Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:

Tree Physiology 17, 291--299
© 1997 Heron Publishing----Victoria, Canada

Diurnal and seasonal changes in the impact of CO2 enrichment on
assimilation, stomatal conductance and growth in a long-term study of
Mangifera indica in the wet--dry tropics of Australia
JOHN GOODFELLOW, D. EAMUS and G. DUFF
School of Biological Sciences, Northern Territory University, Darwin, NT 0909, Australia

Received December 14, 1995

Summary We studied assimilation, stomatal conductance
and growth of Mangifera indica L. saplings during long-term
exposure to a CO2-enriched atmosphere in the seasonally wet-dry tropics of northern Australia. Grafted saplings of M. indica
were planted in the ground in four air-conditioned, sunlit,
plastic-covered chambers and exposed to CO2 at the ambient
or an elevated (700 µmol mol −1) concentration for 28 months.
Light-saturating assimilation (Amax ), stomatal conductance
(gs), apparent quantum yield (φ), biomass and leaf area were
measured periodically. After 28 months, the CO2 treatments
were changed in all four chambers from ambient to the elevated

concentration or vice versa, and Amax and gs were remeasured
during a two-week exposure to the new regime.
Throughout the 28-month period of exposure, Amax and
apparent quantum yield of leaves in the elevated CO2 treatment
were enhanced, whereas stomatal conductance and stomatal
density of leaves were reduced. The relative impacts of atmospheric CO2 enrichment on assimilation and stomatal conductance were significantly larger in the dry season than in the wet
season. Total tree biomass was substantially increased in response to atmospheric CO2 enrichment throughout the experimental period, but total canopy area did not differ between CO2
treatments at either the first or the last harvest.
During the two-week period following the change in CO2
concentration, Amax of plants grown in ambient air but measured in CO2-enriched air was significantly larger than that of
trees grown and measured in CO2-enriched air. There was no
difference in Amax between trees grown and measured in ambient air compared to trees grown in CO2-enriched air but measured in ambient air. No evidence of down-regulation of
assimilation in response to atmospheric CO2 enrichment was
observed when rates of assimilation were compared at a common intercellular CO2 concentration. Reduced stomatal conductance in response to atmospheric CO2 enrichment was
attributed to a decline in both stomatal aperture and stomatal
density.

Keywords: acclimation, apparent quantum yield, down-regulation, elevated CO2, gas exchange.

Introduction

The concentration of atmospheric CO2 has increased since the
industrial revolution and is predicted to increase throughout
the 21st century (Houghton et al. 1992). Because of the central
role of CO2 in the physiology of plants, there is interest in
determining the response of plants to increased atmospheric
CO2 concentrations (Eamus and Jarvis 1989, Ceulemans and
Mousseau 1994, Sage 1994, Eamus 1996a, 1996b).
Trees constitute a major reservoir of carbon (Gifford 1994).
They also have important conservation, aesthetic, economic
and social functions and can have a significant impact on
atmospheric CO2 concentration and local climate (Eamus
1992). Trees within the tropics constitute a major site for the
exchange of carbon between the biosphere and atmosphere.
Furthermore, the tropics are characterized by high temperatures and light flux densities, conditions that may be expected
to enhance the CO2 fertilization effect (Eamus 1996a). However, there are few data on the responses of tropical trees to
CO2 enrichment (Hogan et al. 1991), and in particular, of trees
of the wet--dry tropics where there is distinct seasonality in
atmospheric and soil water contents (Duff et al. 1997). Because of the importance of source--sink relations, which are
themselves highly seasonal, in determining assimilation responses to atmospheric CO2 enrichment (Hilbert et al. 1991,
Eamus 1996b), the relative impact of CO2 enrichment may

vary seasonally. However, the impact of seasonality has rarely
been considered in the context of CO2 enrichment studies for
tropical trees and conflicting results have been obtained for
temperate trees (Eamus 1996b). Gunderson et al. (1993) found
no effect of leaf age or duration of exposure to CO2 enrichment
on assimilation of yellow poplar and white oak. However, in a
comparison of Castanea sativa Mill., a species with a monocyclic growth flush, with Fagus sylvatica L., a species with
indeterminate growth (several flushes per year), Mousseau and
Saugier (1992) showed that, as the growing season progressed,
the impact of CO2 enrichment on assimilation declined with
time for the monocyclic species.
Arp (1991) concluded that, in plants growing in pots, loss of
photosynthetic capacity in response to long-term exposure to

