CryoLetters 24, 213-228 (2003)
Ó CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
A COMPARATIVE STUDY OF ANTIOXIDANT PROTECTION
IN CRYOPRESERVED UNICELLULAR ALGAE
Euglena gracilis AND Haematococcus pluvialis
Roland A. Fleck1#, Erica E. Benson2*, David H. Bremner2 and John G. Day1
1
CCAP, CEH Windermere, Far Sawrey, Ambleside, Cumbria, LA22 0LP.
Conservation and Environmental Chemistry Centre, School of Contemporary Science,
University of Abertay Dundee, Bell Street, Dundee, DD1 1HG. #Current address: National
Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar,
Herts. EN6 3QG. *Corresponding author [email protected].
2
Abstract
Algal culture collections are required to develop robust and broadly applicable cryogenic
storage methods for diverse taxonomic groups and understanding differential responses to
cryoinjury helps to achieve this end. Antioxidant profiles were constructed for cryopreserved
Euglena gracilis (Klebs CCAP 1224/5Z), a freeze sensitive alga, and Haematococcus
pluvialis (Flotow CCAP 34/8), which is a highly freeze-tolerant. H. pluvialis had a
coordinated antioxidant response with respect to catalase, superoxide dismutase (SOD) and
glutathione reductase; it is postulated that this may contribute to freeze tolerance. Formation
(via SOD) and removal (via catalase) of H2O2 were not fully coordinated in freeze-sensitive E.
gracilis and this may exacerbate cryoinjury. Increased SOD activity in the absence of catalase
thus compromises survival due to the formation of hydroxyl radicals (.OH) from H2O2.
Changes in sulfhydryl group (SH) status for non-Protein bound SH groups were greater in
freeze-tolerant H. pluvialis. Therefore, the tolerant organism may have a range of coordinated
protection mechanisms that ameliorate the deleterious effects of oxidative stress during
cryopreservation.
Keywords: cryopreservation, antioxidants, free radicals, oxidative stress, Fenton chemistry.
INTRODUCTION
Cryopreservation is an important means of conserving algae and cyanobacteria; however,
as many organisms are storage recalcitrant (14,15,17,37) understanding the basis of cryoinjury
is important for the development of improved conservation protocols. Oxidative stress has
been observed in plant (3-7), algal (16) and mammalian (19,20,26), tissues exposed to
cryogenic treatments and it is probable that antioxidant protection has a role in post-storage
recovery (16,19,20). This possibility was first considered by Levitt who, over 40 years ago
(27) proposed the sulphide-disulphide theory of freezing damage. Thus, freeze-induced
destabilisation and oxidation of SH groups in macromolecules promotes denaturation and the
conformational changes that impair cellular function.
The objective of this present study is to profile changes in antioxidant protection and
primary metabolism (photosynthesis) in two algae: Euglena gracilis, (Klebs CCAP 1224/5Z),
a freeze-sensitive organism (15,16,) and highly freeze-tolerant Haematococcus pluvialis
(Flotow CCAP 34/8). It has been demonstrated previously (16) that hydroxyl radical (.OH)
activity increases in E. gracilis cells exposed to cryogenic treatments. The production of .OH
via Fenton chemistry in vitro and the Haber-Weiss reaction, in vivo, may be an important
component of cryoinjury in this organism. The addition of iron chelating agents such as
desferrioxamine removes the metal catalyst that results in the production of hydroxyl radicals
as shown in the schematics below.
The Fenton Reaction
H2O2
hydrogen
peroxide
+
.OH
Fe2+
ferrous
ion
Fe3+
+
hydroxyl
radical
+
ferric ion
HO
hydroxide
ion
The Haber-Weiss Reaction
.
.
_ metal ions
+ O2
OH +
hydrogen superoxide
hydroxyl
peroxide
radical
H2O 2
O2
oxygen
+
_
HO
hydroxide
ion
The authors have previously provided evidence for Fenton and Haber-Weiss chemistry
being a potential component of cryoinjury in vitro. Thus, the addition of the potent iron
chelating agent desferrioxamine enhanced post-storage survival in E. gracilis as well as plant
cells (7,16). This investigation builds upon these earlier findings by exploring the role of
antioxidant enzymes involved in O2.- and H2O2 removal and production in cryopreservationinduced stress. Superoxide dismutase (SOD), catalyses the conversion (30,38) of superoxide
(O2×-) to hydrogen peroxide (H2O2) and SOD is induced in plants exposed to drought, chilling,
anoxia and pathogenic injury (3,21,22, 38). H2O2 is also cytotoxic and its removal by catalase
and peroxidases is most important (30,38) as this reduces the possibility of reactions occurring
between O2×-, ×OH, O2×-, H2O2. These reactive oxygen species (ROS) react with lipids to form
cytotoxic, aldehydic lipid peroxidation products that are potentially damaging to stored
germplasm (3). They are also involved in the formation of singlet oxygen 1O2 (4). This study
will investigate the possibility that SH status and antioxidant enzyme protection (specifically,
SOD, catalase, peroxidase and glutathione reductase) have a role in the ability of algae to
recover from cryogenic storage.
