Annals of Botany 79 (Supplement A): 93-96, 1997
Survival and Metabolite Accumulation by Seedlings and Mature Plants of Timothy
Grass during Ice Encasement
BJARNI E. GUDLEIFSSON
Agricultural Research Institute, Modruvellir, IS-601 Akureyri, Iceland
Received: 5 December 1995
Accepted: 7 July 1996
Seedlings often survive ice encasement stress better than mature plants under field conditions. However, in artificial
ice encasement experiments with timothy, no significant difference in survival was detected between these two plant
groups. At -2 C seedlings accumulated respiration metabolites during ice encasement to higher levels than adult
plants on dry weight or protein basis. No specific differences in metabolism between mature and young plants were
detected although mature plants accumulated tartarate and acetate which were not detected in seedlings. Timothy
plants accumulated ethanol, malate, lactate and CO 2 and smaller amounts of propionate and pyruvate while citrate,
fumarate and shikimate were at relatively low levels, and malonate was depleted. Therefore, higher survival of ice
encased seedlings in field conditions cannot be ascribed to slower respiration rates or to different respiration
pathways. Instead, the effect is probably brought about by more open, and thus less compacted soil, generated during
seedbed preparation the previous year. This open soil structure imposes less severe oxygen shortage under ice.
© 1997 Annals of Botany Company
Key words: Timothy, Phlewn pratense, ice encasement, anaerobic respiration, metabolite accumulation, winter
damage.
INTRODUCTION
Fields for hay production in northern areas are often
permanent and only rarely recultivated. The perennial
grasses these fields contain are frequently killed during
winter by prolonged anaerobic conditions under ice cover.
As the leys age, the sown grasses, e.g. timothy (Phleum
pratense), gradually disappear and the fields are invaded by
hardier indigenous species, e.g. Agrostis tenuis, Poa
pratensis and Deschampsia caespitosa (Nesheim, 1986b;
Thorvaldsson, 1994). In grassland surveys in northern
Norway and Finland (Nesheim, 1986a; Ravantti and
Miettinen, 1989) abiotic winter damage (freezing, ice
encasement, frost heaving) was more evident in younger leys
than older ones, presumably because hardier species had
invaded the older fields. However, in most cases, first-year
grassland leys and alfalfa stands are less damaged by ice
encasement than older ones (Graber, 1937; Ravantti, 1960;
Gudleifsson, 1971a; Arsvoll, 1973). In surveys in Iceland
(Gudleifsson, 1971) and Norway (Arsvoll, 1973) 7% of
plants in first-year leys were damaged while, in 2-6 year leys,
39 % of plants were damaged. This difference is not related
to different grass species because relatively young leys, 2-6
year old, are mainly dominated by the sown grasses
(Thorvaldsson, 1994).
Northern cultivars of timothy have slower respiration
rates than southern ones (Sjiseth, 1971). It has been
assumed that this lower aerobic respiration rate helps the
northern cultivars to withstand anaerobic conditions under
ice cover. Thus differences in ice tolerance between seedlings
and old plants might be related to differences in respiration
rate or different pathways of energy metabolism. In addition,
0305-7364/97/0A0093 + 04 $25-00/0
seedlings and adult plants are physiologically different.
Seedlings are much smaller and have usually not been
subject to the cutting or grazing stress experienced by older
plants. The aim of this study was to disover whether
seedlings and old plants differed in ice encasement tolerance
and if this could be related to different respiration rates or
different pathways of respiration under ice. This was
accomplished by measuring accumulation of key respiratory
metabolites during ice encasement.
MATERIALS AND METHODS
Before freeze-up of the soil in the autumn of years
1991-1994, turf containing adult plants of timothy was
placed in plastic trays and kept outdoors at Modruvellir,
Iceland, for growth and hardening. These plants were old
TABLE 1. Weight of experimental plants used in ice
encasement experiments
Winter
Date of icing
1991-92 24
26
1992-93 22
14
1993-94 18
18
1994-95 10
Jan. 1992
Mar. 1992
Mar. 1993
Apr. 1993
Feb. 1994
Mar. 1994
Nov. 1994
Number of
replicates
30
30
35
20
45
25
30
Dry matter per plant (g)
with standard errors
Seedlings
Mature plants
2534+21-6
2232+7-3
70.0+0.4
956+ 15
11.8+0.03
3.8+0004
50+0.004
626-6+39.4
440-8+ 14'1
330.4+ 10.0
4588+28.6
561.8+407
5122+15.0
346-8+6.6
© 1997 Annals of Botany Company
94
Gudleifsson-Survival of Ice Encasement by Timothy Grass
140
120
100
80
>
60
40
220
0
10
0
5
20
10
30
15
40
20
25
50
30
60
0
5
10
15
20
25
30
35
35
0
5
10
15
20
25
30
35
0
5
10
25
30
35
-C
,S
bD
SD
3
tto
l
bb
0
5
10
15
20
25
30
35
Days in ice
15
20
Days in ice
FIG. 1. Plant survival (A) and accumulation of five metabolites (B-F) in seedlings and mature plants of timothy during ice encasement at - 2 C.
