthetic Rubisco capacity perunit leaf area), Jmax (potential rate of electron transport rate perunit leaf area), Rd (mitochondrial respiration in the light per unitleaf area); G*, CO2 photocompensation point. avon Caemmereret al. (1994); bBadger & Collatz (1977); cFarquhar et al. (1980);dJordan & Ogren (1984); eBrooks & Farquhar (1985); fBernacchiet al. (2001).



134 mmol m-2 s-1 averaged by Wullschleger (1993) across109 C3 species. Consequently, the average Jmax : Vcmax ratiowas much lower than that reported in the survey byWullschleger (1993), namely 1.06 and 1.64, respectively.This low value of Jmax and, consequently of Amax, wouldindicate a reduced rate of RuBP regeneration according tothe Farquhar et al. (1980) model. Hence, these findingswould support the hypothesis that environmental stressaffects primarily the mesophyll carbon metabolism, at least

when CO2 is not a limiting factor. Under these conditions,the strong reduction of the photochemical efficiencyobserved in these salt-stressed leaves (Loreto et al. 2003)may effectively indicate the onset of these carbon metabo-lism limitations.

The short-term response of stomatal conductance tostepwise changes of Ci showed that gs reached values as lowas 0.015 mol m-2 s-1 at Ci of ~450 mmol mol-1 in salt-stressedplants (Fig. 2b). To understand whether the photosynthetic

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)

Jmax Vcmax Amax Jmax : Vcmax Rd Ne

(a) in vitro valuesThroubolia 72.0 73.3 10.5 0.98 -0.72 7.37Chalkidikis 81.0 80.4 11.1 1.01 -2.02 8.91Kerkiras 85.6 71.2 11.7 1.20 -2.24 9.05Mean 79.5 ± 3.2 75.0 ± 2.3 11.1 ± 0.3 1.06 ± 0.06 -1.66 ± 0.39 8.44 ± 0.44

(b) in vivo valuesThroubolia 64.30 65.54 10.50 0.98 -0.65 6.53Chalkidikis 76.37 72.73 11.10 1.05 -1.15 7.67Kerkiras 76.73 62.93 11.70 1.22 -1.12 7.25Mean 72.47 ± 3.34 67.07 ± 2.39 11.10 ± 0.28 1.08 ± 0.06 -0.97 ± 0.13 7.15 ± 0.27

Jmax, potential rate of electron transport rate per unit leaf area (mmol m-2 s-1); Vcmax, photosynthetic Rubisco capacity per unit leaf area(mmol m-2 s-1); Amax, maximum photosynthetic rate at saturating PPFD (mmol m-2 s-1), Rd, mitochondrial respiration in the light per unit leafarea (mmol m-2 s-1); Ne, number of electrons consumed per CO2 fixed in non-photorespiratory conditions.

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 mmol 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)

Chalkidikis Kerkiras

MeanControl Salt Control Salt

(a) in vitro valuesJmax 113.29 ± 1.55a 118.21 ± 2.20a 124.87 ± 3.89a 121.19 ± 3.27a 119.39 ± 2.12Vcmax 71.84 ± 0.95a 74.62 ± 0.58ab 77.72 ± 1.65bc 79.16 ± 0.78c 75.84 ± 1.41Amax 20.13 ± 0.47a 21.90 ± 0.76ab 24.10 ± 1.12b 23.38 ± 0.79b 22.38 ± 0.76Jmax : Vcmax 1.58 ± 0.04a 1.58 ± 0.03a 1.61 ± 0.02a 1.53 ± 0.03a 1.57 ± 0.01Rd -1.34 ± 0.02a -1.29 ± 0.27a -0.80 ± 0.12a -1.25 ± 0.14a -1.17 ± 0.11Ne 6.03 ± 0.073a 5.77 ± 0.185a 5.45 ± 0.093a 5.49 ± 0.091a 5.69 ± 0.118

