Journal of General Microbiology (1 983), 129, 2023-2034.
Printed in Great Britain
2023
Effect of Osmotic Stress on the Ultrastructure and Viability of the Yeast
Saccharomyces cerevisiae
By G . J . MORRIS,* L. W I N T E R S , G . E. COULSON A N D K. J . C L A R K E
Institute of Terrestrial Ecology (Natural Environment Research Council), Culture Centre of Algae
and Protozoa, 36 Storey's Way, Cambridge CB3 ODT, UK
(Received 4 November 198.5 ;revised 6 February 1984)
Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating
compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell
volume and osmolality was non-linear; between 1 and 4 OSM there was a plateau in cell volume,
with apparently a resistance to further shrinkage; beyond 4 OSM cell volume was reduced further.
The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in
cell volume in the shrunken state. The plasma membrane is often considered to be the primary
site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted
in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on
mitochondria1 activity and structure were investigated using the fluorescent probe rhodamine
123. The patterns of rhodamine staining were altered only after extreme stress and are assumed
to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure
of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In
shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the
cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic
solutions these structural alterations were reversible, suggesting a considerable degree of
plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified,
indicating that changes in wall structure persist.
INTRODUCTION
The physical stresses to which cells are exposed during freezing are relatively well defined
(Taylor, 1986) whereas the biochemistry and biophysics of cellular freezing injury are poorly
understood. The yeast Saccharomyces cerevisiae has been used to determine the effects of
different rates of cooling on cell volume (Diller & Knox, 1983; Schwartz & Diller, 1980;
Ushiyama & Cravalho, 1979), ultrastructure in the frozen state (Bank & Mazur, 1973; Nei,
1978) and viability upon rewarming (Kruuv et al., 1978; Mazur & Schmidt, 1968). Analysis of
cells on the temperature controlled stage of a light microscope has shown that during slow
cooling a 50% reduction in cell volume, due to freeze-induced dehydration, occurs by - 10 "C
(Diller & Knox, 1983), imposing an osmotic stress at low temperature. In this study we examine
the effects of short-term osmotic stress on the ultrastructure and viability of S. cerevisiae as a
model to investigate freezing injury; a similar approach has provided valuable insights into the
response of higher plant cells during freezing and thawing (reviewed by Steponkus, 1984).
The protoplast of higher plant cells in suspension may separate from the wall when exposed to
hypertonic solutions (plasmolysis), the wall retaining its original shape and dimensions. In
contrast, suspension of yeasts in hypertonic solutions induces shrinkage of the entire cell
envelope (cell wall and plasma membrane) (Corry, 1976; Neidermeyer et al., 1977; Rose, 1975).
Electron microscopy of cells in hypertonic solutions reveals extensive changes in the
ultrastructure of the envelope (Neidermeyer et a!., 1977; Kapecka et al., 1973). In this
investigation we have compared cell viability after osmotic stress with the reduction in cell
volume and surface area previously achieved in hypertonic solutions. During osmotic
0001-3052
0 1986 SGM
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G . J . MORRIS A N D O T H E R S
dehydration of eukaryotic cells the organelles will also be exposed to hypertonic solutions and
will in turn undergo shrinkage followed by rehydration on return of the cell to isotonic
conditions. We examined the morphologyof the mitochondrial network within S. cerevisiue with
the fluorescent probe rhodamine 123. This has been recently used to demonstrate alterations in
the mitochondrial network of the unicellular green alga Chlumydomoms reinhardii during
shrinkage and rehydration (Morris et ul., 1985).
METHODS
Cells and cell culture. All yeasts were obtained from the National Collection of Yeast Cultures (NCYC),
Norwich, UK, whose numbering has been used. Cells of Saccharomyces cerevisiae NCYC 914 were used together
with cells of a range of strains and other species for the studies of volume change. Conical flasks (100 ml)
containing 50 ml yeast nitrogen base (YNB) (Difco) were inoculated with 0.25 ml of a stationary phase culture and
incubated unshaken at 24 "C for 24 h, at which time the cultures were in the late exponential phase of growth. Cells
from liquid culture were used without further preparation. The osmolality of the medium after cell growth was
160 mOSM as determined by vapour pressure osmometry.
