enough to cause significant decreases in the growth and yield of sensitive genotypes in many
parts of the world Heck et al., 1983; Wahid et al., 1995; Fuhrer et al., 1997. Numerous factors are
considered to underlie the decline in productivity induced by O
3
, including reduced photosynthetic capacity Darrall, 1989; Heath, 1994; Pell et al.,
1994, enhanced rates of maintenance respiration Amthor, 1988, and increased retention of fixed
carbon in source leaves Balaguer et al., 1995.
Plant responses to the pollutant vary both within and between species, and may be influ-
enced by the stage of growth andor leaf develop- ment Cooley and Manning, 1987; Fuhrer et al.,
1997; Danielsson et al., 1999. In addition, the impacts of O
3
are profoundly influenced by sev- eral other environmental factors Wolfenden and
Mansfield, 1991; Barnes et al., 1996; Barnes and Wellburn, 1998. Previous work on Plantago ma-
jor has demonstrated that geographically discrete populations exhibit wide variation in O
3
sensitiv- ity Reiling and Davison, 1992a; Lyons et al.,
1997; exposure of sensitive populations to envi- ronmentally relevant concentrations of the pollu-
tant causing decreases in the rate of net CO
2
assimilation Reiling and Davison, 1994, 1995; Whitfield et al., 1996, plant growth Reiling and
Davison, 1992a,b; Pearson et al., 1996; Lyons et al., 1997 and reproductive performance Reiling
and Davison, 1992b; Whitfield et al., 1996; Lyons and Barnes, 1998. However, O
3
-sensitive popula- tions may become more resistant to the pollutant
as plants age Reiling and Davison, 1992b; Lyons and Barnes, 1998, a phenomenon that has also
been documented in other species Blum and Heck, 1980; Walmsley et al., 1980; Lee et al.,
1988; Held et al., 1991; Kasana, 1991; Younglove et al., 1994.
The present study was undertaken to test the hypothesis that plant responses to O
3
are influ- enced by developmental age. An O
3
-sensitive pop- ulation of P. major ‘Valsain’; Lyons and Barnes,
1998; Lyons et al., 1999a was employed to exam- ine the effects of the pollutant on photosynthesis
and assimilate utilization by individual leaves at contrasting stages of plant growth.
2. Materials and methods
2
.
1
. Plant culture and fumigation Seed of P. major L. ‘Valsain’ was germinated in
a propagator containing a standard potting com- post John Innes No. 2 in a controlled environ-
ment chamber
ventilated with
charcoal Purafil
®
-filtered air CFA, B 5 nmol mol
− 1
O
3
. The propagator lid was removed following germi-
nation, and 7-day-old seedlings i.e. three-leaf stage transplanted individually in to 2.5 cm
2
plugs of 10 × 7 modules containing the same com- post. Plants were then transferred to duplicate
controlled environment chambers and exposed to CFA 24 h days
− 1
or CFA plus O
3
CFA plus 15 nmol mol
− 1
O
3
overnight rising to a maximum of 75 nmol mol
− 1
between 12:00 and 16:00 h. De- tails of the chambers are given elsewhere Zheng
et al., 1998. Plants were transplanted in to pro- gressively larger pots 0.45 and 1.74 dm
3
contain- ing the same standard compost after 14 and 28
days, respectively, watered daily and fertilized once every 2 weeks using a medium strength
commercial nutrient solution Phostrogen, Cor- wen, Clwyd, UK.
2
.
2
. Leaf gas exchange After 28 and 42 days of exposure to CFA or
O
3
, the rate of CO
2
and H
2
O exchange of the youngest fully expanded leaf borne on six plants
per chamber was recorded every 2 h over a 24-h period using a portable PP-systems infra-red gas
analysis IRGA system employing a standard Parkinson leaf cuvette PLC-B coupled to a twin-
channel CO
2
H
2
O IRGA CIRAS; PP Systems, Hitchin, Herts., UK. Measurements were per-
formed inside each chamber under ‘growth condi- tions’. Day-time measurements were made at a
PPFD of 200 9 10 mmol m
− 2
s
− 1
, representative of the PPFD at canopy height in the growth
chambers, employing a reference i.e. ambient CO
2
concentration of 365 9 1 mmol mol
− 1
, a cuvette-air temperature of 23 9 0.1°C day and
16 9 0.1°C night and a saturating air vapour pressure deficit of 1.2 9 0.01 kPa and 1.0 9 0.01
kPa, respectively. Gas exchange measurements
were made in rotation on individuals exposed to CFA and O
3
, allowing sufficient time for each leaf to attain a steady-state 5-min equilibration pe-
riod during the day, 20 min at night prior to recording data. Because of the time taken to
attain reliable gas exchange data in the dark, night-time measurements were restricted to three
plants per chamber i.e. six plants per treatment.