One of the major applied objectives of this study is to help develop markers of oxidative
stress that could be used in the future, to detect differential responses to cryoinjury across the
very wide range of algal strains and species that are currently held in working culture
collections. For this reason two different algae have been selected, but ones, which are
maintained under, identical, conditions and that are of similar size. These algae will therefore
be compared on the basis of their differential responses to the previously optimised
cryopreservation protocols to which they best respond. The development of cryopreservation
protocols that can be applied to a diverse spectrum of organisms is highly desirable and this
study will explore differences in antioxidant protection strategies for two diverse algae. This
approach will help elucidate which protection systems contribute to enhanced post-storage
survival and it may therefore lead to the development of improved protocols for
cryopreservation-recalcitrant strains. For example, through the application of targeted
exogenous antioxidants that enhance post-cryogenic storage survival.
Of the two algae selected, one (H. pluvialis) is well adapted to survive environmental
stress such as desiccation and is therefore very likely to recover from cryogenic injury. As
oxidative stress is a generic response in aerobes it may be postulated that H. pluvialis may
have a high capacity for mitigating the effects of free radical injury. The other alga, E.
gracilis, has similar morphogenetic properties to H. pluvialis in that it has similar growth
rates, is of a comparable size and is a flagellate. However, E. gracilis does not manifest
robust, adaptive stress responses in vivo and it is more sensitive to freezing injury. This study
thus compares organisms from two different polarities of stress and tolerance sensitivities. By
doing so it may be possible to “identify” or select algae within a collection, which, based upon
their in vivo responses may be better equipped to survive cryopreservation. Undertaking
comparative physiological studies of diverse organisms may therefore help elucidate the
different patterns of cryoinjury and protection in large-scale international culture collections.
MATERIALS AND METHODS
Routine culture and cryopreservation
Euglena gracilis (Klebs CCAP 1224/5Z) and Haematococcus pluvialis (Flotow, CCAP
34/8) cultures were routinely cultured in Euglena gracilis medium (designated as EG) and
Jaworski’s medium (designated as JM) mixed in 1:1 proportions (37). Cryopreserved cells
were recovered in the same medium. Cultures were maintained at 15ºC under a 12:12h light:
dark regime, illumination was provided by cool white fluorescent lamps with a photon flux
density of 50mmol.m-2.s-1 at the surface of the culture vessel. All cells were harvested for
cryopreservation using centrifugation (1min at 1000rpm, Sanyo-MSE, Cambridge, UK), by
placing in a cryoprotectant (Sigma, USA, Analar/spectroscopic Grade) and transferring to 2ml
NUNC cryovials. Controlled rate cooling was undertaken using a Planer Kryo 10
Programmable Freezer (Planer Select, UK) and cells were maintained for a minimum of 12
hours in liquid nitrogen before thawing. E. gracilis was cryopreserved using the method of
Fleck et al. (16). Cells were cryoprotected using 10% (v/v) methanol (applied at 0ºC on ice
for 15 min.) and exposed to a cooling rate of –0.5ºC min-1 to a terminal transfer temperature
of –60ºC where they were held isothermally for 30 mins before transfer to –196ºC. E. gracilis
was thawed using a two-step protocol in which the vials were maintained in air at room
temperature for 1 min and then rewarmed in a water bath held at 40ºC and agitated until all
the ice had melted. H. pluvialis was cryopreserved as described by Fleck (15). Cells were
cryoprotected using 5% (v/v) dimethyl sulphoxide (DMSO) (applied at 20-22ºC for 15 min.)
and exposed to a cooling rate of –1.0ºC min-1 to a terminal transfer temperature of –35ºC
where they were held isothermally for 30 mins. before transfer to –196ºC. H. pluvialis was
thawed using a single-step protocol in a pre-heated water bath (40ºC) and agitated until all the
ice had melted as described by Day et al. (14). Following thawing, cells were centrifuged (1
min at 2000 rpm, Sanyo-MSE, Cambridge, UK) and the cryoprotectants removed and replaced
with two washes of culture medium to ensure complete removal of the cryoprotective
additive. During recovery, cultures were maintained using initial incubation temperatures of
22ºC, followed by maintenance at 15ºC under light regime described above. Where
treatments are applied in the laboratory at ambient room temperature (designated as RT) this
is in the range of 22-25ºC.
