Physiology of Osmotolerance in Fungi1

Physiology of Osmotolerance in Fungi1

Physiology of Osmotolerance in Fungi' ANDERS BLOMBERG and LENNART ADLER Department of General and Marine Microbiology. University of Goteborg. Carl Sk...

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Physiology of Osmotolerance in Fungi' ANDERS BLOMBERG and LENNART ADLER Department of General and Marine Microbiology. University of Goteborg. Carl Skottsbergs Gata 22. 413 19 Goteborg. Sweden

I . Introduction . . . . . I1. The thermodynamic state of water

. . The concept of water potential .

. . .

. . .

. . . . . . . . . . . . A. . . . . . . B . Componentsofthewaterpotentialofcells . . . . . . 111. Osmotolerance . . . . . . . . . . . . . . A . Cardinal water potentialsof growth . . . . . . . . . IV . Initial osmotic response . . . . . . . . . . . . A . Microscopic observations . . . . . . . . . . B . Boyle-van't Hoff plots and non-osmotic volumes . . . . C . Water loss in relation to cell-wall elasticity and initial turgor pressure V . Osmoregulation . . . . . . . . . . . . . A . Compatible solutes . . . . . . . . . . . B . Inorganic ions . . . . . . . . . . . . . C . Solute compartmentation . . . . . . . . . . D . Regulation of polyol accumulation . . . . . . . . VI . Osmotic hypersensitivity . . . . . . . . . . . A . Osmotichypersensitivitydeterminants . . . . . . . B . Physiological overlap . . . . . . . . . . . values . . . . . VII . Cellular factors involved in determining ymin A . Generation of energy (ATP) at low water potentials . . . .

B . Costofmaintenanceatlowwaterpotentials . . C. Ion transport and accumulation . . . . . D . Production and accumulation of a compatible solute VIII . Conclusion . . . . . . . . . . . IX . Acknowledgements . . . . . . . . . References . . . . . . . . . . .

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148 151 155 156 161 161 163 164 167 167 182 185 186 190 193 196 197 198 199 202 202 204 206 206

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This review is dedicated to Professor Birgitta Norkrans who introduced us to this field of science.

ADVANCESIN MICROBIALPHYSIOLOCY. VOL . 33 ISBN &I24277395

Copyright 0 195'2 .by Academic Press Limited All rights of reproduction in any form reserved

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A . BLOMBERG A N D L. A D I E R

I. Introduction

The cell is, in all its wealth of forms, encapsulated by a membrane, which by its semipermeable properties bestows the phenomenon of osmosis upon the cell. The water molecule permeates the cell membrane more freely than other cellular constituents, implying for most instances a thermodynamic water equilibrium (or quasi-equilibrium) of the cell with its environment. Since active cellular processes occur in water solutions, the physiology of cells inevitably has to cope with these osmotic phenomena and appropriately adjust the cell for survival and growth. Early reports on the relations of fungi to high concentrations of sugar or salt mainly focused on specific effects of the solutes. These studies were of primary economic importance in helping to define water-deficit conditions that extend the shelf life of stored foods. In fact, this interest has followed humans through history. Corry (1987) mentions in her review that grain was preserved in ancient Egypt by drying and stored in granaries (Genesis 41: 3 5 4 ) . However, micro-organisms have frequently been at variance with this strive of humankind for a safe and well-supplied future, which has certainly been a most important incentive for continued studies of fungal relations to solute-rich environments. With the review by Scott (1957), the focus was shifted from the implications of the solute molecules per se to the indirect effect these additives had on water availability, quantitatively expressed as water activity. This unifying way of viewing the effects of high concentrations of solutes directed interest towards the intracellular physiology of cells growing under conditions of low water activity, which led Brown and Simpson (1972) to introduce the concept of “compatible solute”. The concept and its fundamental implications on cell physiology were experimentally solidified by work on yeast (Brown, 1976, 1978), and has today gained general acceptance by scientists working in a wide variety of biological fields (Yancey et af., 1982). However, more recently Pitt (1989) pointed out that water activity as an important factor governing life was outweighed in a number of articles by those relating cellular activities to temperature, pH value and oxygen. He concluded that water activity remained the neglected parameter in ecology as well as physiology. In Fig. 1 the results of a recent database search for articles relating to water-osmosis and fungi are illustrated. Articles published during the period 1987-89 were classified into the indicated categories; studies involving yeasts have been separately plotted. As is evident, the vast majority of articles in the field of physiology have been conducted on yeasts while work on filamentous fungi is mainly directed towards growth-

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Number of articles

FIG. 1. Results of a computer search in the BIOSIS database for articles published during 1987-89 concerning water-osmosis and fungi. Eighty-nine articles (25% of the total) addressed this overlap in their title, and were subsequently classified according to their main focus into the categories indicated in the figure. Solid bars indicate data on filamentous fungi, shaded bars those on yeasts.

survival-related studies. This bias in available information is necessarily reflected in the present review, although efforts have been taken to include physiological information about filamentous fungi whenever relevant studies to our knowledge exist. We are convinced that studies on the fungal physiological response to a changing water availability and the exploration of homeostatic mechanisms that fine tune the cell physiology to its water environment will yield fruitful information relevant to many fields of cell science. Fungi are a heterogeneous group of organisms and many types of vegetative as well as sexual life-cycles are represented, giving the scientist a vast repertoire to choose within, including many species highly amenable for experimentation. Little interest has so far been directed towards genetic studies of fungal water relations (Fig. 1). We believe this avenue with its unparalleled opportunity for applying powerful molecular genetic methods to yeasts and fungi will bring within the range of experimental resolution many of the fundamental mechanisms in the molecular biology of fungal osmotolerance, the physiology of that phenomenon being the main subject of this review.

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11. The Thermodynamic State of Water

A consequence of the small size of the water molecule is its unusually high concentration in solutions. Pure water has a concentration at 20°C of 55.4 M and, even in what is regarded as a highly concentrated solution of any other molecule, the water concentration is not significantly altered, e.g. in a 1 M solution of sodium chloride at 20°C the water concentration is 54.4 M. The water molecules in a solution, however, are “associated” with the dissolved solute, which instigates a change in the state of water, thus giving rise to a dramatic decrease in the amount of thermodynamically available water for living organisms. The state of water can be described by different physicochemical parameters, e.g. osmotic pressure, osmolality, water potential, chemical potential of water, osmotic potential and water activity, their use often being related to the field of science by historical or practical reasons. It should be kept in mind, however, that all of these parameters are strictly related (Wyn Jones and Gorham, 1983).The diversity also holds true for the units of the parameters, where atmospheres (atm), millimetres of mercury (mmHg or torr), bars and pascals (Pa) are in use. A more uniform usage of these parameters and units is certainly recommended and would facilitate communication among scientists in interrelated fields. In this review, water potential (w) will be used because of the mechanistic advantages of its additive nature in describing the overall state of water in the cell. Water potential is the term most frequently in use by plant physiologists and, even though the concept of osmotic or turgor pressure could equally well be applied to most microbes, the generality of water potential because of its gravitational term (see below) should not be overlooked in mycology. Thisis especially valid for those fungi that transport water vertically over great distances, which has been reported for vegetative mycelia of Sepufu lacrimans (reviewed by Jennings, 1983b, 1987). The unit used in this review is the pascal (1 MPa = 10 bars), the recommended unit in the SI system.

