Plant Physiol. (1992) 98, 949-954
Received for publication June 6, 1991
Accepted August 20, 1991
0032-0889/92/98/0949/06/$01 .00/0
Leaf Photosynthesis and Respiration of High CO2-Grown
Tobacco Plants Selected for Survival under CO2
Compensation Point Conditions1
Esteban Delgado, Joaquim Azcon-Bieto*, Xavier Aranda, Javier Palazon, and Hipolito Medrano
Laboratori Fisiologia Vegetal, Departament de Biologia Ambiental, Institut d'Estudis AvanQats-Universitat de les
Illes Balears, 07071 Palma de Mallorca, Spain (E.D., H. M.); Unitat de Fisiologia Vegetal, Departament de Biologia
Vegeta/, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain (J.A.-B., X.A.); and
Unitat de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain (J. P.)
ABSTRACT
Screening procedures based on survival under CO2 compensation point conditions for obtaining lines of C3 plants
with higher photosynthesis and/or lower photorespiration
have not been very successful (10, 21, 24, 26), even though
the simplicity and possibility of large-scale application ofthese
screening methods fueled the hopes of overcoming the main
technical limitations of selection methods based on direct
measurements of photosynthesis rate, as was pointed out by
Nasyrov (22).
Medrano and Primo-Millo (19) developed a screening
chamber in which haploid tobacco (cv Wisconsin-38) plants
were selected by survival after a 45-d period under 60 ppm
CO2 in the circulating air. Haploids were obtained from
ethylmethylsulfonate-treated anthers. These selected haploid
plants showed a higher growth rate, greater leaf area, and
plant production under greenhouse conditions. The floral
buds of surviving haploids were treated with colchicine, and
diploid fertile flowers were developed. After self-pollination,
viable seeds were obtained from five selected lines.
A series of field assays showed consistent advantage of
selected SP lines2 in plant dry matter production in comparison with the unselected source cultivar (20). These field assays
also showed a consistent increase in plant leaf area in those
lines. Nevertheless, CO2 assimilation rate of detached leaves
did not show substantial differences among selected lines and
the control one (12, 13). Recent results suggest that the
specificity factor of Rubisco did not change in these lines in
response to the selection procedure (E. Delgado, M. Parry,
D.W. Lawlor, A.J. Keys, and H. Medrano, unpublished
results).
In the present paper, we report studies of the photosynthetic
and respiratory characteristics of the low CO2 survival-selected
tobacco lines when grown at high CO2 levels. The growth
responses of the selected lines to high CO2 were better than
those of the parental line, and this is remarkable because these
plants were initially selected for survival under low CO2. We
have confirmed the absence of important differences in photosynthesis rates per unit leaf area and CO2 compensation
point, but significant reduction of mature leaf respiration per
unit dry weight was observed in selected lines which could
Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as
haploids by survival in a low CO2 atmosphere, and the parental
cv Wisconsin-38 were grown from seed in a growth room kept at
high CO2 levels (600-700 parts per million). The selected plants
were much larger (especially SP422, SP432, and SP451) than
Wisconsin-38 nine weeks after planting. The specific leaf dry
weight and the carbon (but not nitrogen and sulfur) content per
unit area were also higher in the selected plants. However, the
chlorophyll, carotenoid, and alkaloid contents and the chlorophyll
a/b ratio varied little. The net CO2 assimilation rate per unit area
measured in the growth room at high CO2 was not higher in the
selected plants. The CO2 assimilation rate versus intercellular CO2
curve and the CO2 compensation point showed no substantial
differences among the different lines, even though these plants
were selected for survival under CO2 compensation point conditions. Adult leaf respiration rates were similar when expressed
per unit area but were lower in the selected lines when expressed
per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content
per unit area and were positively correlated with nitrogen and
sulfur content of the dry matter. The altemative pathway was not
involved in respiration in the dark in these leaves. The better
carbon economy of tobacco lines selected for low CO2 survival
was not apparently related to an improvement of photosynthesis
rate but could be related, at least partially, to a significantly
reduced respiration (mainly cytochrome pathway) rate per unit
carbon.
