Plant Physiol. (1982) 70, 97-103
0032-0889/82/70/0097/07/$00.50/0
Relationship between Phospholipid Breakdown and Freezing
Injury in a Cell Wall-Less Mutant of Chiamydomonas reinhardii
Received for publication December 7, 1981 and in revised form March 15, 1982
ANDREW CLARKE, GLYN COULSON, AND G. JOHN MORRIS
British Antarctic Survey, NERC, High Cross, Cambridge CB3 OET (A. C.); and
Culture Centrefor Algae and Protozoa, NERC, Cambridge CB3 ODT (G. C., G. J.M.), England
ABSTRACT
The effects of freezing and thawing on a cell walless mutant (CW15+)
of Chiamydomonas reinhardii were investigated by monitoring enzyme
release, cell viability, cell ultrastructure, and lipid composition. Cells
suspended in Eugkena gracilis medium were extremely susceptible to freezing injury, the median lethal temperature in the presence of extracellular
ice being -5.3°C. Cell damage was associated with a release of intracellular
enzymes and massive breakdown of ceDular organization. Changes in
phosphoUpid fatty acid composition consistent with either a peroxidation
process or phospholipase A2 activity were evident, but the time course of
these changes showed clearly that alterations in phospholipid fatty acid
composition were a secondary, pathological event and not the the primary
cause of freeze-thaw injury in Chlamydomonas reinhardii CW15+.
It is now generally accepted that an early event during freezing
injury is an alteration in the structure and function of cellular
membranes. The precise nature of this damage is, however, far
from understood.
In many plant cell types, alterations in phospholipid composition are observed following freezing and thawing (1 1, 15, 18, 20,
23, 26-30), and in some chilling-sensitive plants a similar degradation of phospholipids accompanies damage (24, 25). These
changes in phospholipid composition are consistent with the activation of intracellular phospholipases. It is not clear, however,
whether the observed changes in cellular lipids are the primary
cause of freezing injury or merely a secondary pathological event,
although Yoshida (27-29) has proposed that activation of phospholipase D is the specific mechanism of freezing injury in woody
plant cells.
In this study, we have examined the relationship between
freezing injury and phospholipid composition in the unicellular
green alga Chlamydomonas reinhardii. We have used a cell wallless mutant (CW15+) because in this organism the plasma membrane is exposed directly to the suspending medium, and this
avoids any complicating effects of an external cell wall (7). The
time course of the alterations in phospholipid fatty acid composition were compared with the patterns of cellular viability, release
of intracellular enzymes, and ultrastructural changes in order to
distinguish primary from secondary events during freezing damage.
MATERIALS AND METHODS
Cell Culture. Chlamydomonas reinhardii, Culture Centre of
Algae and Protozoa, Cambridge, Strain 11/32 CW15+, was isolated and characterized as a cell wall-less mutant by Hymans and
Davies ( 12). It is thus equivalent to a motile, free-living protoplast.
Unless otherwise stated, cells were grown in Euglena gracilis
medium for 7 d at 20°C as previously described for the wild type
(15). This medium contained 0.1% (w/v) Na-acetate. 3H20, 0.1%
beef extract, 0.2% yeast extract, 0.2% bacto t7ptone, and 0.001%
CaCl2. The osmolality was -45 mosm kg- by freezing point
depression.
Freezing and Thawing. Cell suspensions were placed in 12 x 35
mm sterile polypropylene tubes (Nunc) which were frozen at a
rate of cooling of 0.25°C min-' to different subzero temperatures
(13). The thermal history of each treatment was recorded from a
replicate sample with a 29 SWG copper-constantan thermocouple
connected to a Kipp-Zonen potentiometric recorder. Cell suspensions were warmed by rapid agitation of the ampoule in a water
bath at 250C until the last visible crystal of ice had melted. Cell
viability was assayed by an agar plate method (15) and survival
rates below 1% were recorded as zero. In each experiment, there
were five replicates.