A.

THE CONCEPT OF WATER POTENTIAL

The term “chemical potential of water” (p,), by analogy with the chemical potential of any other compound, is defined as follows:

p,

=

p+

+ R T In a, + V,.P + m,gh,

where denotes the chemical potential of water in its standard state (that can be arbitrarily chosen), R and Tare the gas constant and the temperature (K), respectively, a, is the thermodynamic activity of water, V,. the partial molal volume of water and P the hydrostatic pressure. The gravitational term is described by m,gh, where m, denotes the mass per mole of water, g

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acceleration due to gravity and h the height. The gravitational term is of insignificant importance for single-cell micro-organisms, but will, as already stated, be involved in water flow in vertical mycelia since the water potential increases by 0.01 MPa per metre of height. Equation (1) differs from the general formula for the chemical potential of any compound ‘i” by omitting the term ZiFE representing the effect of electrical potential on the chemical potential, since water carries no charge (2, = 0). The expression p W - - ~ has . proven to be of considerable importance for studies on the water relations of plants. It represents the thermodynamic work involved in moving 1 mol of water from some point in a system to a pool of pure water (Nobel, 1974). This chemical potential difference divided by V,. is defined as the water potential and given the symbol w. The osmotic potential (n); (equal in magnitude but opposite in sign to the osmotic pressure) is defined as 71:

= R T In aw/Vw.,

(2)

which transforms the water potential relation into

y~ = n

+ P + 6,gh,

(3)

where 6 , (density of water) equals rn,lV,.. The use of osmotic potential instead of osmotic pressure in evaluating equation (3) is, in our view, preferable since, in that way, the thermodynamic expression of the state of water will have the same sign inside and outside of the cell (see below). Equation (3) thus becomes generally applicable to solutions as well as cells. Sometimes a matrix potential term is included in the expression to account for water bound to surfaces. It is believed, however, that this term is negligible under quite a range of water potentials, and becomes significant only under severe cellular dehydration or in extremely dry soil (Harris, 1981). The validity of a separate matrix term has even been questioned, and it was argued that the term should principally be included in R or P (Passioura, 1980). The water potential of pure water has been given the value zero, and the term will accordingly decrease with increased concentration of a solute, e.g. sea water with a molality of about 0.5 will have a water potential of about -2.5 MPa while a high water-potential basal-growth medium for yeast corresponds to a water potential of roughly -0.4 MPa. The term “water potential” has received little attention by microbiologists over the years, and instead the thermodynamic activity of water (a,) has been used (especially in the field of food preservation). The water activity is experimentally determined as the vapour pressure of the solution divided by the vapour pressure of pure water. Furthermore, the term “osmolality” (Osm) is sometimes used as a concept relating any solution to its “ideal” equivalents

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A . RLOMBERG A N D L. ADLER

by an osmotic coefficient (+) (see the appendix in Harris, 1981). All these physicochemical parameters describing the state of water in a solution are strictly related (if pressure and gravitational terms are excluded from y~), as can be seen in equation (5):

w

=

x

=

R T In awlVw.= RT6, Osmll000,

(5)

which at 20°C yields

y

or

=

135 In a,

y~ = -2.43 Osm.

The water potential is a colligative property of a solution, and is related to other colligative properties such as depression of freezing point, depression of vapour pressure and elevation of boiling point. A solution that is 1 Osm at

0

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

I

1.2

Particle molality ( M 1 FIG. 2. The water potential of solutions in relation to solute “particle” molality, either calculated from data given by Robinson and Stokes (1959) on osmotic coefficients at 25°C (open symbols) or as estimated from the freezing-point depression and chemical data at 20°C (solid symbols) given by Wolf etal. (1979). The solutions were adjusted with sodium chloride (circles) or sucrose (squares), or represent an ideal solution either at 20°C (. . . .) or at 25°C (--).

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its freezing point has a freezing-point depression of 1.86“C. However, as pointed out by Wyn Jones and Gorham (1983), measuring the freezing-point depression strictly only yields the osmolality at the freezing point, since 4 is temperature-dependent. This temperature dependence is most notable for high concentrations and at temperatures near the freezing point (Harned and Owen, 1958). For most practical purposes in experimental physiology, however, the water-potential value of a solution of low particle molality will give essentially the same result irrespective of the method used and will even adhere closely to the ideal van’t Hoff relation (Fig. 2). The two sets of data presented have been recorded at different temperatures. However, a 5°C decrease of a 1 Osm solution will lower the y-value by not more than 0.04 MPa. Most values of the water potential given in this review are calculated for 20°C from the data of Wolf et al. (1979). The water potential of any solution is, at low concentrations where the solution can be regarded as ideal, a function of the concentration of solute “particles”, as stated in the van’t Hoffrelation (e.g. Brown, 1976). It should be noted, however, that this relation is not only applicable to ideal solutions but, furthermore, only for very dilute ideal solutions since it is a special case of the more general equation (2) for osmotic potential (osmotic pressure) (Thain, 1967). The nearly ideal behaviour of dilute solutions breaks down at increased concentrations, where the type of solute molecule becomes increasingly important. Generally, sugars like sucrose or glycerol contribute more (lower water-potential values) than ions at equal solute “particle” molalities (Fig. 3). B. COMPONENTS OF T H E WATER POTENTIAL OF CELLS

Since water is extremely permeable to biological membranes, cells are in most instances in thermodynamic water equilibrium with the solution in their environment, i.e. the water potential of the environment equals the water potential of the cell: yen”= ycell.The water potential of the environment is almost always a function of the osmotic-potential component (including matrix phenomena). Fungi, however, are confined by a more or less rigid cell wall, which restricts swelling of the cytoplasm. The total water potential of the cell is thus the sum of both the osmotic potential of the cytoplasm and the turgor pressure implied by the cell wall. 1 . Osmotic Potential of the Cell

By dissolving the wall of Saccharomyces cerevisiae by treatment with the gut juice of the snail Helix pomatia, Eddy and Williamson (1957) prepared protoplasts which were stable for several hours in solutions of 0.5 M

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-251 0

I

I

I 3.0

1

I

I 6 .O

I

I

I 9.0

Particle rnolality ( M )

FIG. 3. Water potential of solutions at 20°C in relation to solute “particle” molality, calculated from data on freezing-point depression given by Wolf et al. (1979). The solutions were adjusted with sodium chloride (solid symbols) or glycerol (open symbols), or represent an ideal solution (--).

