Journal of Experimental Botany, Vol. 57, No. 10, pp. 2267–2275, 2006
doi:10.1093/jxb/erj199 Advance Access publication 23 June, 2006
RESEARCH PAPER
Anticipated yield loss in field-grown soybean under
elevated ozone can be avoided at the expense of
leaf growth during early reproductive growth stages
in favourable environmental conditions
Maja M. Christ1, Elizabeth A. Ainsworth1,2,3, Randall Nelson2,4, Ulrich Schurr1 and Achim Walter1,*
1
ICG-III (Phytosphere), Research Center Juelich, D-52425 Juelich, Germany
2
USDA-Agricultural Research Service, Photosynthesis Research, and Soybean/Maize Germplasm, Pathology,
and Genetics Research Units, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA
3
Department of Plant Biology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA
4
Department of Crop Sciences, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA
Received 27 January 2006; Accepted 13 March 2006
Abstract
Introduction
Ozone is a powerful oxidizing agent which is responsible for more damage to vegetation than any other air
pollutant. In this study, leaf growth, photosynthesis,
and carbohydrate content were analysed during the
seed-filling growth stage of field-grown soybeans
exposed to ambient air and 1.2 times ambient ozone
concentration using a Free Air Concentration Enrichment (FACE) facility. By contrast to predictions based
on controlled-environment and open-top chamber
studies, final yield did not differ between treatments,
although the cultivar used here was sensitive to ozone
damage: growth and carbohydrate content of upper
canopy leaves was reduced during the seed-filling
stage in which an ozone-induced decrease of photosynthesis was present. However, 2004 was an ideal
growing season in central Illinois and the cumulative
ozone indices were lower than in previous years. Still,
the results indicate that the anticipated yield loss
under ozone concentrations was avoided at the expense of leaf growth, as reserves were diverted from
vegetative to reproductive organs.
Ozone (O3) is a prevalent chemical pollutant in the atmosphere, costing an estimated annual $2–3 billion dollars in
yield losses to major crops in the US (Murphy et al., 1999;
Lorenzini and Saitanis, 2003). Surface O3 concentration
([O3]) has risen from an estimated pre-industrial concentration of 10 parts per billion (ppb) to average summer
concentrations between 30 and 50 ppb in the middle
latitudes of the northern hemisphere, with episodic levels
as high as 50–100 ppb (Pritchard and Amthor, 2005;
Morgan et al., 2006). Surface [O3] is likely to rise by
a further 20% over the next 50 years (Prather et al., 2001).
Sensitive plant species show a reduction in yield when
exposed to O3 concentrations above the threshold of 40 ppb
for extended periods (Fuhrer et al., 1997). During periods
of high radiation, conditions are favourable for photochemical O3 formation (Lorenzini and Saitanis, 2003).
Therefore, temperate crops are especially impacted by O3
because long days and high radiation during the summer
growing season promote the formation of high regional
tropospheric ozone concentrations (Lorenzini and Saitanis,
2003). Hence, there is a clear need for field studies
simulating future O3 scenarios.
Soybean (Glycine max [L.] Merr.) is among the crop
species most sensitive to O3 (Lesser et al., 1990). Current
understanding of its vulnerability to ozone damage is based
on extensive studies conducted in controlled environments
and open-top chambers (Ashmore, 2002). Seed yield is
Key words: Carbohydrates, global atmospheric change,
Glycine max, leaf growth, photosynthesis, stomatal density.
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
2268 Christ et al.
reduced most severely if O3 exposure and reduction
of canopy assimilatory surface occur during early reproductive periods (R3–R5) (McAlister and Krober, 1958;
Begum and Eden, 1965; Shibles and Weber, 1966;
Heagle et al., 1991). Decreases in seed yield are
usually accompanied by reductions in biomass and photosynthesis (Pausch et al., 1996; Fiscus et al., 2005). A
recent meta-analysis of elevated [O3] effects on soybean,
based on enclosure studies from open-top chambers
or growth cabinets, revealed that an average elevated
[O3] of 70 ppb caused a 17% decrease in stomatal
conductance, a 23% decrease in net carbon assimilation
with parallel decreases in leaf total non-structural carbohydrates, and a 24% decrease in seed yield (Morgan
et al., 2003).