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GOODFELLOW, EAMUS AND DUFF

CO2 enrichment, often described as down-regulation, may
result from restricted root volume, juvenility of tissues and

frequent use of growth conditions far removed from ambient.
Recent experiments with large trees growing in the ground in
open top chambers (Gunderson et al. 1993), branch bag experiments (Teskey 1995), or free air carbon enrichment techniques
(Ellsworth et al. 1995) frequently do not reveal down-regulation of assimilation. However, these findings are not universal
and down-regulation has been observed in experiments that
excluded root restriction (Pettersson and McDonald 1992).
Similarly, the hypothesis that atmospheric CO2 enrichment
results in decreased gs has been supported in some studies
(Hollinger 1987, Fetcher et al. 1988, Eamus et al. 1993) but not
others (Bunce 1992, Gunderson et al. 1993, Ellsworth et al.
1995).
Because previous studies of acclimation of assimilation
have involved reciprocal transfer experiments on trees growing
in pots (Hollinger 1987, Fetcher et al. 1988), we carried out a
reciprocal transfer experiment on trees growing in the ground
by reversing the CO2 treatments at the end of a 28-month
period of exposure. We report the results of a long-term
(29 months) study of the assimilation and stomatal conductance responses of the tropical tree species Mangifera indica L. cv Irwin growing in the ground in the wet--dry tropics
of Australia. This species was chosen because it is economically important in Australia and Asia, and because the cultivar
Irwin, which fruits within one year, is amenable to manipulation of source--sink relationships. The specific hypotheses

tested were that: (1) M. indica saplings can maintain an enhanced Amax in response to atmospheric CO2 enrichment over
three growing seasons; (2) time of day and seasonality influence the relative response of assimilation (Amax and φ) and gs
to atmospheric CO2 enrichment; (3) M. indica saplings exhibit
a growth response to atmospheric CO2 enrichment and
biomass accumulation that is correlated with Amax ; and (4)
reciprocal transfer experiments can be used to assess photosynthetic down-regulation in trees grown in CO2-enriched air.

and the number of trees in each chamber. At all times, irrigation and nutrient additions were maintained to minimize any
limitations on growth because of water and nutrient availability. For a full description of chambers, see Berryman et al.
(1993).
Measurements of assimilation and conductance
Assimilation rate (A) and gs were measured with an LI-6200
portable photosynthesis system equipped with a 1 dm3 cuvette
(Li-Cor, Inc., Lincoln, NE). All measurements were made on
three fully mature, upper-canopy, sunlit leaves per tree from
four trees per chamber. Trees were selected from the center and
both ends of each chamber to account for any possible variations in microclimate within the chamber.
Diurnal measurements of A and gs were made in November
1991 (transition period between dry season and wet season
when vapor pressure deficit (VPD) declines substantially, temperature increases but rains are infrequent and intermittent),

March 1992 (wet season) and July 1992 (dry season); these
periods were 3, 21 and 39 weeks, respectively, from the start
of the experiment. Apparent quantum yield (φ) was determined
in the morning (0630--0830 h) and in the afternoon (1630-1830 h) as the slope of the relationship between A and photosynthetic photon flux density (PPFD).
In addition to the diurnal measurements, gs and assimilation
were also measured under light-saturating conditions (PPFD >
800 µmol m −2 s −1) (Amax ). Measurements were made each
morning (0900--1230 h) and afternoon (1230--1600 h) on nine
days between November 1991 and February 1994, during
which there were three wet seasons and two dry seasons.
Stomatal density

Materials and methods

Stomatal density of the abaxial surface of fully expanded
leaves approximately two months old was measured 4 and 25
months after the beginning of the growth experiment. Three
leaves per tree from four trees per chamber were examined by
making a glue print (approximately 1 cm2) mid-way along
each leaf. Stomatal density was calculated by observation of

three view fields per print with a binocular microscope.