rhamnose. Increased concentrations of the sugar decreased the size of the protoplasts while, in more dilute media, the protoplasts became swollen. These results indicate that the cytoplasmic “particle” concentration is of the order of 0.5 M and of an osmotic potential around -1.5 MPa. By applying the same approach a similar value was obtained for protoplasts of Neurospora crassa (Bachmann and Bonner, 1959) and a somewhat lower value of -3 MPa for Zoophthora radicans (Glare et al., 1989). A particle molarity value of around 0.5 M for Sacch. cerevisiae was confirmed by Conway and Armstrong (1961) by use of the microcryoscopic technique, which determines the degree of freezing-point depression. They found a total intracellular molarity of stationary-phase cells corresponding to - 1.5 MPa. However, metabolically active cells fermenting glucose displayed a decreased intracellular osmotic potential of -2.2 MPa. This type of study was extended to other fungi by use of thermocoupled psychometry, where mycelia were growing on solid media to obtain easily material free

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from the solute used, thereby eliminating the need for washing prior to cell disruption (Adebayo et al., 1971). In a high water-potential medium of -0.6 MPa, Mucor hiemalis and Aspergillus wentii exhibited osmotic potentials of -1.1 and -2.1 MPa, respectively. It was found for both fungi that the osmotic potential decreased when the water potential of the growth medium was lowered, finally resulting in an osmotic potential in both species of around -4.3 MPa at an external water potential of -3.1 MPa. Luard (1982a) used the same technique to manifest a decreased osmotic potential for Penicillium chrysogenum and Chrysosporium fastidium proportional to a decreased external water potential. An indirect method for determining the osmotic potential of the cell involves chemical analysis of cell constituents such as ions, sugars, amino acids and other metabolites, in conjunction with measurements of the cytoplasmic osmotic volume. This enables calculations of the osmotic potential as the sum of all of the individual contributions of the cellular constituents. A problem with this approach is that some abundant cell compound might be overlooked in the analysis, resulting in a too high (less negative) value for the osmotic potential. The calculated osmotic potential for P. chrysogenum was found almost to coincide with that experimentally observed by psychrometry for glucose-adjusted media to - 10 MPa, while potassium chloride-adjusted cultures to the same water potential deviated from the calculated value by about 40% (Luard, 1982a). It should be stressed, however, that chemical analysis of cells will yield important information on the balance between different osmotically active substances during growth at different water potentials (Burke and Jennings, 1990; Larsson etal., 1990; Luard, 1982a) (see Section V). A combination of these two techniques is preferable, since a lower osmotic potential obtained by the psychrometric technique will indicate the presence of constituents not analysed in the chemical analysis.

2. Turgor Pressure of the Cell Different means to measure the turgor pressure ( P ) have been applied, most of them being indirect measurements where turgor is estimated from measurements of the osmotic potential of the cell (n) and information on the external water potential (w) ( P = w- n). The requirement for a positive turgor pressure for growth and hyphal extension has been proposed by several authors (e.g. Lockhart, 1965; Reed, 1984), while an alternative hypothesis implicates microtubule-mediated extension. However, inhibitors of microtubule and microfibril function, like colchicine and vinblastine, had no significant effect on hyphal extension in Serpula lacrimans (Jennings, 1983b). Evidence against turgor as a prerequisite for growth comes from a

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study by Walsby (1980) on a square bacterium isolated from saturated brine pools, with an apparently non-existent turgor pressure. Controversy exists as to whether cells regulate turgor, volume or some other unknown parameter. However, from experimental data, species can be classified as either turgor- or volume-regulating (Reed, 1984). From a general point of view, volume seems a more universal variable than turgor, considering the infinitesimally small turgor of wall-less cells. Furthermore, mechano-sensitive ion channels have been identified in the yeast plasma membrane, the activity of the channels reportedly being regulated by the tension in the plane of the membrane (Gustin et al., 1988), which is related to the cell volume and lipid content but not presumably to turgor. Regulation of cell volume has even been attributed to a role in the cell cycle of yeast, as an essential signal governing the start point (Pringle and Hartwell, 1981). However, the turgor pressure is an essential part of the total water potential of the cell, and thus has to be measured in order to understand fully the cellular response to fluctuations in the water status of the environment. Its fundamental importance in growth, however, merits further substantiation. Thermocouple psychrometry has been used to study the water-potential components of fungal mycelia, and it was found that the turgor ranged from 0.4 to 1.1 MPa for Mucor hiemalis (at different external water potentials), and between 1.2 and 1.8 MPa for Aspergillus wentii (Adebayo et al., 1971). While turgor of M . hiemalis increased with a decrease in water potential, the turgor of A . wentii remains relatively constant. Luard and Griffin (1981) extended studies on turgor of fungi to include nine fungal species, representing a wide range of tolerance to growth at low water potentials. It was found that most fungi slightly increased their turgor in response to lower water potentials of the medium, e.g. Phytophthora cinnamomi and Phellinus noxius increased their turgor from 0.9 to 1.4 MPa as a result of a water-potential decrease of the medium from -0.5 to -3.5 MPa. In a recent study on the marine yeast Debaryomyces hansenii, however, the turgor pressure dropped with decreased external water potential. In high waterpotential basal medium, turgor was kept at 2.2 MPa and decreased to just 0.5 MPa during growth in 1.35 M sodium chloride (Larsson et al., 1990). In that study, cells were grown in a chemostat and it was reported that turgor was not significantly altered by rate of growth, at the water potentials examined. The studies reported above indicate complex modes of turgor regulation among fungi during cultivation at low water potentials, some regulating to a constant pressure while yet others tolerate or adjust to deflecting turgor values (higher or lower compared to growth at high water potentials). Using a probe is the only way of directly measuring the turgor of

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eukaryotic cells (Zimmermann, 1978). The probe is an oil-filled microcapillary linked to a pressure transducer, which can be applied to cells with a diameter of more than 20 pm. Besides giving values on the turgor, the technique can be used to determine the elastic modulus of the cells, the hydraulic conductivity of the cell surface and the permeability coefficient of the membrane for any solute. The only study to our knowledge applying the pressure probe for fungal cells is a study on the water relations of the sporangiophores of Phycomyces blakesleeanus (Cosgrove et al., 1987), the study being possible because of the large size of the structure (about 100 pm in diameter and many centimetres in length). The sporangiophore was found to have an average turgor of 0.4 MPa, a value which remained the same irrespective of sporangiophore development. Another direct technique for turgor measurement is the one applied to prokaryotes with gas vesicles, these gas vesicles being used as pressure probes. An externally applied pressure will collapse the vesicles, thereby changing the turbidity of the culture, which by measurements on turgid and plasmolysed cells will yield the turgor pressure of the cell without any assumptions about the cell volume or concentration of constituents (Walsby, 1986). By applying that technique to cells of Ancylobacter aquaticus, it was even possible to measure the turgor of individual cells by observing the gas vesicles of cells in the phase microscope while increasing the pressure. It was found that turgor significantly differed among cells, with a standard deviation of 0.04 MPa and a mean individual turgor of around 0.3 MPa during growth at high water potentials (Pinette and Koch, 1987). The deviating individual turgor pressures in these asynchronous cultures of A . aquaticus could reflect cell cycle-specific turgor values. Experimental data indicating such turgor fluctuations in Sacch. cerevisiae during the cell cycle have been presented (Brown, 1990). 111. Osmotolerance