Free-Air gas Concentration Enrichment (FACE) facilities allow the study of the effects of gases in otherwise
undisturbed conditions in the field, using regional standard
agronomic practices, and hence overcoming several pitfalls
that reduce the significance of experiments from laboratory
or enclosure studies (Rogers et al., 2004). Using a FACE
facility to investigate the response of soybean to elevated
[O3], Morgan et al. (2004) reported that photosynthesis
was not inhibited by O3 (seasonal mean of the maximum
daily 8 h average of 75 ppb) in recently fully expanded
leaves that formed during vegetative development, but O3
exposure resulted in significantly reduced photosynthesis
in the late developmental stages of leaves from the upper
canopy that developed during seed filling. Since this
plant growth stage is of crucial importance for seed
yield and since a high fraction of the carbon that is
translocated to the seeds is supplied by upper canopy
leaves that are about to reach the full-grown state (Thrower,
1962; Gifford and Evans, 1981), it is important to investigate quantitatively the development of those leaves
under chronic O3 exposure.
It was hypothesized that 1.2 times elevated [O3] during
seed filling would lead to reduced growth and photosynthesis in upper canopy leaves and thereby to reduced
yield. In order to test this hypothesis, leaf photosynthesis, stomatal conductance and density, and carbohydrate
content were investigated in upper canopy leaves in
the middle of the reproductive growth stage when the
canopy was reaching maximum leaf area index (Dermody
et al., 2006). For these measurements, the indeterminate
cultivar Spencer, was chosen, based on the sensitivity of
the cultivar to elevated [O3] in 2002 and 2003. Yield was
decreased by 15% and 30%, respectively in 2002 and 2003,
corresponding to years with 90% and 130% increases in
AOT40 levels (accumulated over a threshold of 40 ppb;
Morgan et al., 2006; R Nelson, unpublished data). In
2004, individual leaf growth and leaf area were followed
during the reproductive period, and the effect of elevated
[O3] evaluated on seed yield of this sensitive cultivar of
soybean.
Materials and methods
Site description
This study was conducted at the soybean FACE facility (SoyFACE),
situated on a 32 ha field at the University of Illinois at UrbanaChampaign, USA (408029 N, 888149 W, 228 m a.s.l.) in 2004. The
FACE system and facility is based on the method of Miglietta et al.
(2001) and has been described in detail previously (Ainsworth
et al., 2004; Leakey et al., 2004; Morgan et al., 2004, 2006; Rogers
et al., 2004). Soybean and maize (Zea mays) crops each occupied half
of the field. Since 2001, the crops have been rotated on a yearly basis
according to typical agricultural practices. Four experimental blocks,
each containing one control and one elevated [O3] treatment plot,
were situated in the 16 ha of soybean. Ambient [O3] values were
measured on site and on-line. In the elevated [O3] treatment plots, O3
was added during daytime to a target of 20% above ambient. This
fumigation approach increased both mean and peak [O3] (Morgan
et al., 2006). O3 was not added when leaves were wet, resulting in
times (hours and days) when O3 was not elevated. Therefore, it was
necessary to fumigate to a higher level on dry days in order to reach
a season-long increase of 20%. The maximum increase on any one
day was limited to 50% above the concentration in the control plots
(Morgan et al., 2006).
Fumigation began on 17 June 2004 and continued throughout the
growing season. Arrangement of the plots was randomized, and they
were separated by at least 100 m to avoid cross-contamination of O3
gas (Nagy et al., 1992). Each plot was designed as an octagon
(diameter 21 m) of horizontal pipes that released O3 during daylight
hours from the upwind side of the plot at ;10 cm above the soybean
canopy to ensure a constant increase above the current O3 level.
Measurements for this study were taken during the reproductive
growth phase in August 2004. Total rainfall in June, July, and August
2004 was 347 mm, 11% above the 50 year mean (Leakey et al.,
2006). The daytime temperature during August 2004 averaged
22.8 8C. This was the coolest mean August temperature reported
in Illinois for the past 30 years (NOAA Climatic Data Center,
www.ncdc.noaa.gov/oa/climate/research/cag3/il.html).
Seasonal
[O3], SUM06, and AOT40 for the growing season are shown
in Fig. 1.