E'ecfs of hypertonic exposure on cell viability. The response of cells to hypertonic solutions was determined by
adding 0.25 ml of a cell suspension to 0.25 ml of the experimental solution (prepared in growth medium) to give the
final required concentration. After incubation the cell suspensions were diluted into growth medium (1 :100) and
viability was assessed, after appropriate dilution, by colony formation on YNB agar.
Unless otherwise stated, all experimental procedures were done at room temperature. In experiments to
determine the effects of temperature on the response of cells to hypertonic stress, both cells and experimental
solutions were equilibrated for 15 min to the experimental temperature before being mixed.
Finally, osmolality of the solution was determined in a separate sample. The cell suspension was added to the
experimental solution, centrifuged and the osmolality of the supernatant determined. In each experiment reported
there were at least five replicates, each taken from a different cell culture, for each treatment.
Cell morphology. The dynamic response of individual yeast cells to alterations in the osmolality of the
surrounding medium was examined on a specially developed microscope diffusion stage (McGrath, 1985).
Observations were made with a Leitz Dialux 22 microscope with a 40/0*7NPL fluotar objective combined with a
2 x magnification changer and photographs were taken with a motor driven Olympus OM2 35 mm camera on
Kodak Tri-X 400 ASA film. Data were also recorded on video (Hitachi HV-65 camera, Sony U-matic recorder
model VO-5630), using a video time generator (Panasonic model WJ-810) to record the times of exposure to
solutions.
The recorded data were played back on a video monitor (Hitachi model VM-906A) and the length and width of
individual cells were measured at appropriate times. For each treatment five individual cells were measured, and
there were at least three replicates, each taken from a different cell culture, for each treatment.
For the calculation of cell volume and surface area, yeast cells were assumed to be prolate spheroids, i.e. the
structure formed by rotating an ellipse about its major axis, with length ( I ) = 2 x major axis (a),and width (w)= 2
x minor axis (6).The eccentricity (e)of the ellipse is then defined as :e = 1 (Hale, 1965). The volume (v)
of the prolate spheroid is then: v = nlw2/6. The surface area (A) is: A = n(w* lw sin-'e)/2e.
Rhodamine 123 staining of mitochondria. Cells from 1 ml culture were concentrated by centrifugation,
resuspended in 1 ml experimental solution and incubation at 20 "C for 15 min. After centrifugation the cells were
resuspended in 1 ml rhodamine 123 (Sigma; 10 pg ml-l in distilled water) for 5 min at room temperature and then
washed in growth medium. To examine the effects of inhibitors of mitochondrial function on rhodamine staining,
cells were incubated in growth medium containing either sodium azide, 2,4-dinitrophenol or valinomycin (Sigma).
Cells stained with rhodamine 123 were examined with a blue excitation filter (390490nm) and a 515nm
suppression filter (Ploemopak A). Observations were made with a 40/0-7 NPL fluotar objective combined with a
2 x magnification changer and photographs were taken with a Wild 35 mm camera on Kodak Tri-X Pan film
uprated to 6400 ASA and developed with Kodak HCl 10 developer (dilution B) for 10 min. Exposure times for
fluorescence were 1-2 s and for phase contrast 1/60 s.
Scanning electron microscopy. Cells were washed three times in growth medium and fixed in 2% (w/v) osmium
tetroxide in sodium cacodylate buffer (0.1 M,pH 6.5). Cells suspended in hypertonic NaCl(3 OSM)were fixed in 2%
(v/v) glutaraldehyde in 3 osM-NaC1. Fixed cells were washed in buffer, dehydrated in a graded acetone series and
COz critical point dried on Nuclepore 0.4 pm filters. The filters were mounted on Jeol bulk stubs, sputter coated
with 20 nm gold and examined in a Jeol JEM 100 CX Temscan electron microscope.
