Plant Physiol. (1981) 68, 1156-1160
0032-0889/81/68/1 156/05/$00.50/0
Effects of Freezing on Spinach Leaf Mitochondria and Thylakoids
in Situ and in Vitro'
Received for publication March 18, 1981 and in revised form June 3, 1981
REGINA THEBUD AND KURT A. SANTARIUS
Botanisches Institut, Universitat Dusseldorf, Universitatsstrasse 1, D-4000 Dusseldorf,
Federal Republic of Germany
ABSTRACT
The sensitivity of spinach (Spinacia oleracea L.) leaf mitochondria and
chloroplast membranes to subzero temperature stress was compared after
freezing of the membrane systems in situ and in vitro. Respiratory and
photosynthetic activities were measured polarographically.
When leaves were frozen under controlled conditions for 2 hours to
various minimum temperatures and mitochondria and chloroplasts isolated
after thawing, the membrane systems showed a nearly simultaneous inactivation of respiratory and photosynthetic activities between -5 to -7 C.
At that temperature range in both membrane systems phosphorylation
became only slightly more affected than electron transport, ie. after
freezing in situ conspicuous uncoupling of phosphorylation from electron
transport was not observed.
In contrast, mitochondria and thylakoids isolated from the same preparation of intact leaves under comparable conditions using NaCI as osmoticum exhibited differences in sensitivity towards freezing for 2 to 4 hours
at -25 C in vitro. In the absence of cryoprotectants, photophosphorylation
of isolated thylakoids became completely uncoupled from electron transport which was increased several-fold compared with the unfrozen controls.
Inactivation of respiratory functions of isolated mitochondria followed the
same pattern as observed after freezing in situ. In the presence of sucrose
for protection of thylakoids significantly higher concentrations of the
cryoprotectant were necessary than for preservation ofmitochondria. Thus,
under the conditions used in this study chloroplast membranes proved to
be more sensitive to freezing in vitro than mitochondria.
It is a common view that inactivation of cellular membranes is
the primary cause of frost damage in plant cells (11, 15). In
previous investigations with isolated plant membrane systems,
thylakoids and mitochondria turned out to be highly sensitive to
freezing (4, 6, 7, 24). Inactivation of chloroplast membranes also
took place when intact leaves were killed during extracellular ice
formation (7, 10, 12). Singh et al. (23) found that mitochondria in
situ can retain their normal function even after the cell was killed
by freezing. However, gas exchange measurements on partly frostdamaged spinach leaves have shown that photosynthesis and
respiration decrease almost simultaneously (12).
To clarify these discrepancies, we have investigated the sensitivity of spinach leaf mitochondria and chloroplast membranes to
subzero temperature stress in vitro. Both membrane systems were
isolated from one leaf batch in similar isolation media and subjected to freezing and thawing under identical conditions. Subsequently, respiratory and photosynthetic activities ofthe membrane
systems were tested. In addition, the effect of freezing on mito-
chondria and thylakoids in situ was studied. Intact leaves were
frozen at various temperatures; after thawing, mitochondria and
chloroplasts were isolated and the biochemical activities of the
membrane systems were determined.
MATERIALS AND METHODS
Plant Material. Leaves were harvested from 4- to 6-week-old
spinach plants (Spinacia oleracea L. cv. Monatol) which were
grown in soil culture in green houses at temperatures between 13
and 20 C. During the winter months additional artificial fluorescent light was used for about 10 h per day.
Freezing of Intact Leaves. Frost treatment of intact leaves was
performed as described by Klosson and Krause (12). Detached
leaves were placed into a cryostat and submitted to a freeze-thawcycle between 5 C and -5 to -9 C using cooling and warming
rates of 6 C h-1. Frozen leaves were kept for 2 h at the different
minimum temperatures. Control leaves were stored at 4 C during
the same time.