The relationship between leaf photosynthesis rate and plant
productivity is difficult to establish even though dry matter
accumulation obviously reflects the efficiency of the plant
photosynthetic processes (14, 23). It is well accepted that total
canopy photosynthesis during the growth period is closely
related to yield, as has been reported in several species (4, 29).
However, many attempts to improve leaf photosynthesis per
unit area by genetic selection did not result in any substantial
increase of crop productivity (23, 24, 27).
'Supported by the Programa Nacional de Investigaci6n Agricola,
PLANICYT, Spanish Government, grant No. AGR89-580.
2
Abbreviations: SP lines, self-pollinated lines; CCCP, carbonylcyanide m-chlorophenyl hydrazone.
949
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950
DELGADO ET AL.
help to explain, at least in part, the higher growth performance
of these plants.
MATERIALS AND METHODS
Plant Material
Self-pollinated, doubled-haploid tobacco (Nicotiana tabacum) lines (SP422, SP432, SP435, and SP45 1), selected as
haploids by survival in a low CO2 atmosphere (near the CO2
compensation point [19]), and the parental cv Wisconsin-38
were grown from seed in a growth room kept at high CO2
levels (600-700 ppm). The substrate used was vermiculite.
Plants were watered every day, and nutrients were given two
or three times a week with full-strength Hewitt solution (7).
Photosynthetic quantum flux density was about 300 ,umol.
m2s , and the day/night temperature regimen was 24/
2 1C, with a daylength of 13 h. RH during the day was
between 35 and 50%. Plants used in the experiments were 11
to 12 weeks old after planting, still in the vegetative state.
Analysis of Leaf Components
Specific leaf dry weight was determined by drying the leaves
in an oven at 70 to 80C until their weights were constant.
Carbon, nitrogen, and sulfur contents were determined from
dry biomass with a Carlo Erba NA 1500 elemental analyzer
by the staff of the Serveis Cientifico-T&cnics of the University
of Barcelona. Chl and carotenoid contents were determined
spectrophotometrically from leaf discs according to the
method of Lichtenthaler and Wellburn (17). The content of
alkaloids (mainly nicotine) was determined from dried leaves
according to the method described by Saunders and Blume
(25). Leaves were ground and 500 mg of dry powder was
extracted with 10 mL of 25 mm sodium phosphate buffer (pH
7.8) at room temperature for 24 h under constant agitation.
The aqueous extract was ultracentrifuged and filtered through
a 0.45-tim Millipore filter. The samples were stored in sealed
vials at -20°C. The HPLC system used consisted of an LKB
2158 Uvicord SD detector provided with a 254-nm filter,
2150 HPLC pump, and 2210 recorder. Nicotine was quantitatively separated using a Waters u-Bondapak C18 reversephase column (30- x 0.4-cm), eluted with an isocratic mobile
phase of 40% (v/v) methanol containing 2% (v/v) phosphoric
acid buffered to pH 7.25 with triethylamine, at a flow rate of
0.8 mL/min. The peak areas corresponding to nicotine of the
samples (20,uL injection volume), which had the same retention time of authentic nicotine (Merck), were integrated by
comparison with an external standard calibration curve (r =
0.995). Stomatal density and index were counted from several
randomly chosen fields (400-fold magnification) on epidermal
strips obtained from both the adaxial and abaxial tobacco leaf
surfaces.
Gas Exchange Measurements
Net CO2 exchange rates were measured on attached fully
expanded leaves using a portable gas exchange system (Li-Cor
LI-6200 portable photosynthesis system, Lincoln, NE). Net
CO2 assimilation rate was determined in situ in the growth
room at the C02, light, temperature, and humidity growth
Plant Physiol. Vol. 98, 1992
conditions (see "Plant Material" above). Different external
CO2 concentrations around the enclosed leaf were generated
by mixing the air of the growth room with C02-free air. The
gas exchange system included correction for leaks at the lower
CO2 concentrations to avoid underestimation of the assimilation rate and, thus, overestimation ofthe CO2 compensation
point (18). Measurements were made during the first hours
of the day period. The rate of respiratory CO2 release in the
dark of attached adult leaves was measured at 2 1°C at the end
of the night period, using the same Li-Cor gas exchange
system. 02 uptake rates of leaf slices (1-2 mm thick) in the
dark were measured at the end of the night period using an
02 electrode (Rank Brothers, Cambridge, England) as previously described (7, 8). The use of respiratory inhibitors (potassium cyanide and salicylhydroxamic acid) and uncouplers
for estimating the activity of mitochondrial electron transport
pathways in vivo was described earlier (7, 8).