Cell viability, photosynthetic activity and chlorophyll content
Viability was measured using fluorescein diacetate vital staining as described by Pickup et
al. (33) and Fleck (15) using a FACStar Plus flow cytometer (Becton, Dickinson, UK).
Photosynthesis was assessed as O2 evolution (15) using an Oxygen Electrode (Rank, UK) and
cells were exposed to non-limiting conditions under constant illumination (440mmol.m2.s-1)
using Cool White fluorescent bulbs. Chlorophyll was determined using the methods and
equations as described by MacKinney (28). Viability assessments were undertaken 24 hours
after thawing.
Cell extraction, protein determination and antioxidant measurement
Cells were centrifuged twice (2000 rpm, Sanyo-MSE, Cambridge, UK) post-treatment
ensuring that all cryoprotectant and medium were removed prior to cellular extraction. The
supernatant was discarded and the cells re-suspended in 1ml of extraction buffer (50mM
potassium phosphate buffer (31) at pH 7, comprising: KH2PO4, K2HPO4, 1mM CaCl2, and 1
mM EDTA. Cells were then flash frozen in liquid nitrogen (LN) and transferred to a precooled freeze-dryer (Edwards, UK) and lyophilised in 2 ml Eppendorf (Germany) vials.
Following freeze-drying the vials were resealed and stored under liquid nitrogen until ready
for use. Lyophilised cells were ground for 5min, under LN, with an adapted drill fitted with a
grinding bit (fixed low revolution) polypropylene pestle, (Sigma, USA), in a pre-cooled
(-196ºC), custom-made brass tube holder. Extracts were re-suspended in 2ml of chilled
extraction buffer, vortexed for 1min, centrifuged at 4ºC/14,000 rpm in a Microfuge (SanyoMSE, Cambridge, UK) and supernatants stored under liquid nitrogen until required. Total
soluble protein was measured using the method of Bradford (9) as applied to a protein assay
kit using Coomassie Blue stain (Pierce, USA). Assays were performed following the
manufacturers guidelines and BSA protein standards (range of 5-100mg protein/assay
volume). All assays were performed in triplicate. Protein and antioxidant enzyme assays were
performed in triplicate at 25ºC. SOD activity was determined using the spectroscopic method
of Beauchamp and Fridovich (2), which monitors the inhibition (at 570nm) of
nitrotetrazolium blue (NBT) reduction by light-generated superoxide radicals. Catalase was
determined by the UV spectroscopic method of Aebi (1) as the rate of decrease in ultraviolet
(UV) absorbance of H2O2 at 240nm. Peroxidase activity was measured spectroscopically
using the method of Murphy and Huerta (31) for guaiacol specific peroxidases and monitored
as rate of change in absorbance at 470nm. Glutathione reductase (GR) activity was
determined spectrophotometrically at 334 nm as the oxidation of NADPH using the method
described by Goldberg & Spooner (18).
Sulfhydryl groups were determined
spectroscopically (412 nm) as total and non-protein fractions as described by Chevrier et al.
(12,23) based on the reduction of 5,5¢-dithiobis-(2-nitrobenzoic acid) by SH to 2-nitro-5mercaptobenzoic acid (34). Validation experiments were performed using extraction buffer,
JM media and cryoprotectants to ensure that these components did not cause assay
interference, all tests were negative.
Experimental design and data analysis
The differences between treatment groups, time points, and algae were assessed using 2way or 3-way analysis of variance (ANOVA). Calculations were performed with the SAS
statistical package, using the PROC GLM procedure. The ANOVA approach gives the overall
significance of differences between groups/treatments/time-points. The Duncan’s Multiple
Range method for multiple comparisons was used to assess the significance of differences
between individual group/treatment/time-point means (41).
RESULTS
H. pluvialis (Table 1) is highly amenable to cryopreservation with post-thaw viability levels
approaching 100% with little or no loss of viability associated with freezing, chilling or
DMSO exposure; in contrast E. gracilis had lower post-thaw viability levels of <65% and the
greatest reduction in viability was monitored after exposure to liquid nitrogen.
