rall 1989. In recent years research on pollution- induced changes in cold sensitivity has focused
mainly on conifer trees, notably Picea sitchensis Lucas et al., 1988, Picea rubra De Hayes et al.,
1989; Sheppard et al., 1993, Pinus syl6estris Dueck et al., 1990 or the agricultural grass
Lolium perenne Davison and Bailey, 1982; Davi- son et al., 1988 while less is known of responses
of other semi-natural vegetation. An example of this type of vegetation which forms a large part of
non-agricultural land in Britain is Calluna 6ulgaris heather — dominated heathland. C. 6ulgaris
and related species in the Ericaceae and Empe- traceae are the principal dwarf shrub species of
the small, highly valued areas of lowland heath in north-western Europe. These species also cover
extensive tracts of the upland regions of Europe. In some areas, for example parts of upland
Britain, C. 6ulgaris is an important food source for grazers including sheep and game birds such
as the red grouse Lagopus lagopus scoticus. The predominantly maritime distribution of C. 6ul-
garis Gimingham, 1972 suggests that low tem- perature is one important determinant of its
distribution.
Increased incidence of frost damage, as a result of atmospheric pollutant nitrogen deposition to
C. 6ulgaris, may be one cause of the recent decline in the abundance and health of this species in the
Netherlands Van der Eerden et al., 1991; Power et al., 1998, and could be of widespread signifi-
cance in natural vegetation INDITE, 1994. The aim of this study was to examine the hypothesis
that the frost tolerance of C. 6ulgaris is reduced after long-term exposure to a combination of two
of the most important gaseous pollutants, sulphur dioxide and nitrogen dioxide. The experiments
were designed to answer the following specific questions regarding possible changes to frost tol-
erance as a result of exposure to NO
2
and SO
2
. Firstly, how does the duration of exposure to
NO
2
and SO
2
affect the degree of frost tolerance? Secondly, are plants only affected if exposure to
the pollutants is during the growing season or could they also be damaged if exposure is in the
winter months? Experimental research using both gases simultaneously is justified because these pol-
lutants often occur together in rural and suburban atmospheres and many studies have shown that
NO
2
and SO
2
often exert greater-than-additive effects on various plant growth and physiological
processes Ashenden and Mansfield, 1978. The concentrations used in these experiments 40 nl
l
− 1
are higher than rural levels and were used in order to address the hypothesis. Concentrations
of NO
2
and SO
2
similar to those used in the experiments may be measured in some urban and
suburban regions of Britain Broughton et al., 1997. While these are not the main locations of
Calluna there do exist important pockets of low- land heath close to built-up areas in many parts
of western Europe. The experiments reported here form part of a programme of research into envi-
ronmental influences on the cold hardiness of C.
6 ulgaris Caporn et al., 1994; Foot et al., 1996,
1997; Lee and Caporn, 1998; Carroll et al., 1999.
2. Materials and methods
2
.
1
. Plant material Cuttings 30 mm length of C. 6ulgaris L.
Hull, sampled from an extensive moorland near Ruabon, Clwyd in North Wales altitude 475 m
were rooted in a peat and perlite mixture under mist and transferred into 7 cm square pots con-
taining a mixture of peat soil from the field and a commercial peat compost without added nutri-
ents. The plants were raised in an unheated glasshouse before transfer to fumigation chambers
at the Institute of Terrestrial Ecology, Bangor, North Wales. Continuous exposure to a combina-
tion of NO
2
plus SO
2
, both maintained at a concentration of 40 nl l
− 1
, or to charcoal filtered air during the day and night took place in hemi-
spherical glasshouses ‘Solardomes’. The winter over which the frost hardiness was measured was
characterised by mild climate. Minimum monthly air temperatures at a local meteorological station
Valley, Anglesey during the test period were: October + 5.8; November + 1.2; December −
1.9; January − 3.0; February − 4.5; March − 0.2; April + 1.0°C. The plants in the glasshouse
chambers experienced the same daily and seasonal changes in photo-period, light and thermal envi-
ronment as outdoors but the absolute chamber air temperature was elevated by 1 – 5°C mainly dur-
ing the day and the irradiance was reduced by around 13 – 25. A full description of the fumiga-
tion system is provided by Rafarel and Ashenden 1991. A single chamber only was available for
each gaseous treatment, but the experiments were repeated on six occasions, each time with a differ-
ent set of plants, over 15 months between Febru- ary and April of the following year Table 1. To
remove specific chamber effects, the gaseous treat- ments and plants were switched between cham-
bers at 14-day intervals. After switching of chambers the gas concentrations in the chambers
took : 2 h to stabilise.
2
.
