J. therm. Biol. Vol. 15, No. 2, pp. 141-148, 1990 0306-4565/90 $3.00 + 0.

00
Printed in Great Britain Pergamon Press pie

EFFECT OF HEAT SHOCK ON CELLULAR FUNCTIONS

DIFFERING IN THERMORESISTANCE
V. YA. ALEXANDROV, I. G. ZAVADSKAYAand T. A. ANTROPOVA
Komarov Botanical Insitute of the Academy of Sciences of the U.S.S.R., Leningrad, Russia

(Receit'ed 21 January 1989; accepted in ret'isedform 8 July 1989)

Abstract--l. The dependence of the elevation of thermoresistance of three cell functions on the heat
hardening temperature (heat shock) has been studied using Tradescantia albiflora leaves.
2. The maximum enhancement of thermoresistance of the streaming of cytoplasm, phototaxis of
chloroplasts (thermolabile functions), plasmolises (thermostable function) occurs, after a 30-min harden-
ing, at 35, 36 and 48:C respectively. The maximum enhancement of thermoresistance of these functions
occurs after a 180-min hardening, at 38, 36 and 38C respectively.
3. The convergence in temperature giving the maximum hardening of differing thermostability
functions, under lengthening exposure to heat, is shown to be due to variability in their ability to repair
during hardening at superoptimal temperatures. During 180-rain hardening, repair was most pronounced
for the streaming of cytoplasm, pronounced for phototaxis of chloroplasts, while it was totally absent in
the case of the protoplast plasmolysis.
4. For the understanding of the mechanism of heat hardening it is important to take into account that,
when hardening duration is too short for repair to be accomplished, the dosage of heat required for the
elevation of thermostability of thermoresistant functions is higher than that for thermolabile functions.

Key Word lndex--Tradescantia alb![tora, heat hardening, heat shock, thermoresistance, repair of heat
injury, cytoplasmic streaming, phototaxis of chloroplast, plasmolysis.

INTRODUCTION The history of the problem is as follows. It was

Heat hardening, discovered in our laboratory on
plant cells over 30 yr ago (Alexandrov, 1956) and in
later publications called an "acquired thermotoler-
ance", has attracted attention of a great number
of scientists during the recent decade. However,
physico--chemical mechanisms for reactive elevation
of cell resistance remain unknown. This is mainly due
to the lack of a detailed cytophysiological character-
isation of the process, important both from a theoret-
ical and a practical point of view. Work on heat
hardening reports concern increases or decreases in
thermoresistance of a cell as a whole. In reality, the
investigators deal with thermoresistance of a certain
cell function. In cell cultures it is usually a capacity
for reproduction, cloning. Various functions not con-
nected with cell division are also used, e.g. photosyn-
thesis, resistance to penetration of a dye into the cell.
The streaming of cytoplasm on protoplast plasmo-
lysis in hypertonic solutions are often chosen as
suitable criteria for the thermoresistance of plant
cells. Phototaxis of chloroplasts also appeared to be
convenient index, it is far from being clear whether
different functions behave similarly while both ac-
quiring the hardened state and also after its loss. The
literature on heat shock or heat hardening even
though it is complex, contains little on the compari-
son of the heat shock effect on different cell functions.
The aim of the present paper is to compare the
effect of the temperature and duration of heat
hardening on plant cell functions differing in
thermostability.
found in our laboratory that, for the same rise in
thermoresistance of cytoplasmic streaming, in four
cereals differing in heat-tolerance and in the ther-
moresistance of cell functions, the temperature re-
quired for hardening was higher the more
thermostable the cereals were. The temperature of a
5-min heating required to arrest the protoplasmic
streaming in the cells of Dactylis glomerata was 44'C,
in Phragmites communis was 46~C, in Panicum mili-
aceum was 48.Y>C, and Eleusine indica was 49~C. The
temperature of an 18-h hardening that is needed to
increase thermostability of the protoplasmic stream-
ing by I~C for these cereals was 30, 36, 40 and 40=C,
respectively (Alexandrov and Feldman, 1958). The
question arises whether various hardening tem-
peratures are needed for the development of an
equal thermoresistance in the cell functions differ-
ing in thermostability. To answer the question,
Barabalchuk (1969) in our laboratory hardened
leaves of Tradescantiafluminensis for 3 h at different
temperatures and determined thermoresistance in-
crease of the thermolabile functions: cytoplasmic
streaming and phototaxis of chloroplasts, and ther-
mostable functions: ability for plasmolysis and
retention of antocyan in the vacuole. The results
obtained have shown that the rise in thermostability
of all the four functions starts and reaches its maxi-
mum at a similar temperature for a 3 h hardening:
28-30 and 38~,0 C respectively, however, with the
rise in the hardening temperature, a reduction of the
hardening effect takes place in the thermolabile func-
tions at temperatures significantly lower than for the