RESULTS AND DISCUSSION
Analysis of Leaf Components
The self-pollinated tobacco lines selected as haploids for
low CO2 survival (19) appeared visually much larger (especially SP422, SP432, and SP45 1) than the control parental
genotype Wisconsin-38 nine weeks after growth at 600 to 700
ppm CO2 (Fig. 1). These selected lines also flowered a few
days earlier. The only exception was the line SP435, which
was more like the control in size and flowering time. The
growth parameters were not quantified, but it was evident
that total leaf area per plant was much larger in the lines
SP432 and SP451 (Fig. 1) and SP422 (not shown). Similarly,
the specific leaf dry weight was also higher in these lines
(Table I), suggesting that SP lines had a higher total dry weight
of the aerial part. Earlier growth assays made under field
conditions also showed higher yields of the aerial part (both
dry biomass and leaf area) in SP lines (20).
The carbon content expressed on a leaf area basis was
higher in the selected lines (except SP435; Table I), and the
variation of this parameter mainly explained the higher specific leaf dry weight (Table I). The nitrogen and sulfur levels
per unit area did not significantly differ among lines (Table
I). When expressed on a dry weight basis, the carbon, nitrogen,
and sulfur contents either did not significantly change or
slightly decreased in some selected lines (Table I).
The Chl a/b ratio and the leaf Chl, carotenoid, and alkaloid
contents expressed on either an area or dry weight basis were
similar in all lines studied, although they were slightly lower
in the SP lines (Table II). In this sense, SP451 had significantly
lower values of Chl and carotenoids (Table II). These results
suggested that large quantitative changes in the amount of
some secondary metabolites (e.g. photosynthetic pigments,
alkaloids) did not occur in the SP lines, but perhaps these
small changes might have contributed to their better growth
performance in the long term by saving some energy invested
in the production and maintenance of these molecules (see
"Leaf Respiration in the Dark" for further discussion).
Leaf Photosynthesis and Stomatal Conductance
The leaf net CO2 assimilation rates per unit area measured
in situ in the growth room at high CO2 concentrations ranged
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PHOTOSYNTHESIS AND RESPIRATION IN SELECTED TOBACCO PLANTS
Figure 1. Comparison between the size of two selected tobacco
lines (top, SP432; bottom, SP451) and the control line (Wisconsin38) after growing for 9 weeks in a cabinet at 600 to 700 ppm CO2.
The pots were 10 cm high. SP422 is not shown but was similar in
size to that of SP432 and SP451. SP435 (not shown) was more
similar in size to the control line.
from 8 to 11 ,umol. m-2 * s-', being slightly lower in the selected
SP lines (Fig. 2). However, the global photosynthesis rate of
the selected plants was presumably much higher than in the
control genotype because those plants had a much larger total
leaf area (Fig. 1). On the other hand, the selected lines had
lower values of leaf net photosynthesis per unit dry weight
(data not shown). Similar or lower leaf photosynthesis rates
were obtained when these selected plants were grown at
ambient CO2 levels either in a cabinet or in the field (12, 13).
The shape of the curve relating net CO2 assimilation per unit
951
leaf area and intercellular CO2 pressure showed no substantial
differences among the different lines (Fig. 2). Accordingly,
the CO2 compensation point was about 50 ppm under the
conditions used in all lines studied. Thus, the evidence suggests that the selection procedure based on survival of haploid
plants at CO2 levels slightly higher than the CO2 compensation
point (19) did not yield diploid plants with higher photosynthesis rates and significantly lower CO2 compensation points
(ref. 12 and E. Delgado, D.W. Lawlor, M. Parry, A.J. Keys,
and H. Medrano, unpublished results). Consistently, the specificity factor of Rubisco was unchanged in selected SP lines
(E. Delgado, D.W. Lawlor, M. Parry, A.J. Keys, and H.