Plant material

Growth analyses

In June 1991, root stocks of Mangifera indica cv Kensington,
(planted December 1990) were grafted with scion material
from mature M. indica cv Irwin. Twenty-four grafts were
planted in the ground in each of four chambers in October
1991. Trenches were dug to a depth of 1 meter before the
chambers were constructed and filled with top soil to ensure
uniformity of soil among chambers. All chambers (base area
10 m2 and height 2.4 m) were air-conditioned and ventilated at
a rate of 50 dm3 s −1. Two chambers received CO2-enriched air
(700 µmol mol −1) and two received ambient air. Chambers
were spray irrigated daily with between 9 (at the start) and
127 dm3 of water (at the end) of the experiment. Chambers
received 100--700 g of Osmocote slow-release fertilizer
(14,6,18 N,P,K) and 4--40 g of a commercial water-soluble

fertilizer (23,4,18 N,P,K) per chamber every 10--14 weeks.
Irrigation and fertilizer regimes varied depending on season

We made three destructive harvests of four, six and eight trees
per chamber at weeks 20, 60 and 80, respectively. Leaves were
stripped from each tree and total leaf area was measured with
a leaf area meter (Delta-T Devices, Cambridge, U.K.). Total
leaf dry weight was then determined after drying to constant
weight at 70 °C. The entire aboveground part of the tree
(excluding leaves) was dried to constant weight at 70 °C. Root
mass was estimated by excavating a 300 dm3 volume centered
around the trunk and collecting all roots found.
Whole chamber reciprocal transfer experiment
Twenty-eight months after the start of the growth experiment
(February 1994), CO2 treatments were changed, with chambers that had received air with CO2 at the ambient concentration receiving CO2-enriched air, and chambers that had
received CO2-enriched air receiving air with CO2 at the ambi-

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ent concentration. Both Amax and gs of four leaves per tree and
three trees per chamber were measured 8, 13 and 15 days after
transfer.
Statistical analyses of results
Statistical analyses were performed with the Systat software
package (version 5.0, Wilkinson 1990). Apparent quantum
yields of leaves were compared by analysis of covariance, after
first testing the relationship between the dependent variable
and the covariate for linearity. Data are presented as slopes ±
standard error, number of data points (n) and squared multiple
regression coefficient (r 2). Analysis of covariance was used to
determine whether a significant change in slope of the A versus
PPFD relationship occurred between treatments (CO2; time of
day; season) by determining the significance of the treatment
× PPFD interaction term.
Comparison of means among and between treatments was
performed by a one- or two-factor nested analysis of variance.
The principal factor of analysis was CO2 treatment. The secondary factor was time, either between morning and afternoon
measurements of φ, Amax or gs, or between successive observations of φ, Amax or gs. Nested terms were either trees within

treatment chambers or replicate chambers within CO2 treatments. The probability for rejection of the null hypothesis was
0.05, unless otherwise stated.