141
142 V. YA ALEXANDRO',~'I al.
thermostable functions. This occurs because the ther-
molabile functions are being suppressed in the pro-
cess of hardening itself. On the basis of these data
Barabalchuk concluded that "'reactive elevation of
cellular thermoresistance results from a general reac-
tion of a cell, which leads to an appearance of the
agents or conditions that simultaneously stabilize
various intracell protein systems, irrespectively of
their original thermoresistance'" (pp. 1030-1031).
However, several years ago similar experiments were
performed in our laboratory by Zavadskaya and
Antropova, though not with a 3-h--but with a
30-min--hardening, using leaves of Commelina
a/ricana. These experiments gave opposite results.
In the present paper, effect of 30-rain and 3-h-hard-
ening on two thermolabile functions--cytoplasmic
streaming and phototaxis of chloroplasts, and on a
thermostable function---ability of a protoplast to
respond by plasmolysis to the action of hypertonic
solution, that is, ability of protoplasmic membranes
to prevent penetration of a plasmolytic into the
vacuole, was investigated.
MATERIALS AND METHODS
Object and mode O( hardening
Experiments were performed on the leaves of
Tradescantia albiflora Kunth growing in the green
house of the Botanical Institute of the U.S.S.R.
Academy of Sciences under natural illumination and
at temperature of 18-25 :C. The leaves of the 3rd45th
levels from the top that had completed their growth
were taken for the experiments. The leaves were cut
along the midrib into the halves, one of which was
placed into a humid chamber at room temperature
(20--23 C) and served as a control, the other was
heated in a thermostated humid chamber at a given
temperature kept with a precision of _+0.1 C. The
duration of the hardening was 30 min in one series of
experiments and 3 h in the other.
Determination o[ the heat resistance of cytoplasmic
streaming and ability for plasmoO.sis
To estimate thermostability of the cytoplasmic
streaming, the control and hardened halves of the
leaves were cut into pieces 7 × 4 mm in size. Test
heating was performed in water for 5 min, immedi-
ately after the hardening exposure or 24 h later.
hnmediately after the test heating a piece of leaf was
infiltrated with water and examined under a micro-
scope (objective water immersion - 7 0 ×, aperture
--1.23, ocular ....5 × ). The minimal heating tempera-
ture that completely inhibited spherosome movement
in epidermal cells served as a measure of thermoresis-
tance. A temperature interval between the beatings
was 0.4' C. To estimate thermoresistance of the ther-
mostable function--ability to plasmolysis, the pieces
were infiltrated with 0.8 M glycerol immediately after
the 5-min test heating and 10-15 min later they were
examined under microscope. The minimal tempera-
ture of a 5-rain heating after which no more than 5
plasmolysed cells were observed in the piece served as
a measure of thermoresistance of this function. All
determinations of the heat resistance of cytoplasmic
streaming and plasmolysis are the means from 12
experiments.
Determination of the heat resistance ¢~l chloroplast
phototaxis.
Influence of heat shock on the phototaxis of
chloroplasts was studied by making use of a negative
phototaxis, i.e. transition of the chloroplasts from an
epistroph location which they take under a weak
illumination to the parastroph location to which they
pass in a normal cell under a bright light action. This
translocation was registered using photoelement and
microammeter by enhancement of transmission
through the leaf of blue light which is absorbed by the
chloroplasts. The experiments were performed by a
slightly modified method of Lomagin et al. (1966).
The extent of phototaxis of chloroplasts was calcu-
lated according to the equation
A × 100
Ph. ehl.-- 100
B
where
B is a light transmission through a leaf following
a weak illumination (50 Ix) expressed in divisions
of microammetre;
A is the same but after 10 rain exposure of the
leaf to a bright light (25,000 Ix).
Following hardening, a test heating was carried out
for 5min at 42C. In non-hardened leaves such
heating inhibits chloroplast phototaxis by 80-90%.
In the case of the cytoplasmic streaming and plasmo-
lysis the hardening effect was estimated as a difference
between thermostability of hardened and non-
hardened halves of leaves and expressed in degrees
Celsius. Tbermoresistance of chloroplast phototaxis
was determined by extent of inhibition of this func-
tion, following a 5-min test heating at 42°C; therefore
the hardening effect was estimated as a difference
between phototaxis values of hardened and non-
hardened halves of leaves: Ph.chl. h - Ph.chl. c. All
determinations of the chloroplast phototaxis heat
resistance are the means from 6 experiments.
Determination of the repair from heat damage of three
.(unctions
In the principal experiments evaluation of thermo-
stability of the three functions was performed
immediately after short-term 5-min beatings which
determined their primary thermoresistance with mini-
mum complications induced by destructive post.effect
and repair properties of the cell. In control non-hard-
ened cells the 5-rain heating completely supressed
cytoplasmic streaming at 45.5°C, chloroplast photo-
taxis--at 45"-C, plasmolysis--at 60.5:'C. In the experi-
ments on determination of repair of heat shock
suppressed functions, the test heating was performed
24 h after the heat shock.
RESULTS AND DISCUSSION
Figure ! shows the temperature dependence of the
effect of hardening of the thermolabile function,
cytoplasmic streaming and thermostable plasmolysis
on the leaves Commelina afrieana L. Determination
of the heat resistance of hardened and non-hardened
leaf halves was carried out immediately after the
termination of hardening. Contrary to Barabalehuk's
data, the onset and the end of the rise of the curve of
 Effect of heat shock on cellular functions differing in thermoresistance 143
2.0