(b) in vivo valuesJmax 106.59 ± 3.24a 108.17 ± 4.02a 111.30 ± 4.99a 113.63 ± 5.92a 109.92 ± 1.37Vcmax 66.95 ± 2.51a 66.70 ± 3.28a 68.23 ± 2.56a 70.31 ± 3.21a 68.05 ± 0.71Amax 20.13 ± 0.47a 21.90 ± 0.76ab 24.10 ± 1.12b 23.38 ± 0.79b 22.38 ± 0.76Jmax : Vcmax 1.60 ± 0.05a 1.63 ± 0.04a 1.63 ± 0.03a 1.62 ± 0.02a 1.62 ± 0.01Rd -0.65 ± 0.03a -0.61 ± 0.02a -0.60 ± 0.07a -0.66 ± 0.04a -0.63 ± 0.01Ne 5.48 ± 0.087a 5.08 ± 0.043a 4.74 ± 0.059a 5.00 ± 0.024a 5.07 ± 0.131

Jmax, potential rate of electron transport rate per unit leaf area (mmol m-2 s-1); Vcmax, photosynthetic Rubisco capacity per unit leaf area(mmol m-2 s-1); Amax, maximum photosynthetic rate at saturating PPFD (mmol m-2 s-1), Rd, mitochondrial respiration in the light per unit leafarea (mmol 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.



capacity parameters derived from the A–Ci response curvesshown in Fig. 2a had been affected by such low values of gs,both control and salt-stressed plants of Chalkidikis andKerkiras were exposed to low [CO2] to force stomatalopening and remove the effect of stomatal limitation,before measuring a new set of A–Ci curves (Fig. 1). Sto-matal conductance of both control and salt-stressed plantsresponded positively to low [CO2]. Maximum stomatalopening was reached in about 1.5 h, and gs was increasedto similar values in control and salt-stressed plants of bothcultivars: gs of control and salt-stressed plants was increasedto an average of 0.22 and 0.20 mol m-2 s-1, respectively, inChalkidikis (Fig. 1b), and to an average of 0.19 and0.18 mol m-2 s-1, respectively, in Kerkiras (Fig. 1b). After gs

had approached the maximum value, the A–Ci responsewas again measured by progressively increasing [CO2] upto 350 mmol mol-1, then [CO2] was suddenly increased upto 1500 mmol mol-1, and in subsequent steps progressivelydecreased back to 350 mmol mol-1.

These new A–Ci curves, made on pre-conditioned plants,showed that photosynthesis reached a maximum in allleaves at Ci values between 800 and 900 mmol mol-1 (Fig. 3).In general, Kerkiras plants (Fig. 3b) had higher photosyn-thetic capacity than Chalkidikis plants (Fig. 3a). Likewise,salt-stressed plants had a slightly higher photosynthetic

capacity than control plants. However, analysis of the datausing the Farquhar et al. model (Farquhar et al. 1980; dePury & Farquhar 1997) fitted to individual A–Ci shows thatthe best-fit of Vcmax, Jmax, Amax and Rd were not statisticallydifferent in control and salt-stressed plants of both cultivars(Table 3). This indicates that the biochemical capacityof photosynthesis was similar between cultivars and salttreatment.

Interestingly, by comparing the A–Ci curves shown inFigs 2 and 3, it is evident that whereas mean Vcmax wasalmost identical in the two sets of A–Ci curves, the esti-mates of the best-fit Jmax and Amax were much higher in theA–Ci curves of plants pre-conditioned at low [CO2](Table 3) than in standard A–Ci responses (Table 2). TheJmax of pre-conditioned plants had a mean value, averagedacross salt-stressed and control saplings of both Chalkidikisand Kerkiras cultivars, of 119.39 mmol m-2 s-1 (Table 3a)which was similar in magnitude to the average valuereported in the survey by Wullschleger (1993).