Different terms are used in the literature to describe the overall response of organisms to low availability of water, such as “osmotolerance”, “xerotolerance”, “halophilism”, “osmophilism” and “desiccation tolerance” (Onishi, 1963; Pitt, 1975; Brown, 1976, 1978; Tilbury, 1980; Corry, 1987; Hoekstra and Van Roekel, 1988). As far as fungi are concerned, the first two terms are the most generally used. Attempts have been made to classify species according to their ability to grow below some defined threshold water potential. It should be pointed out, however, that the water relations of organisms distribute over a range rather than being restricted to two main classes such as tolerant and non-tolerant. Such a classification is thus

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prone to be artificial, but can of course be used as a relatively qualitative indicator. To determine experimentally the degree of osmotolerance, different approaches have been applied, growth-related studies being the most frequent. However, Koppensteiner and Windisch (1971), besides studying growth, included both survival criteria and a study of gas production, and found that the investigated yeasts exhibited different critical water potentials depending on the method used. Generally, the survival criteria gave the lowest y-values while growth was reported to be the most osmosensitive feature. Similarly, respiration and fermentation by yeasts isolated from the sea were reported by Norkrans (1968) to be generally more tolerant than growth to low water potentials. A.

CARDINAL WATER POTENTIALS OF GROWTH

Since the ultimate manifestation of life is multiplication, the rate of the increase in biomass seems to be a relevant parameter for studies on how much the cell is affected by water-potential change. Several growth-related investigations have appeared over the years. Most of these studies present results which are basically similar to those reported by Scott (1957) and Anand and Brown (1968). The latter authors measured growth rates of 16 yeasts at 30°C in media adjusted to different water potentials with polyethylene glycol 200. Examples of osmotolerant yeasts included in the study were Zygosaccharomyces rugosus, Zygosacch. rouxii, Torulopsis halonitratophila and Saccharomyces mellis, while the non-osmotolerant isolates were either Sacch. fragilis o r different strains of Sacch. cerevisiae. All strains more or less fitted the generalized graph depicted in Fig. 4, where one can identify values for ymax, yoptand yminfor growth. The graph indicates that these yeast strains grew faster at high water potentials, with their yoptvalues being closer to ymax than ymin. Thus, these strains merely tolerated low water potentials, which is why the term “osmotolerant” is a more preferable description of the water relations of these organisms than “osmophilic”. 1 . Values for ymax

For all of the yeasts but one used in the study by Anand and Brown (1968), the ymax value was close to 0 MPa, with this limit probably determined by the availability of carbon and energy sources in extremely dilute solutions and not by the high water potential per se. However, some fungi are unable to grow in high water-potential basal media, and seem to require addition of some solutes to lower the y-value. Two representatives for this seemingly

157

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

/

8

80

' f

'\

u

f

3

/

60

.4-

/

0

a, *

0 [L

/

/ /

40

0 -25

/

/ I/

- 20

I -15

I -10

I -5

1

0

Water potential ( MPa 1

FIG. 4. Generalized graph of the relative rate of growth in relation to the water potential of the growth medium. Indicated are the cardinal water potentials of growth, namely Wrnax, Wept and Wrnin.

osmophilic response are Xeromyces bisporus and Chrysosporiumfastidium, with reported vmax values at 25°C of -5.6 and -2.8 MPa, respectively (Pitt and Hocking, 1977). The osmophilic character of these species determined at 25°C may, however, change at lower temperatures since not only is the water potential of a solution dependent on the temperature but the water relations of organisms are also affected. This was pointed out by Onishi (1960b), who reported isolation from old soy-mash of Torulopsis halonitratophila, which at first sight seemed to be an obligate halophilic yeast. The halophilic phenotype was, however, lost by decreasing the temperature from 30 to 20°C since, at the lower temperature, the strain grew in high water-potential medium. Inversely, the cardinal temperatures for growth, namely Tmin,Toptand T,, for an organism, are dependent on the water potential of the medium. For osmotolerant yeasts, a decrease in the water potential from -1.5 to -15 MPa by addition of glucose resulted in about a 5°C increase in all three cardinal temperatures (Jermini and Schmidt-Lorentz, 1987). The interrelation between water potential and temperature has been manifested

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also for temperature-conditional mutants auxotrophic for amino acids and nucleotides, which with a decreased water potential in the medium at the restrictive temperature reverted to a prototrophic phenotype (osmotic remedial mutants; Hawthorne and Friis, 1964). The increase in the cardinal temperatures, as well as the permissive temperature of conditional mutants, is possibly brought about by an increased accumulation of compatible solutes during growth at low water potentials (see Section V.A). These intracellular solutes have been shown to stabilize proteins against thermal denaturation in vitro (Back et al., 1979), thus opposing the detrimental effects on the cells of raised temperatures. Thus, species unable to grow at high water potentials might suffer from denaturation of proteins at the prevailing temperature, and growth will be made possible either by a decreased water potential or a diminished temperature. An alternative physiological explanation for an osmophilic response comes from a mutant of Zygosacch. rouxii unable to grow at high water potentials. This defect seemed to be caused by a decrease in the mechanical strength of the cell wall (Koh, 1975). 2. Values for yopt

Anand and Brown (1968) reported values that differed for different strains of yeasts but were around -1 to -5 MPa in media adjusted with polyethylene glycol, with the osmotolerant yeasts exhibiting a broader optimum range; for example, the rate of growth for one of the strains of Zygosacch. rouxii was nearly unaffected by water potentials between -1.0 and -5.6 MPa. The non-osmotolerant strains, however, generally displayed sharper optima at water potentials around -1.5 MPa. In his studies on the fungus Eurotium amstelodami, Scott (1957) postulated that the optimum water potential for growth of osmotolerant fungi was independent of the predominant solute of the medium. This hypothesis has subsequently been substantiated by work by Pitt and Hocking (1977), Luard and Griffin (1981) and Andrews and Pitt (1987), extending the study to include at least 20 species of fungi. Among those species adhering to the hypothesis were: Eurotium chevalieri, yoptabout -7 MPa; Aspergillus jlavus, yopt-3.5 MPa (Pitt and Hocking, 1977); Phytophthora cinnamomi, yopt - 1.2 MPa; Aspergillus restrictus, yopt -5 MPa (Luard and Griffin, 1981); Aspergillus ventii, Vopt -7 MPa; Exophiala werneckii, yopt-7 MPa (Andrews and Pitt, 1987). Some other species exhibited more specialized solute tolerance, exemplified by Basipetospora halophila for which the yoptvalue in glycerol- or glucose-fructose-adjusted media was - 14 MPa, while in sodium chloridecontaining media the voptvalue was -21 MPa (Andrews and Pitt, 1987). As