Results
Assimilation rate increased curvilinearly with increasing
PPFD in both treatments (data not shown). On all sampling
dates, the elevated CO2 treatment significantly increased mean
daily apparent quantum yield (φ) (mean of morning and afternoon values (P < 0.001)) (Table 1). The percentage increase in
φ in response to CO2 enrichment was largest in the dry season
(July, 50%), smallest in the wet season (March, 17%) and
intermediate in the transition period (November, 17%). In
addition, φ was significantly smaller (P < 0.001) in the dry
season than in the wet season or the transition period, irrespective of treatment (Table 1).
In both treatments, φ was significantly higher in the afternoon than in the morning in both the wet and dry seasons, but

293

significantly lower in the afternoon compared with the morning in the transition period (Table 1). Within a season, significant differences in φ between CO2 treatments occurred only in
the afternoon during the transition period and the wet season
with increases of 33 and 30%, respectively. In contrast, in the

dry season, there were increases in φ in response to CO2
enrichment in both the morning (67%) and the afternoon
(38%).
On all but one measurement day, Amax was significantly
increased by atmospheric CO2 enrichment (Figure 1a). In both
treatments, Amax was significantly greater in the wet season
than in the dry season; however, the relative increase in Amax in
response to CO2 enrichment was significantly (P < 0.01) larger
in the dry season (25%) than in the wet season (16%).
The elevated CO2 treatment significantly decreased gs for all
but one measurement period (Figure 1b). In both treatments, gs
was larger in the wet season than in the dry season, and the
relative difference in gs between CO2 treatments was also
larger in the wet season than in the dry season. The elevated
CO2 treatment not only reduced gs at all times of the year but
also moderated the change in gs between the wet and dry
seasons (Figure 1b).
In six of seven measurements between November 1991 and
December 1993, Amax was significantly less (typically 20--30%)
in the afternoon than in the morning, irrespective of treatment
(data not shown). For one dry season (August 1993) and one
wet season (December 1993), the decline in Amax between
morning and afternoon was accompanied by an increase in the
the intercellular CO2 concentration/atmospheric CO2 concentration ratio (C i /Ca) in the wet season but a decline in this ratio
in the dry season (Figure 2a). In the dry season, the decline in
gs in the afternoon was associated with a decline in Ci /C a
irrespective of treatment (Figure 2b); however, in the wet
season, both gs and C i /Ca increased in the afternoon in both
treatments (Figure 2b).
In both treatments, Amax declined linearly (r 2 = 0.73) with
increasing leaf to air vapor pressure difference (LAVPD) (Figure 3a), whereas gs declined curvilinearly over the same range
of LAVPD values (Figure 3b). The elevated CO2 treatment
decreased stomatal density by approximately 17%. There was
no difference between stomatal density measured early in the

Table 1. Apparent quantum yield (φ mol mol −1) of Mangifera indica in CO2-enriched or ambient air during three observation periods: November
1991 (transition), March 1992 (wet season) and July 1992 (dry season). For daily mean φ, values followed by a different letter within a row are
significantly different (P < 0.05). For morning and afternoon observations, means followed by a different letter within a measurement period are
significantly different (P < 0.05).
Transition (November 1991)

Wet season (March 1992)

Dry season (July 1992)

Ambient air

CO2-enriched

Ambient air

CO2-enriched

Ambient air

CO2-enriched

Daily mean φ ± SE
n (r 2)

0.019 ± 0.001 a
187 (0.79)

0.023 ± 0.001 b
165 (0.75)

0.018 ± 0.001 a
396 (0.74)

0.021 ± 0.001 b
362 (0.66)

0.006 ± 0.001 c
270 (0.45)

0.009 ± 0.001 d
198 (0.43)

Morning φ ± SE
n (r 2)

0.023 ± 0.002 a
89 (0.64)

0.023 ± 0.001 a
87 (0.77)

0.016 ± 0.001 a
183 (0.68)

0.014 ± 0.001 a
127 (0.63)

0.003 ± 0.001 a
131 (0.47)

0.005 ± 0.001 c
121 (0.27)

Afternoon φ ± SE
n (r 2)

0.015 ± 0.001 b
89 (0.75)

0.020 ± 0.001 c
77 (0.71)

0.020 ± 0.001 b
218 (0.80)

0.026 ± 0.001 c
235 (0.72)

0.008 ± 0.001 b
139 (0.67)

0.011 ± 0.001 d
100 (0.61)