1.6
o o 2

1.2

"C
0.8

0.4 '-

I
0 34 36 38 40 42 44 46 48 50 52 54

°C
Fig. I. Effect of a 30-min hardening on cytoplasmic streaming (I) and plasmolysis (2) of the epidermal
cells from Commelina africana leaves as a function of hardening temperature. Abscissa: temperature of
hardening; ordinate: a difference between thermoresistance of the cytoplasmic streaming and plasmolysis
in hardened and control cells.

temperature-dependent effect of hardening for the
thermolabile function occurs at a temperature about
10°C lower than that for the thermostable function.
Figure 2 shows the dependence of a hardening
effect of three cell functions on temperature for a 3-h
hardening. The result is similar to that described by
Barabalchuk, when using Tradescantia flumensis.
Maximum increase in thermoresistance of the cyto-
plasmic streaming and of the plasmolysis occurs at
the same temperature--38°C; however, after the 3h-
heating, the streaming stops at 41°C, and plasmolysis
is suppressed at 47°C. Optimum temperature of
chloroplast phototaxis hardening is somewhat lower.
Effect of the plasmolysis hardening can be also found
after the heatings, that completely inhibit both the
cytoplasmic streaming and chloroplast phototaxis
i.e, at 41-44°C. However, the hardening of plasmoly-
sis starts at a temperature, several degrees higher than
at which a thermoresistance of the thermolabile
functions starts to rise.
The curves depicting a 30-min hardening have a
different form (Fig. 3). In accordance with the data
obtained using Commelina africana (Fig. 1) the opti-
mum temperature of hardening for plasmolysis ap-
peared to be 13° higher than that for the streaming
of cytoplasm and 12°C higher than that for chloro-
plast phototaxis. The optimum temperatures of the
hardening of the three functions were 48, 35 and
36°C, respectively. The maximum rise in thermoresis-
tance of plasmolysis following 30 min hardening was
registered at a temperature 6 '~ higher than that which
completely suppressed the streaming of cytoplasm

