ratios in tissues of C
3
-plants have been shown to increase considerably Conroy, 1992; Cotrufo
et al., 1998. This is not caused by a simple ‘dilution’ due to higher carbohydrate concentra-
tions, but is rather due to a decreased demand for nitrogen in green tissues. Under CO
2
enrichment, optimization of resources within the photosyn-
thetic apparatus may occur Webber et al., 1994 since ribulose-bisphosphate- RuBP and phos-
phate- P
i
regeneration rather than carboxylation by RubisCO will limit the rate of CO
2
assimila- tion Harley and Sharkey, 1991. Decreased con-
tent of RubisCO Moore et al., 1999 which comprises up to 60 of soluble leaf protein Ja-
cob et al., 1995 will reduce total nitrogen demand of green tissues. Additionally, the depression of
the photorespiratory pathway approximetaly half at doubled CO
2
concentrations, Sharkey, 1988 will also decrease the leaf nitrogen demand be-
cause of smaller contents of enzymes of the glyco- late pathway Webber et al., 1994; Fangmeier and
Ja¨ger, 1998.
In previous studies with cereal crops, we could demonstrate that nitrogen uptake by the crops
was not affected by CO
2
enrichment, but was dependent on nitrogen supply Fangmeier et al.,
1997. At the same time, grain yield was signifi- cantly increased under CO
2
enrichment. We also observed a faster progress of senescence, and an
earlier remobilisation of proteins, in flag leaves of wheat crops under CO
2
enrichment Vermehren et al., 1998. Similar observations have been made
by Sicher and Bunce 1998. We assume that enhanced flag leaf senescence
during grain filling in cereals under CO
2
enrich- ment may be triggered by the different effects of
elevated [CO
2
] on grain production which is thought to be increased on the one hand, and on
the acquisition and storage of nitrogen in vegeta- tive pools used during grain filling which are
thought to be not affected, on the other hand. By this means, CO
2
enrichment might accelerate senescence via increased grain nutrient sink capac-
ity. We also speculate that the effect of elevated CO
2
on flag leaf senescence will not be mitigated by additional nitrogen fertilization as additional
N will lead to increased biomass and yield rather than to higher nitrogen pools that would be avail-
able per unit grain yield. To test these hypotheses, we used open-top field
chambers to expose spring barley H. 6ulgare cv. Alexis crops to CO
2
enrichment at two nitrogen supplies and assessed the effects on growth and
yield, on nitrogen acquisition and redistribution by the crops, and on the progress of senescence in
barley flag leaves.
2. Materials and methods
2
.
1
. Plant culture On May 3, 1997, Julian Date JD 123 spring
barley H. 6ulgare L. cv. Alexis was sown into circular containers with a volume of 90 l diame-
ter 61 cm, depth 40 cm which were placed in open-top field chambers OTC. Sowing density
was 500 plants per m
2
. One week after seedling emergence JD 127 the stands were thinned to
250 plants per m
2
. Soil was taken from an agricul- tural field site with vega fluvisol as prevailing
soil type and mixed with sand 1:1 vol:vol to get a substrate low in organic matter B 1. The
crops were supplied with two levels of NPK-fertil- izer, also containing micronutrients, correspond-
ing to 140 kg N ha
− 1
= 14 g N m
− 2
or 80 kg N ha
− 1
= 8 g N m
− 2
split into three applica- tions of 20, 60 and 60, or 20, 30 and 30, kg N
ha
− 1
on JD 140, 148, and 163, respectively, which corresponded to growth stages according to
Tottman and Broad, 1987 13, 22 and 34. OTC were equipped with a rain exclusion top. The
plants were regularly irrigated using deionized water to avoid any drought stress. Green side
shading panels, which reduced diffuse radiation by 50, were raised as the crop grew. The pots
were covered with white isolating panels to avoid high soil temperatures.
2
.