Moreover, in the pre-conditioned plants, the value of 1.57of the mean Jmax : Vcmax ratio (Table 3a) was similar to thatof 1.64 averaged by Wullschleger (1993) across 109 C3 spe-cies. In the pre-conditioned plants a positive linear correla-tion was observed between the best-fit estimates of Jmax andVcmax (Fig. 4), which indicates a tight co-ordination betweenthe activities of thylakoid proteins (photochemistry) andsoluble proteins (Calvin cycle) to match each other. ThisFigure 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 mmol m-2 s-1) in salt-stressed plants of Chalkidikis (�), Kerkiras (�) and Throubolia (�).

A( m

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0 100 200 300 400 500

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(a)

(b)

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

A( m

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relationship was not observed in salt-stressed plants beforepre-conditioning. The ability to maintain a constant ratiobetween the carboxylation and light-harvesting activitiesacross a wide range of environmental conditions, originatesfrom a functional balance between RuBP consumption andRuBP regeneration. Thus, the A–Ci curves on salt-stressedleaves after pre-conditioning at low [CO2] clearly indicatethat (1) salt stress did not affect the biochemical capacityto assimilate CO2, and (2) the estimates of photosyntheticcapacity without removing stomatal limitation are artifi-cially low and may lead to a wrong interpretation of theactual limitation of photosynthesis. Moreover, because gs

was strongly increased also in control plants pre-condi-tioned at low [CO2] (Fig. 1), it is reasonable to speculatethat at least in sclerophytic leaves the A–Ci curves shouldbe assessed after pre-conditioning plants in low [CO2].Finally, the path-dependent reduction in Vcmax and gs, putforward by Prioul, Cornic & Jones (1984) and then revisedby Jones (1985), Assmann (1988), and more recently byWilson, Baldocchi & Hanson (2000), has not been found inour study, because Vcmax was almost identical in the two setsof A–Ci curves (Figs 2a & 3) despite the remarkable differ-ences in gs between non-pre-conditioned (Fig. 2b) and pre-conditioned (Fig. 1) plants.

Comparing the model parameterization

The parameterization of the model developed by de Pury& Farquhar (1997) showed that Jmax, Vcmax, Rd, and Ne (thenumber of electrons consumed per CO2 fixed in non-photorespiratory conditions, that is, Ne = Jmax/(A + Rd))

were lower using Rubisco kinetics parameters measured invivo (Tables 2b & 3b) than those obtained using in vitro-measured parameters (Tables 2a & 3a). In contrast, theJmax : Vcmax ratio was increased by parameterizing the modelwith the in vivo kinetics parameters estimated by Bernacchiet al. (2001). However, these differences were not signifi-cant at the 5% level in salt-stressed plants of Chalkidikis,Kerkiras, and Throubolia (Table 2). Whereas in control andsalt-stressed plants of Chalkidikis and Kerkiras pre-conditioned at low [CO2], these differences were signifi-cant. In fact, applying the Bernacchi et al. (2001) in vivokinetics parameters caused a significant decrease atP < 0.05 in Jmax, Rd, and Ne, and at P < 0.01 in Vcmax, and asignificant increase at P < 0.001 in the Jmax : Vcmax ratio(Table 3b) with respect to the values estimated by usingRubisco kinetics parameters measured in vitro (Table 3a).The mean number of electrons consumed per CO2 fixed innon-photorespiratory conditions was reduced from 5.69(Table 3a) to 5.07 (Table 3b) by applying the Bernacchiet al. (2001) in vivo parameters. This latter value is not farfrom the theoretical values of four electrons per CO2 fixedin non-photorespiratory conditions [i.e. Jmax = 4(A + Rd)](Farquhar et al. 1980), assuming that light were equally dis-tributed between the photosystems I and II, and that alter-native electron sinks were absent (i.e. electrons were notused for processes other than photosynthesis, such aspseudocyclic electron transport and nitrogen assimilation),and is similar to values obtained in other experiments bothin C3 and C4 plants (Genty & Harbison 1996). The reducedvalue in Ne obtained with the Bernacchi et al. (2001) in vivoparameters, along with the increase in the Jmax : Vcmax ratioand, above all, the reduction in Rd, indicates a betterparameterization of the photosynthetic model.