2
. Har6est and frost testing Batches of C. 6ulgaris, aged 6 – 9 months from
rooting, were fumigated over different dates Table 1. Frost hardiness of C. 6ulgaris was
tested following
various fumigation
periods. These were designed to permit comparison of i
pre-winter and post-winter exposures and ii fu- migation of varying duration. In addition to stud-
ies of frost tolerance other measurements were made of growth and nutrient contents in order to
assess general physiological responses of the whole plant to the pollutants. The effect on the
growth of C. 6ulgaris of exposure to NO
2
and SO
2
for 8 months was examined in October. At har- vest, measurements were made of the dry mass of
shoots and roots and the foliar concentrations of nitrogen, sulphur and potassium. Growth and
nutrient data were analysed using a 2-tailed t-test. For the assay of frost hardiness, intact plants
were transferred to the laboratory for immediate harvest and frost hardiness tests. The freezing
treatments were given on the same night and following night after transfer from the exposure
chambers i.e. after : 6 and 30 h. Those plants frosted on the second night were stored for 24 h
outside. Full details of the methods of frost test- ing are given by Caporn et al. 1994; these were
based on similar methods described by Murray et al. 1989 and Cape et al. 1991. A brief descrip-
tion only is provided below. The distal ca. 40 mm of the recent seasons shoot growth were excised.
Ten replicate shoots per treatment were used at each test temperature. These were mounted hori-
zontally on clipboards in dark, fan-ventilated freezing cabinets. Freezing cycles started at 10°C,
and the temperature was then lowered at 5°C per hour to the minimum test temperature. This was
held for 3 h after which the temperature was raised at 10°C per hour to 5°C; the shoots were
then removed. Two cabinets were used on succes- sive nights, while other shoots, stored overnight in
a cold room 5°C, were used as un-frozen con- trols. Cabinet air temperatures were recorded with
thermistors linked to a data logger Delta T, Cambridge, England. The frost test temperatures
used on different dates were altered to match the likely range of frost tolerance at different times of
Table 1 Details of pollutant exposures, frost test temperatures and overall results of two-way analysis of variance of ion leakage results from
each series of experiments series 1–6
a
Exposure series Temp×treatment
Duration Effect of
Freezing test temperature Effect of NO
2
°C months
SO
2
temperature nteraction
8 +
4, −5, −10, −15 1 Feb–Oct
+ 4, −10, −5, −20
9 2 Feb–Nov
11 +
4, −15, −20, −25 3 Feb–Jan
5 +
4, −15, −20, −25 4 Aug–Jan
5 +
4, −10, −15, −20 5 Nov–April
+ 4, −15, −20, −25
0.198 n.s. 0.837 n.s.
6 Nov–Jan 2
a
Calluna 6ulgaris was fumigated for different periods with charcoal-filtered air with or without the addition of 40 nl l
− 1
of both NO
2
plus SO
2
. Excised shoots were then exposed to controlled frost tests. The significance of effects of test temperature and pollution on ion leakage is indicated in the right-hand columns PB0.001; PB0.05.
Table 2 Effect of an 8 month exposure to 40 nl l
− 1
NO
2
+ SO
2
on the mean value of a dry mass g of plant parts and b the concentrations of nutrients mg g
− 1
in shoots
a
a Root mass
Treatment Total mass
Shoot mass Ratio rootshoot
0.801 Control
1.252 0.451
1.815 40 nl l
− 1
NO
2
+ SO
2
0.618 0.820
1.434 1.359
t-value, after 2-tailed t-test 3.71
0.23 1.84
2.51 0.823
0.083 0.02
0.002 Probability P
b Sulphur
Potassium Treatment
Nitrogen 0.30
3.47 6.54
Control 8.76
40 nl l
− 1
NO
2
+ SO
2
0.81 7.53
4.65 t-value, after 2-tailed t-test
7.23 7.48
0.0009 B
0.0001 B
0.0001 Probability P
a
Plants were harvested in October after 8 months treatment.
the year. For example the test temperatures in January were − 15, − 20 and − 25°C, while in
October they were − 5, − 10 and − 15°C.
2
.
3
. Assessment of frost injury After the overnight freezing cycle, the elec-
trolyte leakage from the shoots was determined from the increase in conductivity C of the
bathing solution measured using a platinum elec- trode at 20°C, after briefly shaking the vial by
hand. The first measurement C
was : 1 h after the shoots were placed in the vial while the next
was after a 5 h interval C
5
during which vials remained static at 20°C. Finally, the vials were
autoclaved at 105°C for 4 min, and after cooling, measured again. This final value, C
8
i.e. C at ‘infinity’, was an estimate of total electrolyte
content. Using the three conductivity measure- ments C
, C
5
and C
8
the electrolyte leakage coefficients k, units h
− 1
were calculated for each shoot using the following equation:
k = ln C
8
− C
C
8
− C
t
t where t = 5 h.
The electrolyte leakage coefficient k was shown previously to be a sensitive and reliable
method of assessment of frost injury in this spe- cies Caporn et al., 1994. The electrolyte leakage
results are shown in the tables as k × 1000. Statistical one and two-way analysis of variance
with frost temperature and pollution treatment as main effects in the latter was performed on loga-
rithmically-transformed values i.e. ln k × 1000. Transformation was required to normalise the
distribution of the data. All the data was analysed using the SPSS Statistical Package.
3. Results
