Journal of Experimental Botany

JXB Advance Access originally published online on September 12, 2006
Journal of Experimental Botany 2006 57(14):3659-3667; doi:10.1093/jxb/erl113

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RESEARCH PAPER

Acclimation of Rubisco specificity factor to drought in tobacco: discrepancies between in vitro and in vivo estimations

Jeroni Galmés, Hipólito Medrano and Jaume Flexas^*

Laboratori de Fisiologia Vegetal, Grup de Biologia de les Plantes en Condicions Mediterrànies, Universitat de les Illes Balears. Carretera de Valldemossa Km 7.5, E-07122 Palma de Mallorca, Balears, Spain

^* To whom correspondence should be addressed. E-mail: jaume.flexas{at}uib.es

Received 30 June 2006; Accepted 5 July 2006

	Abstract

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
In studies about the photosynthesis response to environmental stresses, such as drought, the Rubisco specificity factor () is assumed to be constant or derived indirectly from gas exchange measurements. However, an analysis of the acclimation of to drought using in vitro determinations is lacking. The aim of the present work was to analyse the acclimation of to different drought intensities in tobacco (Nicotiana tabacum L.). Potted tobacco plants were subjected to three different water regimes (100%, 40%, and 15% of field capacity) and new leaves were allowed to develop. When acclimated leaves were fully developed, they were sampled for gas exchange and chlorophyll fluorescence measurements, as well as for the in vitro analysis of Rubisco kinetic properties. Relative water content and gas exchange decreased with increasing water shortage. The apparent Rubisco specificity factor as estimated in vivo by gas exchange decreased with water stress. However, in vitro estimates of were identical among treatments, as were Rubisco specific initial activity and activation state. The reasons for the observed discrepancy between in vitro and in vivo estimates are profusely discussed. It is suggested that the Rubisco specificity factor does not acclimate to water stress in the short term (weeks or months) in tobacco, and the validity of the so-called Laisk gas exchange method to estimate under drought is questioned.

Key words: Acclimation, drought, Nicotiana tabacum, Rubisco, specificity factor, water stress

	Introduction

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39 [EC] ) plays a central role in plant photosynthesis since it is involved in the uptake of CO₂ by photosynthetic organisms. Besides its carboxylase activity, Rubisco also acts as an oxygenase in a reaction involving competition between O₂ and CO₂ for reaction with ribulose-1,5-bisphosphate (RuBP). Thus, while photosynthesis is initiated by the carboxylase activity, the oxygenase activity catalyses the first reaction in the photorespiratory pathway (Ogren and Bowes, 1971; Laing et al., 1974). The balance between the two competitive reactions is determined by the kinetic properties of Rubisco and the CO₂ and O₂ concentrations at the site of the enzyme (Laing et al., 1974):

where v_c and v_o are the velocities of carboxylation and oxygenation, respectively, V_c and V_o are the maximal velocities of the two reactions, and K_c and K_o the Michaelis constants for CO₂ and O₂. The substrate specificity factor, V_cK_o/V_oK_c (), determines the relative rates of the two reactions at any given CO₂ and O₂ concentrations.

Current models of leaf photosynthesis assume constant values among C₃ plants (Long and Bernacchi, 2003) or derive them indirectly from the CO₂ compensation point in the absence of mitochondrial respiration (*) after gas exchange measurements (Bernacchi et al., 2001). However, may not be constant under varying environmental conditions, such as light quality and quantity or water stress, although there has been no attempt to assess this possibility. However, the molecular basis and/or structural features determining Rubisco are still poorly understood (Spreitzer and Salvucci, 2002; Andersson and Taylor, 2003; Spreitzer, 2003). The Rubisco holoenzyme of higher Rubisco is a hexadecamer described as four dimers of large subunits surrounded by two tetramers of small subunits. Two active sites are found at the interface of the large subunits in each dimer, i.e. eight catalytic sites per holoenzyme (Andersson and Taylor, 2003). In higher plants, the Rubisco large subunit is encoded by multiple identical copies of rbcL in the chloroplast genome (Eilenberg et al., 1998), whereas an rbcS gene family having 5–12 nuclear genes encodes small subunit peptides (Dean et al., 1989; Spreitzer, 2003). Thus, while the copies of the large subunit would probably be the same, the differential expression of rbcS genes may depend on the environment. For instance, transcription of specific rbcS genes appears to be dependent on light quality in the fern Pteris vittata (Eilenberg et al., 1990). Although large subunits have the main influence on catalytic properties, the Rubisco small subunits have also been hypothesized to affect key kinetic characteristics like (Roy and Andrews, 2000; Parry et al., 2003). Hence, in principle, any environmental condition capable of modulating rbcS gene expression could putatively induce changes in Rubisco .

Water stress, in particular, induces stomatal closure and a decrease in leaf internal CO₂ concentration, which results in increased oxygenase over carboxylase activity, thus increasing the ratio of photorespiration to photosynthesis (Flexas and Medrano, 2002). As a consequence, Delgado et al. (1995) and Kent and Tomany (1995) hypothesized that in hot environments associated with water stress, stomatal closure and low CO₂ concentrations at the site of Rubisco may impose increased selection pressure on Rubisco for improved specificity. Indeed, recent results by Galmés et al. (2005) showed that, in a survey of Mediterranean plant species, the highest values of were observed in Rubisco from plant species growing and adapted for growth in the driest environments. Despite these facts, when applying photosynthesis models to study the effects of water stress on photosynthetic limitations and/or on mesophyll conductance to CO₂ (g_m), Rubisco (or *) is usually assumed to be constant (Roupsard et al., 1995; Wilson et al., 2000; Xu and Baldocchi, 2003; Peña-Rojas et al., 2004; Warren et al., 2004). However, Bota et al. (2002) and Warren et al. (2004) have reported an apparent increase of * under water stress. This fact merits some attention, since it may change substantially the interpretations of water stress on photosynthesis coming from studies in which * was assumed as constant. Fluorescence estimates of g_m, in particular, are very sensitive to * (Harley et al., 1992). However, estimations of * under water stress include a number of assumptions that may not be correct and, as noted by Warren et al. (2004), it would be preferable to determine Rubisco (or *) independently using an alternative method. The aim of the present study is to determine whether Rubisco acclimates to water stress in the short term (weeks) in tobacco plants, by determining in vitro, and comparing it with in vivo estimates of *.