Plant material
The soybean crop was planted on 28 May 2004 [day of the year
(DOY) 148] in four rows 2.7 m long with row spacing of 0.38 m. At
maturity, 2.1 m of the centre two rows were harvested to measure
seed yield. Three out of four blocks were chosen for this study (n=3)
and, within each plot, four plants were selected as sub-replicates
for each leaf developmental stage. In soybean, growth stage R3 is
‘beginning pod’ when pods on the upper four nodes initiate
(McWilliams et al., 2004). Rapid pod growth occurs during stage
R4 and seed filling begins at R5 (Fehr et al., 1971). At the beginning
of August when plants were just entering the R5 growth phase (DOY
217), a cohort of leaves was marked. These leaves were situated
three leaves below the latest unfolded leaf and were already fully
expanded. Throughout this paper, this cohort of leaves is referred
to as ‘leaf 1’. Thus, the latest unfolded leaf at the beginning of
the measurements was ‘leaf 4’. Leaves 3 and 4 were still growing at
the beginning of the measurements. This method of marking and
measuring leaves was chosen because leaves of the same size
and developmental stage were not always the same trifoliate number
on different plants; leaf 1 ranged between the 7th and the 9th
trifoliate.
Photosynthesis
Midday net CO2 exchange rate in saturating light conditions (Asat)
and stomatal conductance (gs) were measured at the beginning and
Ozone effects on leaf growth, photosynthesis and yield
2269
middle leaflets can be used as a proxy for leaf area measurements
(Ainsworth et al., 2005). Leaflet area (LA) was estimated from the
relationship: LA=L3W30.74 (species-specific factor determined
from the measured area of ;50 leaves of different ages and
developmental stages; data not shown). As there was no effect of
ozone on leaflet shape (data not shown) the same length/width
relationship was used to determine leaflet area for leaves from
ambient and O3 treatments, respectively. The relative growth rate
(RGR) was calculated by:
RGRð% d1 Þ = lnðLAt2 =LAt1 Þ 3 Dt 3 100
where LAt1 and LAt2 are the leaflet areas at the points in time t1 and
t2, respectively (Walter and Schurr, 1999). Four sub-replicates were
used to determine a mean value for each plot.
Stomata density and elemental analysis
Discs (10 mm diameter) from mature (leaf 1) to rapidly growing (leaf
5) leaflets were sampled at the onset of R5 for stomatal density
measurements and elemental analysis. For stomatal density measurements, two sub-replicates from each plot were bleached as described
by Walter et al. (2003). Stomata from five randomly selected areas of
each leaflet were counted under a microscope (Olympus BX 40,
Olympus America, Inc., Melville, NY, USA; field of view 0.12 mm2),
and average stomatal density per leaflet was determined. Elemental
analysis of carbon and nitrogen content of dried leaflet discs was
determined on a mass basis with an elemental combustion system
(Costech Analytical Technologies, Inc., Model 4010, Valencia, CA,
USA), standardized with acetanilide (Costech Analytical Technologies). Values were divided by the specific leaf area (dry mass basis)
in order to express the C and N content on a relative area basis.
Fig. 1. Ozone treatment for the 2004 growing season at the SoyFACE
facility. (A) Daily 12 h mean ozone concentration; the solid line shows
the ambient treatment and the dotted line the elevated treatment. (B)
AOT40 ozone index for 2004 and (C) SUM06 ozone index, calculated
according to Mauzerall and Wang (2001). Open circles show ambient
treatment and filled circles show elevated treatment.
end of R5 (DOY 227 and 243, respectively) with an open gasexchange system (LI-6400; Li-Cor, Lincoln, NE, USA) incorporating
an integrated fluorescence chamber head (LI-6400-40 leaf chamber
fluorometer; Li-Cor). Light was set to the maximum light intensity
of the day (1500 and 1800 lmol m2 s1, respectively). Chamber
CO2 concentration was set to 370 ppm and leaf temperature was
held constant at 26 8C and 28 8C, respectively. At least two leaves
from each developmental stage in each ring were measured
on DOY 227 and DOY 243, and sub-replicates were averaged to
give one mean value per ring.