2
.
3
. Carbohydrate analyses and the construction of carbon budgets
Leaves employed for diurnal gas exchange mea- surements were harvested immediately prior to
the end of the photoperiod ‘dusk’, 21:00 – 22:00 h and an equivalent set of leaves harvested from
five additional plants in each chamber i.e. ten plants per treatment immediately prior to the
beginning of the next photoperiod ‘dawn’, 06:00 – 07:00 h. Plants were sampled after 28 and
42 days exposure to CFA or O
3
, the same day that diel gas exchange measurements were per-
formed. The projected area and weight of each leaf was recorded, then ethanol-soluble and water-
soluble carbohydrates separated according to Far- rar 1980, 1993 by successive extractions in hot
80°C 95 vv ethanol ethanol-soluble carbo- hydrates and acetate buffer pH 4.5 water solu-
ble carbohydrates. Starch was extracted from the residual leaf material, following grinding, auto-
claving and the addition of 10 U a-amylase EC 3.2.1.1 and 10 U amyloglucosidase EC 3.2.1.3
to the cooled extracts.
Carbohydrate concentrations were determined using the phenolsulphuric acid assay described by
Dubois et al. 1956. Borland and Farrar 1985 have previously shown that the fractions contain:
i ethanol soluble-fraction – neutral sugars; su- crose, glucose, fructose and low d.p. fructans
d.p. B 5, ii water-soluble fraction – high d.p. fructans, and iii starch a-amylaseamyloglucosi-
dase extracts.
Daily carbon budgets were constructed as de- scribed by Borland and Farrar 1985. Assimilate
export in the light was calculated by subtracting the amount of non-structural carbohydrate accu-
mulated during the day i.e. stored in the light from the total amount of CO
2
assimilated over the same period. Carbohydrate losses associated
with dark respiration R
d
were calculated from night-time measurements of the rate of CO
2
efflux from leaves under the assumption that respiratory
processes are fuelled solely by hexose carbohy- drates at night.
2
.
4
. Growth and dry-matter partitioning Thirty seedlings were randomly harvested when
plants were transferred to fumigation chambers, and this process was repeated on five plants per
chamber i.e. ten plants per treatment after 28 and 91 days exposure to CFA or O
3
. Plants were separated in to root and shoot, and then dried to
constant weight in an oven at 70°C. Plant relative growth rate R and rootshoot allometry K
were calculated according to Hunt 1990. At the final harvest 91 days, the number of flower
spikes were recorded, before separating from each plant and drying to constant weight in an oven at
70°C. The number of capsules per plant, and the number of seeds per capsule, number of seeds per
plant and the dry weight of 50 seeds were subse- quently recorded based on determinations for five
capsules per spike and five spikes per plant. Re- productive effort was calculated from the ratio of
total dry weight of seeds per plant:total dry weight of the whole plant Pearson et al., 1996.
2
.
5
. Statistical analyses Statistical analyses were performed using SPSS
SPSS Inc., Chicago, IL, USA. Data were first subjected to analysis of variance ANOVA inves-
tigating the influence of chamber on all measured parameters. No significant chamber to chamber
variation was found within treatments, so data for individual plants were reanalyzed using a reduced
multivariate ANOVA MANOVA model to test the effects of O
3
under the assumption that plants in duplicate chambers were as likely to be as
similar, or as different from, plants within an individual chamber. Significant differences be-
tween means were established using the least sig- nificant difference calculated at the 5 level or by
using one-way ANOVA.
3. Results