Freeze-fracture electron microscopy. Unfixed and uncryoprotected cells were concentrated to a paste by
centrifugation, thinly coated onto a titanium electron microscope grid and rapidly cooled by plunging into a liquid
nitrogen slurry at - 210 "C. Freeze-fracture replication was done in a modified Bullivant apparatus (Bullivant &
+
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Osmotic stress in yeasts
g
n
100
Y
&
1
0
2
4
Osmolality (OSM)
Fig. 1
6
0
50
Time of exposure (rnin)
Fig. 2
100
Fig. 1. Viability of S. cerevisiue NCYC 914 after exposure to hypertonic solutionsof NaCl (a),glycerol
( 0 )or methanol (m), for 15 min at 20 "C and dilution (1 : 100) into isotonic medium. Each point is the
mean of at least five replicates, each taken from a different cell culture, k 1 SE if not too small to depict.
Fig. 2. Viability of S. cerevisiue NCYC 914 after exposure for various times to hypertonic NaCl
solutions (2 OSM) at either 40 "C (0)or 20 "C (0).Each point is the mean of at least five replicates, each
taken from a different cell culture, 1 SE.
Ames, 1966), using platinum/carbon as shadowing material and carbon as replicant. Replicas were cleaned with
sulphuric acid (70%, v/v) at 50 "C for 6 h and washed for 5 min in distilled water six times. The replicas were
examined in the electron microscope.
Resistance of cell walls to enzyme digestion. The rate of enzyme digestion of yeast cell walls was determined in a
lysis assay similar to that described by Diamond & Rose (1970). Cells were suspended to initial ODbb00.4 in 0.1 Mpotassium phosphate buffer (pH 6.5) containing 50 mg ml-1 of the enzyme preparation Novozym 234 (Novo
Industry, Copenhagen). Cell suspensions were incubated at 30°C and OD660
read at intervals. No osmotic
stabilizers are present in this system and it is assumed that the reduction in OD is due to osmotic lysis of the
unprotected spheroplasts and is proportional to the rate of cell wall digestion. To allow comparison of different
treatments, the time taken for SO% reduction in OD was determined graphically.
RESULTS
Cell viability
There was an initial loss of cell viability after exposure to 1 osM-NaC1 or glycerol with a
smaller reduction in viability between 1 and 4 0 s ~(Fig. 1). Exposure to more concentrated
solutes further decreased viability. By contrast, methanol was non-toxic at concentrations up to
6 OSM.After exposure to solutions of NaCl an initial dilution (1 :100) into either distilled water or
sorbitol (1 M) for 15 min before a second dilution (1 :100) into growth medium did not
significantly modify cell viability.
The temperature at which the cells were exposed to hypertonic NaCl solution (2 OSM final
concentration) did not significantly affect cell viability within the range 0 to +40 "C (Fig. 2).
The data presented here correspond to exposures at both 40°C and 20"C, the other
temperatures examined (0, 10 and 30 "C) resulted in cell viability. At each temperature cell
viability was independent of the period of incubation from 1 to 15 min; longer times of exposure
resulted in progressive loss of cell viability.
Cell morphology on exposure to hypertonic solutions
Direct observation on a diffusion stage of cells during exposure to hypertonic solutions of
NaCl or glycerol showed that the whole cell shrank (Fig. 3). There was no plasmolysis
(separation of the protoplast from the cell wall). During shrinkage, the vacuole decreased in size
and in the shrunken state yeasts were phase bright and contained optically dense granules. Upon
resuspension in isotonic medium cells swelled and the vacuole become apparent. In contrast, the
rapidly permeating additive methanol induced no change in cell volume. Other strains of S.