	Materials and methods

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Plant material and treatments
Thirty seeds of Nicotiana tacabum var. White Burley were germinated and grown individually in pots (20 cm height, 4.1 l volume) containing a mixture of clay–calcareous soil, horticultural substrate, and perlite (40:40:20 by vol.). The experiment was performed during spring 2004 inside a greenhouse located at the University of the Balearic Islands (Mallorca, Spain). Plants were randomly distributed for growing and spread out to avoid mutual shading and to give similar light and temperature.

All seedlings were well-watered until they had four fully expanded leaves and, presumably, an adequate root system to cope with water constraints. Irrigation treatments were started on 22 April 2004. Ten such plants were randomly selected for each of the irrigation treatments: (i) maintained at field capacity during all experiment (control treatment, C), (ii) maintained at 40% field capacity (moderate drought treatment, MD), and (iii) maintained at 15% field capacity (severe drought treatment, SD). The desired moisture levels were attained by allowing the soil to dry until close to the selected moisture level, as determined gravimetrically by weighing pots on alternate days and, from then on, compensating their daily water losses with the addition of an equal amount of 50% Hoagland's solution.

New leaves were allowed to develop and expand under the three irrigation treatments until 15 June 2004. Then, leaves developed during the irrigation treatments (i.e. acclimated to different water availability) were sampled to determine the leaf mass area (LMA), the relative water content (RWC), and , total leaf soluble protein, Rubisco activity, gas exchange, and chlorophyll fluorescence.

Rubisco purification and specificity factor measurement
Leaves (30–50 g) of each treatment (C, MD, and SD) were collected and immediately frozen in liquid nitrogen. The leaf material was ground to a powder in a mortar, buffer was added and grinding continued from time to time as the mixture thawed. The protein extraction media used contained: 0.1 M Bicine, 10 mM Na-DIECA, 6% PEG (polyethylene glycol) 4000, 3% (w/v) PVP (polyvinylpyrrolidone) 25 000, 1 mM DTT (dithiothreitol), 1 mM benzamidine, 1 mM -amino-n-caproic acid, and 1 mM PMSF (phenylmethylsulphonylfluoride), at pH 8.

All the purification steps were carried out at 0–4 °C. Fully thawed but still cold homogenates were filtered through butter muslin and centrifuged at 18 000 g for 20 min. The supernatant liquid was decanted through 50 µm nylon mesh and PEG 4000 was added as a 60% aqueous solution to the supernatant liquid to produce a final concentration of 20% w/v. Also, 1 M MgCl₂ was added to a final concentration of 20 mM followed by gentle mixing. After standing for 10 min the mixture was centrifuged again at 18 000 g for 20 min. The pellet was resuspended in 6 ml of column buffer (10 mM TRIS pH 8.0 with 10 mM MgCl₂, 10 mM NaHCO₃, 1 mM EDTA, and 1 mM KH₂PO₄) containing 1 mM each of DTT, PMSF, benzamidine, and -amino-n-caproic acid. The suspension was then centrifuged to remove insoluble material. The supernatant liquid was layered onto step gradients from 1.2 M to 0.4 M in sucrose in the column buffer. Gradients were centrifuged at 50 000 rpm for 120 min in a 70.1Ti rotor (Beckman, High Wycombe, UK). Fractions with a high protein concentration were combined and applied to two 1 ml HiTrap Q HP columns (Amersham Biosciences) connected in series previously equilibrated with column buffer and operated at 1 ml min^–1. The proteins were eluted using a step gradient from 0 M to 0.8 M NaCl in column buffer and fractions were collected in 1 ml intervals. Total soluble protein content in fractions was confirmed using the Bradford assay (Bradford, 1976). Those fractions with high protein (Rubisco) concentration were combined and stored at –70 °C.

Rubisco-rich fractions were used to make 8–10 measurements of Rubisco specificity factor () per treatment at 25 °C as follows. Rubisco solutions with a high protein concentration were desalted by centrifugation through G25 Sephadex columns (Helmerhorst and Stokes, 1980) previously equilibrated with CO₂-free 0.1 M bicine pH 8.2 containing 20 mM MgCl₂. The desalted solutions were made 10 mM with NaH¹⁴CO₃ and 0.4 mM with orthophosphate. These mixtures were incubated at 37 °C for 40 min to activate the Rubisco. Reaction mixtures were prepared in an oxygen electrode (Dual digital Model 20, Rank Brothers Ltd., Cambridge, UK) by first adding 0.93 ml of a solution of 100 mM bicine pH 8.2, 10 mM MgCl₂ containing 1.5 mg (7000 W-A units) per 100 ml of carbonic anhydrase and equilibrated with CO₂-free air at 25 °C. After adding 0.02 ml of 0.1 M NaH¹⁴CO₃ the plug was fitted to the oxygen electrode vessel. Enough activated Rubisco was then added to 40 µl for the reaction to be completed in 5 min. When the signal from the electrode was steady, the reaction was started by the addition of 10 µl of 15 mM RuBP. The final volume of the reaction mixture was 1 cm³. RuBP oxygenation was calculated from the oxygen consumption and carboxylation from the amount of ¹⁴C incorporated into PGA when all the RuBP had been consumed (Parry et al., 1989). A sequence of reaction mixtures containing pure wheat Rubisco were interspersed with those containing Rubisco from the tobacco plants and the results were normalized to the average value obtained from wheat Rubisco (102.5 at 25 °C).

Rubisco carboxylase activity and total soluble protein
For Rubisco carboxylase activity, 3–4 samples per treatment were ground to a fine powder in a mortar, previously chilled with liquid nitrogen and homogenized in 1 ml of an ice-cold extraction medium. The extraction medium was the same as that used for Rubisco purification for measurements. Extracts were clarified by centrifugation (12 000 rpm at 4 °C for 2 min) and the supernatant immediately assayed at 25 °C for Rubisco activity. The initial and total activities were determined according to Parry et al. (1997). Total soluble protein was determined according to the method of Bradford (1976).