Growth
Leaflet growth was analysed continuously by measuring leaflet length
(L) and width (W) of the middle leaflet approximately every second
day on four plants per plot with a ruler. Leaflet growth rate does not
differ between middle and side leaflets, hence area measurements of
Leaf biochemical analysis
Samples for chlorophyll and carbohydrate analysis were taken on
growing (leaf 4) and mature leaflets (leaf 1) every 3 h throughout
24 h on DOY 217 to test for O3 effects on diel cycles of carbohydrate content. Leaflets 4 (mature) to 7 (growing) were sampled in
a second harvest on DOY 237 at 16.00 h. Two 10 mm discs per leaflet were frozen in liquid nitrogen and stored at –80 8C for further
extraction. Soluble sugars and chlorophyll were extracted from
frozen leaflet material as described in Walter et al. (2005).
For chlorophyll analysis 200 ll of the supernatant were diluted in
600 ll ethanol, centrifuged at 16045 g for 4 min, and the content
was measured with a spectrophotometer (Ultrospec II, Pharmacia,
LBK, Freiburg, Germany) at 652 nm (Arnon, 1949). Glucose,
fructose, and sucrose were analysed with a coupled enzyme assay
(Jones et al., 1977) using a multiplate spectrophotometer (ht II; Anthos
Mikrosysteme GmbH, Krefeld, Germany).
The remaining leaflet material was prepared for starch analysis as
described by Walter et al. (2005) and the starch content was
determined enzymatically as glucose concentration using the same
procedure described above for soluble sugars.
Statistical analysis
Growth, gas exchange, and leaf biochemical variables were statistically analysed with a mixed model analysis of variance (PROC
MIXED version 8.02; SAS Institute, Cary, NC, USA). Each day was
analysed independently due to differences in leaf age and developmental state. For biochemical data, time of day, O3 treatment, and the
interactions of those variables were fixed effects of the model. A
repeated-measures analysis was performed for growth data (PROC
MIXED) with Julian day of year as the repeated measure. For all
variables of interest, a priori pairwise linear comparisons were made
between ambient and elevated [O3] (a=0.05).
2270 Christ et al.
Results
The final leaf area of soybean was reduced by elevated
ozone in leaves formed after the onset of pod filling (Fig. 2).
At the beginning of stage R5, there was no significant
difference in total area of upper canopy leaves, but later in
the pod-filling stage, the area of upper canopy leaves in
elevated [O3] was significantly reduced (Fig. 2). The
reduction of total leaf area in elevated [O3] was mainly
due to a reduction in growth and final size of leaves which
emerged during the R5 growth phase. Leaf area of leaves 3
and 4 was reduced by 3% and 7%, respectively, at DOY
237, but the reduction was not statistically significant.
Reduction in leaf area of leaves 5 and 6 was significant at
DOY 237 with 18% and 30% less area, respectively. In
total, the area of upper canopy leaves investigated here was
decreased by 14% due to the ozone treatment.
Light-saturated photosynthesis (Asat) was significantly
reduced in elevated [O3], averaged over all leaves, by 28%
Fig. 2. Effect of elevated [O3] on the development of leaf area of
Glycine max cv. Spencer. Leaves 3–6 (A–D) and sum of upper canopy
leaves (E) in August 2004 at SoyFACE, IL, USA in control (open
symbols) and elevated (1.23 control, filled symbols) [O3] (n=3
6standard error). The results from the analysis of variance (F test
and statistical significance, P) for O3 treatment, leaf age, and the interaction between O3 treatment and leaf age are shown for each variable,
with bold numbers indicating statistical significance (P <0.05).
(F=38.25, P <0.05) in the middle of stage R5 (DOY 227,
Fig. 3A). During this time, there was also a significant
developmental gradient in photosynthetic rates (Fig. 3A;
r2=0.95 and 0.89 for ambient and elevated [O3], respectively; linear fit lines not shown). Fully expanded leaves
had significantly higher photosynthetic rates than growing
leaves (Fig. 3A). Stomatal conductance (gs) was significantly reduced by 41% in elevated [O3] (Fig. 3B, F=9.11,
P=0.039). This reduction was not caused by changes in
stomatal density on either the abaxial or adaxial leaf surface (Table 1). At the end of stage R5 (DOY 243), a second
measurement of leaf gas exchange was conducted (Fig. 4).
By contrast to DOY 227, all leaves were fully expanded and
there was no gradient in photosynthetic rates associated
Fig. 3. Effect of elevated [O3] on photosynthesis of Glycine max cv.