cerevisiae (NCYC 74, 79, 207, 366, 506, 679, 686, 738, 1341 and 1392) and 12 other species of
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G . J . MORRIS AND OTHERS
Fig. 3. Light micrographs of the same field of S. cereuisiae NCYC 914 during exposure to and removal
from hypertonic NaCl solutions (2 OSM). (a) Control and after exposure to NaCl for (b) 10 s, (c) 20 s and
( d ) 50 s, or during resuspension in isotonic medium for (e) 20 s and (s)60 s. Bar, 10 pm.
yeasts (Candida curvata NCYC 476, C. humicola NCYC 818, C. utilis NCYC 707, Cryptococcus
laurentii NCYC 1462, Kluyveromyces marxianus NCYC 587, Pkk-#Gmbranaefaciens
NCYC 44, Rhodosporidium toruloides NCYC 979, Rhodotorula glutinis NCYC 61, Saccharomyces dairensis NCYC 1477, S. exiquus NCYC 1476, Schizosaccharomyces octosporus
NCYC 427, Schizosaccharomyces pombe NCYC 1430) responded in a similar manner upon
exposure to hypertonic NaCl (2 OSM) on the diffusion stage.
The reduction in cell volume was evidently osmotic and not simply an electrochemical effect
of concentrated solutionson the structure of the cell wall, as occurs in isolated bacterial cell walls
(Marquis, 1968), for cells permeabilized with Triton X-100 (0.1%, vlv) for 30 min at 37 "C did
not shrink on addition of 4 osM-NaC1. Pretreatment of cells with dithiothreitol(l0 mM) to reduce
the thiol bonds of the wall polymers did not modify the osmotic response. Cells that were
exposed to and resuspended from 5 osM-NaC1 shrank if immediately re-exposed to 5 osM-NaC1.
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Osmotic stress in yeasts
2027
Rehydration
120
100
50
I
1
I
1
I
3
5
1
3
Time of exposure (min)
Fig. 4. Cell volume of S. cereuisiue NCYC 914 after exposure to either 1 (O), 3 ( 0 )or 5 (B)osM-NaC1
and return to isotonic medium. Each point is the mean of 25 individual cells taken from at least three
different cultures 1 SE.
0
1
1
2
5
25
50
l/(Osmolality of NaCl, OSM)
Percentage viability
Fig. 6
Fig. 5
Fig. 5. Boyle Van't Hoff plot of cell volume (0)and surface area (0)of S.cereuisiue NCYC 914. Cells
were exposed for 5 min at 20 "C to NaCl solutions. Bars & 1 SE.
Fig. 6. Relationship between the loss of viability after shrinkage and rehydration as a function of the
cell volume in hypertonic solution. Bars + 1 SE.
O r
100
Quantitative analysis of cells during exposure to hypertonic solutions of NaCl at 20 "Cshowed
that shrinkage occurred within the first minute and that the cell volume remained stable for up
to 5 min, with no reswelling of cells in the hypertonic solution (Fig. 4). Cells did not shrink
isotropically, for example after 5 min exposure to 2 osM-NaC1 the diameter decreased by 23%
but the length by only 11% (n = 25). After resuspension in isotonic medium cells regained their
original volume within 1 min, with no significant swelling beyond the initial isotonic values. The
subsequent cell volume was stable for at least 15 min. Similar results were also obtained with
hypertonic solutions of glycerol. However, because of the higher viscosity of these solutions,
practical problems were encountered in using the diffusion stage, which limited all such
experiments with glycerol to osmolalities below 1.
A Boyle Van? Hoff plot with respect to cell volume was non-linear (Fig. 5 ) with no reduction
in cell volume on transfer from growth medium (160 mosM) to solutions of 500 mOSM.
Subsequent doubling of the osmolality to 1 OSM reduced the volume by 40%. A further increase
in external hypertonicity to ~ O S Minduced only an additional 10% reduction in cell volume;
beyond 4 OSM cell volume was further reduced. If the reduction in cell viability after shrinkage
and rehydration was plotted against cell volume in the hypertonic solution there was a direct
correlation between cell volume and viability (Fig. 6).