Isolation Procedure. Mitochondria and thylakoids were isolated
in a single procedure. Freshly harvested leaves (100 g, or 10 g
frozen-thawed and the respective control leaves) were homogenized for 3 s in a Waring Blendor in 400 ml (40 ml) of a medium
containing 300 mm mannitol, 20 mm Hepes-NaOH, 20 mm Mops2NaOH, 1 miM MgCl2, 1 mm KH2PO4, 1 mm EDTA, 4 mM cysteine,
0.6% insoluble PVP, and 0.4% BSA (pH 7.3). The homogenate
was filtered through 8 layers of cheesecloth and the filtrate was
centrifuged for 90 s at 1600g. The pellet contained the chloroplasts.
The mitochondria were isolated from the supernatant similar to
the procedure described by Douce et al. (2). After 10 min centrifugation at 12,000g, the mitochondria were resuspended in media
containing either 200 mm NaCl (for subsequent freezing in vitro)
or 300 mm mannitol (when leaves were already subjected to
freezing) and in addition 15 mi Hepes, 15 mm Mops, 1 mM
MgCl2, 1 mm KH2PO4, 1 mM EDTA, and 0.4% BSA; the pH was
adjusted to 7.2 with KOH in the case of NaCl and with NaOH in
the case of mannitol as osmotic agents. The mitochondria were
centrifuged again 10 min at 12,000g. The pellet was resuspended
in a small volume of the respective medium used for the washing
procedure and stored at 0 C. The chloroplasts were resuspended
in a medium containing 20 mm Hepes-NaOH, 5 MM MgCl2, 5 mM
KCI, and 5 mm KH2PO4 (pH 7.0) leading to rupture of the
envelopes. To restore tonicity, an equal amount of the same
medium containing additionally 400 mm sorbitol and 0.8% BSA
was added. After 3 min centrifugation at 250g to remove debris
the supernatant was centrifuged for 3.5 min at 3000g. The sedimented thylakoids were treated as follows: for freezing experiments in vitro, thylakoids were resuspended in the medium used
2 Abbreviations: Mops, 2-N-morpholino-propane sulfonic acid; PC,
photosynthetic control.
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1156
1 This research was supported by the Deutsche Forschungsgemeinschaft.
Copyright © 1981 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 68, 1981
FREEZING OF MITOCHONDRIA AND THYLAKOIDS
for the washing procedure of mitochondria (with 200 mnm NaCl,
see above); after centrifugation for 3.5 min at 3000g thylakoids
were again resuspended in a small volume (2-4 ml) of the same
medium and stored at 0 C or frozen as indicated below. When
chloroplasts were isolated from frozen-thawed leaves and from
the corresponding control material, thylakoids were resuspended
in 1 ml of the washing medium but with 300 mm mannitol instead
of NaCl.
Chlorophyll and Protein Determination. Chl was determined
according to Amnon (1). The protein content of the mitochondria
was evaluated by a method described by Lowry et al. (14);
correction for contamination with thylakoid protein was performed as proposed by Douce et al. (2).
Freezing of Isolated Membrane Systems. Frost treatment of
isolated mitochondria and thylakoids took place for 2 to 4 h at
-20 to -25 C in the absence and presence of various concentrations of sucrose. Controls were kept at 0 C for the same time. The
membrane systems which were suspended in a medium described
above were diluted either with 200 mm NaCl or with sucrose
dissolved in 200 mM NaCl in order to keep the NaCl concentration
at 200 mM.
Measurements of Respiratory and Photosynthetic Activities.
Biochemical functions of the membrane systems were estimated
polarographically with a Clark-type 02 electrode at 20 C. 02
consumption of mitochondria was measured as described by
Douce et al. (2); either 40 mm malate plus 25 mm glutamate or 10
mM glycine were used as substrates. ATP synthesis, RC, and
ADP/O ratios were calculated according to Estabrook (3). Photosynthetic activities of thylakoids were measured similar to Klosson and Krause (12) by light-dependent O2 uptake in the presence
of 20 ILM methylviologen and 1 mM KCN; the reaction medium
contained 300 mM sorbitol, 50 mM Hepes-NaOH, 10 mm KH2PO4,
5 mM MgCl2, 0.2% BSA (pH 7.5). The reaction was started by
illumination with saturating light intensity (550 w/m2) using a 150
w halogen lamp and a filter combination of two IR-absorbing
filters Calflex C (800-1000 nm; Balzers, Liechtenstein) and WG
4 (> 1000 nm; Schott & Gen., Mainz) and a 3-mm cutoff filter
RG 630 (Schott & Gen.). ATP formation, photosynthetic control
and ADP/O ratios were calculated according to Robinson and
Wiskich (16).