2
. CO
2
exposure Plants were exposed to CO
2
for 24 h per day in circular 3.15 m diameter OTC as described by
Fangmeier et al. 1992, as soon as seedlings emerged on JD 127 until canopy maturity on
JD 231 at final harvest. CO
2
mean target concen- trations were set at 365 ambient and 650
elevated mmol mol
− 1
see Fig. 1. Air samples for CO
2
monitoring were taken 5 cm above the top of the canopies. The air sampling lines were
moved up as the canopies grew. Microclimatic conditions in OTC were monitored continuously
throughout the season and logged as hourly means.
2
.
3
. Assessments
2
.
3
.
1
. Har6ests for biomass and yield estimations An inner circle of the canopies 50 cm in diame-
ter was used for final harvest at maturity to gain biomass and yield data. Samples were separated
into main stem ear, tiller ears, and straw leaves and stems. Because appropriate sampling of
roots from cereal crops is extremely time-consum- ing and only restricted man-power was available,
roots were not harvested. Straw samples were dried at 70°C, and ears at 35°C, until weight
constancy. Ears were threshed to estimate yield, grain number, thousand grain weight, and glume
weight, separately for main stem ears and for tiller ears. Nitrogen concentrations were estimated in
these samples by Kjeldahl.
2
.
3
.
2
. Chlorophyll content Chlorophyll contents of flag leaves were esti-
mated non-destructively at the mid position of flag leaf blades using a SPAD 502 chlorophyll
meter Minolta, Japan on dates when, and just before, flag leaf harvests took place see below.
2
.
3
.
3
. Har6ests for estimations of protein degradation during flag leaf senescence
Flag leaf harvesting and SPAD measurements took place on 7 occasions over a period of 20
days from full maturity of flag leaves JD 189 until nearly complete senescence JD 209. Flag
leaf ligules were visible on JD 166 growth stage 39, Tottman and Broad, 1987, and anthesis
growth stage 65 was on JD 178 in all treatments. Twenty two border plants per pot, growing out-
side the 50 cm diameter circle, were used for the intermediate harvests. Only plants from high nu-
trient supply treatments were analysed. Harvests were done between 1:00 and 2:00 p.m. Then, fresh
weight and length of flag leaves were measured immediately and samples were frozen in liquid
nitrogen within one minute after harvest. Samples were stored at − 80 °C until analysis.
Data on leaf blade length were used to calculate leaf area by regression analysis of 30 flag leaves
harvested from sparse plants in ambient plots. Best fit adjusted R
2
= 0.982 was achieved using
the formula: y = 0.02944 + 0.2907x + 0.03651x
2
where x is the length of flag leaf blade cm and y is the leaf area cm
2
. Proteins were extracted from flag leaf samples
as described earlier Humbeck et al., 1996. Protein concentrations were estimated using the
method of Lowry et al. 1951. Aliquots of 30 mg protein were subjected to SDS – PAGE, and incu-
bated
with antibodies
directed towards
cy- tochrome b559 Cyt b559, and large subunit
LSU and small subunit SSU of RubisCO. Gel photographs were analysed for protein contents
using a Kodak Electrophoresis Documentation and Analysis System 120 in order to yield relative
contents of Cyt b559, LSU, and SSU, based on flag leaf area.
Fig. 1. A Daily mean air temperature and cumulative daily global radiation, and B CO
2
concentrations daily means in six OTC chambers used to grow spring barley crops in the
growth period, 1997
2
.
4
. Statistical design and data e6aluation CO
2
treatments were carried out in three repli- cate OTC, respectively. For each nutrient supply,
one barley pot was exposed within each OTC. OTC means served as input data for analysis of
variance, using
SPSS
8.0 for Windows SPSS Inc., Chicago. Biomass and nitrogen data represent
CO
2
treatment means 9 standard deviation based on three chamber replicates.
SPAD measurements were taken both on plants in the inner circle left for final harvest and on
border plants. There were no significant differ- ences between these data-sets. SPAD data given
for any particular date represent means of eight to 15 single measurements per OTC and each nutri-
ent supply. Standard deviation bars shown in the graph were calculated using means of three repli-
cate OTC, respectively.
Because of restricted number of border plants available for intermediate flag leaf harvests, no
harvests in replicate OTC could be carried out. Rather, at any harvest date four flag leaves taken
from all three replicate OTC were combined to yield one sample.
3. Results