Relative water content and leaf mass area
RWC at mid-morning was determined as follows: RWC=(Fresh weight–Dry weight)/(Turgid weight–Dry weight). To determine the turgid weight of the leaves, these were kept in distilled water in darkness at 4 °C to minimize respiration losses, until they reached a constant weight (full turgor, typically after 24 h). Their dry weight was obtained after 48 h at 70 °C in an oven. Six replicates per treatment were obtained.

LMA was calculated, in six replicates per treatment, as the ratio of dry mass to leaf area. Leaf area was determined in fresh leaves using an AM-100 leaf area meter (ADC, Herts, UK).

Gas exchange and chlorophyll fluorescence measurements
Leaf gas exchange parameters were measured simultaneously with measurements of chlorophyll fluorescence using an open gas exchange system (Li-6400; Li-Cor, Inc., Nebraska, USA) with an integrated fluorescence chamber head (Li-6400–40 leaf chamber fluorometer; Li-Cor, Inc.).

In light-adapted leaves, the actual photochemical efficiency of photosystem II (_PSII) was determined by measuring steady-state fluorescence (F_s) and maximum fluorescence during a light-saturating pulse of c. 8000 µmol m^–2 s^–1 (F'_m) following the procedures of Genty et al. (1989):

The electron transport rate (ETR) was then calculated as:

where PFD is the photosynthetically active photon flux density and is a term which includes the product of leaf absorptance and the partitioning of absorbed quanta between photosystems I and II. was previously determined for each treatment as the slope of the relationship between _PSII and _CO2 obtained by varying light intensity under non-photorespiratory conditions in an atmosphere containing less than 1% O₂ (Valentini et al., 1995). resulted as 0.357, with no difference between treatments.

All measurements were started at 25 °C and at 1000 µmol m^–2 s^–1 to ensure light saturation, with 10% blue light. Cuvette CO₂ concentration (C_a) was set at 400 µmol CO₂ mol air^–1 and the vapour pressure deficit was maintained between 1.0 and 1.5 kPa. After inducing photosynthesis under the above conditions and once steady-state was reached, photosynthesis response curves to varying substomatal CO₂ concentration (C_i) were performed. First, the C_a was lowered stepwise from 400 to 50 µmol mol^–1 and then fixed again at 400 µmol mol^–1 until reaching a steady-state value similar to that obtained at the beginning of the curve. Then, C_a was increased stepwise from 400 to 1500 µmol mol^–1. Gas exchange measurements were determined at each step after maintaining the leaf for at least 5 min at the new C_a. Measurements consisted of 12–13 measurements for each curve. Leaf respiration in the dark (R_D) was measured at the same temperature and CO₂ concentration in the same leaves after keeping them for 30 min in darkness.

The rate of non-photorespiratory CO₂ evolution in the light (R_L) and the substomatal CO₂ compensation point in the absence of mitochondrial respiration (C_i^*) were estimated according to the method of Laisk (1977) as described by Brooks and Farquhar (1985), i.e. from the response of A_N to C_i at three different irradiances (750, 150, and 50 µmol m^–2 s^–1). Four to five replicates were made for each treatment. Finally, C_i^* was converted to the chloroplast CO₂ compensation point (*) considering the effects of the internal diffusion conductance and R_L as in von Caemmerer (2000):

The apparent Rubisco specificity factor operating in vivo (*) was determined by the properties of Rubisco (Brooks and Farquhar, 1985) as:

where O represents the oxygen molar fraction at the oxygenation site.

Cuticular conductance to H₂O was measured in four replicates per treatment as described by Boyer et al. (1997). The results showed decreasing values as drought intensified (10 mmol m^–2 s^–1 in C, 13 mmol m^–2 s^–1 in MD, and 16 mmol m^–2 s^–1 in SD). These values were used to correct all C_i values and subsequent estimations.

Net photosynthesis and C_i estimations are also affected by lateral CO₂ diffusion through the IRGA's chamber gaskets, particularly when CO₂ concentrations inside and outside the chamber are substantially different, as during measurements of A_N-C_i curves (Long and Bernacchi, 2003). To establish the magnitude of gasket leakage and correct A_N and C_i values, CO₂ response curves were made using an inert tobacco leaf thermally killed by submerging it in boiling water. The leaf was submerged in boiling water for some seconds, and then its variable chlorophyll fluorescence in the dark-adapted state was measured using a PAM-2000 (Walz, Effeltrich, Germany). This procedure was repeated until no variable chlorophyll fluorescence was detected (typically a few minutes after initiating boiling), which was taken as an evidence for total photosynthesis impairment and leaf death (Schreiber et al., 1998). Using the relationship between C_a and the ‘apparent’ photosynthesis of a chamber filled with a dead leaf, the actual A_N of live leaves was obtained at each C_a by simple subtraction of the leak flow to the obtained value. Using the corrected A_N values, C_i was recalculated using the manufacturer formulas (Long and Bernacchi, 2003).

Estimation of C_c and g_m
From combined gas-exchange and chlorophyll fluorescence measurements, the CO₂ concentration in the chloroplasts (C_c) was calculated according to Epron et al. (1995). This model works on the assumption that all the reducing power generated by the electron transport chain is used for photosynthesis and photorespiration, and that chlorophyll fluorescence gives a reliable estimate of the quantum yield of electron transport. Thus, the electron transport rate (ETR) measured by chlorophyll fluorescence can be divided into two components: ETR=ETR_A+ETR_P, where ETR_A is the fraction of ETR used for CO₂ assimilation, and ETR_P is the fraction of ETR used for photorespiration. ETR_A and ETR_P can be solved from data of A_N, R_L, and ETR, and from the known stochiometries of electron use in photosynthesis and photorespiration, as follows (Epron et al., 1995; Valentini et al., 1995): ETR_A=1/3[ETR+8(A_N+R_L)]; ETR_P=2/3[ETR–4(A_N+R_L)].