Enzyme Assay. Loss of membrane integrity following freezing
and thawing was determined by measuring the release of the
cytoplasmic enzyme GOT.' The activity of GOT in cell-free
supernatants and cell sonicates was assayed by the method of
Schmidt and Schmidt (17). Enzyme loss to the supernatant was
expressed as a percentage of the total intracellular enzyme activity.
Electron Microscopy. Thin-section electron microscopy was
carried out as previously described (10).
Lipid Extraction and Analysis. Cells were harvested by centrifugation and the total lipids extracted with excess methanol-chloroform (2). Purified lipids were stored in chloroform containing
0.0 1% 2,6-di-tert-butyl-p-cresol (BHT) as antioxidant, under nitrogen at -50°C.
Phospholipids and other polar lipids were separated from neutral lipids, sterols, carotenoids, and porphyrins by chromatography
on prepacked silica gel columns (Sep-paks; Waters Associates).
Purity of this polar lipid fraction was checked by TLC and only
minimal contamination by a polar porphyrin fraction was found.
Fatty acid methyl esters were prepared by mild alkaline deacylation (4) and taken up in n-hexane.
Fatty acid methyl esters were separated on a 25 m SPIOOO
WCOT glass capillary column using helium carrier gas (1 ml
min-') and either splitless (Grob) or 12:1 split injection. During
analytical experiments, column temperature was programmed
from 180 to 230°C at 2°C min-' following a 15-min period at
room temperature. Precision isothermal chromatography for identification was at 190°C. Peak areas were integrated electronically
(HP 3390A; Hewlett Packard), and data are presented as percentage total uncorrected integrator counts in the range C14 to C20.
Precision and accuracy are discussed elsewhere (4). Unsaturation
'Abbreviation: GOT, glutamic-oxaloacetic transaminase; ECL, equivalent chain length.
97
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98
CLARKE ET AL.
parameters were calculated by computer program after conversion
to molar percent compositions. Statistical analyses were performed
using the subprogram Nonpar corr of the Statistical Package for
the Social Sciences (SPSS).
Identification of fatty acids was by coinjection with standards,
where available (16:0, 16:1w7, 18:0, 18:1w7, 18:1X9, 18:2o3,
18:2w6, 18:3w6; Sigma), semilog plot graphical techniques, Ag+TLC, and quantitative hydrogenation in methanol with PtO2 as
catalyst (3).
RESULTS
Viability following Freezing and Thawing. At the rate of cooling
used in these experiments (0.25°C min-'), Chlamydomonas reinhardii CW15+ could be undercooled to -7.5°C with no loss of
viability; at temperatures below this, spontaneous nucleation occurred in the suspending medium. In the presence of extracellular
ice, however, cell recovery was reduced, and at a rate of cooling
of 0.25°C min-' the median lethal temperature was -5.3°C (Fig.
1). This loss of viability was time dependent, for in cells maintained isothermally at -5°C in the presence of extracellular ice,
survival fell dramatically with time (Table I).
Cooling the cells to -7.5°C in the presence of extracellular ice
resulted in a release of the cytoplasmic enzyme GOT, and this
release reached a plateau about 5 min after thawing (Fig. 2).
Neither the amount of GOT released, nor the time course of this
release was altered by incubating the cells at 4 or 37°C after
thawing of the cell suspension.
Electron Microscopy. Following freezing of the cell suspension
to -5°C and then thawing, numerous alterations in the cellular
ultrastructure were visible by thin-section electron microscopy
0
a,
OS
0
a)
0
-5
-10
Temperature (IC)
FIG. 1. Recovery (%) of Chiamydomonas reinhardii CW15+ following
cooling at 0.25°C min-' to different final temperatures. Cell suspensions
were cooled either in the presence of extracellular ice (0) or undercooled
(-). Each point is the mean of five replicates.
Table I. Recovery (%) of C. reinhardii CW15+ Maintained at -5°Cfor
Different Times in the Presence of Extracellular Ice
Control value obtained at -5°C in absence of extracellular ice. Value
at 0.1 min in parentheses since thermal equilibrium at -5°C may not have
been achieved within this time.