Medrano, unpublished results). Other attempts to select C3
plants with a much lower CO2 compensation point have been
unsuccessful (10, 21, 24, 26). Small differences in the CO2
compensation point associated with the component of respiration in the dark (6) may have occurred (see later) but could
not be detected with our gas exchange system.
The leaf stomatal conductance measured in plants kept in
the growth cabinet was lower in the lines SP422, SP432, and
SP451 compared with both the control Wisconsin-38 and
SP435 (not shown). Changes in conductance were not apparently related to changes in the relative water content and
stomatal number: the relative water content was about 78 to
80% in all lines (not statistically different) and the stomatal
density and index of the adaxial and abaxial leaf surfaces were
very similar or slightly higher in SP45 1 compared with the
parental cv Wisconsin-38 (results not shown). The possibility
of an improved water economy in the selected lines, which
could help the plant to survive under low CO2 conditions
(which promote stomata opening), should be further
investigated.
Leaf Respiration in the Dark
The rates of CO2 release and 02 uptake per unit area of
adult tobacco leaves in the dark were about 0.5 ± 0.05 Atmol.
m-2s and did not significantly differ among the studied
lines (results not shown). However, leaf 02 uptake rates in the
dark were lower in the SP lines (especially in SP432 and
SP45 1) when expressed on a dry weight basis (Fig. 3) and
were negatively correlated with the specific leaf dry weight
(Fig. 3A) and with the carbon content per unit area (Fig. 3B).
A similar negative relationship between respiration (CO2 release) and specific dry weight was found when these same
lines were grown in the field (12). A strong negative relationship between 02 uptake of whole young seedlings (including
Table I. Specific Dry Weight and Carbon, Nitrogen, and Sulfur Contents of Adult Tobacco Leaves
Values are means ± SE of 11 to 22 replicates in the case of specific dry weight and of three to four replicates for the other parameters.
Asterisk, statistical comparison between the selected and the control (Wisconsin-38) lines is significantly different (P < 0.05).
Carbon
Sulfur
Line
Specific Dry
Nitrogen
% of dry wt
% of dry wt
% of dry wt
9.m-2
9 .m2
9 .m2
9 .m-2
0.16 ± 0.008
0.52 ± 0.02
39.50 ± 0.20
0.86 ± 0.10 2.81 ± 0.32
30.9 ± 0.9
12.07 ± 0.15
Wisconsin 38
2.67 ± 0.22
0.16 ± 0.011
0.46 ± 0.02
0.90 ± 0.10
13.11 ± 0.64
39.06 ± 1.03
32.6 ± 1.4
SP435
0.17 ± 0.004
0.95 ± 0.05 2.65 ± 0.15
0.47 ± 0.02
14.70 ± 0.36*
40.91 ± 0.41*
34.6 ± 1.1*
SP422
2.44 ± 0.30
0.16 ± 0.009
0.39 ± 0.01*
40.09 ± 0.46
0.98 ± 0.14
16.11 ± 0.59*
39.7 ± 1.0*
SP432
1.83 ± 0.12*
0.16 ± 0.007
0.37 ± 0.02*
17.41 ± 0.51*
0.78 + 0.05
40.76 ± 0.51
42.9 ± 1.0*
SP451
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Plant Physiol. Vol. 98, 1992
DELGADO ET AL.
952
Table II. Photosynthetic Pigments and Alkaloid Content of Adult Tobacco Leaves
Values are means ± SE of four to seven replicates. See Table I legend for other details.
Chi
Line
Carotenoids
Wisconsin38
SP435
SP422
SP432
SP451
a+b
a+b
mg.m-2
330±10
361 ±13
346±11
340±28
300 ± 7*
mg-g-' drywt
mg.m-2
mg 9-1 dry wt
mg.r-2
mg-g1' drywt
10.0±0.8
11.7±0.4
11.0±0.6
9.0±0.7
7.6 ± 0.6*
3.78±0.11
3.72±0.08
3.57±0.03
3.55±0.04
3.64 ± 0.06
49±2
52±2
47±2
44±2
42 ± 1*
1.48±0.12
1.66±0.07
1.51 ±0.09
1.16±0.05
1.06 ± 0.07*
51.4±4.4
45.6±2.8
51.8±4.0
57.3±6.7
63.2 ± 4.4
1.64±0.12
1.45±0.05
1.42±0.10
1.42±0.14
1.48 ± 0.11
the root) and the total plant dry weight in the dark was also
found (X. Aranda and J. Azc6n-Bieto, unpublished results).