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an approximate rule of thumb, however, Scott's idea hypothesis of a soluteindependent yoptvalue seems to be valid. Despite the solute independence on yoptvalues, Scott (1957) noted that the absolute rate of growth was related to the nature of the solute. This growth-rate discrepancy seems to be maximal at or around the yoptvalue, e.g. the rate of radial growth of Xeromyces bisporus at yopt-22 MPa was in glycerol-containing media reported to be about 20 pm h-', while in glucose-fructose-containing media that brought about the same yoptvalue the growth rate was increased three-fold (Pitt and Hocking, 1977). Even though the yoptvalue seems to be relatively unaffected by the type of stress solute, it is reasonable to believe that it should be related to growth temperature; detrimental effects by increased temperature will be opposed by increased accumulation of a compatible solute. Thus, water potential and temperature have appropriately to balance each other for optimum growth to occur. This was shown for Wallemia sebi, which at 20°C exhibited a yopt value of -5.5 MPa while, at 34"C, the value decreased substantially to -18 MPa (Wheeler et al., 1988). Another interesting and apparently general finding by Anand and Brown (1968) was that the most osmotolerant yeasts had a maximal growth rate substantially lower than that of the non-osmotolerant strains. This was shown to be an intrinsic property of the organisms since it was not caused specifically by polyethylene glycol, but the trend still persisted in solutions of glycerol or sucrose. The mean generation times for the osmotolerant species were about four to eight hours while those of the non-osmotolerant species were in the range of 1.5-2 hours. No relevant explanation has been given so far for this interesting finding.

3. Values for v/min Among filamentous fungi and yeasts, there are representatives with an extreme capacity for growth at low water potentials. In an overview of microbial tolerance to low water potentials (including bacteria, fungi and algae), the majority capable of growth below -25 MPa were fungi (Harris, 1981). Pitt (1975) listed fungi with ymin values below -22 MPa (a, 0.85) and found that only 11 genera of filamentous fungi were represented, all being ascomycetes. The ascomycetes constitute a large group of fungi with roughly 3000 genera. Clearly, a very small number of highly specialized variants with high osmotolerance have evolved. Some representative species with ymin values below -45 MPa are Aspergillus conicus, Chrysosporium fastidium, Eurotium amstelodami and Xeromyces bisporus. Three species of yeast were listed, namely Deb. hansenii, Sacch. (now Zygosacch.) bailii and Zygosacch. rouxii. Barnett et al. (1983) reported in their taxonomic

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classification of 469 species of yeasts data on the growth response of yeasts in glucose media adjusted to either -11 or -15 MPa. It was found that about 56% of the species were unable to grow at water potentials below -11 MPa, 28% grew at -11 MPa but not at -15 MPa and the remaining 16% had ymin values lower than -15 MPa. It can thus be concluded that, even if both filamentous fungi and yeasts encompass extremely osmotolerant species, the majority of fungal species exhibit a moderate tolerance to growth at low water potentials. It has furthermore been shown that the water potential of a solution is sometimes not the single parameter determining the response of an organism, but that solute-specific properties have to be taken into account. This statement can be exemplified by the study of growth of yeasts in media containing fructose or polyethylene glycol 200 (Anand and Brown, 1968). The osmotolerant strains had a ymin value in high concentrations of fructose of at least -36 MPa while the non-osmotolerant strains ceased growing at roughly - 12 MPa. This great difference in ymin value among strains during growth in the presence of fructose was substantially diminished when the strains were grown in polyethylene glycol-adjusted media, where growth was restricted to water potentials above about - 10 MPa, with no real evident difference in yminvalues between osmotolerant and non-osmotolerant strains. The solute-specific effect in this case might be related to uptake of the stress solute to various extents by the different organisms. Polyethylene glycol 200 seems readily to penetrate the plasma membrane of some yeasts, since Rose (1975) found that the cell volume of some strains did not respond to increased concentrations of this solute. However, polyethylene glycols of higher molecular weight are sometimes used to impose matrix waterpotential stress on organisms, assuming that these bulky compounds cannot penetrate the cell wall (Adebayo and Harris, 1971; Smith et a f . , 1990). Andrews and Pitt (1987) found ymin values for some filamentous fungi to be solute-invariant (e.g. Aspergifluspenociffoides),while the ymin values for others like Exophiafa werneckii and Geomyces sp. were more solutedependent. For species exhibiting different growth responses depending on the stress solute applied, more solute-specific terms like “salt tolerance” or “sugar tolerance” are sometimes used instead of osmotolerance. However, even these terms are found to be too simplistic since different salts exhibit marked differences in growth inhibition (Onishi, 1957a). One of the extreme cases reported was the difference between sodium chloride and lithium chloride, the latter salt inhibiting growth completely at 0.5 M (about -2.5 MPa) while growth was sustained in the presence of sodium chloride even at 3 M (-15 MPa). We believe that the term “osmotolerance” can still be used as an overall description of the phenomenon, since all media will inevitably have an

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osmotic effect on the cells, an effect that usually extends to the whole growth period but may differ in magnitude depending on solute-specific interactions with organisms. However, the specific effects should never be overlooked, and any phenomenon being classified as “osmotic” in nature should have been shown to apply for both salts and sugars. Many studies have reported that fungi show greatest tolerance to low water potentials when other environmental conditions are close to their high water-potential optimum for the species (Corry, 1987). Ayerst (1969) showed ymin values for Aspergillus chevalieri to be lowest at Topt.It was furthermore shown by Onishi (1957b) that pH value strongly influenced the ability of the cells to grow at low water potentials. While Zygosacch. rouxii grew well over a wide range of p H values at high water potentials (pH 3-7), the pH range allowing growth was narrowed to p H 4-5 in media adjusted with sodium chloride to roughly - 15.0 MPa. In conclusion, if cardinal water potentials for growth are to be reported, care should be taken in accurately describing the side factors involved, e.g. ymin-20.0 MPa (sodium chloride, 3WC, pH 4.5). For further discussions about cellular factors involved in determining the yminvalue, see Section VII. IV. Initial Osmotic Response A . MICROSCOPIC OBSERVATIONS