Time
Recovery
0 (control)
%
100
(0. 1)
(54)
min
1
5
10
50
100
7.7
0
0
0
0
Plant Physiol. Vol. 70, 1982
100
a)
0J
U)
C-
@ 50 I
0
GO
E
N
c
o
r
0
50
Time (min)
100
FIG. 2. Time course of the release (expressed as % total mtracellular
enzyme) of glutamic-oxaloacetic transaminase from Chlamydomonas reinhardii CW15+ following freezing of the cell suspension to, and thawing
from, -7.5°C. Horizontal axis is time since thawing, with point at to
representing the (unfrozen) control cells. Each point is the mean of five
replicates.
(Fig. 3), and these pathological changes became more pronounced
the longer the cells were held at -5°C.
Control cells (e.g. Fig. 3A) showed granular cytoplasm, prominent mitochondria with well-defined cristae, and distinct evenly
spaced thylakoid membranes in the chloroplasts. After 5 min at
-5°C, some cells were apparently undamaged and some markedly
damaged, but the majority appeared as in Figure 3B. After 15
min, there was little obvious cytoplasm, the thylakoid system was
disorganized, and there were no discernable mitochondria. Cells
exposed to -5°C for 30 min had greatly disorganized thylakoids
and no mitochondria, and the remaining cytoplasmic ground
matter was less granular, containing densely staining bodies which
were probably lipid. After this, evagination of the plasma membrane occurred and there was such a loss of cell definition that
only membrane vesicles were discernable in the electron micrographs. On thawing samples from - 10°C a similar but more rapid
sequence of events occurred.
Phospholipid Fatty Acid Composition. Analysis of Chlamydomonas reinhardii CW15+ phospholipid fatty acids by capillary
GC revealed many previously unreported minor components. The
overall pattern was, however, similar to that found in wild-type C.
reinhardii (15), although the CW15+ strain contained a slightly
greater proportion of monoenoic fatty acids. The absence of a cellwall in CW15+ thus has little effect on phospholipid fatty acid
composition.
In the previous study, several of these minor components remained unidentified. Of particular interest was a peak (CR8)
which ran between 16: 1 7 and 16:2o6, a peak (CR21) between
18:2X6 and 18:3X3, and a peak (CR27) of retention time >18:4X3.
Inasmuch as at least two of these fatty acids were unsaturated,
and hence liable to affect any calculated unsaturation parameters,
they were examined in detail.
CR8 had an ECL of 16.62, markedly greater than 16:1X7, but
did not run with the dienoic acids on Ag+-TLC plates; it was
therefore assumed to be a C16 monoenoic acid of unusual structure.
CR21 separated with the 3-6 double bond fraction in Ag+-TLC
and was shown by hydrogenation to be a C18 acid. No trace of
this component could be detected in the dienoic fraction after
Ag+-TLC, and it had an ECL of 18.94, compared with 18.72 for
standard (and C. reinhardii) 18:2w6. It was therefore assumed to
be an octadecatrienoic acid of unusual structure, possibly 18:3X9
or 18:3X7. CR27 had an ECL of 20.19 and was shown to be a C18
polyenoic acid by hydrogenation. This component is very likely
the octadecapentaenoic acid (18:5w3, A3.69'12 5) tentatively identified by precision capillary GC and structural element retention
data in the unicellular marine alga Prorocentrum minimum (1).
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FREEZING INJURY IN CHLAMYDOMONAS
99
food
D
D~~~~t ':~~~f~N
FIG. 3. Thin-section electron micrographs of Chlamydomonas reinhardii CWI5+. A, Unfrozen control, x 9,900; B, frozen to -50C for 5 min,
x 12,500; C, frozen to -50C for 30 min, x 9,900; D, frozen to -100C for 5 min, x 8,750.