On the other hand, a positive relationship was found between 02 uptake per unit dry weight and leaf nitrogen (Fig.
4A) and sulfur (Fig. 4B) contents (expressed as percentage of
dry weight) in the dark.
These results suggest that selected SP lines were able to
maintain rates of respiration per unit area similar to the
control genotype, despite the fact that these lines accumulated
more carbon (i.e. carbon compounds) per unit leaf area.
Consequently, the rates of respiration expressed on a dry
weight or carbon basis were lower in the SP lines. The fact
that these plants were grown at high CO2 undoubtedly facilitated carbon accumulation and, perhaps, exaggerated possible
genotypic differences in this character. The rate of respiration
of leaves in the dark may depend on the carbohydrate content,
as shown in several species (2, 5, 6, 8, 16), and this dependence
may have varied in the selected tobacco lines. The leaf respiration of selected SP lines was apparently less sensitive to
carbon content, allowing a greater carbon accumulation per
unit area and, perhaps, more efficient carbon storage for
subsequent use in growth. The use of the uncoupler of oxidative phosphorylation CCCP suggested that leaf respiration
of the studied lines was controlled by adenylates in a similar
A
U
0
C
70
60
50
40
* o.
1o V
*A 0~
0
a,n '(A
_)
o3
.
In
0
* t
._
U)
(0
4t
._
)
80
70
60
50
A
5
30
35
45
40
Specific dry weight (g.m
0
0)
co
WISCONSIN 38
SP 422
El SP 432
0 SP 435
A SP 451
U)
-
80
\2~
15
C\)
Alkaloids
a/b
ratio
AA
A
AA
40
A
z4
aV)
0
_
O
1 00
,.-
200
3
300
C. (,ubar CO2
4
400
bar
0
500
6
600
air)
Figure 2. Net CO2 assimilation rates of adult tobacco leaves at
different intercellular C02 levels. Quantum flux density (400-700 nm)
was 300 Mmol. m-2. s- . Leaf temperature was 240C.
12
14
16
18
Carbon content (g.m )
Figure 3. Relationship between 02 uptake per unit dry weight (DW)
and either specific dry weight (A) or carbon content per unit area (B)
in adult tobacco leaves in the dark.
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PHOTOSYNTHESIS AND RESPIRATION IN SELECTED TOBACCO PLANTS
80
70
60
50
40
2.0
2.5
3.0
Nitrogen content (% DW)
0
aD
0
I6
0
cQ
It
80
B
70
60
50
40
0.35
0.45
0.55
Sulphur content (% DW)
Figure 4. Relationship between 02 uptake per unit dry weight (DW)
and either the nitrogen (A) or sulfur (B) content (as percentage of dry
weight) in tobacco leaves in the dark.
because CCCP stimulated the rate of 02 uptake of leaf
slices by about 35 to 45% (results not shown).
It is generally considered that some reduction of total plant
respiration would offer the possibility of a small, but significant, increase in yield (1, 15, 16). In this sense, several authors
(1, 11, 15, 16, 28) suggested that selection of plants for lower
mature leaf respiration per unit dry weight results in significantly higher yields. Bugbee and Salisbury (9) discussed the
limits of crop potential productivity and suggested that carbon
use efficiency, which is inversely related to respiration, can
still be significantly increased in plants. However, it is still a
way,
953
matter of discussion among physiologists and biochemists
whether a true "wasteful" component of respiration really
exists (3, 5), and this question should be clarified.
A possible wasteful component of respiration is the existence of the alternative oxidase, which is the terminal end of
a nonphosphorylating mitochondrial electron transport pathway (16). The capacity of the alternative pathway in tobacco
leaves, estimated by the cyanide-resistant rate (corrected for
any residual respiration), was rather small, being significantly
higher in the lines SP422 and SP45 1 (Table III). The percentage of cyanide resistance of total respiration generally increased in the selected lines (see values in parentheses in Table
III). However, the alternative pathway was not normally
active in these leaves in the absence of cyanide (Table III),
even when respiration was stimulated by the uncoupler CCCP
(not shown), suggesting that the cytochrome pathway was
predominantly used in respiration. Thus, the alternative oxidase of adult leaves seems not to play a significant role in the
determination of the carbon balance of the selected tobacco
lines in the growth conditions used, but this pathway might
have been important for survival during the low CO2 selection
period (19).