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GOODFELLOW, EAMUS AND DUFF

Figure 1. (a) Light-saturating assimilation (Amax ) and (b) stomatal
conductance (gs) determined for grafted Mangifera indica grown at
ambient (s) or an elevated (d) CO2 concentration. Measurements
were made between 0900 and 1600 h over a 28-month period. Data are
means ± standard error. N/S indicates a nonsignificant difference
between data points where P > 0.05.

growth experiment (January 1992) and late in the growth
experiment (February 1994) (data not shown).
The elevated CO2 treatment increased mean daily Ci by
approximately 78% (Figure 4a), and all but two measurements
of Ci /C a were significantly smaller for leaves grown at the
elevated CO2 concentration than for leaves grown in ambient
air (Figure 4b). In both treatments, Ci and C i /Ca were significantly larger in the wet season than in the dry season. Minimum Ci /C a ratios were observed at the time that LAVPD was
maximal (Figure 4c); however, the difference in Ci /C a between
treatments was significantly larger in the wet season than in the
dry season (Figure 4b).

Reciprocal transfer experiment
By Day 13 after transfer, assimilation had equilibrated to the
new CO2 regime (data not shown). Figure 5a shows that Amax
of leaves grown and measured in elevated CO2 was significantly larger than Amax of trees grown and measured in ambient
air. On transfer of trees from ambient air to CO2-enriched air,
Amax increased by 76%, which was 46% larger than the Amax of
leaves grown and measured in CO2-enriched air. On transfer
from CO2-enriched air to ambient air, Amax decreased by 16.5%
compared to leaves grown and measured in CO2-enriched air.
The Amax of leaves grown in CO2-enriched air and measured in

Figure 2. (a) Light-saturating assimilation (Amax ) and (b) stomatal
conductance (gs) as a function of C i /C a for grafted Mangifera indica
grown at ambient (s) or an elevated (d) CO2 concentration. Measurements were made in the morning (0900--1230 h) and afternoon (1230-1600 h). Means and the greatest two-way standard error bars are
shown.

ambient air was similar to that of leaves grown and measured
in ambient air (Figure 5a).
Stomatal conductance of leaves grown and measured in
CO2-enriched air was significantly less than that of leaves
grown and measured in ambient air. There was a slight but
insignificant decrease in gs of leaves grown in ambient air and
measured in CO2-enriched air compared to leaves grown and
measured in ambient air (Figure 5b). However, gs of leaves
grown in ambient air and measured in CO2-enriched air increased by 134% compared to leaves grown and measured in
CO2-enriched air. The gs of leaves grown in CO2-enriched air
and measured in ambient air increased by 32% compared to
leaves grown and measured in ambient air, and by 245%
compared to leaves grown and measured in CO2-enriched air
(Figure 5b).
Leaves grown and measured in CO2-enriched air had a
significantly higher C i compared to leaves grown and measured in ambient air (Figure 6a), resulting in a decreased Ci /C a
ratio (Figure 6b). Compared with all other measurement conditions, C i was significantly larger in leaves grown in ambient

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Figure 3. (a) Light-saturating assimilation (Amax ) as a function of
LAVPD, and (b) stomatal conductance (gs) as a function of LAVPD
for grafted Mangifera indica grown at an elevated (d) or ambient (s)
CO2 concentration. Data are means of Amax or gs measured in the
morning (0900--1230 h) and afternoon (1230--1600 h).

air and measured in CO2-enriched air; however, the C i /Ca ratio
was not significantly different from that of leaves grown and
measured in ambient air. Although C i in leaves grown in
CO2-enriched air and measured in ambient air was not significantly different from that of leaves grown and measured in
ambient air, Ci /C a ratio was significantly larger (Figures 6a and
6b).
Total tree dry weight, shoot dry weight and estimated root
dry weight were all significantly increased by CO2 enrichment
(Figure 7) and the treatment differences appeared to increase
with time. In contrast, there were no significant treatment
effects on total canopy area and total canopy weight by the end
of the experiment. However, throughout the growth experiment specific leaf area was significantly less by 14% with CO2
enrichment (data not shown).