The ratio ETR_A to ETR_P is related to the C_c/O ratio in the chloroplast through the Rubisco specificity factor (), as follows (Laing et al., 1974): =(ETR_A/ETR_P)/(C_c/O). Using the values of previously determined in vitro for each treatment, and assuming O to be equal to the molar fraction in the air, the above equation was solved for C_c. The mesophyll conductance to CO₂ was then calculated as:

The estimated C_c values were used to convert A_N–C_i curves into A_N–C_c curves (Terashima and Ono, 2002; Manter and Kerrigan, 2004). From A_N–C_c curves, the maximum carboxylation capacity (V_c,max) was calculated (Long and Bernacchi, 2003).

	Results and discussion

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Acclimation to water stress: water relations and photosynthetic parameters
Despite the acclimation of the leaves to water deficit, a decreased water supply resulted in large decreases in RWC, from 83.4% in C to 75.0 and 65.1% in MD and SD treatments, respectively (Table 1). As expected, A_N and g_s also decreased with decreasing water availability (Table 1). A_N decreased from 23.3 µmol CO₂ m^–2 s^–1 in C to 16.9 µmol CO₂ m^–2 s^–1 in MD and 7.9 µmol CO₂ m^–2 s^–1 in SD, while the stomatal conductance to CO₂ (g_s) decreased from 230 mmol CO₂ m^–2 s^–1 in C to 119 mmol CO₂ m^–2 s^–1 in MD and 46 mmol CO₂ m^–2 s^–1 in SD. ETR decreased to a much lesser extent than A_N, from 191 in C to 139 µmol m^–2 s^–1 in SD, possibly indicating an increased electron partitioning towards sinks other than photosynthesis, mainly photorespiration (Cornic and Massacci, 1996; Flexas and Medrano, 2002). Acclimation to drought resulted in an increased LMA and a somewhat increased total soluble protein content (Table 1). However, the Rubisco specific initial activity and its activation state were similar in all treatments (Table 1), as usually observed in water stress experiments, except when stress is very severe (Flexas et al., 2004). The procedure used to extract Rubisco resulted in a certain loss of the enzyme during extraction, which could explain the low in vitro Rubisco carboxylation activities compared with the maximum rate of carboxylation estimated in vivo (V_c,max), as already discussed by Bota et al. (2004) and Rogers et al. (2001). R_D remained similar (2.4±0.2 µmol CO₂ m^–2 s^–1) for all treatments.

View this table: [in this window] [in a new window]   Table 1 Fluorescence, gas exchange and Rubisco assays of tobacco treatments

 
Water stress also resulted in significantly different A_N–C_i responses (Fig. 1). The operational C_i (i.e. the C_i at atmospheric C_a) declined as water stress intensified, from 261 µmol mol^–1 in C to 170 µmol mol^–1 in MD and 53 µmol mol^–1 in SD, pointing to an increased stomatal limitation to photosynthesis (Cornic and Fresneau, 2002). However, light- and CO₂-saturated photosynthesys (A_SAT) also decreased apparently from 27.7 in C to 19.3 and 13.0 in MD and SD treatments, respectively, which suggested an increased non-stomatal limitation to photosynthesis in the stressed leaves (Lawlor, 2002). In addition, both in MD and SD leaves the maximum A_N value was attained at an intermediate C_i, suggesting that photosynthesis at high C_i was limited by triose phosphate use (Long and Bernacchi, 2003), a situation sometimes observed under water stress (Flexas et al., 2004). The maximum carboxylation capacity did not differ significantly (P> 0.05) among treatments (Table 1). All the plants maintained their operational C_i at the breakpoint between CO₂-limited and RuBP- and/or TPU-limited photosynthesis, which has been interpreted as an acclimation to optimize both the photochemical and biochemical photosynthetic reactions (von Caemmerer and Farquhar, 1984).

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Relationship between net photosynthesis (AN) and the internal CO2 concentration (Ci). Filled symbols as control treatment, empty symbols as mild drought treatment, and filled triangles as severe drought treatment. Data are given by means and standard deviation.

 
However, the validity of A_N–C_i analysis has been questioned, particularly in water-stress experiments where g_m usually decreases in parallel with g_s (Flexas et al., 2002, 2004; Centritto et al., 2003; Warren et al., 2004), as actually occurs in the present study (Fig. 2). These results are very similar to those often encountered using other methods for the estimation of g_m that do not rely on previous knowledge of the in vitro , which supports the validity of our estimations (Loreto et al., 1992; Evans and Loreto, 2000; Flexas et al., 2004). It may be argued, however, that g_m estimations (and A_N–C_i curves) are not reliable under drought due to the incorrect estimations of C_i associated with heterogeneous stomatal closure (Terashima, 1992; Buckley et al., 1997; Mott and Buckley, 1998). However, from combined gas-exchange and chlorophyll fluorescence measurements, A_N–C_c curves can be obtained that are totally independent of C_i estimations (Sánchez-Rodríguez et al., 1999).

View larger version (6K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Relationship between mesophyll conductance (gm) and stomatal conductance (gs). Measurements were made at light saturation, 400 ppm CO2 and 25 °C in plants under control, mild drought, and severe drought treatments. Data are given by means and standard deviation.

 
Clearly, all the differences observed between treatments in the A_N–C_i curves disappeared in the A_N-C_c curves (Fig. 3), as already shown by Sánchez-Rodríguez et al. (1999) in water-stressed Casuarina equisetifolia. This is consistent with the constancy of Rubisco V_c,max and activation state, determined in vitro (Table 1). The only difference between treatments is the fact that MD and SD leaves attain, for the same range of C_a, maximum C_c values much lower than C leaves, due to the superimposed decreasing stomatal and mesophyll conductances to CO₂. It is interesting to note that, in A_N–C_c curves, CO₂-limited and RuBP-limited regions can be differentiated only in well-watered plants, which maintain their operational C_c at the breakpoint between these two limitations. By contrast, MD and SD operate at the CO₂-limited region. This is due to the superimposition of two different effects, a decrease of average g_m due to water stress and a more dynamic decrease of g_m with increasing C_i during A_N–C_i curves measurements, as already demonstrated by Centritto et al. (2003).