Spencer. Light-saturated photosynthetic rate, Asat, in lmol m2 s1 (A)
and stomatal conductance, gS, in mol m2 s1 (B) of Glycine max grown
in control (open columns) and elevated (1.23 control, filled columns)
[O3] at SoyFACE, IL, USA. Measurements were performed on leaves
with a range of developmental stages at 1500 lmol m2 s1 PAR on 15
August 2004 (DOY 227, n=3 6standard error). The average relative
growth rates (RGR) measured on 15 August for each leaf and treatment are
given below the figure. The ANOVA table is shown below the figure,
with bold numbers indicating statistical significance (P <0.05). Mean
control and elevated [O3] values for each leaf were separated using preplanned linear contrasts. *P <0.05, **P <0.01.
Ozone effects on leaf growth, photosynthesis and yield
2271
Table 1. Effect of elevated [O3] on specific leaf area (SLA), stomatal density, and elemental analysis of Glycine max cv. Spencer in
leaves of a range of developmental stages with the respective relative growth rate (RGR, % d1) in control and elevated (1.23
control) [O3] (DOY 224, n=3 6standard error)
The results from the analysis of variance (F test and statistical significance, P) for O3 treatment, leaf age, and the interaction between O3 treatment and
leaf age are shown for each variable. Bold figures indicate significant differences.
Leaf
Treatment
1
Ambient
O3
Ambient
O3
Ambient
O3
Ambient
O3
Ambient
O3
2
3
4
5
RGR
(% d1)
0
0
0
0
0.760.2
0.560.1
6.560.6
4.360.8
33.860.7
34.561.2
Main effects
O3
Leaf
O33leaf
SLA
(cm2 g1)
Abaxial stomatal
density (mm2)
Adaxial stomatal
density (mm2)
%C
%N
67.061.0
75.661.9
66.962.5
70.062.5
71.461.4
72.963.2
67.960.5
70.663.9
47.962.7
45.962.6
F, P
1.05, 0.319
2.58, 0.072
0.03, 0998
361.1610.6
386.7612.6
371.169.5
390.0620.1
428.9620.8
407.862.2
330.0627.1
371.1625.0
–
–
F, P
1.15, 0.396
5.64, 0.012
1.25, 0.334
144.4612.4
147.862.9
127.8612.4
133.3610.7
138.964.4
141.1611.8
122.568.5
114.4621.6
–
–
F, P
0.01, 0.929
2.49, 0.110
0.16, 0.924
10.460.2
12.461.1
10.960.3
11.060.4
14.260.6
14.261.0
13.760.1
14.660.6
8.260.6
8.261.2
F, P
1.76, 0.316
26.1, <0.001
0.85, 0.514
1.0260.05
1.2360.21
1.1260.08
1.0660.08
1.6060.09
1.5560.16
1.7160.06
1.7560.04
0.6560.06
0.6960.10
F, P
0.22, 0.687
31.19, <0.001
0.59, 0.678
with different leaf ages or position along the stem (F=0.85,
P=0.513). Although photosynthesis was consistently lower
in elevated [O3] (Fig. 4A), the average 16% decrease was
statistically not significant (F=0.98, P=0.379). The 19%
reduction in gs in elevated [O3] was also statistically
not significant (Fig. 4B; F=0.97, P=0.384).
Carbohydrate and chlorophyll concentrations of leaves
were largely unaffected by elevated [O3] at the onset of
R5 (Fig. 5). A clear diel course was present in soluble
sugars and starch, but a significant difference between
ambient and elevated O3 was only detectable at some
points in time for fructose (Fig. 5). Growing leaves had
significantly lower concentrations of fructose, sucrose, and
especially starch, but there was no difference between
growing and mature leaves in glucose concentration. There
was no detectable O3 treatment effect on leaf carbon or
nitrogen content or specific leaf area (Table 1), but growing
leaves had significantly lower concentrations of both C
and N compared with fully expanded leaves.
Carbohydrate concentration was measured again later
during the pod-filling stage (DOY 237) during the late
afternoon (Fig. 6), when all leaves were photosynthetically
mature (Fig. 4A). Hexose content was lower in leaves
exposed to elevated [O3]. Sucrose levels also decreased
in the older leaves (4 and 5), and sucrose concentration
was significantly lower in leaves exposed to elevated
[O3] (Fig. 6). Starch and total non-structural carbohydrate
concentrations decreased with leaf age, but were unaffected by [O3].