Discussion
Apparent quantum yield----daily and seasonal variations
Apparent quantum yield (φ) increased in response to CO2
enrichment (cf. Ziska et al. 1990, Long and Drake 1991, Ziska

295

Figure 4. Values of (a) C i, (b) C i /C a and (c) LAVPD for grafted
Mangifera indica grown at an elevated (d) or ambient (s) CO2
concentration. Measurements were made between 0900 and 1600 h on
nine days over a 28-month period. N/S indicates a nonsignificant
difference between data points where P > 0.05.

et al. 1991). Because assimilation is limited under low PPFD
by the rate of regeneration of ribulose bisphosphate (RuBP),
and decreased photorespiration results in increased availability
of RuBP for carbon fixation (Stitt 1991), we postulate that the
increase in φ in response to CO2 enrichment is associated with
a decrease in photorespiration. Such an increase in φ may have
significant implications for total daily carbon gain in tree
species such as M. indica that have low light or shade habits
(Eamus et al. 1993).
The response of φ to CO2 enrichment was larger in the dry
season than in the transition period and wet season. The proportional impact of CO2 enrichment on assimilation may be
enhanced when water stress is imposed (Chaves and Pereira
1992), because as gs declines during water stress, the benefit of
CO2 enrichment in maintaining a high C i becomes proportionally larger. Furthermore, the decline in sucrose phosphate
synthase associated with drought stress can be reversed by CO2
enrichment (Vassey et al. 1990), which may also contribute to
the enhancement of assimilation that occurs in response to CO2
enrichment during drought.
Both φ and gs (for all values of PPFD < 300 µmol m −2 s −1)
were lower in the dry season than in the wet season, irrespec-

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GOODFELLOW, EAMUS AND DUFF

Figure 5. (a) Light-saturating assimilation (Amax ) and (b) stomatal
conductance (gs) in grafted Mangifera indica grown for 28 months at
an elevated (Enr.) or ambient (Amb.) CO2 concentration and then
transferred from one CO2 concentration to the other. Measurements
were made before (left-hand columns) and after 8 to 15 days at the new
CO2 concentration. Data are means ± standard error. Bars with different letters are significantly different (P < 0.05).

Figure 6. Values of (a) C i and (b) C i /C a for grafted Mangifera indica
grown for 28 months at an elevated (Enr.) or ambient (Amb.) CO 2
concentration and then transferred from one CO 2 concentration to the
other. Measurements were made before (columns at left) and after 8 to
15 days at the new CO2 concentration. Data are means ± standard
error. Bars with different letters are significantly different (P < 0.05).

tive of treatment. Based on the observation that Ci decreased
between wet and dry seasons for all measurements of assimilation when PPFD was less than 300 µmol m −2 s −1, we postulate that the decline in gs contributed to the decline in φ.
In the transition and the wet season, differences in φ between
treatments only occurred in the afternoon, whereas in the dry
season there were differences between treatments for both
morning and afternoon measurements. This pattern may reflect
differences between seasons in the magnitude of the effect of
LAVPD on gs, and hence φ. In the transition period and wet
season, gs and C i /Ca increased in the afternoon because atmospheric and soil water content were high and LAVPD was low
because of cloud cover and reduced solar radiation load (Duff
et al. 1997, Eamus and Cole 1997). However, gs was considerably smaller throughout the day in the dry season than in the
wet season or the transition period, and was always smaller in
the afternoon than in the morning. Because the proportional
impact of CO2 enrichment increased as gs declined, the differences in gs between seasons and between morning and afternoon may explain why the elevated CO2 treatment