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Relationship between net photosynthesis (AN) and the chloroplastic CO2 concentration (Cc). Filled symbols as control treatment, empty symbols as mild drought treatment, and filled triangles as severe drought treatment. Data are given by means and standard deviation.

 
In vivo and in vitro estimations of Rubisco specificity
The rate of respiration in the light (R_L) was estimated following the method described by Laisk (1977) and Brooks and Farquhar (1985). R_L was strongly reduced compared with R_D, as usually observed (Villar et al., 1994, 1995). In addition, significant differences (P< 0.05) were observed in R_L between plants subjected to SD (0.2 µmol m^–2 s^–1) and to MD and C (0.5 µmol m^–2 s^–1) (Table 2). It is worth noting that R_L estimations are strongly affected by lateral leakage through the IRGA's chamber gaskets as well as by cuticular conductance to CO₂ and water. Thus, without correcting A_N and C_i values to account for these effects, in C plants R_L resulted in 1.1 µmol CO₂ m^–2 s^–1 but decreased to 0.5 µmol CO₂ m^–2 s^–1 when corrections were taken into account. Similar reductions were observed in MD and SD plants (not shown). The same method yields a value of C_i at the intersection of the three A_N–C_i curves (C_i^*), which has been used as a proxy for the CO₂ compensation point in the absence of mitochondrial respiration (Brooks and Farquhar, 1985; Villar et al., 1994, 1995). As for R_L, C_i^* estimations are also strongly affected by lateral leakage through the IRGA's chamber gaskets as well as by cuticular conductance to CO₂ and water. For instance, C_i^* resulted in 39.5 µmol mol^–1 in C plants before corrections, while it was only 36.5 µmol mol^–1 after corrections. The magnitude of these differences was even larger in water-stressed plants. In SD, for instance, a value of 73.4 µmol mol^–1 was obtained prior to corrections compared with 56.7 µmol mol^–1 after corrections. Hence, when using the Laisk method to estimate both R_L and C_i^*, special care needs to be taken to determine leakage and cuticular conductance accurately, particularly in water-stressed plants.

View this table: [in this window] [in a new window]   Table 2 Non-photorespiratory rate of respiration in the light (RL), Ci at the the CO2 compensation point in the absence of mitochondrial respiration (Ci*), the apparent Rubisco specificity factor operating in vivo on a Ci basis (Ci*), Cc at the CO2 compensation point in the absence of mitochondrial respiration (*), the apparent Rubisco specificity factor operating in vivo on a Cc basis (Cc*), and the in vitro Rubisco specificity factor at 25 °C () for the control (C), mild drought (MD), and severe drought (SD) treatments

 
Even taking into account the corrections, C_i^* strongly differed among treatments (Table 2), from 36.5 µmol mol^–1 in C to 39.8 µmol mol^–1 in MD and up to 56.7 µmol mol^–1 in SD. This pattern is in accordance with that observed by Bota et al. (2002) and Warren et al. (2004) in grapevines and Douglas-fir, respectively, and may be interpreted as an indication that water stress induces changes in the Rubisco specificity factor (see estimations of in vivo apparent * from C_i^* values in Table 2). However, it has been pointed out that a more precise estimation of the CO₂ photocompensation point in the absence of respiration (*) should take into account respiratory CO₂ flux and mesophyll conductance to CO₂ (von Caemmerer, 2000; Peisker and Apel, 2001). Warren et al. (2004) suggested that the fact that C_i^* estimations do not take into account variable g_m could be on the basis of the observed discrepancies between in vivo and in vitro estimations. However, when * was estimated using the reported values of C_i^*, R_L, and g_m, the discrepancies between treatments were even larger (Table 2).

In vivo apparent (_Cc^*) estimated from * according to Brooks and Farquhar (1985), resulted in the following values: 103.1 in C, 92.4 in MD, and 62.1 in SD (Table 2). However, when was measured in vitro there were no significant differences between treatments (Table 2). The in vitro averaged 99.3, which did not differ significantly from that of wheat. Therefore, _Cc^* largely underestimated in SD, and resulted in being similar to it in C and MD. An improved Rubisco specificity has recently been reported for Mediterranean species inhabiting the driest and hottest areas (Galmés et al., 2005), however, the present study demonstrates that in vitro Rubisco is stable during acclimation to drought, which is in accordance with the use of stable values of * (Roupsard et al., 1996).

Possible causes to explain the observed discrepancies between in vivo and in vitro estimations of Rubisco specificity
The large difference observed between the in vitro and the in vivo and the fact that in vivo estimations show a water-stress effect of Rubisco specificity factor, while in vitro estimations show a constant value, suggests that one of the two methods is not reliable, particularly under water-stress conditions. While, a priori, the misleading method could either be the in vitro or the in vivo one, the former is a well-established enzymatic method while the latter is very sensitive to small errors in the calculation of A_N, C_i, R_L, or g_m, of which a number has been described (Long and Bernacchi, 2003). Therefore, the authors believe that the differences come from the fact that the Laisk method, although yielding quite approximate estimates in control plants, is unable to cope with the large number of different small errors that occur in the calculation of the above parameters in drought-stressed plants. The differences between C_i^* and * for a given treatment are relatively small compared with the large differences in both parameters between treatments (Table 2). Hence, misleading estimations of R_L or g_m cannot explain the observed discrepancies between the in vivo and in vitro methods. Rather, these arise from differences in C_i^*, which is strongly dependent on the correct estimations of A_N and, particularly, C_i. Among the reported limitations that could affect the estimations of A_N and C_i using commercial gas-exchange systems, therefore possibly inducing the observed discrepancies between the in vivo and in vitro methods, are (i) interference of cuticular conductance (Boyer et al., 1997), (ii) leaks through the IRGA chamber gaskets (Long and Bernacchi, 2003), (iii) heterogeneous (‘patchy’) stomatal closure (Terashima, 1992; Buckley et al., 1997; Mott and Buckley, 1998), (iv) edge effects (Pons and Welschen, 2002), and (v) lateral flux of CO₂ through the internal air space (Jahnke and Krewitt, 2002; Pieruschka et al., 2005).