Semilog plots of precision capillary GC data showed that 18:4
and 16:4 were probably of similar structure, most likely 03, and
also that the minor octadecatrienoic (CR21) and major 16:3
components were also of similar structure. Quantitative hydrogenation indicated that C16 and C18 acids accounted for -98% total
acids and that there were no detectable branched-chain, cyclopropane, cyclopropene, or hydroxy-acids present. The percentage
chain length composition of the hydrogenated and unhydrogenated samples were very similar, indicating that chain length and
unsaturation identification were largely correct and that losses of
polyenoic acids during analysis totaled 3% at maximum.
The overall composition reported here (Tables II to V) is similar
to our previous analyses (15) and that of Eichenberger (5) for
wild-type C. reinhardii. In contrast, Gealt et al (6) reported high
levels of 16:3 with no evidence of 16:4. This is an unusual result,
as in most other unicellular algae 16:4X3 is a prominent component and 16:3 a minor constituent.
As with many microorganisms grown in batch culture, fatty
acid composition varied with the stage of culture (Table II) and
it is thus not possible to present a 'definitive' lipid composition for
C. reinhardii CW15+. These differences were particularly pronounced at early stages of culture (days 1 to 3) when cell density
was low and cells were dividing rapidly. At later stages (days 4 to
7), the culture was in the late exponential/early stationary phase,
and the fatty acid composition was more uniform. All cultures
used in the following experiments were of the same age (7 d), but
even so, batch to batch variations were observed. To minimize
this effect, the results of any one experimental treatment were
obtained with the same algal culture; each table is therefore
internally consistent.
Effects of Freezing on Fatty Acid Composition. When cells were
frozen to -50C and then held for between 5 and 100 min at
+250C following thawing, all of the frozen samples had an altered
fatty acid composition when compared with the control, though
not in all cases was there a statistically significant trend with time
(Table III). The major difference was a loss of polyunsaturated
acids, particularly 16:4M3 and 18:3X3 (both p < 0.02), with a
corresponding increase in the proportion of the more saturated
acids, and a significant reduction in both the mean number of
double bonds per molecule of fatty acid and the unsaturated to
saturated ratio. The extent of this alteration became greater the
longer the cells were held at 250C.
When the time that the cells were held in frozen medium at
-50C was varied, but the incubation period after thawing kept to
5 min, there was again a difference between the treated and
control cells (Table IV). A reduction in 16:4X3 content occurred
with increasing time at -50C, but there was no indication of any
loss of 18:3X3 until cells had been frozen for 600 min. The results
here are less clear cut than with the previous treatment, indeed
only in the proportion of 16:4w3 was there a significant correlation
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Plant Physiol. Vol. 70, 1982
CLARKE ET AL.
100
Table II. Changes in Phospholipid Fatty Acid Composition of C. reinhardii CWJ5+ during Batch Culture at
200 C
Data are % total uncorrected integrator counts in range C14 to C20 and represent single analyses of parallel
cultures initiated with inocula from a 7-d-old culture at day 0.
Day of Culture
r^a
No. of cells ml-'
x i0-5
Fatty acid
16:0
16:2o,6
16:3c
16:3c
16:403
18:0
1
2
3
0.102
0.30
1.9
12.62
7.89
15.23
0.40
1.85
19.32
1.64
1.90
2.63
17.83
1.83
1.27
4.75
9.88
7.33
26.49
0.61
1.33
6.58
3.19
20.45
2.89
1.83
1.76
11.39
5.01
18:1w9
O
O12.15
18:17
18:2w6
18:3w6
18:3w3
18:4X3
18:5c
4
5
ph
7
74
25.0
34.0
22.64
4.48
1.61
2.87
12.62
5.49
3.46
5.06
16.32
6.77
16.24
0.10
0.40
15.29
2.01
2.07
1.73
16.43
2.49
0.81
5.44
13.35
10.61
25.40
1.04
0.05
17.79
2.40
3.05
1.14
14.50
1.99
0.64
5.68
13.18
9.50
22.72
1.65
0.93
20.76
2.54
3.18
1.08
15.61
2.43
0.58
4.72
11.51
9.39
21.57
2.35
1.28
1.94
3.28
5.43
3.00
-0.086
-0.086
0.829
-0.771
0.257
-0.257
-0.829
-0.429
0.086
0.543
0.200
0.829
-0.486
0.872
0.872
0.042
0.072
0.623
0.623
0.042
0.397
0.872
0.267
0.704
0.042
0.329
Unknowns and
minor compo-
nentsd
0.704
0.200
2.24
2.27
2.35
1.90
2.32
0.468
0.371
3.18
3.86
4.43
2.48
3.60
a Spearman rank correlation coefficient for proportion of fatty acid against time.