Diffusional limitations

We have suggested that the sum of stomatal and mesophyllresistances set the photosynthesis rates of salt-stressedleaves in ambient [CO2] (Loreto et al. 2003). With theseexperiments we have proved that both these additive resis-tances can be removed by pre-conditioning salt-stressedleaves to low [CO2] (Table 4). Short-term changes in [CO2]not only affected gs, but also affected gm. In fact, gm mea-sured at [CO2] of 350 mmol mol-1 was not constant, but waslinearly related (r2 = 0.68) to gs (Fig. 5). The factors settingmesophyll conductance were believed to be constitutiveand essentially dependent on leaf anatomy (Evans et al.1994; Syvertsen et al. 1995). However, a recent study hasdemonstrated that the water-deficit effect of salinity did notaffect photochemistry, but induced a reversible decrease ofgm which, in turn, was responsible of transient reduction ofphotosynthesis in an herbaceous species (Delfine et al.1999). Our current experiments also suggest that changesof gm can be as fast as those of gs (Fig. 5); and also thatthe sum of the diffusional resistances set the limit to pho-tosynthesis rates. This is the reason why there was no rela-tionship between A measured at CO2 concentration of~350 mmol mol-1 and the CO2 drawdown caused by

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 sym-bols) and of salt-stressed and control plants of Chalkidikis and Kerkiras (closed symbols) pre-conditioned at [CO2] of ~50 mmol mol-1 for about 1.5 h to force stomatal opening (see Fig. 1). The linear relationship (r2 = 0.78) between these two com-ponents of photosynthetic capacity of the pre-conditioned plants had a slope of 1.57 (i.e. Jmax/Vcmax).

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stomatal resistance, whereas a clear relationship was foundbetween A and CO2 drawdown when this was caused by thesum of stomatal and mesophyll resistances (Fig. 6, andLoreto et al. 2003).

Recently Wilson et al. (2000) applied the path-dependentmethod (Prioul et al. 1984; Jones 1985; Assmann 1988) toquantify stomatal versus non-stomatal limitations to PPFD-saturated A of chestnut oak and sugar maple in responseto drought. They estimated that stomatal limitations to pho-tosynthesis accounted for about 75%, whereas non-sto-matal limitations did not exceed 25%. However, they didnot measure gm and did not pre-conditioned plants at[CO2]. Consequently, they could not separate diffusionaland non-diffusional limitations of photosynthesis. There-fore, if the linear relationship between gm and gs found inour 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-conditioningleaves at [CO2], the size of the stomatal limitations tophotosynthesis estimated by Wilson et al. (2000) wouldhave to be consistently reduced. It is well known that sto-mata open in response to low [CO2]. The reasons why mes-ophyll conductance also recover after pre-conditioningleaves at low [CO2] is however, unknown and deservesfurther investigation.

CONCLUSION

The assessment of reduced photosynthetic efficiency understress conditions in terms of stomatal and non-stomatal

Table 4 Assimilation rate (A, mmol 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 mmol mol-1 in control and salt-stressed plants of Kerkiras and Chalkidikis during leaf exposure to the changes in [CO2] shown in Fig. 1

Control Salt-stressed

A gs gm A gs gm

Kerkiras:Pre 7.34 ± 0.57 0.08 ± 0.011 0.085 ± 0.008 2.65 ± 0.48 0.02 ± 0.005 0.048 ± 0.007Post 12.87 ± 1.89 0.17 ± 0.004 0.120 ± 0.012 12.92 ± 1.45 0.15 ± 0.023 0.162 ± 0.018End 8.04 ± 0.97 0.08 ± 0.011 0.079 ± 0.010 7.70 ± 1.45 0.07 ± 0.013 0.034 ± 0.011