Cuticular conductance may have a great effect on C_i at low g_s, but this was measured and the values corrected accordingly, as was leakage (see Materials and methods). This leaves patchy stomatal closure, edge effects, and lateral diffusion in the mesophyll as the most likely causes for the observed bias.

Patchy stomatal closure is known to impair C_i calculations and to occur under certain water-stress situations (Terashima, 1992; Buckley et al., 1997; Mott and Buckley, 1998). However, it occurs mostly when stress is applied rapidly, and not under progressive water stress (Gunasekera and Berkowitz, 1992). Hence, it is unlikely that the leaves used here, which have been stress-acclimated for their entire life, suffer patchy stomatal closure. Even if some was present, its effects on C_i estimations would be relatively minor, since these are important only when g_s is lower than 0.03 mol H₂O m^–2 s^–1 (Buckley et al., 1997; Flexas et al., 2002), but perhaps important for the determination of *, where the observed range of values was small (about 20 µmol mol^–1 of difference between C and SD plants, compared with a difference in operational C_i of about 200 µmol mol^–1).

Edge effects come from the fact that, due to design of leaf chambers, the gaskets cause the photosynthesizing surface to be surrounded by tissue in darkness that is respiring. This respired CO₂ will decrease the measured net flux, i.e. lead to underestimated A_N and, consequently, erroneous C_i. Pons and Welschen (2002) showed that the effect was important at low light (about 25% underestimation of A_N) but negligible at high light. Therefore, this effect may change the slope of the A_N–C_i curve at low light but not at high light, hence probably modifying the interception point and thus affecting *. However, an estimation using the data from Pons and Welschen (2002) reveals that * is almost unaffected by this effect either in C, MD, or SD plants (data not shown). Therefore, it is unlikely that edge effects account for the observed differences between treatments.

Finally, lateral diffusion through air space in the mesophyll has been shown to occur, particularly in homobaric species such as tobacco (Jahnke and Krewitt, 2002; Pieruschka et al., 2005). This occurs due to the fact that illuminated mesophyll patches inside IRGA's leaf chamber and the surrounding mesophyll patches darkened under the leaf chamber gaskets have different C_i, since the cells in the former patches are photosynthesizing while those in the latter are respiring. This gradient causes a lateral flux of CO₂ from the darkened air spaces to the illuminated air spaces. This leads to underestimations of the true photosynthesis (since a part of the CO₂ used by illuminated cells comes from the surrounding internal spaces of the leaves, not from the IRGA chamber) and the corresponding errors in the estimation of C_i. While it is difficult to estimate the magnitude of such an effect and to use its knowledge to correct the values, it is reasonable to think that this may be most important at high light (i.e. high photosynthesis leading to decreased C_i in the illuminated patches, hence maximizing the lateral CO₂ gradient). This would lead to an almost parallel displacement of the A_N–C_i curve at high light with a small effect on that at low light, hence displacing * to lower values. Using similar reasoning, it is likely that this effect is much larger in water-stressed plants, since they do have a much lower C_i than the controls in the illuminated mesophyll with similar C_i under the gaskets, i.e. a much larger lateral CO₂ gradient. Therefore, lateral diffusion through the air spaces in the mesophyll is likely to cause an important part of the observed differences in * between treatments.

Together, all these possible errors lead to misleading estimations of *, particularly under water stress. In contrast to all the uncertainties and discrepancies regarding technical problems when using the Laisk method, calculating * from the linear relationship at low C_c between gross photosynthesis (i.e. the sum of A_N and R_L) and C_c (Long and Bernacchi, 2003), resulted in a single value for all treatments (39 µmol mol^–1) which, obviously, reflected a value (99) very close to that determined in vitro.

	Concluding remarks

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
In summary, the present results clearly show that the Rubisco specificity factor does not acclimate to water stress in the short term (weeks or months) in tobacco. The observations by Galmés et al. (2005) that the highest values of are observed in Rubisco from plants adapted to the driest environments may reflect an adaptation mechanism operating over a much longer time-scale (i.e. generations).

The comparison of in vitro and in vivo estimated values clearly support the conclusion by Warren et al. (2004) that, in plants with restricted photosynthesis (e.g. water stressed), it would be preferable to determine independently of gas-exchange measurements, using an alternative method. In vitro determinations proved to be a suitable means. An alternative would be to estimate * in control plants using the Laisk method as corrected for R_L and g_s (von Caemmerer, 2000), and assume that it is not affected by the applied treatments, as our data suggest.

	Acknowledgements

 
This work is part of projects BFI2002-00772 and BFU2005-03102/BFI (Plan Nacional, Spain). JG was supported by an FPU grant from the University of the Balearic Islands. We are indebted to Drs O Atkin, F Loreto, AJ Keys, M Ribas-Carbó, and TD Sharkey for stimulating discussion of the data.

	Abbreviations

 
A_N, light-saturated net photosynthesis; A_SAT, light- and CO₂-saturated photosynthesis; C_a, atmospheric CO₂ concentration; C_c, CO₂ concentration at the carboxylation site; C_i, substomatal CO₂ concentration; C_i^*, C_i at the the CO₂ compensation point in the absence of mitochondrial respiration; ETR, electron transport rate; g_m, CO₂ mesophyll conductance; g_s, CO₂ stomatal conductance; LMA, leaf mass area; PGA, phosphoglyceride acid; R_D, leaf respiration in the dark; R_L, leaf respiration in the light; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; RWC, relative water content; , Rubisco specificity factor; *, the apparent Rubisco specificity factor operating in vivo; _Cc^*, the apparent Rubisco specificity factor operating in vivo on a C_c basis; _Ci^*, the apparent Rubisco specificity factor operating in vivo on a C_i basis; *, C_c at the CO₂ compensation point in the absence of mitochondrial respiration; TPU, triosephosphate utilization; V_c,max, maximum rates of carboxylation.