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G . J . M O R R I S AND O T H E R S
Fig. 7. Phase contrast (u, c, e) and fluorescence (b, d , n electron micrographs of the same fields of
S. cereoisiue NCYC 914 stained with rhodamine 123. Cells were either in growth medium (a, b) or
exposed to 5 ow-glycerol(c, d ) or 5 osM-methanol ( e , f )for 15 min at 20 "C and resuspended in growth
medium before addition of rhodamine 123. Bar, 10 pm.
Rhodamine 123 staining
After rhodamine 123 treatment of control cells of S. cerevisiae NCYC914 an intense,
fluorescent mitochondrial network was evident in the majority of cells (Fig. 7 6). The addition of
sodium azide
M), 2,4-dinitrophenol
M) or valinomycin (5 pg ml-I) to cells for 15 min
before the addition of rhodamine 123 completely inhibited this pattern of staining. With cells
initially treated with rhodamine 123 the subsequent addition of inhibitors of mitochondrial
function abolished the specific staining and induced a general fluorescence within the cell. From
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100 r
50
0
4
6
8
Osmolality (OSM)
Fig. 8. Effects of exposure to and removal from hypertonic solutions of NaCl on the pattern of
rhodamine 123 staining in S . cereuisiae NCYC 914. Staining by rhodamine 123 was classified into four
patterns : normal mitochondria (O), fragmented or beaded mitochondria (I),
general cellular
fluorescence (0)
and unstained cells (0)-For each hypertonic treatment at least 100 cells were
observed.
0
2
Fig. 9. Scanning electron micrographs of S. cereuisiae NCYC 914. (a) Control cells, (6)cells suspended
and fixed in 3 osM-NaC1, (c) after exposure to 3 osM-NaC1 and resuspension in isotonic medium for
1 min. Bar, 10pm.
these data it was assumed that in S. cereuisiae, as in other cell-types (Johnson et al., 1980, 1981;
Morris et al., 1985), rhodamine 123 is a useful probe for examining mitochondrial morphology
and activity.
After exposure to hypertonic solutions this pattern of staining was greatly modified (Fig. 7d,
f).In some cells the mitochondria apparently fragmented into small structures, which often had
a necklace or beaded morphology, in others an unspecific overall fluorescence was apparent
(Fig. 7 4 . The relative proportions of the various patterns of rhodamine 123 staining were
dependent on the hypertonic stress (Fig. 8), equivalent results being obtained with solutions of
both glycerol and NaC1. The alteration in the pattern of normal mitochondrial staining occurred
only at osmolalities > ~ O S M . At lower osmolalities, even though there was a significant
reduction in viability (Fig. l), no alteration in rhodamine staining was apparent. Generalized
staining is assumed to be pathological, reflecting loss of cell viability and autolysis.
Fragmentation may be an intermediate state of mitochondrial injury but such morphology was
observed after exposure to hypertonic methanol (Fig. 7f) and is compatible with survival.
Scanning electron microscopy
The surface structures of cells shrunken and fixed in hypertonic NaCl were not different from
those of cells fixed in isotonic medium (Fig. 9); there was no crenation of the outside of the
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G . J . MORRIS AND OTHERS
Fig. 10. Freeze-fracture electron micrographs of S.cerevisiue NCYC 914. (a) Control cells; bar, 1 ym.
(b) Cells in 3 osM-NaC1; bar, 0.5 ym. (c) Cells in 5 om-NaC1; bar, 10 ym. (d) After exposure to 2.5
osM-NaC1 and resuspension in isotonic medium for 1 min; bar, 0.5 pm.
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Osmotic stress in yeasts
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Incubation time (min)
Fig. 1 1 . Decrease in OD,,o of cell suspensions incubated with Novozym 234 (see Methods for details).
Cells were either untreated ( 0 )or exposed to 5 osM-NaC1 and resuspended in isotonic medium (0).
Bars represent k 1 SE, if not too small to depict.
envelope. Cells resuspended in isotonic medium were identical to control cells; bud scars were
retained both in the shrunken state and upon resuspension.