b Two-tailed probability of this result.
c Structure unknown.
d
Minor components includes up to 15 fatty acids (all <1% total).
e Mean number of double bonds per fatty acid molecule.
f
Ratio of unsaturated to saturated fatty acids. All fatty acids identified (not just major components reported
in Tables II to V) were used in computing unsaturation parameters.
db/mole
Unsat/satf
1.93
2.70
with increasing time at -5°C (p < 0.001; Table IV). The difference
in the timing of changes in phospholipid acid composition in
Tables III and IV suggest that although damage to the cell
(assessed as alterations in lipid composition) is induced initially
by exposure to extracellular ice, significant alterations in phospholipid fatty acids do not appear until some time has elapsed
after thawing. This is confirmed by the data in Table V.
When cells were cooled in the presence of extracellular ice to
various temperatures in the range -1 to -15°C and lipids extracted from cell suspensions within 5 min of thawing, there was
a small but significant loss of polyunsaturated fatty acids compared with the control (Table V). However, only in the case of
16:1 (CR8), 18:3o3, and 18:5 was there a strong correlation of
composition with fimal temperature. These variations resulted in
a significant correlation between unsaturation ratio and final
temperature, but there was no evidence of any systematic variation
in the mean number of double bonds per fatty acid molecule with
final temperature.
DISCUSSION
Injury of C. reinhardii during freezing and thawing was associated with the formation of ice. Although respiration and photosynthesis were both reduced in undercooled cells when these were
rewarmed to 20°C (8), there were no long-term effects on viability
for cells could be undercooled to -7.5°C with no reduction in
recovery (Fig. 1). Direct cryomicroscope observation of C. reinhardii CW15+ at the rate of cooling and the temperatures used in
this study showed that all ice formation was extracellular, indicat-
ing that the damaging stress was freeze-induced dehydration (16).
However, should damage occur to the plasma membrane, this
may then no longer act as a barrier to the penetration of ice into
the cell, and secondary injury to organelles may follow. The
recovery of C. reinhardii CW15+ following freezing and thawing
was similar to that of the wild-type organism (15). Because the
mutant CW15+ lacks the cell wall found in the wild-type, this
indicates that disruption of the plasma membrane/cell wall interaction was not a primary cause of freezing injury in C. reinhardii.
Similar conclusions have resulted from studies of isolated protoplasts of higher plant cells (19).
As with many other cell types, the reduction in cell viability
was associated with a loss of membrane integrity, as demonstrated
here by the release of intracellular enzymes upon thawing (Fig.
2). Both the total amount of enzyme released and the kinetics of
this release, however, differed from that observed with the wildtype (15). More enzyme was released from the cell wall-less
mutant, this loss occurring within 5 min of thawing, and there was
no evidence of the two-phase kinetics observed with the wild-type.
These differences merely reflect the presence of a cell wall (which
in the wild-type will retain the cellular constitutents even when
the plasma membrane is damaged) rather than multiple mechanisms of injury. In addition, rates of diffusion will be altered as
the cell wall matrix will behave as a molecular sieve (22). Cell
disintegration was observed during thawing of CW15+, whereas
in the wild-type, although morphological alterations were apparent (e.g. vesiculation of the cytoplasm), the cells remained intact
(16).
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101
FREEZING INJURY IN CHLAMYDOMONAS
Table III. Phospholipid Fatty Acid Composition of C. reinhardii CW15+ following Freezing to -5C, Thawing,
and Incubation for Various Times at 250C
Data reported as for Table II, and represent replicates from the same culture.