Mean of three replicates with standard error. A, Plant survival; B, ethanol; C, malate; D, citrate; E, lactate; F, carbon dioxide accumulation.
(---) Mature plants, (-) seedlings.
mature plants from leys at least 4 years old, that had been
subject to cutting and grazing. In 1991 and 1992, seedlings
were also taken into trays from first year fields but in 1993
and 1994 seedlings (Icelandic cultivar Adda) were sown
directly into trays containing peat soil. The size of mature
plants was similar in all experiments, but seedlings from the
field were smaller, and those sown directly in trays were
1). Plant roots were washed clean of soil under cold tap
water, top and root-trimmed to about 3 cm. Groups of five
or ten plants were placed in 200 ml plastic beakers containing
cold tap water and crushed ice for cooling. The beakers were
then placed in a modified domestic freezer and frozen to
-2 °C. Samples (beakers) of ice encased plants were
collected weekly from the freezer, thawed and analysed for
smaller still (Table 1). Mature plants were of similar dry
mass in all experiments and about twice as heavy as field
survival and accumulation of metabolites. Survival was
evaluated by transplanting thawed plants into peat soil in
grown seedlings and 100 times heavier than seedlings from
plastic trays for growth at 15 °C and surviving plants
trays. In Nov.-Mar. (Table 1) trays were taken to the
laboratory for thawing and plants withdrawn from soil for
treatment. Each winter, except 1994-1995, plants were
taken to the laboratory for treatment on two dates (Table
counted and evaluated 3 weeks later. In most cases, survival
was evaluated on each plant on a 0-10 scale indicating %
survival. In some cases, only the proportion of surviving
plants was registered. Plants for chemical analysis were
Gudleifsson-Survival of Ice Encasement by Timothy Grass
ground in a mortar in thaw water, the liquid volume
measured and plant residue dried in an oven at 70 C for dry
matter determination. Liquid samples for chemical analysis
were filtered through 045 um HA membrane filters for
determination of acids and ethanol but not for CO2 analysis.
Ethanol was quantified enzymatically with a UV-test kit
(Boehringer Mannheim) at 340 nm using a Vitatron universal photometer. Organic acids were measured by high
performance liquid chromatography (HPLC HewlettPackard 1050 series) with a solvent flow rate of 035 ml min-'
and the UV-detector at 210 nm. The analytical column was
an Aminex HPX-87H (Bio-Rad) ion exclusion column,
300 x 87 mm stabilized at 50 C. The mobile phase consists
of 3 % isopropylalcohol 1, 1% 08 N H2 SO4 and 96 % distilled
water. The separation c)f some metabolites was incomplete.
As a check, L-lactic a cid, D-lactic acid, formic acid and
malic acid were someti. mes quantified enzymatically with a
UV-test kit as described I for ethanol. CO 2 was analysed with
a carbon dioxide electr ode (Orion, Model 95-02 see Orion
Research, 1982). Durin g ice melting the CO 2 was retained in
solution by addition ol f NaOH and acidified to pH 48-5-2
during analysis. In all cases two or more replicates were
analysed.
RESULTS
Absolute levels of plant survival varied from one experiment
to another. In general, ice tolerance of the plants decreased
throughout the winterr. For example, after 23 d of ice
encasement, mean surviival of mature plants encased in Nov.
in these seven experime nts was 100 %. The value declined to
54% in plants encaseed in Jan.-Mar. while only 40%
survived when plants were encased in Apr. Comparable
seedling survival levels were 95, 38 and 5 %, respectively.
The larger, mature plan ts survived slightly but insignificantly
better than seedlings (Fig. 1A). Figure 1A shows, how
increased length of enc:asement time raised the mean plant
mortality for all seven experiments.
Metabolite analysis in the first 2 years of experiments
revealed accumulation of ethanol and lactate under ice.
Tissue concentrations of these metabolites were considerably
higher in seedlings tha n adult plants (Table 2).