Freeze-fracture electron microscopy
Freeze-fracture electron microscopy of shrunken cells identified major alterations in the
organization of the cell envelope. Invaginations typical of the yeast plasma membrane (Fig. 10a)
disappeared and novel structures possibly originating from these invaginations were observed
(Fig. lob). Structural alterations that may represent a double cylindrical folding of the envelope
into the cytoplasm were observed after exposure to solutions of either NaCl or glycerol
of > 2.5 OSM at both 20 and 0 "C (Fig. 1Oc). Upon rehydration, these changes in cellular
ultrastructure appeared to be reversible (Fig. 10d). After exposure to hypertonic solutions of less
than 2 OSM, i.e. with a surface area >80% of that in isotonic solution (Fig. 5), these structures
were not apparent and plasma membrane morphology was normal. With the rapidly permeating
additive methanol, changes of this sort in cellular ultrastructure were not observed even at
concentrations of 5 OSM. In the shrunken state there was no evidence of vesiculation of the
plasma membrane or release of membrane material.
Cell wall digestion
Practical difficulties were encountered in removal of walls from cells fixed in hypertonic
solutions. These changes in the structure of the cell wall may also persist upon rehydration, for
enzyme treatments that digest cell walls of untreated cells were ineffective against cells that had
been exposed to hypertonic NaCl(5 OSM) and resuspended in isotonic medium (Fig. 11). This
effect was dependent on both the concentration and the nature of the solute (Table I), e.g.
treatment with hypertonic glycerol increased the rate of enzyme digestion. The alterations in the
organization of the cell wall induced by exposure to hypertonic NaCl(5 OSM) were apparently
reversed by treatment with dithiothreitol (0.1mM).
DISCUSSION
The loss of viability of S . cerevisiae after hypertonic stress was directly related to the reduction
in cell volume in the shrunken state (Fig. 6). Further, viability was determined by the minimum
volume attained and within limits was independent of the rate of shrinkage and rehydration, the
nature of the impermeant solute or the temperature at which the cell was osmotically stressed.
The plasma membrane is often considered to be the primary site of such osmotic injury
(Steponkus, 1984) but after exposure to and removal from 5 osM-NaCl, a stress which would
have resulted in a major loss of viability, all cells were osmotically responsive. A similar response
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G . J . MORRIS AND OTHERS
Table 1. Eflect of exposure to hypertonic solutions of NaCl or glycerol on the rate of
cell wall digestion of S . cerevisiae NCYC 914
Additive
None
NaCl
Glycerol
Concn
(OSM)
Treatment with
dithiothreitol*
-
1
3
5
5
1
3
5
-
+
Cell wall
digestion (min f sD)t
115 &
68 k
105 f
122 &
76
96
64
40
a3
&
f
&
&
5
13
5
6
7
4
6
4
* After resuspension in isotonic medium the cells were incubated with dithiothreitol(O.1 mM) for 1 h at 30 "C.
t The time taken for a 50% reduction in OD66@
to a second osmotic challenge has been observed in mammalian hepatocytes after a lethal
hypertonic stress (Fuller et al., 1984). In liposomes and isolated chloroplasts exposed to
hypertonic solutions, the properties of the bilayer are modified so that it becomes permeable to
small molecules and, upon return to isotonic solutions, reseals (Morris, 1981; Schmitt et al.,
1985). Similar processes may occur in the plasma membrane of S. cerevisiae, resulting in
cytotoxic redistribution of ions but retention of osmotic semi-permeability.