1157
lesser extent. In contrast, under identical freezing conditions isolated thylakoids completely lose photosynthetic control and phosphorylating capacity, whereas, electron transport not only is uninhibited but even becomes strongly stimulated. Thus, in isolated
thylakoids uncoupling is a primary effect of freezing as already
outlined by Heber and Santarius (7).
Freezing of isolated mitochondria and thylakoids in the presence of various concentrations of sucrose resulted in partial or
complete protection of the membrane systems (Fig. 1). In mitochondria the protective effect of sucrose was already conspicuous
when only low concentrations of the cryoprotectant were present
during freezing irrespective of whether malate/glutamate or glycine were used as substrate. Under those conditions photophosphorylation of thylakoids was fully inactivated and electron transport was stimulated. At higher sugar concentrations, chloroplast
membranes also became protected during freezing. Here it should
be noted that the extent of protection for both thylakoids and
mitochondria and the minimal sucrose concentration at which
photosynthetic control is preserved vary slightly with each preparation. The fact that under the same conditions thylakoids need
higher amounts of sucrose for a comparable degree of protection
than mitochondria again demonstrates that under the conditions
used here the latter are more sensitive to freezing in vitro than
isolated plant mitochondria.
In earlier investigations on the effect of freezing on thylakoid
membranes, chloroplasts were isolated in Tris/NaCl media. To
elucidate whether the course of freeze inactivation and protection
is affected by the isolation procedure, thylakoids obtained with
different preparation techniques were compared. In Figure 2 it is
shown that chloroplast membranes isolated in Tris/NaCl media
turned out to be more sensitive to freezing than thylakoids used
for the experiments in Table I and Figure 1. This is evident from
the much stronger stimulation of electron transport and the fact
that a still higher sucrose concentration is necessary for the
preservation of ATP synthesis of chloroplasts isolated in Tris/
NaCl media. However, the general inactivation pattern of thylakoids frozen in vitro is the same irrespective of the way of isolation.
The question arises whether differences in the sensitivity between thylakoid and mitochondrial membrane systems can be
observed when freezing takes place in situ, i.e. when intact leaves
are submitted to subzero temperatures and organelles are isolated
from the partially or completely damaged tissue. In Figure 3 it is
shown that this is not the case. Both mitochondria and thylakoids
became inactivated nearly simultaneously in a narrow temperature
range, i.e. at minimum temperatures between -5 to -7 C. It was
conspicuous that thylakoid membranes isolated from partially
frost-damaged leaves never showed a stimulation of methylviologen-mediated electron transport as was observed during freezing
in vitro in the absence of cryoprotectants (Table I, Figs. 1 and 2;
see also lc. 12). Obviously, exposure of intact leaves to subzero
temperatures which caused partial or complete damage of the
tissue led to an almost simultaneous inactivation of electron
transport and ATP synthesis in both mitochondria and thylakoids.
This is also evident from the finding that the ADP/O ratio
remained rather unchanged up to freezing temperatures at which
electron transport and oxidative and photosynthetic phosphorylation were already considerably influenced. In these cases the
organelles were isolated from leaves which proved to be almost
completely water-infiltrated after thawing. The small residual
electron transport remaining in both membrane systems even after
strong frost damage of the leaves was not stimulated by addition
of ADP, i.e. respiratory and photosynthetic control mechanisms
became inactivated in the course of freezing.