Another possible wasteful component of respiration is a
portion associated with "maintenance" processes, not in the
sense that reflects an uncoupled respiration (normally the
cytochrome path is involved in maintenance respiration) but
in the sense that it may support excessively high rates of
protein turnover in relation to the growth rate (1, 15). The
lines of Lolium perenne selected for low respiration of mature
leaves seem to have a reduced maintenance component of
respiration (1 1, 15, 28). The respiration rate of the tobacco
lines selected by survival to low CO2 seem to differ also in the
maintenance component, because the respiration rate was
correlated with the nitrogen and sulfur content (Fig. 4), which
reflects the protein content. The fact that the maximum
differential growth responses in the field between selected and
control tobacco lines mainly occur during later growth stages
(H. Medrano, unpublished data) is consistent with reduced
maintenance respiration, because the biomass that needs to
be maintained is larger in older plants.
Table l1l. Capacity and Degree of Engagement (p) of the Alternative
Pathway in Adult Tobacco Leaves
Values are means ± SE of four to 12 replicates (Vf) and of two to
three replicates (p). The parameter p is equal to Vaft/Va.,, where Vft is
the activity of the alternative pathway. The values in parentheses are
the percentage of cyanide-resistant and salicylhydroxamic acid-sensitive rate of total 02 uptake. 02 uptake was measured in thin slices
(1-2 mm thick) submerged in a solution containing 10 mM Mes buffer
(pH 6.6) and 0.2 mm CaC12. Concentrations of KCN and salicylhydroxamic acid used were 1 and 10 mm, respectively. See "Materials
and Methods" and Table I legend for other details.
Line
pMOl 02 *m21
*
Wisconsin 38 0.06 ± 0.01 (12)
0.06 ± 0.005 (14)
SP435
0.14 ± 0.015' (28)
SP422
SP432
0.08 ± 0.03 (19)
SP451
0.18 ± 0.02* (38)
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Va,t
AMol 02 *g dry wt * h
7.8 ± 1.5
9.4 ± 0.9
15.5 ± 2.4*
9.7 ± 2.8
16.4 ± 1.4*
p
ratio
0
0
0
0
0
954
DELGADO ET AL.
Interestingly, the selected tobacco lines have similar respiratory characteristics to those of the selected Lolium lines of
Wilson (28), e.g. a lower respiration per unit dry weight (but
not per unit area) of mature leaves, predominance of the
cytochrome pathway and no engagement of the alternative
oxidase in leaves (even in the presence of uncouplers [11]),
and similar adenylate control of respiration (1 1).
In conclusion, the apparently improved carbon economy
of the tobacco lines selected by survival under CO2 compensation point conditions could be related, at least partially, to
a reduced leaf respiration rate (mainly cytochrome pathway)
per unit carbon.
The reduced respiration rate per unit carbon (see above)
could have significantly contributed to the saving of carbohydrates and other substrates during the initial selection
period with very low CO2 availability for growth (19), helping
to keep a more positive carbon balance and, hence, increase
the survival chance under such drastic starvation conditions.
The optimization of the carbon balance in the selected
tobacco lines compared with the parental genotype Wisconsin-38 could have presumably accentuated the growth differences when grown in conditions favoring large carbon accumulation in the plant, such as the high atmospheric CO2 levels
used in this study.
ACKNOWLEDGMENTS
We thank the Programa Nacional de Investigaci6n Agricola,
PLANICYT, Spanish Government, for financial support. We also
thank Prof. A. Caballero and Dr. B.G. Drake for critical reading of
the manuscript; Maria Reixach and Isidre Casals of the Serveis
Cientifico-Tecnics of the University of Barcelona for determinations
of carbon, nitrogen, and sulfur; Roser Matamala for stomata number
determinations; Xavier Labrafia and Jordi Bort for pigment analysis;
and Miquel A. Gonzalez-Meler and Miquel Ribas-Carbo for assistance with the figures.
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