There was no significant effect of elevated [O3] on seed
weight or final harvestable yield (F=1.18; P=0.39), which
was determined at DOY 280. Seed yield under ambient
conditions was 3125 kg ha1 and 2985 kg ha1 in elevated
[O3] (Fig. 7E).
Fig. 4. Effect of elevated [O3] on photosynthesis of Glycine max cv.
Spencer. Light-saturated photosynthetic rate, Asat, in lmol m2 s1 (A)
and stomatal conductance, gS, in mol m2 s1 (B) of Glycine max grown
in control (open columns) and elevated (1.23 control, filled columns)
[O3] at SoyFACE, IL, USA. Measurements were performed on leaves
with a range of developmental stages at 1800 lmol m2 s1 PAR on 31
August 2004 (DOY 243, n=3 6standard error). The ANOVA table is
shown below the figure, with bold numbers indicating statistical
significance (P <0.05). Mean control and elevated [O3] values for each
leaf were separated using pre-planned linear contrasts.
2272 Christ et al.
Fig. 5. Effect of elevated [O3] on diel chlorophyll and carbohydrate concentrations (c) of Glycine max cv. Spencer. Diel concentration of glucose (A),
fructose (B), sucrose (C), and starch (D) in lmol cm2 and of chlorophyll in mg cm2 (E), of growing and mature leaves on 5 August 2004 (DOY 217) at
SoyFACE, IL, USA in control (open symbols) and elevated (1.23 control, filled symbols) [O3] (n=3 6standard error). The ANOVA table is shown
below the figure, with bold numbers indicating statistical significance (P <0.05).
Discussion
In this field experiment, O3 was applied under open-air
conditions at a level predicted for the middle of the 21st
century (Morgan et al., 2004, 2006). The aim was to test,
in an O3-sensitive soybean cultivar, if O3 decreased yield
potential by (i) reducing photoassimilatory area and (ii)
decreasing the photosynthetic carbon gain. Elevated [O3]
led to a decrease in growth of upper canopy leaves
throughout the seed-filling period from DOY 217 to DOY
237 (Fig. 2). At DOY 217, leaf size was still comparable
between treatments in the upper canopy while, at DOY
237, leaf area in plants from 1.2 times elevated [O3] was significantly smaller compared with plants from the ambient
treatment. This suggests that part of the late-season decrease
in leaf area index in soybean exposed to elevated [O3] is
caused by a decrease in individual leaf size, as well as
potential decreases in leaf number (Dermody et al., 2006).
There was no effect of O3 treatment on stomatal density on
either leaf surface. Abaxial stomatal density was approximately three times greater than adaxial stomatal density,
which is consistent with values reported for field-grown
soybeans (Liu-Gitz et al., 2000). Although the observed
high stomatal density indicates a potentially high susceptibility to O3 damage (Dean, 1972; Ferdinand et al., 2000),
stomatal density alone is not necessarily a clear indicator
for O3 susceptibility (Saitanis and Karandinos, 2002).
The observed decrease in photosynthesis is well within the
range reported in the literature (Morgan et al., 2003) and in
FACE treatments. Morgan et al. (2004) reported a decrease in
photosynthesis in field-grown soybean only in upper canopy
leaves late in the season, i.e. leaves with accumulated damage
from prolonged O3 exposure. Experiments conducted in
enclosed facilities showed that O3 decreases the activity and
amount of Rubisco and hence assimilation (Farage et al.,
1991; Pell et al., 1997; Zheng et al., 2002), as well as the
maximum rate of carboxylation progressively over the
growing season (Reid and Fiscus, 1998). Plants exposed to
chronic O3 accumulate damage, resulting in a progressive
decrease of photosynthesis over the growing season (Morgan
et al., 2003). The present study indicated that photosynthesis
Ozone effects on leaf growth, photosynthesis and yield
can be reduced by elevated [O3] in leaves of different
developmental stages, in combination with decreased stomatal conductance (Fig. 3). However, in the present experiments,
by the time all leaves reached maximum light-saturated
Fig. 6. Effect of elevated [O3] on carbohydrate concentrations (c) of
Glycine max cv. Spencer. Concentration of hexoses (A), sucrose (B),
starch (C), and total non-structural carbohydrates (TNC) (D) in lmol
cm2 of leaves of a range of developmental stages on 25 August 2004
(DOY 237) at SoyFACE, IL, USA in control (open symbols) and elevated
(1.23 control, filled symbols) [O3] (n=3 6standard error). The results
from the analysis of variance (F test and statistical significance, P) for O3
treatment, leaf age, and the interaction between O3 treatment and leaf age
are shown for each variable, with bold numbers indicating statistical
significance (P <0.05).