significantly increased φ only in the afternoon during the wet
season, but significantly increased φ in both the morning and
the afternoon during the dry season.
Amax ----daily and seasonal trends, and growth
On the first measurement date (November 1991), Amax increased in the afternoon compared to the morning, in both
treatments, perhaps indicating that the trees were acclimating
to the transfer from pots to the ground.
For eight of the nine measurements, Amax increased in response to CO2 enrichment, a result ascribed to decreased
photorespiration and increased CO2 concentration for Rubisco
(Eamus and Jarvis 1989). In both treatments, Amax was larger
in the morning than in the afternoon for the majority of observations (Figure 2a). In the dry season, this decline was likely
the result of the large increase in LAVPD, which caused a large
decline in g s and hence in the supply of CO2 to the chloroplast
(Figures 4a and 4c). This hypothesis is supported by the observation that Ci /C a declined in the afternoon compared to the
morning (Figures 2a and 2b) in the dry season, as would be

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297

Figure 7. (a) Tree dry weight, (b)
root dry weight, (c) leaf dry weight
and (d) canopy area for grafted
Mangifera indica grown in CO2-enriched (d) or ambient (s) air. Measurements were made at intervals up
to 80 or 113 weeks. Data are means
± standard error. N/S indicates a nonsignificant difference between data
points where P > 0.05.

expected if g s limited assimilation. A similar decline in g s and
C i /Ca in response to a large increase in LAVPD has been shown
in a range of tropical tree species growing in the wet--dry
tropics of Australia (Berryman et al. 1993, Eamus and Cole
1997, Prior et al. 1997).
There was no treatment difference in the decline in Amax
between the morning and the afternoon in the wet season. In
contrast, the decline in Amax in the afternoon in the dry season
was significantly larger with CO2 enrichment compared with
controls. This is because trees were sink limited in the dry
season with a seasonal decline in growth, yet Ci remained
enhanced despite a reduction in g s, with the result that Amax
remained enhanced in the dry season. An increase in the
source--sink strength ratio results in end point inhibition of
photosynthesis (Sage 1994). In the wet season, the decline in
Amax in the afternoon was associated with increased g s and
C i /Ca (Figure 3), indicating that stomatal closure in response
to increased LAVPD cannot explain the decline. A decrease in
Amax despite increased gs was also reported by Eamus et al.
(1993) who suggested a depletion of phosphate supply to the
chloroplast could result in a lower Amax. Phosphate requirement
is increased by CO2 enrichment as a result of an increase in
Amax (Conroy 1992).
The significant decline in Amax in the dry season compared
to the wet season in both treatments was the result of a decreased gs caused by increased LAVPD (Figure 4). Leaf water
potential did not differ between seasons indicating that soil
water availability was not a limiting factor (data not shown)
and so was unlikely to account for the decline in g s in the dry
season. Therefore, we attribute the greater enhancement of
Amax in response to CO2 enrichment in the dry season than the

wet season to the proportional increase in benefit of CO2
enrichment as stomatal limitation increased, i.e., increased C i
was maintained at a reduced gs in the dry season.
By the end of the experiment, trees grown in the CO2
enriched atmosphere had approximately 60% more biomass
than trees grown in ambient air. The increase in biomass was
was more than the 15% estimated increase in carbon fixed over
the 28-month study (estimated from the integrated sum of
Amax from Figure 1); however, this was based on rates of
light-saturated assimilation. In M. indica, which has a dense
closed canopy, a large and significant fraction of daily carbon
gain occurs in self-shaded leaves.
Stomatal conductance was significantly decreased in leaves
grown in CO2-enriched air on most measurement dates. This
has been observed previously in many studies (for review see
Eamus and Jarvis 1989, Sage 1994) and is the result of decreases in both stomatal density and stomatal aperture. The
percentage decline in gs between wet and dry seasons was
significantly smaller for leaves grown at an elevated CO2
concentration than for leaves grown at the ambient CO2 concentration. Carbon dioxide enrichment in the wet season (December 1993) also resulted in a larger difference between
morning and afternoon values of gs. A partial explanation for
this may be that, in the morning, stomatal opening is at a
maximum for trees grown in ambient CO2 if VPD is low. In
contrast, the stomata of trees grown in elevated CO2 are not
fully open (g s increases when trees grown in CO2-enriched air
are transferred to ambient air), therefore, increased stomatal
opening can occur in the afternoon and does so under favorable
conditions.