RESULTS
An exact comparison of the sensitivity of mitochondria and
thylakoids to freezing appears possible only if both membrane
systems are isolated from leaf material under comparable conditions, e.g. in a single isolation procedure using similar isolation,
washing and suspension media. Small differences in the composition of the media used during the course of isolation were
allowed in order to maintain high functional integrity of the
respective membrane systems. In the course of varying of the
isolation procedure it was found that 300 mm mannitol is a suitable
osmoticum for both mitochondria and thylakoids. For freezing of
isolated membrane systems the cryoprotective sugar alcohol was
replaced by 200 mM NaCl; up to 4 to 5 h the decrease in the
respiratory and photosynthetic activities was relatively small when
the membrane systems were stored at 0 C in the presence of 200
mM NaCl (see also lc. 21, 24). Freezing of the isolated mitochondria and thylakoids and storage of the unfrozen controls was
always performed at the same temperature conditions and in
identical media.
Table I shows that both biomembrane systems are sensitive to
freezing in vitro in the absence of a cryoprotectant. However,
mitochondria and thylakoids are influenced in a different way. In
isolated mitochondria both electron transport and oxidative phosDISCUSSION
phorylation become only partially inactivated and these memThe data presented in this paper clearly indicate that both
brane systems still exhibit RC. As ATP synthesis is more affected
than 02 consumption the ADP/O ratios decrease, too, but to a mitochondria and thylakoids are very susceptible to freezing stress.
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Plant Physiol. Vol. 68,1981
THEBUD AND SANTARIUS
1158
Table I. Respiratory and Photosynthetic Activities of Isolated Mitochondria and Thylakoids after Freezingfor 2-4
h at -20 to -25 C in a Medium Containing 200 mM NaCI
in
cent of the activity of membrane systems kept at 0 C. Rates of the unfrozen controls
per
Data are expressed
are given in nmol 02 consumed (electron transport) and ATP synthesized mg-' protein min-' (mitochondria;
substrate: malate/glutamate) and,mol 02 consumed and ATP synthesized mg-' Chl h-' (thylakoids in the
presence of methylviologen), respectively. Averages of 7 experiments.
Thylakoids
Mitochondria
Rates of the
unfrozen
controls
Respiratory/photosynthetic control
50-90
140-300
2.3-3.4
ADP/O ratio
1.6-2.0
Electron transport (State 3)
ATP synthesis
Activities after
Activities after
Rates of the
freezing in % of
freezing in % of
unfrozen
controls
the unfrozen
controls
36 ± 3.7
the unfrozen
controls
376 ± 39
40-70
80-115
1.8-2.8
0.7-1.1
19 ± 2.6
29 ± 6.4
52 ± 7.9
0
0
0
la~~~~~~~~~~~~~~~~
0
c-
p
c
p
4CL
I
I
2 \
200
]
~~~~~~~~~~505
°
1
sucrose concentration (mM)
sucrose
concentration (mM)
u}
-EI
c
>1
,0
tlI
Q;
sucrose concentration
(mM)
sucrose concentration
(mM)
FIG. 1. Respiratory and photosynthetic activities of isolated mitochondria (-0-) and thylakoids (--A--) after freezing 2-4 h at -20 C in
washing medium containing 200 mM NaCl and various concentrations of
sucrose. Abscissae: sucrose concentration in mm. Ordinates: activities in
per cent of the unfrozen controls. Activities of the unfrozen controls (=
100%o): mitochondria (in nmol mg-' protein min-'): 69 02, 220 ATP; RC
3.11, ADP/O ratio 1.56, substrate glycine (10 mM); thylakoids (in iLmol
mg-' Chl h-1): 46 02, 71 ATP; PC 2.23, ADP/O ratio 0.79.
AU results were obtained from greenhouse material without subjecting it to a specific hardening procedure, since it was shown
that the course of inactivation was very similar in frost-hardened
and unhardened spinach leaves with only the temperature at
which injury occurred being some degrees lower in cold-acclimated leaves (12).