2.4 - 60 • o
/
2.0 - 50 e/ /

1.6 - 40 2

°C 1.2- 30 x 3

/
0.8 - 20

/
0.4 - 10
i I 3

0 I- 0
28 30 32 34 36 38 40 42 44 46
.¢
Fig. 2 Effect of a 3-h hardening on cytoplasmic streaming (1), chloroplast phototaxis (2) and plasmolysis
(3) of Tradescantia albiflora leaves as a function of hardening temperature. Abscissa: temperature of
hardening; ordinate: left--cytoplasmic streaming and plasmolysis: a difference between thermostability of
hardened and control cells. Right--hardening effect of chloroplast phototaxis: Ph.chl. h - Ph.chl.c, see text.
Arrows indicate minimum temperature which arrests the streaming of cytoplasm (I) and plasmolysis (3)
after 3-h heating.
 144 V. YA. ALEXANDROVet al.

2o I
2.4 -- 30
x

2.0
x x

1.6 - 20

"c
1.2 --

o
x
0.8 -- 10 x

2 1
0.4 -
x

0 - 0 I I I I I
30 32 34 36 38 40 42 44 46 48 50 52

Fig. 3. The same as in Fig. 2, but with 30-min hardening.

and chloroplast phototaxis. On the other hand, the
temperature of a 30-min hardening, that gives the
maximum rise in thermoresistance of cytoplasmic
streaming and of chloroplast phototaxis is not suf-
ficient for the thermoresistance of the plasmolysis to
be affected; this requires a hardening temperature
6-7°C higher. The idea that the hardening is a general
reaction of a cell in which, concomitantly, resistance
of the cellular functions increases independently of
the level of their thermostability, can be discounted
by considering the 30 min hardening experiments.
In order that the reasons underlying the differences
in the results of 30-rain and 3-h hardenings can be
understood, the way in which changing the depen-
dence of the effect on hardening temperature at
different exposure periods should be examined
for each cell function studied. Figure 4 gives the
curves of the plasmolysis hardening. The maximum