Incubation at 25°C (min)
Fatty
Acid
Unfrozen
a
ra
ph
Fatty Acid
Control
30
50
100
5
15
Controle
16:0
16: 1 7
16:2w6
16:3c
16:403
18:0
18: 1w9
18:17
18:246
13:3w6
18:303
18:4X3
18:5C
Unknowns
and minor
components
14.54
4.75
2.56
6.18
13.07
2.06
4.96
3.83
10.48
8.60
21.33
2.54
1.61
19.17
6.07
2.96
5.68
10.35
1.55
6.23
4.79
10.72
6.89
18.27
2.06
1.76
18.07
5.71
2.94
5.65
10.67
1.80
6.08
4.69
10.46
6.93
17.89
1.89
3.40
23.11
7.20
3.24
4.30
7.26
2.02
7.94
5.61
10.51
6.33
14.36
1.68
2.71
23.25
7.97
3.23
4.03
7.24
1.86
8.28
6.23
10.35
6.37
13.81
1.54
1.89
23.03
7.72
3.27
3.94
6.39
1.81
8.80
6.02
10.13
6.43
12.74
1.66
3.64
3.49
3.50
3.82
3.73
3.95
4.77
0.600
0.730
0.730
-1.000
0.091
0.039
0.039
0.005
-0.867
-0.067
0.867
0.733
-0.600
-0.467
-1.000
-0.867
0.600
0.015
0.851
0.015
0.039
0.091
0.188
0.005
0.015
0.091
1.75
1.69
1.72
-0.886
0.019
2.22
1.98
2.05
db/mol
4.78
3.51
3.80
2.84
2.82
2.50
-0.943
0.005
Unsat/sat
a Spearman rank correlation coefficient for proportion of fatty acid against incubation time at 25°C.
a Two-tailed probability of this result.
'Structure unknown.
Electron microscopy revealed extensive alterations to the celTable IV. Phospholipid Fatty Acid Composition of C. reinhardii CW15+ lular ultrastructure associated with freeze-thaw damage. These
following Freezing to -5°Cfor Diferent Times, Thawing, and Holding at changes were apparent immediately upon thawing and so numer250Cfor 5 Minutes
ous that is not possible to speculate on a specific primary site of
Data reported as for Table II and represent replicates from the same injury. However, one pathological feature of interest was the
appearance in the cytoplasm of densely staining material (Fig. 3,
culture.
C and D) which was probably lipid.
Unfrozen Incubation at -5°C (min)
Changes in the phospholipid fatty acid composition of C.
r8a
Fatty Acid
Control
ph
reinhardii CW15+ were observed both with prolonged incubation
500
5
50
following freezing and thawing (Table III) and to a lesser extent
0.200 0.800
17.97 17.74 26.07
20.76
16:0
during long-term maintenance in frozen medium (Table IV). In
2.71
0.800 0.200
2.14
2.59
2.18
16: 1w7
both cases, the major alteration was a reduction in the proportion
0.200 0.800
2.40
1.84
3.00
2.54
16:2w6
of the more highly unsaturated fatty acids, as has been reported
2.46
2.68 -0.200 0.800 for wild-type C. reinhardii (15) and Chlorella emersonii (14). The
2.05
3.18
16:3c
1.14
0.94
1.91
1.08
0.400 0.600 most likely explanation for these changes in fatty acid composition
16:3c
15.61
14.50 13.09 10.85 -1.000 0.001
16:403
is either the activation of intracellular phospholipases or peroxi1.99
2.43
1.78
2.87
0.200 0.800 dation.