Comparison of the sensitivity of photosynthetic and respiratory
membrane systems towards ice formation in situ and in vitro
0
400
200
sucrose concentration (mM)
0
400
200
sucrose concentration (mM)
FIG. 2. The effect of the isolation procedure on the activity of electron
transport and photophosphorylation of thylakoids obtained from the same
leaf material and frozen in vitro 2-4 h at -20 C in the presence of various
concentrations of sucrose. --A--: isolation technique as described in "Materials and Methods"; thylakoids were suspended and frozen in the same
washing medium as used for mitochondria containing 200 mM NaCl as
osmoticum. Activities of the unfrozen controls (= 100O) in ,umol mg-' Chl
h-1: 66 02, 114 ATP; PC 2.43, ADP/O ratio 0.85. -A-: an aliquot of the
chloroplasts pelleted after the first centrifugation step were washed in a
medium containing 50 mM Tris-HCI (pH 8.0) and 350 mM NaCl under
conditions described by Santarius (20). Washed chloroplasts were shocked
in distilled H20 and thylakoids were stored and frozen in 200 mm NaCl.
Activities of the unfrozen controls (= 100%) in pmol mg-' Chl h-': 56 02,
97 ATP; PC 2.36, ADP/O ratio 0.88. Sucrose concentrations present
during freezing are indicated on the abscissae.
showed conspicuous differences in the course of inactivation.
During freezing of intact leaves, ice formation occurred at about
-4 to -5 C (12). Freezing to temperatures which cause partial or
complete damage of the tissue demonstrated that inactivation of
both membrane systems occurred nearly simultaneously within a
narrow range at minimum temperatures of-5 to -7 C (Fig. 3; see
also lc. 12). This is in agreement with measurements on gas
exchange, Chl fluorescence, and light-scattering reactions of intact
leaves under comparable freezing conditions (12, 13). Thus it
seems established that photosynthesis and respiration are equally
affected by freezing in vivo.
In contrast, mitochondria and thylakoids isolated from intact
leaves differ in sensitivity towards ice formation in vitro. However,
it must be taken into consideration that comparison of the sensitivity of cellular membrane systems towards freezing in vitro are
problematic because of the fact that all isolations are artifactual.
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Plant Physiol. Vol. 68,1981
FREEZING OF MITOCHONDRIA AND THYLAKOIDS
I
I
0
n 10 10
0
1159
in
VI
B3 O '°v
I0
'a
-a
-Y
a
E
0
0-cc
0
2
0.
CL
Ln
C
0'
-i
5
0
~~~~~~~~~~~~~00a
0
o;
AL
in
a-
£~
0
4)
'k
21
w
o
4
0
-4
0
-8
temperature (C)
temperature (C)
temperature
temperature (C)
(C)
FIG. 3. Respiratory and photosynthetic activities of mitochondria (->-) and thylakoids (--v--) isolated from spinach leaves after freezing in situ
function of the freezing temperature. On the abscissae the minimum temperature is indicated at which the leaves were kept for 2 h during frost
treatment. Rates are expressed in nmol 02 consumed (ATP synthesized) mg-' mitochondrial protein min-' (mitochondria; substrate: malate/glutamate)
and umol 02 consumed (ATP synthesized) mg-' Chl h-' (thylakoids; methylviologen).
as a
It is well known and outlined in Figure 2 that the effects of
freezing on membrane systems are highly dependent on the media
in which they were isolated. In addition, variations in freezing
conditions affect membranes differently. In general, thylakoids
isolated from spinach leaves are relatively insensitive to storage
conditions and even retain their function when suspended in
distilled H20 (9, 18). In contrast, preservation of respiratory
functions of mitochondria is possible only in media of defined
composition and under conditions which prevent extreme swelling
of the organelles (24). Nevertheless, under comparable conditions
used in this study mitochondria became markedly, but not completely inactivated (Table I, Fig. 1, ic. 24), while freezing of
isolated thylakoid membranes in the absence of cryoprotectants
always resulted in uncoupling of photophosphorylation from electron transport (Table I, Fig. 1) irrespective of the manner of
isolation and the composition of the storage medium (Fig. 2; lc.