2273
photosynthetic rates, inhibition of photosynthesis due to O3,
although consistent, was no longer significant (Fig. 4).
The diel carbohydrate analyses indicate that at an early
stage of leaf expansion (DOY 217) upper canopy leaves
of soybean contain high concentrations of glucose, but
low concentrations of fructose and sucrose, compared with
fully expanded leaves (Fig. 5). Potentially diagnostic of sink
leaves, this phenomenon was not altered by elevated [O3],
but at some times of the day, fructose, sucrose, and starch
content of mature leaves were higher in elevated [O3].
Twenty days later, all upper canopy leaves had lower
hexose and sucrose concentrations in elevated [O3]
(Fig. 6), indicating that these smaller leaves were now
stronger sources of soluble carbohydrates for seed-filling.
Changes in C translocation from sources to sinks have
been linked to developmental stage of soybean and O3
dosage (Pausch et al., 1996), and in soybean would depend
strongly upon growth habit, i.e. indeterminate versus
determinate growth form (Fiscus et al., 2005).
The final yield did not differ between treatments. The time
series summary of all the above data in Fig. 7 shows that the
smaller assimilatory area in the upper canopy and reduced
photosynthesis need not necessarily lead to yield loss.
Evidently, plant development and metabolism was sufficiently flexible to divert carbohydrate fluxes selectively to
the reproductive organs. In this situation, photoassimilates
may have been directed away from vegetative growth
towards reproductive growth, and smaller leaves became
stronger sources for seed-filling. This was a surprising result given the yield losses from elevated [O3] in previous
years of the SoyFACE experiment were 15% and 30%
(R Nelson, unpublished results). However, 2004 had lower
seasonal O3 concentrations in both ambient and elevated
treatments compared with 2002 and 2003 (Morgan et al.,
2006). Still, O3 levels were above critical thresholds for
soybean (Nali et al., 2002), and the response of yield to
increased O3 is reportedly linear, so some yield loss was
anticipated (Ashmore, 2002). The 2004 growing season
Fig. 7. Summary of the effects of elevated [O3] on photosynthesis, growth, carbohydrates, and final yield of Glycine max cv. Spencer in August 2004
at SoyFACE, IL, USA. Mean net CO2 exchange rate (Asat) of Fig. 3 at DOY (day of the year) 227 (A), sum of upper canopy leaf area of Fig. 2 at DOY
237 (B), sum of soluble carbohydrate concentration (c) of Fig. 6 at DOY 237 (C), mean net CO2 exchange rate of Fig. 4 at DOY 243 (D), and final
yield at DOY 280 (E) (n=3 6standard error) in control (open bars) and elevated (1.23 control, filled symbols) [O3].
2274 Christ et al.
was ideal for soybean, with a distinct lack of drought
stress throughout the season (www.usda.gov/oce/waob/
jawf; Leakey et al., 2006). Therefore, it is possible that the
interaction of O3 with other stresses had a significant impact
on yield in 2002 and 2003, and that in the ideal growing
season of 2004, the stress of O3 alone was not enough to
negatively impact yield. This supports earlier observations
that O3-induced yield decreases were exacerbated in a year
with significant hail damage (Morgan et al., 2006).
In conclusion, this study shows that an indeterminate
soybean cultivar is able to maintain yield or reproductive
growth at the cost of leaf growth under elevated [O3] in
otherwise favourable environmental conditions. The results
also imply that it might be important to select for cultivars
that have enough reserves to foster seed growth at the cost
of vegetative growth under chronic O3-exposure to cope
with future climate scenarios.
Acknowledgements
We thank Steve Long, Tim Mies, and Andrew Leakey for assistance
at SoyFACE, and Barry Osmond for helpful comments on this
manuscript. SoyFACE is possible through funding by the Illinois
Council for Food and Agricultural Research, Archer Daniels Midland Company, and USDA/ARS. MMC was supported by a DAAD
Grant (project no. D/03/36755) and acknowledges support for her
PhD thesis at the Heinrich-Heine University Düsseldorf. EAA was
supported by the Alexander von Humboldt Foundation.
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