Table 1. Effects of different cryopreservation treatments on the viability (as fluorescein
diacetate vital staining) of E. gracilis and H. pluvialis
Treatment
Organism/Viability (%)
E. gracilis
H. pluvialis
100 ± 0
100 ± 0
Chilling (0 C)
97 ± 1
95 ± 2
Cryoprotectant exposure
98 ± 1
92 ± 2
Freeze/Thaw from Holding
78 ± 2
92 ± 3
Freeze/Thaw from LN
64 ± 4
94 ± 1
Non-cryoprotected exposure to LN
0±0
0±0
Control
o
Viability is expressed as a percentage of the untreated control. Holding isothermal
temperatures were –60°C for E. gracilis and –35°C for H. pluvialis. Liquid nitrogen is
designated as LN. n = 3, replicates and error terms are expressed as standard errors of
mean. Viability assessments were undertaken 24 hours after thawing.
Photosynthetic capacity in E. gracilis was impaired (Fig. 1) following a range of different
cryogenic treatments, whereas oxygen evolution was only reduced in H. pluvialis after the
freeze/thaw cycle, with reduction in photosynthetic activity correlating with viability loss
following cryopreservation (compare Fig. 1, Table 1). In the case of some treatments using
cryoprotective additives, photosynthetic activity was enhanced in H. pluvialis. Sulfhydryl
groups (total, protein and non-protein) were detected in both algae and their distribution
varied with respect to organism and treatment (Figs. 2 and 3, noting differences in scale of the
y-axis). Total SH levels (as combined non-protein and protein SH groups) were higher in E.
gracilis as compared to H. pluvialis. Similarly, significantly higher levels of non-protein SH
groups were detected in E. gracilis than in H. pluvialis with respect to both treatment and time
(F14, 83 = 15.76, P < 0.0001; Figs. 2 and 3). Exposing E. gracilis to cryogenic treatments
affected non-protein SH group status, showing a significant interaction with recovery time (F2,
41 = 235.70, P < 0.0001; Fig. 2). Freeze-thaw stresses initially reduced non-protein bound SH,
but a significant increase in non-protein bound SH occurred as recovery progressed. Chilling
and cryoprotection significantly reduced non-protein bound SH groups in E. gracilis with a
significant interaction between recovery period and treatment (F12, 41 = 7.88, P < 0.0001, Fig.
2). H. pluvialis did not exhibit changes in non-protein SH groups with the exception that an
increase in non-protein SH groups was detected at 48h (Fig. 3).
Photosynthesis as O2 Evolution
(as % of untreated control,
-1
mmol O2.mg Chl a.h )
140
120
100
80
60
40
20
0
a
Euglena gracilis
Haematococcus pluvialis
b
c
d
e
f
g
h
i
Treatment
Figure 1. Effects of cryopreservation treatments on photosynthetic capacity (as O2 evolution)
in E. gracilis and H. pluvialis.
Cell treatments designated as: (a) untreated controls (20°C), (b) cooled to 0°C and held for
15min, (c) removed from cryoprotectant after 15min exposure, (d) exposed to cryoprotectant
for 15min, (e) plunged directly into LN, no cryoprotectant present, (f) control-cooled to a
holding temperature (-60°C for E. gracilis and –35°C for H. pluvialis) and held for 30min (g)
plunged into LN from holding temperature, (h) control-cooled to holding temperature for
30min, 24h post treatment, (i) plunged directly into LN from holding temperature, 24h posttreatment. n = 3, errors are expressed as standard errors of mean. H. pluvialis exposed to
5% (v/v) DMSO at 25°C. E. gracilis exposed to 10 % (v/v) methanol at 0°C. H. pluvialis
-1
control-cooled from 22°C at –1°C min to –35°C. E. gracilis control-cooled from 0°C at
-1
–0.5°C.min to –60°C. Photosynthesis determined on the basis of O 2 evolution (as a ratio of
chlorophyll content) and calculated as a percentage of the non-treated control.
The distribution of antioxidant enzymes in the two algae varied considerably. Peroxidase
was not detectable in either organism and was at the limits of assay detection, catalase was not
detected in E. gracilis but was present in H. pluvialis. SOD and glutathione reductase were
detected in both algae but at different levels. E. gracilis had a higher SOD activity as
compared to H. pluvialis and in contrast, H. pluvialis had a higher glutathione reductase
activity.
Protein SH
200
180
160
140
120
100
Sulfhyrdryl content
4
(expressed as nmol per 10 Cells)
80
60
40
20
0
a
b
c
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
20
Non-protein SH
18
16
14
12
10
8
6
4
2
0
a
b
c
Time zero
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
Treatment
24h post-treatment
48h post-treatment
Figure 2. Effects of cryopreservation treatments on protein and non-protein sulfhydryl group
(SH) status of E. gracilis.