In 1994, plants were encased on 18 Feb. and 10 Nov. for
up to 35 d and twelve mMetabolites were analysed at intervals.
During prolonged ice encasement, timothy accumulated
high concentrations of ethanol, malate and citrate and
TABLE 2. Accumulationi of metabolites (mg g-1 dry matter) in
timothy plants during ice encasement at -2 C. Mean of
three replicates with standarderrors. Plants were encased on
26 Mar. 1992 and 22 .Mar. 1993 and the results combined
12 dI in ice
Seedlings
Mature
36 d in ice
Seedlings
plants
Ethanol
Lactate
192+41
76 + 42
92+34
13+ 13
Mature
plants
430+95
262+49
191+4.7
113+28
95
lower levels of lactate and CO2 (Fig. 1) In all cases,
concentrations were higher in seedlings than adult plants.
The remaining seven metabolites analysed were in lower
concentrations (Table 3). Of these, timothy accumulated
pyruvate and propionate during ice encasement, maintained
fumarate and shikimate, while malonate was depleted. In all
cases, levels were higher in seedlings than mature plants.
Tartarate and acetate were present only in trace amounts
and were found in adult plants but not in seedlings.
DISCUSSION
In these experiments, repeated over 4 years, no significant
differences in plant survival were detected between seedlings
and mature plants (Fig. 1A). This fails to verify field
observations showing that seedlings survive better than old
plants (Gudleifsson, 1971 a; Arsvoll, 1973). One possible
explanation is that the seedlings in current laboratory
experiments were too small to withstand the stress imposed
by the experimental procedure (Table 1). Alternatively,
greater damage to old leys caused by ice encasement under
field conditions could be related to greater compaction of
the soil leading to poor root growth, poor hardening and/or
greater anaerobic stress during winter than in newly
established swards. In organic soil, the ice encasement
damage increases as the soil porosity decreases (Gudleifsson,
1971 b). The adult plants used in the current experiments
were taken from farmers' fields and had therefore been
subject to natural growth and hardening conditions and had
also been cut and grazed. In the experimental icing, soil had
been removed and replaced by solid ice and stress on
seedlings and mature plants was similar.
Generally, mature plants and seedlings of timothy produce
the same metabolites under ice. Thus, differences in ice
tolerance cannot be ascribed to different respiratory
pathways. Tartarate and acetate were only accumulated in
adults (Table 3). These metabolites have been detected from
ice encased field plants and could indicate bacterial
fermentation occurring in these whole plant experiments
(Gudleifsson, 1994). Working at tissue or cellular level
would be more appropriate in such experiments.
The accumulation of different metabolites increases slowly
during the ice encasement in mature plants. In seedlings, the
accumulation is more erratic than in adult plants (Fig. 1,
Tables 2 and 3). The conventional metabolites produced
through the glycolytic pathway in anaerobic respiration are
ethanol, malate, lactate and CO2. These are accumulated
gradually throughout the experimental period (Fig. 1). This
is similar to results obtained with winter wheat (Andrews
and Pomeroy, 1979; 1983). Pyruvate accumulation is
notably small (Table 3). Metabolites of the citrate cycle,
fumarate and citrate are maintained at their original level or
increase slightly (Fig. 1D, Table 3). In addition, small
amounts of shikimate were detected, a metabolite also
produced by iris and lily roots (Crawford, 1978). It is
interesting to note that propionate is accumulated to
considerable concentrations while malonate is depleted
both in adults and seedlings. Malonate is regarded as a
useful carbon-skeleton reserve in higher plants (Ranson,
Gudleifsson-Survival of Ice Encasement by Timothy Grass
96
TABLE 3. Accumulation of seven metabolites (mg g- dry
matter) in timothy during ice encasement at -2 C. Mean of
three replicates with standarderrors
No icing
Pyruvate
Fumarate
Shikimate
Propionate
Malonate
Tartarate
Acetate
34 d in ice
ACKNOWLEDGEMENTS
The author thanks Birna J. Olafsd6ttir and Lilja
Valdimarsd6ttir for analytical work and the Icelandic
Research Council for economical support.
Seedlings
Adults
Seedlings
Adults
LITERATURE CITED
1.49 + 078
0.91 +050
2.46 +1.52
1.77 + 177
462+ 13.5
000
0.00
024 + 011
008 +005
010 +006
0.09 + 009
2.67+267
0.16 + 016
0.20 + 020
4.08 + 0.99
184+0-07
233 + 109
8.38 + 458
000
000
0-00
056 + 020
0.11 +003
0.20+0.11
3.65+ 169
0.00
0.58 + 049
5.00 + 212
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