18:0
0.64
0.60
0.82
0.800 0.200
0.58
18: 1w9
Phospholipase A2 cleaves fatty acids from the sn-2 position of
5.89
0.800 0.200
4.72
5.68
5.00
18: 1w7
the glycero-phosphate backbone, whereas phospholipase B cleaves
0
0.999 acids from both the sn-l and sn-2 positions (12). Plant fatty acids
11.51
13.18 11.50 12.50
18:2w6
9.39
9.50
8.56
8.18
0.800 0.200 are not generally randomly attached to the glycerophosphate
18:3w6
21.57
22.72 24.19 17.77 -0.200 0.800 backbone, but have a tendency for saturated acids to occupy the
18:3w3
1.65
7.19
1.22 -0.400 0.600 sn- 1 position, and polyunsaturated acids to occupy the sn-2 posi2.35
18:403
0.93
1.1-6
2.19
1.28
0.400 0.600 tion (10). Phospholipase A2 activity is thus liable to result in just
18:5C
those alterations of fatty acid composition reported in Tables II to
V. Also, release of free fatty acids has been demonstrated by TLC
in freeze-damaged wild-type C. reinhardii (15) and Chlorella emer0.86
3.06
1.77
1.34
nents
sonii (14).
Phospholipase D, which is activated following the freezing and
2.21
2.23
2.33
1.93 -0.200 0.800 thawing of woody twigs (27-29), removes the head group and thus
db/mol
3.99
3.27
4.07
2.44 -0.200 0.800 liberates phosphatidic acid but not free fatty acid. It is also possible
unsat/sat
a Spearman rank correlation coefficient for proportion of fatty acid that a peroxidation process may occur following production of
against incubation time at -5°C.
free radicals; such a reaction would selectively degrade polyunb
Two-tailed probability of this result.
saturated fatty acids and be autocatalytic. The relatively clean
c Structure unknown.
capillary GC traces, with no indication of partially oxidized
Unknowns and
minor compo-
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Copyright © 1982 American Society of Plant Biologists. All rights reserved.
102
Plant Physiol. Vol. 70, 1982
CLARKE ET AL.
Table V. Phospholipid Fatty Acid Composition of C. reinhardii CW15+ following Freezing to D/foerent Final Temperatures, Thawing, and Holding at
25°Cfor 5 Minutes
Data reported as for Table 11 and represent replicates from the same culture. All statistics exclude data for the control.
Fatty Acid
16:0
16: 1 7
16:1 (CR8)C
16:2w6
16:3c
16:403
18:0
18:1w9
18: 1 7
18:2w6
18:3w6
18:303
18:403
18:5c
Unknowns
and minor
components
Final Temperature (°C)
5
7
9
oUnfrozen
Control
~1
3
12.12
7.38
1.32
2.26
10.37
12.69
1.06
3.79
2.56
11.63
4.93
22.35
1.10
4.06
13.69
7.76
1.83
2.50
9.98
12.02
1.01
4.23
2.59
12.26
4.63
21.91
1.05
2.22
14.24
7.99
1.79
2.30
9.64
11.68
0.96
4.18
2.59
12.04
4.17
21.63
0.98
3.44
14.23
7.03
1.72
2.47
10.05
12.29
0.90
3.86
2.45
11.98
4.39
21.61
1.23
3.22
14.82
7.06
1.82
2.53
9.72
11.92
0.90
3.73
2.83
12.14
4.42
21.30
0.99
3.65
2.38
2.32
2.37
2.57
2.17
12
15
14.69
6.56
1.68
2.53
9.79
11.92
0.89
3.51
2.83
11.81
4.34
20.93
0.94
5.26
14.23
7.45
1.45
2.47
9.63
11.39
1.01
4.08
2.75
11.93
4.40
20.86
0.96
4.94
15.59
7.23
1.67
2.56
9.54
11.09
0.98
4.00
2.85
11.98
4.29
20.73
0.95
3.96
2.32
2.45
2.55
2.21
2.24
2.33
2.20
2.22
2.27
2.25
2.18
db/mol
5.17
6.02
5.41
5.22
5.00
5.12
5.00
4.66
Unsat/sat
a Spearman rank correlation coefficient for proportion of fatty acid against final temperature.
b Two-tailed probability of this result.
c Structure unknown.