(a) Untreated control cells (20°C), (b) cells cooled to 0°C and held for 15min, (c) cells
exposed to cryoprotectant at 20°C (10% (v/v) methanol) for 15min, (d) cells exposed to
cryoprotectant at 0°C (10% (v/v) methanol) for 15 min., (e) cells control-cooled from 0°C at
–0.5°C.min-1 to –60°C and held for 30min, (f) cells plunged into LN from –60°C, (g) cells
plunged directly into LN, without cryoprotectant. n = 3, error terms are expressed as
standard errors of mean.
25
Protein SH
20
15
Sulfhyrdryl content
4
(expressed as nmol per 10 Cells)
10
5
0
a
b
c
d
e
f
g
a b
c
d e
f
g
a b
c
d e
f
g
5
Non-protein SH
4
3
2
1
0
a
b
Time zero
c
d
e
f
g
a b
c
d e
f
g
a b
c
d e
f
g
Treatment
24h post-treatment
48h post-treatment
Figure 3. Effects of cryopreservation treatments on protein and non-protein sulfhydryl group
(SH) status of H. pluvialis
(a) Untreated control cells (20°C), (b) cells cooled to 0°C and held for 15min, (c) cells
exposed to cryoprotectant at RT (5% (v/v) DMSO) for 15min, (d) cells exposed to
cryoprotectant at 22°C (5% (v/v) DMSO) for 15min, (e) cells control-cooled from 22°C at
–1°C.min-1 to –35°C and held for 30min, (f) cells plunged into LN from –35°C, (g) cells
plunged directly into LN, without cryoprotectant. n = 3, errors are expressed as standard
errors of mean.
350
Euglena gracilis
300
250
200
150
50
-1
SOD activity.100mg Protein
100
0
a
b
c
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
b
c
d
e
f
g
a
b
c
d
e
f
g
350
Haematococcus pluvialis
300
250
200
150
100
50
0
a
b
c
Tim e Zero
d
e
f
g
a
Treatment
24h post-treatm ent
48h post-treatm ent
Figure 4. Effects of cryopreservation treatments on SOD activities in E. gracilis and H.
pluvialis
(a) Untreated control cells (20°C/RT), (b) cells cooled to 0°C and held for 15min, (c) cells
exposed to cryoprotectant at RT for 15min, (d) cells exposed to cryoprotectant at 0°C for
15min, (e) cells control-cooled to a predetermined intermediate temperature and held for
30min, (f) cells plunged into LN from the intermediate holding temperature, (g) cells plunged
directly into LN, without cryoprotectant. n = 3, errors are expressed as standard errors of
mean. H. pluvialis exposed to 5% (v/v) DMSO. E. gracilis exposed to 10 % (v/v) methanol. H.
-1
pluvialis control cooled from 22°C at –1°C.min to –35°C. E. gracilis control cooled from 0°C
-1
at –0.5°C.min to -60°C.
0.03
Euglena gracilis
0.01
-1
Glutathione reductase activity.100mg Protein
-1
as change in A339.min
0.02
0.00
0.03
a
b
c
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
Haematococcus pluvialis
0.02
0.01
0.00
a
b
Time Zero
c
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
Treatment
24h post-treatment
48h post-treatment
Figure 5. Effects of cryopreservation treatments on glutathione reductase activities in E.
gracilis and H. pluvialis
(a) Untreated control cells (20°C/RT), (b) cells cooled to 0°C and held for 15min, (c) cells
exposed to cryoprotectant at RT for 15min, (d) cells exposed to cryoprotectant at 0°C for
15min, (e) cells control-cooled to a predetermined intermediate temperature and held for
30min, (f) cells plunged into LN from the intermediate holding temperature, (g) cells plunged
directly into LN, without cryoprotectant. n = 3, errors are expressed as standard errors of
mean. H. pluvialis exposed to 5% (v/v) DMSO. E. gracilis exposed to 10 % (v/v) methanol.
-1
H. pluvialis control cooled from 22°C at –1°C.min to –35°C. E. gracilis control cooled from
-1
0°C at –0.5°C.min to –60°C.
Exposure to freezing caused substantial and significant increases in SOD activity in E.
gracilis and this continued during recovery with a synergistic interaction (F12, 39 = 27.51, P <
0.0001) between treatment and recovery period (Fig. 4). Individual steps of the
cryopreservation protocol affected levels of SOD activity in H. pluvialis and these treatments
had a significant interaction (F12, 39 = 28.25, P < 0.0001) with respect to recovery period. SOD
activity increased in all algal samples exposed to stress and highest SOD activities were in
cells exposed to subzero treatments (F2, 33 = 115.93, P < 0.0001). SOD activity in E. gracilis
changed dramatically after exposure to freezing and were significantly higher (F6, 33 = 834.83,
P < 0.0001) than those detected in H. pluvialis (Fig. 4). Interestingly, non-cryogenic
treatments also influenced SOD activity.