Direct microscopic observations on the initial response of yeast cells subjected to low water-potential media revealed cell shrinkage for both the osmotolerant Zygosucch. rouxii and the less osmotolerant Candidu utilis. This decrease in cell size was reported to be a result of non-isotropic shrinkage, the short axis being the major axis of decrease in length (Corry, 1976). This type of study was extended to include species of the yeast genera Candida, Cryptococcus, Kluyveromyces, Pichia, Rhodosporidium, Rhodotorulu, Saccharomyces and Schizosaccharomyces, which all were shown to respond qualitatively similarly to 2 Osm sodium chloride (-5.0 MPa), that is, by non-isotropic cell shrinkage. A quantitative measure of the non-isotropicity was also given for Sacch. cerevisiae, with the long axis being decreased by 11Yo while the short axis was decreased by 23% (Morris et al., 1986). The decrease in cell volume was shown to be osmotic in nature and not just an electrochemical effect of concentrated solutions on the structure of the cell wall, since treatment of intact yeast cells with Triton X-100 (a procedure that will impair the semipermeability of the plasma membrane) failed to cause a reduction in cell volumes even in 4 Osm sodium chloride (-10.0 MPa). The response of the cell size to changes in water potential was reported to

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be reversible, since osmotically dehydrated cells of Sacch. cerevisiae resuspended in high water-potential media immediately increased in volume. This cycle of dehydration-hydration could be repeated without loss in cellular osmotic responsiveness (Morris et af., 1986). When exponentially growing Sacch. cerevkiae (basal medium, -1.2 MPa) was transferred to media adjusted to -2.8 MPa by addition of glycerol or sorbitol, cells immediately decreased in volume to about 60% of their pretransfer size (Niedermeyer et al., 1977). These shrunken cells were studied by freeze-fracture electron microscopy, which revealed that large depressions of the plasma membrane appeared in response to cellular dehydration. These depressions were always associated with the typical plasma-membrane invaginations seen on cells during growth at high water potentials. In contrast to the plasma membrane of intact cells, however, that of protoplasts retained its normal ultrastructure in response to dehydration, and depressions could not be seen. Vacuoles of Sacch. cerevisiae were reported to decrease in size in vivo in response to cellular dehydration (Niedermeyer et a f . , 1976; Morns et a f . , 1986). In contrast to whole cells, the tonoplast response was reported to be irreversible; cells once shrunk could not attain their original vacuolar volume upon cellular rehydration at high water potentials (Niedermeyer et a f . ,1976). This irreversibility could also be mimicked in vitro for isolated tonoplasts, and it was furthermore proposed that the degree of elasticity of the tonoplast membrane was dependent on the protein components of the membranes (Niedermeyer, 1976). The alga Dunaliefla safina exhibited organelle shrinkage upon osmotic cell dehydration. The surface area of the endoplasmic reticulum increased, however, leading to the suggestion that it acted as a reservoir for membrane material present in temporary excess when the organelles shrink (Einspahr et af., 1988). The effect of osmotic stress on mitochondria1 structures in yeast was studied by using the fluorescent probe rhodamine 123, the pattern of staining being severely altered only at water potentials below - 14.5 MPa (Morris et a f . , 1986). Taken together, not only does the total cell volume respond to, but also the organelles of the cell will be affected by, dehydration. A well-known and well-characterized phenomenon in plant cells is plasmolysis, which is the physical separation of the plasma membrane from the cell wall as a consequence of substantial protoplast shrinkage. By definition, the turgor pressure at the point of plasmolysis is zero. For microbial cells, this phenomenon is less well documented, and conflicting data and views appear in the literature as to its occurrence. Older articles and textbooks describe the phenomenon, while in more recent publications the information is absent. However, in Salmonella typhimurium, substantial plasmolysis has been documented both by electron-microscopic examination and solute-distribution measurements (Stock et a f ., 1977).

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Recent reports on severe osmotic dehydration of Sacch. cerevisiae have not been able microscopically to identify plasmolysed cells (Morris et al., 1986; Meikle et al., 1988). Saccharomyces cerevisiae, stained with saffranin and subsequently osmotically dehydrated by treatment with 2.6 M sodium chloride, revealed no plasmolysis but the cell wall appeared rough (A. Blomberg, unpublished result). This is in accordance with studies on cells dehydrated by slow freezing resulting in extensive cell-volume decrease, where plasmolysis could not be observed either in the electron microscope (Bank, 1973; Bank and Mazur, 1973) or in the light microscope (Morris et al., 1988). The reported reduction in cell volume at water potentials of -10 MPa and lower is a puzzle (Morris et al., 1986), since turgor should at that point be zero and the cell wall be in its most relaxed state, i.e. constant cell size. However, the above-discussed depressions of the plasma membrane upon cell dehydration might indicate plasma-membrane anchorage with restricted protoplast shrinkage beyond incipient plasmolysis. A consequence of this might be protoplast-driven total cell-volume shrinkage, with the wall attaining a “negative” tension and the whole cell being subjected to a negative turgor. A negative turgor pressure has been proposed for Escherichia coli as a transient state prior to the plasmolysed state, resulting in a contraction of the cell wall below its most relaxed state (Koch and Pinette, 1987). The initial cell-shrinkage of Sacch. cerevisiae in response to low water potentials is a rapid process and, at 24”C, dehydration is completed in less than a minute, irrespective of the final water potential (Morris et al., 1986). The rapid response is a consequence of the high water permeability of biological membranes, the water permeability coefficient being around 2X cm s-l for the osmotolerant alga D.salina (Degani and Avron, 1982), human erythrocytes (Shporer and Civan, 1975) and artificially produced vesicular lipid membranes (Lipschitz-Farber and Degani, 1980). The permeability for solutes is generally much lower, as reflected in the permeability coefficient for sugars and sodium ions of around lo-’ and lo-” cm s-l, respectively (Stein, 1986). B. BOYLE-VAN’T

HOFF PLOTS AND NON-OSMOTIC VOLUMES

The Boyle-van’t Hoff relation states that nV,,,, is constant, where V,,, denotes the osmotically active volume. On rearrangement one obtains the more usually applied form for plotting volume-related data, which states that, for non-turgid cells, volume is inversely related to the external water potential. The non-osmotic volume of cells is extrapolated out of the plot as the intercept with the y-axis. By use of the Coulter-counter technique in determining average cell volume and, by plotting the data in conjunction

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with the Boyle-van’t Hoff relation, the non-osmotic volume (V,,) of cells of Sacch. cerevisiae was reported to be 35% of the original protoplast volume (Meikle et al., 1988). Whole cells had a larger V,, value than protoplasts (48%), indicating that some of the V,, value consists of cell-wall material. The nature of the remaining part of the V,, value might be organelles and storage granules or even accumulated glycerol, as indicated by the increased V,, values during growth at low water potentials (Reed et al., 1987). Even though incipient plasmolysis could not be visually observed as already discussed, it could be extracted from the Boyle-van? Hoff plot as the external water potential instigating non-linear osmotic behaviour of cells, and was reported to be -2.48 MPa (corresponding to roughly 1 Osm) (Meikle et al., 1988).