	References

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Andersson I and Taylor TC. (2003) Structural framework for catalysis and regulation in ribulose 1,5-bisphosphate carboxylase/oxygenase. Archives of Biochemistry and Biophysics 414:130–140.[CrossRef][Web of Science][Medline]

Bernacchi CJ, Singsaas EL, Pimentel C, Portis AR, Long SP. (2001) Improved temperature response functions for models of Rubisco-limited photosynthesis. Plant, Cell and Environment 24:253–259.[CrossRef]

Bota J, Flexas J, Keys AJ, Loveland J, Parry MAJ, Medrano H. (2002) CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase in grapevines (Vitis vinifera L.): first in vitro determination and comparison to in vivo estimations. Vitis 41:163–168.[Web of Science]

Bota J, Medrano H, Flexas J. (2004) Is photosynthesis limited by decreased Rubisco activity and RuBP content under progressive water stress? New Phytologist 162:671–681.[CrossRef][Web of Science]

Boyer JS, Wong SC, Farquhar GD. (1997) CO₂ and water vapour exchange across leaf cuticle (epidermis) at various water potentials. Plant Physiology 114:185–191.[Abstract]

Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72:248–254.[CrossRef][Web of Science][Medline]

Brooks A and Farquhar GD. (1985) Effect of temperature on the CO₂/O₂ specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light: estimate from gas-exchange measurements on spinach. Planta 165:397–406.[CrossRef][Web of Science]

Buckley TN, Farquhar GD, Mott KA. (1997) Qualitative effects of patchy stomatal conductance distribution features on gas-exchange calculations. Plant, Cell and Environment 20:867–880.[CrossRef]

Centritto M, Loreto F, Chartzoulakis K. (2003) The use of low [CO₂] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive samplings. Plant, Cell and Environment 26:585–594.[CrossRef]

Cornic G and Fresneau C. (2002) Photosynthetic carbon reduction and carbon oxidation cycles are the main electron sinks for photosystem II activity during a mild drought. Annals of Botany 89:887–894.[Abstract/Free Full Text]

Cornic G and Massacci A. (1996) Leaf photosynthesis under drought stress. In Baker NR (Ed.). Photosynthesis and the environment(Kluwer Academic Publishers, The Netherlands) pp. 347–366.

Dean C, Pichersky E, Dunsmuir P. (1989) Structure, evolution and regulation of rbcS genes in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 40:801–808.

Delgado E, Medrano H, Keys AJ, Parry MAJ. (1995) Species variation in Rubisco specificity factor. Journal of Experimental Botany 46:1775–1777.[Abstract/Free Full Text]

Eilenberg H, Beer S, Gepstein S, Geva N, Zilberstein A. (1990) Red or blue light induced different Rubisco small subunits in fern gametophytes affecting carboxylation rates. In Baltscheffsky M (Ed.). Current research in photosynthesis(Kluwer Academic Publishers, The Netherlands) pp. 411–414.

Eilenberg H, Hanania U, Stein H, Zilberstein A. (1998) Characterization of rbcS genes in the fern Pteris vittata and their photoregulation. Planta 206:204–214.[CrossRef][Web of Science][Medline]

Epron D, Godard G, Cornic G, Genty B. (1995) Limitation of net CO₂ assimilation rate by internal resistances to CO₂ transfer in the leaves of two tree species (Fagus sylvatica and Castanea sativa Mill.). Plant, Cell and Environment 18:43–51.[Medline]

Evans JR and Loreto F. (2000) Acquisition and diffusion of CO₂ in higher plant leaves. In Leegood RC, Sharkey TD, von Caemmerer S (Eds.). Photosynthesis: physiology and metabolism(Kluwer Academic Publishers, The Netherlands) pp. 321–351.

Flexas J, Bota J, Escalona JM, 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 29:461–471.[CrossRef][Web of Science]

Flexas J, Bota J, Loreto F, Cornic G, Sharkey TD. (2004) Diffusive and metabolic limitations to photosynthesis under drought and salinity in C₃ plants. Plant Biology 6:269–279.[CrossRef][Medline]

Flexas J and Medrano H. (2002) Energy dissipation in C₃ plants under drought. Functional Plant Biology 29:1209–1215.[CrossRef][Web of Science]

Galmés J, Flexas J, Keys AJ, Cifre J, Mitchell RAC, Madgwick PJ, Haslam RP, Medrano H, Parry MAJ. (2005) Rubisco specificity factor tends to be larger in plant species from drier habitats and in species with persistent leaves. Plant, Cell and Environment 28:571–579.[CrossRef]

Genty B, Briantais JM, Baker NR. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990:87–92.[Web of Science]

Gunasekera D and Berkowitz GA. (1992) Heterogeneous stomatal closure in response to leaf water deficits is not a universal phenomenon. Plant Physiology 98:660–665.[Abstract/Free Full Text]

Harley PC, Loreto F, Di Marco G, Sharkey TD. (1992) Theoretical considerations when estimating the mesophyll conductance to CO₂ flux by the analysis of the response of photosynthesis to CO₂. Plant Physiology 98:1429–1436.[Abstract/Free Full Text]

Helmerhorst E and Stokes GB. (1980) Microfuge desalting. A rapid quantitative method for desalting small amounts of protein. Annals of Biochemistry 104:130–135.

Jahnke S and Krewitt M. (2002) Atmospheric CO₂ concentration may directly affect leaf respiration measurement in tobacco, but not respiration itself. Plant, Cell and Environment 25:641–651.[CrossRef]

Kent SS and Tomany MJ. (1995) The differential of the ribulose 1,5-bisphosphate carboxylase/oxygenase specificity factor among higher plants and the potential for biomass enhancement. Plant Physiology and Biochemistry 33:71–80.[Web of Science]

Laing WA, Ogren WL, Hageman RH. (1974) Regulation of soybean net photosynthetic CO₂ fixation by the interaction of CO₂, O₂ and ribulose-1,5-bisphosphate carboxylase. Plant Physiology 54:678–685.[Abstract/Free Full Text]

Laisk AK. (1977) Kinetics of photosynthesis and photorespiration in C₃-plants. , Moscow [In Russian] Nauka,.