Exposure of S. cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell
shrinkage, without plasmolysis (Fig. 3). In contrast, exposure of higher plant cells to hypertonic
solutions may induce the protoplast to separate from the cell wall, the wall retaining its shape
and dimensions. The cell wall of higher plants is composed predominantly of rigid polymers and
under normal turgor the wall is only slightly strained (Zimmerman, 1978). The walls of Gramnegative bacteria and fungi are composed of more elastic polymers and are under considerable
tension at isotonic equilibrium (Koch, 1984). The volumetric elastic modulus VAP/AV has been
calculated to be 4.7 MPa for isotonic yeast cells (Levin et al., 1979), i.e. l-lO% that of higher
plants (Zimmerman, 1978). As the volume of the protoplast decreases, due to the osmotic water
loss, the wall will shrink with the plasma membrane until the tension in the wall is zero, when the
plasma membrane may separate.
In S. cerevisiae the relationship between cell volume and osmolality was non-linear (Fig. 5).
Previous workers have calculated the isotonic intracellular osmolality to be approximately
600 MOSM (Levin et al., 1979). Cell volume did not change on transfer from growth medium
(1 60 mosM) to 500 mosM-NaC1 or glycerol. As the extracellular concentration was increased to
1 OSM cell volume was reduced by 40%. The corresponding reduction in surface area of about
20% may have resulted in changes in the structure of the cell wall and plasma membrane. These
compensatory changes may include alterations in the density and/or thickness of the wall, elastic
compressibility of membrane lipids of 2-3% (Wolfe & Steponkus, 1981), vertical movements
(Neidermeyer et al., 1976) and release of membrane proteins (Heber et al., 1981). However, any
alterations that occurred were not detectable by scanning and freeze-fracture electron
microscopy. Between 1 and 4 OSM there was a plateau in cell volume with apparently a resistance
to further shrinkage. This type of osmotic response has been observed with a number of plant
cell-types (Meryman & Williams, 1985), from which it has been argued that this form of nonideality is a protective mechanism preventing the cell volume from reaching a damaging
minimum. Within the range of extracellular tonicities there were major alterations in the
ultrastructure of the cell envelope of S. cerevisiae. The cell wall increased in thickness and the
outer surface remained unaltered (Fig. 9) whilst the cytoplasmic side buckled with irregular
projections into the cytoplasm (Fig. 10). These sites of buckling appeared to be initiated from the
invaginations within the cell wall. After osmotic shrinkage, changes in the organization of the
cell wall were evident by the increased resistance to enzyme digestion. Other workers have
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Osmotic stress in yeasts
2033
reported similar structural changes after exposure of S. cereuisiue to hypertonic solutions of
glycerol (Neidermeyer et al., 1977; Kapecka et al., 1973). Analogous structures exist in an
envelope mutant of S. cerevisiae (Ghosh et ul., 1973) and are induced after the binding of sexspecific glycoproteins (Nakagawa et al., 1983). Membranes are not significantly elastic and a
reduction in surface area may be expected to result in a release of plasma membrane material
(see Steponkus, 1984). Buckling of the cell envelope may be an efficient strategy for the
conservation of the plasma membrane surface area. On the return of cells to isotonic solutions
these structural alterations were reversible, suggesting a considerable degree of plasticity of the
wall. However, the rate of enzyme digestion of the wall may have been modified (Table 1; Fig.
1 l), indicating that the wall structure changed.
The effects of shrinkage and rehydration on organelles within eukaryotic cells are frequently
ignored. In the unicellular green alga Chlamydomonas reinhardii, fragmentation of the
mitochondria1network is apparent after hypertonic stress within potentially viable cells (Morris
et al., 1985). In contrast, in S.cerevisiae hyperosmotic stress that reduces viability by 50%, i.e.
1 to 5oshf-NaC1, had no effect on the pattern of rhodamine 123 staining (Figs 1 and 8).
Mitochondria1 morphology was changed only after extreme stress, i.e. > 6 osM-NaCl, and is
assumed to be a gross pathological feature.
There is a good correlation between the presence of invaginations in the cell envelope and
resistance to hypertonic stress in unicellular green algae (Clarke 8z Leeson, 1985). The
physiological function of invaginations in S. cereuisiue and their role in determining the response
of cells to hypertonic solutions merit further consideration.
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CLARKE,K. J. & LEESON,E. A. (1985). Plasmalemma
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