C. WATER LOSS IN RELATION TO CELL-WALL ELASTICITY AND INITIAL TURGOR PRESSURE

The amount of water leaving the cell, as a response to a decrease in the extracellular water potential, is not only related to the water potential difference but also a function of the elastic properties of the cell wall and the initial turgor pressure. The constant relating the relative change in cell volume to the pressure generated is called the volumetric elastic modulus (E):

AP

= E

AVIV,,

where Vdenotes the total cell volume and V, the reference volume, which is usually the volume at incipient plasmolysis (Dainty, 1976). Thus, the responses of walled and wall-less cells to a perturbation of the external osmotic potential may be considered as formally identical, the relative changes in the pressure and volume components being related via the volumetric elastic modulus (Wyn Jones and Gorham, 1983). The yeast cell wall is fairly elastic (De Bruijne and Van Steveninck, 1970) and reported to have an &-valueof 2-5 MPa (Levin, 1979; Meikle et al., 1988), with stationary phase cells being most elastic (Meikle et al., 1988). A consequence of this cell-wall elasticity is a 20% decrease in total cell size before the point of incipient plasmolysis. Meikle et al. (1988) obtained an &-value that was constant over the turgor range studied. Pressure-probe studies of plant cells have indicated that the value of E is strongly dependent on turgor and cell volume at low pressure, whereas at high pressures E seems to approach a constant (Steudle et al., 1977). A theoretical treatment of cellular water loss subsequent to a sudden exposure to low water potentials is depicted in Fig. 5. Cells being absolutely

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rigid ( E = w ) will in principle not lose water until the point of incipient plasmolysis (Fig. 5(a)). On the contrary, cells with elastic walls will face a decreased water content even after minor fluctuations in the water potential of the environment, the magnitude of the water loss being related to the &-value.Furthermore, the water potential at incipient plasmolysis (yplasm) will be altered, giving rise to steadily decreasing yplasm values for more elastic cells. Thus, for the theoretical cell with the lowest &-value(3 MPa) in is -6 MPa. To minimize water loss in an environment with Fig. 5(a), yplasm only slightly fluctuating water potentials, a fairly rigid cell wall would be beneficial. However, if plasmolysis is to be avoided during sudden exposure to low water potentials, the cell should have an elastic wall. At water potentials below yplasm, however, the amount of water lost is independent of E. An alternative strategy for minimizing the water loss would be decreased intracellular osmotic potential (x), which results in an increased turgor pressure that will cushion the cell against water loss in a fluctuating environment (Harris, 1981). In Fig. 5(b) are depicted three cell types with different turgor pressures at y = 0, all cells having the same &-value. Even more pronounced than a decreased water loss in relation to initial cells with highest initial turgor turgor is its consequence on yplasm, not being plasmolysed until a water potential of -18 MPa. Thus, in order to avoid plasmolysis or to retain a positive turgor pressure upon severe dehydration, cells ought to adjust their intracellular osmotic potential to enhance turgor. As reported by Meikle et ul. (1988), Sacch. cerevisiae is weakly buffered against water loss on sudden osmotic dehydration, since, at a water potential of -10 MPa (sodium chloride), only about 10% of the initial V,,, value remained. Similar results were presented by Rose (1975) for osmotic dehydration in sucrose solutions, where the cell volume of Sacch. cerevisiae at -17 MPa almost exclusively consisted of V,,,. On the contrary, it was found that the highly osmotolerant species Zygosacch. rouxii subsequent to a sudden stress at -17 MPa retained almost 77% of its V,,, value. A conservative osmotic response was also exhibited by the marine yeast Deb. hansenii, which at -13 MPa retained about 40% of the osmotically active volume (Norkrans and Kylin, 1969). Thus, the osmotolerant species were not only able to grow at low water potentials but also resisted severe dehydration on sudden exposure. The low degree of water loss might partly be explained by both a low n-value and a high &-value.However, in order to understand the remarkable resistance of Zygosacch. rouxii, additional factors have to be included like rapid solute penetration or some kind of cellwall imposed negative turgor (see above).

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-10

I -8

I -6

I

-4 Water potential (MPa)

I -2

1

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V. Osmoregulation

By osmoregulation is understood the ability of cells to adjust their intracellular solute content in the face of an external water stress. The term has been considered misleading (Cram, 1976; Reed, 1984) in not recognizing the importance of cellular control of turgor or volume in this process. It is also argued that the term “regulation” should be reserved for a stricter meaning than is usually the case in biology. Although it appears logical that turgor or volume is the controlled parameter, evidence for this is still only indirect. We therefore adhere to the more commonly adopted term “osmoregulation”, which will be used here to describe the ability of the cell to adjust the total number of intracellular solute molecules, without making any assumptions concerning the mechanisms by which the process is controlled. The discussion will be concerned with the types of solutes used by fungi to adjust their intracellular osmotic potential during either adaptive osmoregulation, by which the cells respond to a changed water potential by increasing or decreasing their internal solute . content, or steady-state osmoregulation, by which the growing cells maintain an intracellular solute content which is appropriate to the prevailing water potential. A . COMPATIBLE SOLUTES

A general mechanism by which micro-organisms counteract the dehydration effects of diverse and fluctuating external solute compositions is by excluding the stress solute and by compensating through intracellular accumulation of one o r more specific solutes called compatible solutes (Brown and Simpson, 1972) or osmolytes (Yancey et al., 1982). These compounds can be accumulated by endogenous production or by uptake from the medium to high concentrations without giving rise to appreciable enzyme inhibition or inactivation (Brown, 1976, 1978, 1990). Thus, this FIG. 5. Theoretical predictions of the initial decrease in the osmotic volume upon a sudden osmotic dehydration at indicated water potentials. Calculations have been based on equation (8) and the Boyle-van’t Hoff relation. The value of E was considered to be independent of pressure and volume. (a) Initial water loss in relation to cell-wall elasticity. Values are depicted for a model fungal cell with n = -2 MPa and P = 2 MPa at an external y-value of 0 MPa, but with different elasticities: (A)E = 3 MPa, (0)E = 10 MPa and (W) E = m MPa (rigid cell wall). (b) Initial water loss in relation to cell turgor when y = 0 MPa. Values are depicted for a model fungal cell with E = 3 MPa, but with various P-values at y = 0 MPa: (0) P = 1 MPa, (A)P = 2 MPa and (W) P = 3 MPa. In all cases a V,, of 40% has been used. Arrows indicate predicted water potentials for the model cell at incipient plasmolysis.

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allows cellular processes to operate at low intracellular osmotic potentials without the requirement for structural modification of sensitive cellular enzymes or structures. Some compatible solutes, sometimes called osmoprotectants, are not only highly innocuous to protein structure and function but also alleviate some of the inhibitory effects of high ionic strength (Pollard and Wyn Jones, 1979; Yancey et al., 1982). Only a limited number of organic compounds are used as compatible solutes. These compounds can be considered to fall into two main groups: (a) polyhydroxy compounds and (b) amino acids or amino-acid derivatives (Yancey et al. , 1982). As will be discussed below, glycerol and other polyhydroxy alcohols (polyols) are the main organic compatible solutes in fungi. The biophysical and biological properties of compatible solutes have been discussed by Wyn Jones and Pollard (1982), Yancey et al. (1982) and Low (1985). They all share an ability to raise the denaturation temperature and lower the solubility of codissolved globular proteins. Density measurements by Timasheff and his coworkers have demonstrated that glycerol (e.g. Gekko and Timasheff, 1981) is excluded from the vicinal hydration sphere of proteins in protein-glycerol solutions. This property promotes minimal protein-solvent interactions, which accounts for observed consequences such as subunit association and stabilization against denaturation. The preferential hydration and the concomitant solute exclusion from the immediate vicinity of the proteins appear to be general mechanisms by which compatible solutes stabilize proteins (see Low, 198.5, and references therein).