3.2

x
2.8

2.4
x

2.0
o x

°C 1.6-

0.8

0.4
1
1 A

o l I I I I l I I I l
34 36 38 40 42 44 46 48 50 52
"C
Fig. 4. Effect of hardenin$ on plasmolysis of T. albiflora leaf cells in dependence on temperature of a
30-rain (I) and 3-h (2) hardening. Absc/~: unnperature of hardening; ordinate: a difference between
thermostability of plasmolysis in ~ and control cells. Arrows indicate the minimal temperature
at which ab. plasmolysis inhibited after 30-min (I) and 3-h (2) heating.
 Effect of heat shock on cellular functions differing in thermoresistance
hardening effect is reached after 3h at 38°C (curve 2).
If the hardening effect is achieved as a response to
some shift from normal in the cellular state, which
needs a certain heat shock dose then to obtain the
same dose, with exposure to heat being shortened
from 3 h to 30 min, a higher temperature will be
required. In full accordance with this, the curve for
the hardening response shifted sharply to the right
(curve 1) for the 30min hardening. This simple
consideration, however, cannot be applied to two
thermolabile functions, for they respond quite differ-
ently to the shortening of hardening duration from
3 h to 30 min. Figure 5 depicts curves obtained for
chloroplast phototaxis. The optimum temperature of
the hardening effect is attained after 3-h and 30-min
exposure at the same temperature, 36~C; but the
hardening effect after the short exposure is less
pronounced. Cytoplasmic streaming responded to the
changes in duration of hardening in an even more
paradoxal manner. The optimum temperature of the
hardening effect was lower for the shorter period of
heating (Fig. 6). Once again, the level of the harden-
ing effect after a 30-min exposure was lower than
after a 3 h exposure. To provide a plausible hypoth-
esis to explain these apparently conflicting results the
present experimental data must be carefully consid-
ered. Figure 3 shows that a significantly increased
thermostability of the cytoplasmic streaming and
chloroplast phototaxis, can be seen in the cell after
30 min at temperatures as low as 3Y~C. These condi-
tions are, however, quite insufficient for enhancement
of thermostability of plasmolysis. Hardening of this
function can be elicited only by a hardening temper-
ature 8-9 C higher. If a rise in thermoresistance of a
cellular function requires a hardening factor to shift
the functional state from normal by a certain value,
then this may explain why more intensive heating is
needed for a 30-min hardening of the thermostable
plasmolysis than for the same effect in the more
heat-sensitive cytoplasmic streaming and chloroplast
phototaxis. However, this point does not explain the
divergence in behaviour of these three functions on
moving from 30-rain to 3-h hardening exposure.
Why, in order to get the same hardening effect after
an increase of hardening time does the hardening tern-
60 x quickly, others more slowly, and some do not move
2O
O a 30-min exposure than after a 180-min one. Then, it
l x
I I I 1 I I I a 180-min hardening should be higher than that of a
30 32 34 36 38 40 42
"c
Fig. 5. Effect of hardening of chloroplast phototaxis of T.
albiflora cells as a function of temperature of 30-min (i) and
3-h (2) hardening. Abscissa: temperature of hardening;
ordinate: hardening effect on chloroplast phototaxis:
Ph.chl.- Ph.chlc, see text.
TB 15.'2--D
145
2.0
1.6 •
=C 1.2
1 2
0.8
0.4 -- ~ i 2f
O I I I = , •
32 34 36 3a 40 42
"c
Fig. 6. Effect of hardening on cytoplasmic streaming of the
cells from T. albiflora leaves in dependence on temperature
of 30-min (1) and 3-h (2) hardening. Abscissa: temperature
of hardening; ordinate: a difference between thermoresis-
tance of cytoplasmic streaming in hardened and non-
hardened cells. Arrows indicate minimal temperature at
which the streaming of cytoplasm stops after 30-min (I) and
3-h (2) heating.
perature need to be lowered in the case of plasmolysis,
elevated in the case of protoplasmic streaming and be
unaltered in the case of chlorophyll phototaxis?
To answer the question, it is necessary to consider
ability of cells to repair heat injury. Restoration of
function, complete or partial, may occur not only
after heating has stopped, but also during the action
of heat. This phenomenon was called a reparatory
adaptation (Alexandrov, 1956, 1964, 1979). Thus, for
example, if Campanula persifolia leaf is exposed to
41°C, cytoplasmic streaming in the epidermal cells
stops after about 40 min, however, it resumes 1.5-2 h
later and continues for several hours, after which it
dies away, this time irreversibly. The ability of the
cytoplasmic streaming to repair during inhibiting
action of heating is very well expressed in the cells of
Tradescantia albiflora. Unfortunately, it is difficult to
measure the cytoplasmic streaming in this organism
because in the normal cells some spherosomes move
at all. The following observation on behaviour of the
cytoplasm in Tradescantia leaves placed into water at
380C can be made. After 30 min heating the move-
ment of the spherosomes sharply slows down, after
90rain movement ceases completely, but after
180 min the spherosomes move quickly. Thus, at
38°C movement is inhibited much more strongly after
is clear why in order to get the same shift from
normal for cytoplasmic streaming, the temperature of
30-rain hardening (Fig. 6).
The case with chloroplast phototaxis is less compli-
cated as we can measure precisely alteration in a
paraenchyme cell of the ability to respond with a
negative chloroplast phototaxis to intensive illumin-
ation during various exposures to the same super-
optimal temperature. The data are presented in Fig. 7.
146 V YA. ALEXANI)ROVet al.