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GOODFELLOW, EAMUS AND DUFF

Acclimation of assimilation
The enhancement of assimilation in response to the elevation
of CO2 concentration did not diminish over the 28-month study
(cf. Gunderson et al. 1993, Idso and Kimball 1993). In contrast, the enhancement of assimilation in response to the elevation of CO2 concentration declined within 30 days in Castanea
sativa (Mousseau and Saugier 1992), suggesting that determinate species with monocyclic growth flushes show a gradual
decline in the response of assimilation to CO2 enrichment as
the growing season progresses. Kohen et al. (1993) compared
a monocyclic determinate species (C. sativa) with Fagus sylvatica, an indeterminate species with several growth flushes,
and found that F. sylvatica maintained an enhanced assimilation rate in response to CO2 enrichment throughout the growing season. Mangifera indica is relatively indeterminate in its
growth, and during the dry season, large amounts of carbon are
temporarily stored in the bole of the tree (E. Chacko, CSIRO,
Darwin, Australia, personal communication) to be utilized
later during flowering and fruit growth. Consequently, major
sinks for carbon are maintained throughout the dry season. The
larger percentage increase in assimilation rate in response to
CO2 enrichment in the dry season than in the wet season may
be associated with sink strength.
Acclimation or down-regulation of assimilation in response
to long-term exposure to an elevated atmospheric CO2 concentration is often inferred from a comparison of assimilation rates
at a common Ca (Hollinger 1987, Ceulemans et al. 1995);
however, as indicated by Sage (1994), a comparison of assimilation rates at a common Ci is needed to compare photosynthetic efficiencies. We found that comparisons at a common Ca
need not result in the same C i for trees exposed to both the
ambient and an elevated CO2 concentration and therefore comparisons of their respective assimilation rates may be misleading. It was found that the assimilation rate of trees grown and
measured in an elevated CO2 concentration was significantly
lower than that of trees grown in ambient air and measured at
an elevated CO2 concentration (Figure 5a). Such a result is
frequently thought to be indicative of a loss of photosynthetic
potential at an elevated CO2 concentration. However, because
g s of leaves grown in ambient air but measured in CO2-enriched air was more than double that of leaves grown and
measured in CO2-enriched air (Figure 5b), C i will have been
larger. Thus, it was to be expected that assimilation would be
larger if it was CO2 limited. Thus, there may have been no loss
of photosynthetic potential in the trees grown in CO2-enriched
air. This view is supported by the observation that the assimilation rate of leaves grown in CO2-enriched air and measured
in ambient air was not significantly different from that of trees
grown and measured in ambient air. Furthermore, because Ci
did not differ, we conclude that no loss of assimilation potential occurred. A similar lack of down-regulation has been
observed in mature trees (Gunderson et al. 1993, Ellsworth
et al. 1995, Liu and Teskey 1995, Teskey 1995).
Stomatal conductance of leaves grown in CO2-enriched air
and measured in ambient air was larger than gs of leaves grown
and measured in ambient air (Figure 5b). However, Ci and
Amax were similar for both sets of plants (Figures 5a and 6a).

Based on our findings and the observation that as mesophyll
conductance (gm) of evergreen perennials (such as M. indica)
declines, stomatal cavity CO2 concentration declines (Lloyd
et al. 1992), we propose that, at an elevated CO2 concentration,
stomatal density and specific leaf area decline, and that the
average path length for diffusion of CO2 from the bulk air to
the furthest chloroplast increases. Consequently, gm may decline, and hence a larger gs be required to maintain the same
Ci and assimilation rate.

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