5, 7, 8, 25; Klosson and Krause, unpublished). Freezing injury in
vitro supposedly is caused by increase in the concentration of
potentially membrane-toxic solutes, e.g. NaCL in the surroundings
of the biomembrane systems resulting in loss of the semipermeability of the membranes (4-8, 11, 17, 21). If cryoprotectants such
as sugars were also present during freezing the deleterious effect
of salts could be overcome (6-8, 19). However, under identical
freezing conditions, for protection of mitochondria significantly
lower concentrations of sucrose were necessary than for preservation of thylakoids (Fig. 1). Moreover, the latter showed ATP
synthesis and photosynthetic control after freezing and thawing
only if a certain threshold concentration of sucrose was exceeded
(Figs. 1, 2). This again points to a higher sensitivity of the
chloroplast membranes in vitro at least during freezing in the
of 200 mM NaCl.
In addition to differences in the susceptibility of mitochondria
and thylakoid membranes to freezing in vitro and in situ, there are
also differences in the inactivation pattern of photosynthetic and
respiratory activities dependent on whether the membrane systems
were exposed to subzero temperature stress within their natural
surroundings, ie. in intact leaf cells, or after an isolation procedure. Whereas mitochondrial functions became equally impaired
during freezing in situ and in vitro, photosynthetic activities of
thylakoids turned out to be affected differently when intact leaves
or isolated chloroplast membranes were subjected to subzero
temperatures (compare Table I and Fig. 1 with Fig. 3). As mentioned already, freezing of isolated thylakoids in the absence of
cryoprotectants resulted in uncoupling of photophosphorylation
from electron transport. In contrast, when membrane inactivation
took place during freezing of intact leaves, both photophosphorylation and electron transport became affected nearly simultaneously, i.e. uncoupling was not observed (Fig. 3, lc. 12; see also lc.
10). Decrease in RC and PC ratios (Fig. 3) was caused primarily
by the inhibition of state 3 electron transport. The other possibility
presence
for lowering the RC and PC values, an accelerated state 4 02
consumption, is in terms of Mitchell's chemiosmotic hypothesis,
supposedly due to a decrease of the pH gradient. A relative
increase of state 4 electron transport could not be observed here.
So it may be concluded that in both mitochondria and thylakoids,
membrane inactivation in vivo seemed to be primarily the result
of inhibition of electron transport.
The occurrence of freezing injury to mitochondrial and chloroplast membranes is not understood at present. Results of other
laboratories (22, 26, 27) point in the direction that freezing damage
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1160
THEBUD AN]D SANTARIUS
of intact tissue is possibly due to breakdown of the compartmentation ofthe cells, maybe by damage of the tonoplast. For instance,
release of toxic compounds from the vacuole into the cytoplasm
could lead to inhibitions on biomembrane systems. In frost-damaged tissue, vacuolar compounds could act on membrane systems
in concentrations comparable with those in the vacuoles themselves. In contrast, during the isolation of the cell organelles the
toxic materials from the vacuole become highly diluted within the
isolation medium containing buffering substances, PVP and BSA
and, therefore, are no longer effective on the membrane systems.
The hypothesis mentioned above could explain the simultaneous
inactivation of mitochondria and thylakoids during freezing of
intact leaves.
Acknowledgments-The authors thank Prof. Dr. G. H. Krause for critical reading
of the manuscript. The study contains part of the thesis work of R. Thebud.
1.
2.
3.
4.
5.
6.
7.
LITERATURE CITED
ARNON DI 1949 Copper enzymes in isolated chloroplasts. Plant Physiol 24: 1-15
DOUCE R, AL MooRE, M NEUBURGER 1977 Isolation and oxidative properties of
intact mitochondria isolated from spinach leaves. Plant Physiol 60: 625-628
ESTABROOK RW 1967 Mitochondrial respiratory control and the polarographic
measurements of ADP:O ratios. In RW Estabrook, ME Pullman, eds, Methods
Enzymol vol X. Academic Press, New York, London, pp 41-47
GARBER MP, PL STEONKUS 1976 Alterations in chloroplast thylakoids during
an in vitro freeze-thaw cycle. Plant Physiol 57: 673-680
HEBER U 1967 Freezing injury and uncoupling of photophosphorylation from
electron transport in chloroplasts. Plant Physiol 42: 1343-1350
HEBER U 1968 Freezing injury in relation to loss of enzyme activities and
protection against freezing. Cryobiology 5: 188-201
HEBER U, KA SANTARIUS 1964 Loss of adenosine triphosphate synthesis caused
by freezing and its relationship to frost hardiness problems. Plant Physiol 39:
712-719
8. HEBER U, KA SANTARIUS 1976 Water stress during freezing. In OL Lange, L
Kappen, ED Schulze, eds, Water and Plant Life, Ecological Studies, Vol 19.