0.045
-1
Catalase activity.100mg Protein
-1
as change in A240.min
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
a
b
Time Zero
c
d e
f
g
a b
c
d e
f
g
a
b c
d e
f
g
Treatment
24h post-treatment
48h post-treatment
Figure 6. Effects of cryopreservation treatments on catalase activity in H. pluvialis
a) Untreated control cells (20°C/RT), (b) cells cooled to 0°C and held for 15min, (c) cells
exposed to cryoprotectant at RT for 15min, (d) cells exposed to cryoprotectant at 0°C for
15min, (e) cells control-cooled to a predetermined intermediate temperature and held for
30min, (f) cells plunged into LN from the intermediate holding temperature, (g) cells plunged
directly into LN, without cryoprotectant. n = 3, errors are expressed as standard errors of
-1
mean. Exposed to 5% (v/v) DMSO. Control cooled from 22°C at –1°C.min to –35°C.
E. gracilis had significantly higher (F6, 65 = 5.37, P < 0.0001) level of glutathione
reductase activity than H. pluvialis (Fig. 5). Exposing E. gracilis and H. pluvialis to cryogenic
and non-cryogenic stresses significantly increased (F6, 35 = 12.26, P < 0.0001 and F6, 30 =
13.95, P < 0.0001 respectively) glutathione reductase activities (Fig. 5). Treatments had a
greater effect on glutathione reductase activity in E. gracilis than in H. pluvialis following
exposure to subzero temperatures (F14, 65 = 4.22, P < 0.0001) and this was concomitant with
increased glutathione reductase activity during the 48h recovery period (Fig. 5).
In considering glutathione reductase activities, it is interesting to note that for the
Euglenoid there was a significant interaction (F 12,40 = 18.75, P < 0.0001) between, treatment
and recovery period for Protein-SH groups. This was noted for cells that had been exposed to
cryogenic temperatures and was observed as an increase at 24 hours. A similar effect was
noted for H. pluvialis (Figs. 2 and 3) at 24-48 hours (F 12,41 = 4.27, P < 0.0001). However, the
highest levels of Protein-SH were detected in cultures that had not been exposed to cooling
below 0oC (Fig. 3). Consideration of Protein-SH and non-Protein SH evaluations (Total SH)
at 48 hours (see Fig. 3) indicates that effects of cryogenic treatments are minimised as
recovery progresses, as compared to time zero and 24 hour evaluations.
Catalase activity was absent, or at the limits of detection, for all E. gracilis treatments, but
was detectable in H. pluvialis. Treatment stresses significantly (F2,41 = 75.58, P < 0.0001)
affected levels of catalase activity in H. pluvialis and this changed significantly (F12,41 = 6.72,
P < 0.0001) over recovery time (Fig. 6). Catalase activity in H. pluvialis increased during
post-treatment recovery with maximum levels observed in those cells exposed to chilling
stress.
DISCUSSION
General Observations
The algae displayed differential responses to cryopreservation, exemplified by the
contrasting protocols applied to each organism. E. gracilis is particularly sensitive to DMSO
(15) applied at the levels required for cryoprotection and a slow rate of cooling must be used
to ensure post-freeze survival. In contrast, H. pluvialis is more robust, able to tolerate DMSO,
and will survive cryogenic storage following a rapid rates of cooling. Post-cryopreservation
survival approaches 100% in H. pluvialis whereas in contrast it is ca. 65% in E. gracilis.
That these organisms respond differently to cryogenic storage is demonstrated in Figure 1,
which shows that photosynthesis is particularly impaired in E. gracilis exposed to different
cryogenic and non-cryogenic treatments.
The potential for free radical production can be increased if primary metabolism and
electron transfer processes become uncoupled. It would thus be expected that those organisms
which display metabolic perturbation on freezing and chilling might also have a propensity
for increased free radical production. It is interesting to note that non-cryoprotected cells
exposed to liquid nitrogen exhibit O2 evolution, however this may be due to O2 production
from non-coupled photosynthesis and, or photoxidation events involving singlet oxygen (4).
Previous studies performed on E. gracilis have demonstrated differences in response to
cryopreservation, including photosynthetic inhibition, loss of osmotic responsiveness (15) and
hydroxyl radical injury following cryopreservation (15-17). Cold stress and freezing injury in
chloroplasts may promote oxidative injury by uncoupling electron flow, incurring membrane
damage and perturbing the proton pump involved in photophosphorylation. Low temperatures
will also reduce the activities of enzymes in the Calvin-Benson cycle thereby causing an
uncoupling of the regulation of the light and dark reactions (40). In contrast to E. gracilis,
present and previous investigations did not indicate a major inhibitory effect on
photosynthetic capacity in cryotolerant H. pluvialis following exposure to subzero
temperatures (15).