Lawlor DW. (2002) Limitations to photosynthesis in water-stressed leaves: stomata versus metabolism and the role of ATP. Annals of Botany 89:871–885.[Abstract/Free Full Text]

Long SP and Bernacchi CJ. (2003) Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. Journal of Experimental Botany 54:2393–2401.[Abstract/Free Full Text]

Loreto F, Harley PC, Di Marco G, Sharkey TD. (1992) Estimation of mesophyll conductance to CO₂ flux by three different methods. Plant Physiology 98:1437–1443.[Abstract/Free Full Text]

Manter DK and Kerrigan J. (2004) A/C_i curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance. Journal of Experimental Botany 55:2581–2588.[Abstract/Free Full Text]

Mott KA and Buckley TN. (1998) Stomatal heterogeneity. Journal of Experimental Botany 49:407–417.[Abstract]

Ogren WL and Bowes G. (1971) Ribulose diphosphate carboxylase regulates soybean photorespiration. Nature New Biology 230:159–160.[Web of Science][Medline]

Parry MAJ, Andralojc PJ, Mitchell RAC, Madgwick PJ, Keys AJ. (2003) Manipulation of Rubisco: the amount, the activity, function and regulation. Journal of Experimental Botany 54:1321–1333.[Abstract/Free Full Text]

Parry MAJ, Andralojc PJ, Parmar S, Keys AJ, Habash D, Paul MJ, Alred R, Quick WP, Servaites JC. (1997) Regulation of Rubisco inhibitors in the light. Plant, Cell and Environment 20:528–534.[CrossRef]

Parry MAJ, Keys AJ, Gutteridge S. (1989) Variation in the specificity factor of C₃ higher plants Rubiscos determined by the total consumption of ribulose-P₂. Journal of Experimental Botany 40:317–320.[Abstract/Free Full Text]

Peisker M and Apel H. (2001) Inhibition by light of CO₂ evolution from dark respiration: comparison of two gas exchange methods. Photosynthesis Research 70:291–298.[CrossRef][Web of Science][Medline]

Peña-Rojas K, Aranda X, Fleck I. (2004) Stomatal limitation to CO₂ assimilation and down-regulation of photosynthesis in Quercus ilex resprouts in response to slowly imposed drought. Tree Physiology 24:813–822.[Web of Science][Medline]

Pieruschka R, Schurr U, Jahnke S. (2005) Lateral gas diffusion inside leaves. Journal of Experimental Botany 56:857–864.[Abstract/Free Full Text]

Pons TL and Welschen RAM. (2002) Overestimation of respiration rates in commercially available clamp-on leaf chambers. Complications with measurement of net photosynthesis. Plant, Cell and Environment 25:1367–1372.[CrossRef]

Rogers A, Ellsworth DS, Humphries SW. (2001) Possible explanation of the disparity between the in vitro and in vivo measurements of Rubisco activity: a study in loblolly pine grown in elevated pCO₂. Journal of Experimental Botany 52:1555–1561.[Abstract/Free Full Text]

Roupsard O, Gross P, Dreyer E. (1996) Limitation of photosynthetic activity by CO₂ availability in the chloroplasts of oak leaves from different species and during drought. Annales des Sciences Forestieres 53:243–254.

Roy H and Andrews TJ. (2000) Rubisco: assembly and mechanism. In Leegood RC, Sharkey TD, von Caemmerer S (Eds.). Photosynthesis: physiology and mechanism(Kluwer Academic Publishers, The Netherlands) pp. 53–83.

Sánchez-Rodríguez J, Pérez P, Martínez-Carrasco R. (1999) Photosynthesis, carbohydrate levels and chorophyll fluorescence-estimated intercellular CO₂ in water-stressed Casuarina equisetifolia Forst. and Forst. Plant, Cell and Environment 22:867–873.[CrossRef]

Schreiber U, Bilger W, Hormann H, Neubauer C. (1998) Chlorophyll fluorescence as a diagnostic tool: basics and some aspects of practical rellevance. In Raghavendra AS (Ed.). Photosynthesis. A comprehensive treatise(Cambridge University Press, Cambridge) pp. 320–336.

Spreitzer RJ. (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Archives of Biochemistry and Biophysics 414:141–149.[CrossRef][Web of Science][Medline]

Spreitzer RJ and Salvucci ME. (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annual Review of Plant Physiology and Plant Molecular Biology 53:449–475.[CrossRef][Medline]

Terashima I. (1992) Anatomy of non-uniform leaf photosynthesis. Photosynthesis Research 31:195–212.

Terashima I and Ono K. (2002) Effects of HgCl₂ on CO₂ dependence of leaf photosynthesis: evidence indicating involvement of aquaporins in CO₂ diffusion across the plasma membrane. Plant and Cell Physiology 43:70–78.[Abstract/Free Full Text]

Valentini R, Epron D, De Angelis P, Matteucci G, Dreyer E. (1995) In situ estimation of net CO₂ assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Quercus cerris L.) leaves: diurnal cycles under different levels of water supply. Plant, Cell and Environment 18:631–640.[CrossRef]

Villar R, Held AA, Merino J. (1994) Comparison of methods to estimate dark respiration in the light in leaves of two woody species. Plant Physiology 105:167–172.[Abstract]

Villar R, Held AA, Merino J. (1995) Dark leaf respiration in light and darkness of an evergreen and a deciduous plant species. Plant Physiology 107:421–427.[Abstract]

Von Caemmerer S. (2000) Biochemical model of leaf photosynthesis(CSIRO Publishing, Canberra).

Von Caemmerer S and Farquhar GD. (1984) Effects of partial defoliation, changes of irradiance during growth, short-term water stress and growth at enhanced p(CO₂) on the photosynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160:320–329.[CrossRef][Web of Science]

Warren CR, Livingston NJ, Turpin DH. (2004) Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings. Tree Physiology 24:971–979.

Wilson KB, Baldocchi DD, Hanson PJ. (2000) Quantifying stomatal and non-stomatal limitations to carbon assimilation resulting from leaf aging and drought in mature deciduous tree species. Tree Physiology 20:787–797.

Xu L and Baldocchi DD. (2003) Seasonal trends in photosynthetic parameters and stomatal conductance of blue oak (Quercus douglasii) under prolonged summer drought and high temperature. Tree Physiology 23:865–877.

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