Springer, Berlin, Heidelberg, pp 253-267
9. HEBER U, L TYANKOVA, KA SANTARMIUS 1971 Stabilization and inactivation of
biological membranes during freezing in the presence of amino acids. Biochim
Biophys Acta 241: 578-592
10. HEBER U, L TYANKOVA, KA SANTAIUUS 1973 Effects of freezing on biological
membranes in vivo and in vitro. Biochim Biophys Acta 291: 23-37
Plant Physiol. Vol. 68, 1981
11. HEBER U, H VOLGER, V OVERBECK, KA SANTARIUS 1979 Membrane damage
and protection during freezing. In 0 Fennema, ed, Proteins at Low Temperatures. Advances in Chemistry Series No. 180. Am Chem Soc Washington DC,
pp 159-189
12. KLoSSON RJ, GH KRAUSE 1981 Freezing injury in cold-acclimated and unhardened spinach leaves I. Photosynthetic reactions of thylakoids isolated from
frost-damaged leaves. Planta 151: 339-346
13. KLoSSON RJ, GH KRAUSE 1981 Freezing injury in cold-acclimated and unhardened spinach leaves II. Effects of freezing on chlorophyll fluorescence and
light-scattering reactions. Planta 151: 347-352
14. LowRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275
15. LYONS JM, JK RAISON, PL STEPONKUS 1979 The plant membrane in response to
low temperature: an overview. In JM Lyons, D Graham, JK Raison, eds, Low
Temperature Stress in Crop Plants. Academic Press, New York, pp 1-24
16. ROBINSON SP, JT WISKICH 1976 Factors affecting the ADP/O ratio in isolated
chloroplasts. Biochim Biophys Acta 440: 131-146
17. SANTARIUS KA 1969 Der Einflul3 von Elektrolyten auf Chloroplasten beim
Gefrieren und Trocknen. Planta 89: 23-46
18. SANTARIUS KA 1971 The effect of freezing on thylakoid membranes in the
presence of organic acids. Plant Physiol 48: 156-162
19. SANTARIUS KA 1973 The protective effect of sugars on chloroplast membranes
during temperature and water stress and its relationship to frost, desiccation
and heat resistance. Planta 113: 105-114
20. SANTARIUS KA 1980 Membrane lipids in heat injury of spinach chloroplasts.
Physiol Plant 49: 1-6
21. SANTARIUS KA, U HEBER 1970 The kinetics of the inactivation of thylakoid
membranes by freezing and high concentrations of electrolytes. Cryobiology 7:
71-78
22. SENSER M, E BECK 1977 On the mechanism of frost injury and frost hardening
of spruce chloroplasts. Planta 137: 195-201
23. SINGH J, AI DE LA ROCHE, D SIMINOVITCH 1977 Relative insensitivity of
mitochondria in hardened and nonhardened rye coleoptile cells to freezing in
situL Plant Physiol 60: 713-715
24. THEBUD R, KA SANTARIUS 1981 Effects of freezing on isolated plant mitochondria. Planta. 152: 242-247
25. VOLGER H, U HEBER, RJ BERZBORN 1978 Loss of function of biomembranes and
solubilization of membrane proteins during freezing. Biochim Biophys Acta
511:455-469
26. YOSHIDA S 1979 Freezing injury and phospholipid degradation in vivo in woody
plant cells. III. Effects of freezing on activity of membrane-bound phospholipase D in microsome-enriched membranes. Plant Physiol 64: 252-256
27. ZIEGLER P, 0 KANDLER 1980 Tonoplast stability as a critical factor in frost injury
and hardening of spruce (Picea abies L. Karst.) needles. Z Pflanzenphysiol 99:
393-410
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