x
80
60
40
20
0 l I I l l
o 30 60 90 12o
Fig. 7. Influence of duration of heating at 37~C (1), 38~C (2)
and 40°C (3) on the level of chloroplast phototaxis in the
cells of T. albiflora. Abscissa: duration of heating (min);
ordinate: chloroplast phototaxis
Measurements of chloroplast phototaxis values were
carried out 30, 90 and 180 min after the beginning of
the leaves exposure to a given temperature. At a
temperature of 40°C this function is rapidly sup-
pressed and after 3 h chloroplasts lose completely
their ability to respond to a strong light, apparently
making no resistance to an injuring action of heating.
During heating at 37 and 38°C, inhibitory effect of
heating developed rapidly within the first half hour,
chloroplast phototaxis value diminishes by 45 and
65%, respectively. However, after this time, in spite
of continued heating, no further lowering of chloro-
plast phototaxis occurs, while a tendency to some
increase of this function can be noticed. Repair of
chloroplast phototaxis is weaker than that for cyto-
plasmic streaming, but the level of suppression of this
function after 30min and 180rain of heating is
approximately the same. This makes it clear why the
same hardening temperature is required to get an
optimal effect of hardening of chloroplast phototaxis
after 30-min and 180-rain exposure (Fig. 5). From the
results showing that, with a change from 30- to
180-min hardening for plasmolysis, the maximum
hardening effect is reached at a significantly lower
temperature (Fig. 4). it can be concluded that this
function is incapable of repairing a heat-induced shift
from normal, at least during action of heat. It is
1 ° known that in damaged cells some functions, after
their suppression, are capable of restoration, while
x
the others are injured irreversibly (Alexandrov. 1979.
1985).
This it could be that being heat-damaged during
the process of hardening, the three functions of
Tradescantia cells studied show different repair ca-
3 pacities. Comparison of the dependence of hardening
I effect on temperature immediately after hardening
1so 18o
with that 24 h later provides support for that sugges-
tion. Figure 8 shows such two curves for 30-min
hardening of the cytoplasmic streaming. When the
thermostability increase is determined immediately
after hardening (curve I). the optimum effect ( I . T C
increase) is attained at 35 C. No increase is seen in
thermostability 38 C or at higher temperatures. This
is owing to a strong suppression of the function
during the process of hardening by itself. Hardening
for 30min inhibited the streaming completely at
4 2 C . If the effect of 30 min hardening on the ther-
mostability of cytoplasmic streaming is estimated
after 24 h, the optimum temperature for hardening
was between 41~t4 C. This is because the hardened
leaves at the room temperature were able to restore
the cytoplasmic streaming that was inhibited by
hardening at 42 C and higher temperatures.
An important conclusion can be drawn from
Fig. 8. The lack of a hardening effect after a 30-min
hardening at 41 C and its appearance after 24-h
exposure to room temperature does not suggest that
the development of tolerance requires time. It ap-
pears, that time is needed for the repair of cytoplas-
mic streaming which has been inhibited by the
hardening itself. If hardening at 35 C is considered,
the hardening effect is found to diminish in 24 h.
Figure 9 shows two similar curves for the effect
of 3-h hardening dose on cytoplasmic streaming. The
difference between hardening temperature optimum

2.0

1.6 "f°\

/ \' I \'

0.8 I ~ o

0.4 -

0 ° I I ~""~" b ° t [ [ I
32 54 36 38 40 42 44 46 48
"C
Fig. 8. Effect of 30-rain hardening of the cytoplasmic streaming in the cells of T. albiflora leaves in
dependence on temperature o f hardening, thermostability being recorded irnmediatcly (1) and over 24 h
(2) after hardening. A b ~ s s a : tempetntture of hardeningq ordimtte: a difference between thermoresistance
of the streaming in hardened and non-hardet~ cells. Arrows indicate minimal temperature of a 30-rain
heating stopping cytoplasmic streaming immediately after heating.
 Effect of heat shock on cellular functions differing in thermoresistance 147
2.4

2.0

1.6

°C 1.2

0.8

0.4
!
0 I I I I I / I
f I I I
32 34 38 40 42 44 46 48

"C
Fig. 9. The same as in Fig. 8, but after 3-h hardening.