Chalkidikis:Pre 6.12 ± 1.20 0.10 ± 0.028 0.062 ± 0.011 2.92 ± 0.42 0.02 ± 0.003 0.028 ± 0.009Post 11.83 ± 0.55 0.21 ± 0.006 0.107 ± 0.016 12.90 ± 0.67 0.18 ± 0.016 0.141 ± 0.011End 6.37 ± 0.70 0.11 ± 0.009 0.088 ± 0.010 7.93 ± 0.63 0.07 ± 0.007 0.064 ± 0.006

These parameters were measured three times per plant at [CO2] of 350 mmol mol-1: before (pre) and after (post) exposing the leaves at[CO2] of ~50 mmol mol-1, and after progressively decreasing [CO2] from 1500 mmol mol-1 (end). Data are means of three to four plants pertreatment ± 1 SEM.

Figure 5. Linear relationship between the mean data of meso-phyll conductance to CO2 (gm) and stomatal conductance (gs) shown in Table 4 (when [CO2] = 350 mmol 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.

gs (mol m-2s-1)

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g m(m

olm

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Figure 6. Relationship between photosynthesis and CO2 draw-down 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 mmol mol-1: before and after exposing the leaves at [CO2] of ~50 mmol mol-1, and after progressively decreasing [CO2] from 1500 mmol mol-1 (see Fig. 1).

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limitations has been a standard practice over the last30 years (Jones 1973; Bradford & Hsiao 1982; Cornic, Pri-oul & 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 beenoften regarded as quite separate levels of control as if therewere no reciprocal influences. To complicate matters,although it is clear that stomatal limitations to photosyn-thesis describe the diffusional resistances in the gas phaseof the CO2 transport pathway between the ambient air andthe carboxylation sites, the interpretations of non-stomatallimitations to photosynthesis are somehow confusing. Non-stomatal limitations include both physical limitations,namely internal (or mesophyll) resistances to CO2 diffusionin 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 differ-ent role played by these two components of the mesophyllproperties in determining the non-stomatal limitations tophotosynthesis have been ignored or confused. This cangive misleading indications on the response of photosyn-thesis to environmental stresses, because it can lead to theconclusion that photosynthesis capacity is directly reducedby environmental stresses, even under circumstances inwhich there may be no real damage to the photosyntheticapparatus. However, during the last decade, three methodshave been developed to estimate in vivo the internal con-ductance to CO2 diffusion between the intercellular spacesand the chloroplasts (Loreto et al. 1992; Evans & Loreto2000). This allows the separation of the physical and bio-chemical components of non-stomatal limitations to photo-synthesis. Based on this methodology, we have presented anon-destructive-in vivo method to estimate the photosyn-thetic capacity in stressed plants by separating the diffu-sional limitations from the non-diffusional limitations. Ourfindings clearly indicates that: (1) salt stress did affect gs andgm but not the biochemical capacity to assimilate CO2.Therefore, in these conditions the sum of the diffusionalresistances set the limit to photosynthesis rates; (2) therewas a linear relationship between gm and gs, and, thus,changes of gm can be as fast as those of gs; (3) the estimatesof photosynthetic capacity based on A–Ci curves madewithout removing diffusional limitations are artificially lowand lead to incorrect interpretations of the actual limita-tions of photosynthesis; and (4) the analysis of the photo-synthetic properties in terms of stomatal and non-stomatallimitations should be replaced by the analysis of diffusionaland non-diffusional limitations of photosynthesis. Finally,applying in vivo-measured Rubisco kinetics parametersinstead of the in vitro-measured parameters results in abetter parameterization of the Farquhar et al. (1980) C3

photosynthesis model.

ACKNOWLEDGMENTS

This work has been supported by the Italian and GreekMinistry of Foreign Affairs within the frame of the Italian– Greek Bilateral Cooperation for Research and Develop-

ment (Contract no. GSRT-18345). The assistance of Ms.Maria Moutsopoulou during gas-exchange measurementsis gratefully acknowledged.

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

the use of low [co2] to estimate diffusional and non-diffusional limitations of photosynthetic...

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