Carotenoids may also confer antioxidant protection to H. pluvialis, cultures which may
contain aplanospores. These are resting structures which accumulate carotenoids at >1% of
their dry biomass (11). The antioxidant profiles of E. gracilis and H. pluvialis are very
different in terms of the detectable presence of the antioxidants monitored in this study, the
quantitative distribution of which varies with respect to both cryogenic and non-cryogenic
stresses. Thus, guaiacol specific peroxidases were not detected in either alga. However, it is
important to note that these peroxidases are associated with substrate level peroxidation
reactions rather than the removal of H2O2.
It is also possible that ascorbate peroxidase and glutathione peroxidase (24,25,32) may be
implicated in algal antioxidant protection during cryogenic stress and future studies will aim
to elucidate if this is the case. Catalase was not detected in E. gracilis although it is
considered to be present in all aerobic organisms including algae (29, 36) and that it was not
detectable in this study of E. gracilis may be due to the fact that the enzyme was either not
expressed or expressed at non-detectable levels. Profiles of SH and glutathione reductase were
very different between the organisms, particularly with respect to cryogenic treatments.
Euglenoid cells are not only bound by the plasmalemma but also a helical proteinaceous,
pellicle, comprising up to 80% protein (8). In contrast, the cells of fresh-water chlorophyte
species may only contain low levels of protein (8,13). These differences may in part explain
the higher levels of total (non-protein and protein SH, see Fig. 2) detected in E. gracilis as
compared to H. pluvialis (Figure 3). Antioxidant enzyme activity was detected in noncryoprotected cells exposed to liquid nitrogen. Lethally damaged cells may be expected to
display residual enzyme activity as abiotic (chemical as opposed to metabolic) free radicalmediated oxidative reactions will occur in dead cells. Oxidative processes may well be
different, in non-viable cells, as it will not be mediated by active metabolism and the
uncoupling of primary pathways from electron transfer reactions. Comparisons of lethal, sublethal and non-lethal treatments must therefore take into account the dynamics of pro- and
antioxidant pathways in the recovery processes of mixed populations of non-viable, viable and
recovering cells.
Sulfhydryl Group Status
Levitt’s theory (27) pertaining to SH groups and freezing injury provides an interesting
basis on which to study the involvement of SH status and glutathione reductase in algal
cryotolerance. Recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), has
an important function in protecting membranes from oxidative damage as GSH protects
oxygen-sensitive proteins by providing a preferential substrate for S-H oxidation (29,31).
GSH is thus one of the most important components of the non-protein bound SH pool.
Duncan multiple range comparisons clearly identified significant differences between
alga/treatment and recovery-period. E. gracilis had a significantly higher (F14, 83 = 15.76, P <
0.0001) level of non-protein SH than H. pluvialis (Figs. 2 and 3). However, exposing E.
gracilis to cryogenic and non-cryogenic treatments, affected levels of non-protein SH groups
such that they were significantly reduced after freezing (Figs. 2 and 3). The greatest reduction
was detected in cells exposed to liquid nitrogen (F12, 41 = 7.88, P < 0.0001) with recovery
period as non-protein bound SH groups increased after 48h (Fig. 2). This may be due to the
preferential oxidation of GSH within the cells and as the algae recover, enzymatic recycling
(21,22,39) of non-protein SH groups occurs. It may be possible that non-protein bound SH
groups in H. pluvialis also offers protection against oxidative stress as this organism showed a
significant increase in non-protein bound SH groups at 48 h. Overall, however, cryogenic and
non-cryogenic treatments did not affect levels of non-protein SH groups in H. pluvialis.
Comparisons of different types of cryogenic treatments reveals that for E. gracilis, nonprotein bound SH group content declined after exposure to freezing, indicating that the
preferential oxidation of GSH was likely. Oxidation of GSH to GSSG and the efficient
recycling of GSSG is important as the oxidized S-S form can be cytotoxic (3,21,22, 29).