for a 30 min hardening dose estimated from the
results recorded immediately and those estimated
24h later was 8°C (43-35°), but at a 3-h hardening
the difference was smaller, 4°C (42-38 °). This occurs
because during a 3-h hardening, a cell has passed
a part of the repair process within the period of
hardening.
Analogous experiments on the hardening of
chloroplast phototaxis show similar influence on
acquisition of thermoresistance after a 24-h period
between hardening and recording of the results.
However, if we compare the behaviour of chloroplast
phototaxis (Fig. 10) to that of cytoplasmic streaming
(Fig. 8) after 30-min hardening, the ability to repair
damage reparation is weaker in chloroplast photo-
taxis than in cytoplasmic streaming. The shift in the
hardening optimum after 24 h is only 2°C (38-36 °) in
the former case and 8°C in the latter one. Figure 10
also demonstrates that values of acquired thermore-
sistance for chloroplast phototaxis, when recorded
24 h later, are in all cases from 34°C, significantly
higher (curve 2) than those recorded immediately
after hardening (curve 1). To explain this phe-
nomenon, additional experiments are needed.
Figure 11 shows the dependence of a 3-h hardening
of plasmolysis on hardening temperature being eval-
uated immediately and 24h after the hardening.
Comparison of these data with the data from Figs
9-11 clearly demonstrates that the hardened leaf
halves which had been placed into a humid chamber
at room temperature for 24 h show no sign of repair
of plasmolysis function. It is clear why repair of the
suppressed plasmolysis is impossible. The potential

x
100 -

80 -
1.2 --
1
60
x °C 0.8 / "~~ ~_ ~~

40 x 2 0.4

20 O,

0 I I I I I 1 I
34 36 38 40 42 44 46
"c
1 I I I I I I J
30 32 34 36 38 40 42 44 Fig. l 1. Effect of 3-h hardening on plasmolysis in T.
"c albiflora cells in dependence on temperature of hardening,
thermostability being determined immediately (I) and over
Fig. 10. Effect of 30-rain hardening on chloroplast photo- 24 h (2) after hardening. Abscissa: temperature of harden-
taxis of T. albiflora leaves as a function of temperature, ing; ordinate: difference between thermoresistance of plas-
thermostability being determined immediately (l) and over molysis in hardened and non-hardened cells. Arrow
24 h after hardening (2). Abscissa: hardening temperature; indicates minimal temperature of 3-h heating suppressing
ordinate: hardening effect Ph.chl h - Ph.chlc, see text. plasmolysis.
148 V. YA. ALEXANDROVet al.
for restoration of a suppressed function is set not only
by the extent of the shift from normal of the function
itself, but also by the value of shift from normal of
the cell as a whole. Plasmolysis is very thermostable
so to impair it, a high heating dose is required such
that the cell, as a whole, appeared to be seriously
damaged.
Therefore, the lack of repair of heat injury in
plasmolysis, the repair of chloroplast phototaxis and
even the more pronounced ability of cytoplasmic
streaming to recover from thermal injury were ob-
served both during heating and after its termination.
These results fully explain the difference found in
reaction of the three functions under study to the
increase in hardening from 30 min to 3 h. To obtain
the maximum increase in thermostability, the harden-
ing temperature should be lowered from 48 to 38:C
for plasmolysis; unchanged (36=C) for chloroplast
phototaxis; and increased from 35 to 38'C for cyto-
plasmic streaming.
The description of the data on behaviour of the
three functions under study, in response to a change
in the length of the heat shock period, together with
the conclusions given suggest that the reaction of the
cell to heat shock presents a combination of separate
reactions of its components which differ in ther-
mostability and capacity for repair. It is possible,
however, that the matter is more complicated still.
It cannot be excluded that in response to a stress
a situation in the cell is induced, which stabilizes
protein molecules and their complexes. However, in
order to increase the stability of a given cellular
function, some change from its normal state is needed
and only then the function becomes susceptible to
stabilization. A mechanism for acquisition of in-
creased stability induced by heat shock (and as it
is known heating may be replaced by a number
of other agents (see Alexandrov, 1985) is at present
time completely unknown. There are many reasons
to doubt that a simple casual relationship exists
between acquisition of elevated thermostability and
appearance of heat shock proteins. This is supported
by the data of Hall, 1983; Alexandrov, 1985; Xiao
and Mascarenhas, 1985; Landry and Lamarche,
1985; Carper et al., 1987 and many others. To explain
physico-chemical basis of the capacity of cells to
increase resistance, in response to the action of
superoptimal heating or other damaging agents, re-
quires further cytophysiological work.
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