d Control value outside the 95% confidence limits of the experimental data.
br8
mean
14.50
7.30
1.71
2.48
9.76
11.76
0.93
3.94
2.70
12.02
4.38
21.28
1.01
3.81
0.23
0.18
0.05
0.03
0.07
0.15
0.02
0.10
0.06
0.06
0.05
0.17
0.04
0.39
2.22
5.08
0.01
0.08
SE
+d
+
+
+
+
+
+
+
+
+
+
0.631
-0.393
-0.857
0.546
-0.679
-0.703
-0.036
-0.464
0.764
-0.685
-0.286
-1.000
-0.714
0.821
0.129
0.383
0.014
0.205
0.094
0.078
0.938
0.294
0.046
0.090
0.535
0.001
0.071
0.023
0.179
-0.847
0.702
0.016
components, and the large amounts of free fatty acid released Changes in lipid composition under these conditions were, how(14,15), however, suggest strongly that damage is enzymic rather ever, slight (Table IV).
The consistent small differences between control and treated
than by peroxidation.
Whereas the specific mechanism of these alterations in lipid cells following either short periods of incubation at 25°C after
structure requires further elucidation, the nature of the relation- freezing to -5°C (Table IV) or after freezing to different tempership between those changes we have observed and the reduction atures (Table V) are interesting. The method used for freezing the
in cellular viability is of critical importance for an understanding cell suspension (13) involves a reduction in temperature to -1I C
when the sample is nucleated, and then holding at -1C as the
of freezing injury.
Yoshida has proposed that activation of phospholipase D is the latent heat of fusion is dissipated, before cooling at a rate of
primary mechanism of freeze-thaw damage in woody twigs (27- 0.25°C min-' is continued. This sequence takes 20 min to produce
29). Although phospholipase D activity was not monitored in this a frozen sample in equilibrium at -1°C, and it is possible that
study, the relationship between alterations in the fatty acid com- either a rapid acclimation process occurs or, more likely, that some
position, indicative of the activity of phospholipase A or B, and lipid is lost from the protoplast membrane is response to exposure
experimental treatments suggests that lipid alterations are a sec- to the hypertonic solutions formed during freezing (21). It is also
possible that some enzymic damage occurs during the short time
ondary event, and not the primary cause of freezing injury.
Several independent parameters, including loss of motility, interval between thawing the cell suspension at 25°C and lipid
release of intracellular enzymes (Fig. 2), alterations in cellular extraction, or even while frozen (9).
It is clear that the time course of alterations in the phospholipid
ultrastructure (Fig. 3), reduction in cell surface charge (7), and
loss of viability (Table I), all occurred more or less immediately fatty acid composition of Chlamydomonas reinhardii CW15+ folupon thawing from -5°C. Also, cryomicroscopy (16) and electron lowing freezing and thawing of the cell suspension and the time
microscopy (Fig. 3) showed that cell disruption occurred during course of changes in membrane integrity and cellular ultrastructhawing rather than the initial freezing. In contrast, the changes ture are very different. Damage as assessed by motility, enzyme
in lipid composition were only slight at first, becoming more release, and loss of ultrastructure occur rapidly; significant altermarked with increasing time the cells were maintained at 25°C ations in lipid composition only appear after incubation at 25°C.
(Table III). If lipids were extracted within 5 min of thawing after We conclude that in C. reinhardii CW15+ alterations in phosphofrom
exposure to extracellular ice at various temperatures, with the lipid fatty acid composition are a separate phenomenon
in
these
that
and
of
loss
freeze-induced
changes
lipid
viability,
exception of 16:1 (CR8) and 18:3w3, there were no systematic
variations in fatty acid composition with temperature (Table V), composition are not the primary cause of freezing injury but
despite the range of final temperatures achieved and survivals secondary, pathological events.
varying from 0 to 100%.
LITERATURE CITED
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Copyright © 1982 American Society of Plant Biologists. All rights reserved.
103
FREEZING INJURY IN CHLAMYDOMONAS
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