Glutathione reductase activity was detected in both algae and is likely to function, within
the chloroplasts (22). E. gracilis had significantly (F6,65 = 5.37, P < 0.0001) higher levels of
glutathione reductase activity as compared to H. pluvialis (Fig. 5). Exposure to chilling (0°C),
cryoprotectants (at non-chilling temperatures) and freezing resulted in significant increases in
glutathione reductase activities immediately after thawing. Initial variability in responses to
these stresses may be due to the cells being in a state of flux immediately following the stress
treatment. As recovery progressed
(i.e. at 24-48h) glutathione reductase activities
significantly increased (F14, 65 = 4.22, P < 0.0001) in those cultures which had been exposed to
freezing and was particularly evident for cells that had been cooled using a two-step protocol
and exposed to liquid nitrogen. Furthermore, glutathione reductase activity in E. gracilis after
exposure to freezing was greater than in H. pluvialis (Fig. 5).
Freezing induced a significant (F12, 40 = 18.75, P < 0.0001) increase in E. gracilis protein
SH during recovery (Fig. 2), peaking at 24h of recovery and returning to pre-treatment status
after a further 24h (Fig. 2). In contrast, H. pluvialis had the highest protein SH group content
in cultures which had not been exposed to cooling to below 0°C, with significantly (F6, 41 =
5.08, P = 0.0006) lower levels of protein SH groups in cultures which had been exposed to
subzero temperatures (Fig. 3). Lower protein SH in H. pluvialis exposed to subzero
temperatures may be due to preferential oxidation, although no decrease in non-protein bound
SH groups was detected (Fig. 3). Elevated levels of protein SH groups in both H. pluvialis
and E. gracilis detected in cultures 24h post-treatment indicates that both organisms were able
to respond to increased stress by altering the status of their protein-SH (Figs. 2 and 3).
H2O2 and O2.- and antioxidant protection
Enhanced SOD activity in E. gracilis exposed to cryogenic treatments will result in
increased H2O2 which, if allowed to accumulate in the cell will become toxic (especially if
catalase activity is low) leading to the production of the .OH (11) via Fenton chemistry.
Although SOD was detected in the cryotolerant, H. pluvialis SOD activity was much lower
than that of E. gracilis (F2, 33 = 115.93, P < 0.0001; Fig. 4). Therefore, H. pluvialis had a
lower potential for the accumulation of H2O2 via SOD and H 2O2 so produced would be
removed by catalase, which was detected in this alga but not in E. gracilis. The fact that
catalase was either at the limits of detection or absent in E. gracilis, even following low
temperature stress, confirms previous reports that E. gracilis lacks (or more likely only has
low levels) of catalase (10). In E. gracilis it has also been reported that H2O2 produced in the
mitochondria and chloroplasts immediately diffuses into the cytosol where ascorbate
peroxidase is primarily located in this alga (24,25,35). Rapid diffusion of H2O2 is thus
considered important in enabling E. gracilis to regulate intracellular H2O2. However, this may
be less effective in H2O2 regulation during freezing stress. The peroxidase enzymes; ascorbate
and GSH peroxidase, may therefore function as the main defence in E. gracilis against
oxidative damage by H2O2. Moreover, increases in both glutathione reductase and non-protein
bound SH groups in E. gracilis may be related to the concomitant rise in SOD activity
detected in cultures after freezing (Fig. 4). Catalase was detected in H. pluvialis, the activity
of which changed in response to freezing and chilling and during post-treatment recovery after
48 h (F12, 41 = 6.72, P < 0.001; Fig. 6). Exposure to the cryoprotectant and liquid nitrogen
caused a minimal increase in catalase activity in H. pluvialis, reaching a maximum after 48h
in cultures, which had not been exposed to freezing (Fig. 6). Catalase activity in H. pluvialis
was largely confined to cells, which had been exposed to non-cryogenic stresses (Fig. 6).
Conclusions
Antioxidant profiles of cryopreserved H. pluvialis and E. gracilis are different, especially
for those enzymes involved in H2O2 formation and removal. Stress-induced increases in SOD
activity in E. gracilis may result in an accumulation of H2O2 in the absence of catalase. Fleck
et al. (11) hypothesised that ×OH radicals (generated by Fenton chemistry from H2O2) may
contribute to cryoinjury in E. gracilis and this is further substantiated by the present study.
Sulfhydryl group status may have an important part in conferring protection and future studies
should examine the role of ascorbate peroxidase in algal cryo-tolerance. This study offers
potential for cryopreservation protocol development by designing strategies that apply
exogenous antioxidants, free radical scavenging cryoprotectants and inhibitors of Fenton
chemistry. These approaches may be especially useful for developing cryostorage methods for
algae such as the Euglenoids that have, to date, proved difficult to cryopreserve.
Acknowledgements: The Esmée Fairbairn Charitable Trust is acknowledged for financial
support. The authors also thank Alan Heath for his statistical advice and Dr Keith Harding for
help in the formulation of the paper.
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Accepted for publication 11/6/03