I . Polyols Polyols are widely distributed in fungi (Lewis and Smith, 1967; Rast and Pfyffer, 1989). Onishi (1960a, b) surveyed 119 strains of yeasts for polyol production and found that most species produced glycerol and arabinitol, and a few of them also small amounts of erythritol. Analysis of the pattern of polyol production by some 450 fungal species for the purpose of chemotaxonomy (Pfyffer et al., 1986, 1990; Rast and Pfyffer, 1989) detected polyols (the main polyols being glycerol, threitol, erythritol, ribitol, arabinitol, xylitol, sorbitol, mannitol and galactitol) in all fungi examined except among the Oomycetes. All other fungal taxa fell into two groups with respect to polyol production: (a) those that contained various polyols except mannitol (Zygomycetes and Hemiascomycetes) and (b) those that contained mannitol as well as other polyols (Chytidriomycetes, Euascomycetes, Basidiomycotina and Deuteromycotina). Work by Onishi and his coworkers, reviewed by Onishi (1963), showed that high concentrations of sugars or salts in the growth medium changed the

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pattern of fermentation in many osmotolerant yeasts, resulting in increased production of polyols. An intracellular role for these polyols was indicated by Brown and his colleagues, who demonstrated that osmotolerant yeasts, in contrast to non-tolerant species, contained arabinitol as a major intracellular component when grown in dilute basal medium (Brown and Simpson, 1972). The significance of polyols in yeast-water relations was further emphasized by the observation that the intracellular polyol content in Zygosacch. rouxii and Sacch. cerevisiae increased when the external water potential was lowered by addition of polyethylene glycol 200, glucose (Brown, 1974) or sodium chloride (Brown, 1978) and that the internal concentrations at low water potentials could reach molal levels. Similar conclusions were reached by Norkrans who emphasized the role of glycerol in the salt relations of yeasts. It was first reported that the total salt content in the marine yeast Deb. hansenii did not balance the water potential of the growth medium when the cells were cultured at high salinities (Norkrans and Kylin, 1969). Glycerol was suggested as an osmoregulator (Gezelius and Norkrans, 1970) and subsequent studies confirmed that the intracellular level of glycerol in growing Deb. hansenii increased in parallel with the external concentration of sodium chloride (Gustafsson and Norkrans, 1976; Gustafsson, 1979). It was later demonstrated that isotonic concentrations of sodium chloride, potassium chloride and sucrose promoted glycerol accumulation to similar levels, while return of cells to more dilute media resulted in rapid release of glycerol in amounts that were proportional to the decrease of external solute concentration (Andre et al., 1988). This work also provided more direct evidence for a role for glycerol in osmoregulation by the finding that externally supplied glycerol re-established ability to grow in the presence of high salinity for a mutant of Deb. hansenii with a lowered capacity to produce glycerol (see Section VII1.D). The importance of glycerol as a compatible solute in yeast has been further confirmed by natural-abundance 13C nuclear magnetic resonance (NMR) spectroscopy, which allows identification and quantification of all classes of organic compounds that attain a significant concentration within the intact cell (e.g. Norton, 1980). Analysis by NMR of yeasts grown in media containing concentrations of sodium chloride from 0 to 0.86 M showed that glycerol was the major osmoresponsive organic solute in exponentially growing Sacch. cerevisiae, Zygosacch. rouxii and Deb. hansenii (Reed et al., 1987). Simultaneous measurements of the non-osmotic volume allowed an estimate to be made of the intracellular glycerol concentrations, which were calculated to counterbalance up to 95% of the total external osmotic pressure. In a more detailed NMR study by Bellinger and Larher (1987), glycerol, arabinitol and the disaccharide trehalose were detected as the main internal organic solutes in Hansenula anornala, while glycerol and trehalose

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prevailed in Sacch. cerevisiae. Of these solutes only glycerol responded to growth-medium salinity. One of the problems involved in evaluating the importance of polyols in yeast osmoregulation is the dynamics of the individual pol yo1 concentrations during the growth cycle in batch culture. In saline media, the glycerol content of Deb. hansenii increases during the lag and early exponential phases and decreases again during the late exponential phase of growth. The level of arabinitol increases slightly but progressively during exponential growth and reaches its highest value in the early stationary phase (Adler and Gustafsson, 1980). On a water-potential basis, this increase does not compensate for the decrease in glycerol. Thus, there has been a requirement for studies using continuous culture to establish an osmotic budget of cells grown under steady-state conditions and to separate clearly the effects of the external water potential from those due to changes in growth rate, pH value and nutrient composition. Larsson et af. (1990) examined the intracellular solute composition of Deb. hansenii in glucose-limited chemostat cultures containing 0, 0.7 (-3 MPa) and 1.4 M (-6.5 MPa) sodium chloride. To determine intracellular solute concentrations, the osmotic volume of the cells was determined by a radiotracer technique. The results showed that arabinitol responded little to salinity but appeared to be slightly adjusted relative to growth rate, at high salinity, as the levels increased with decreased rate of growth (Fig. 6). Glycerol, on the other hand, behaved as a

(b)

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1

. . .. .... .. n riu. 0. inrraceiiuiar concenrrarions OT \a) araoiniroi ana (0)glycerol in ueoaryornyces hansenii grown in a glucose-limited chemostat at four different dilution rates in media containing 4 mM (triangles), 0.7 M (squares) and 1.4 M (circles) sodium chloride. Redrawn from Larsson et al. (1990). -I-

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PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

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typical osmoresponsive solute; the contents rose dramatically with medium salinity and also appeared adjusted relative to arabinitol such that the overall polyol content remained relatively constant at the various growth rates. The total polyol pool counteracted about 75% of the external water potential in media containing 0.7 and 1.4 M sodium chloride. It was concluded from this work and a similar study by Burke and Jennings (1990) that Deb. hansenii adjusted to the prevailing water potential using primarily potassium chloride at low external salinity and glycerol and sodium chloride at high salinity. Nobre and DaCosta (1985b) followed the content of arabinitol in Deb. hazsenii during growth in batch culture adjusted to low water potential by various stress solutes. Low water potential promoted an up to two-fold increase in the arabinitol content of stationary phase cells, but the final content was more influenced by the type of stress solute used than by its concentration. The authors concluded that osmotic control of the arabinitol level appears imprecise and that the primary control may be exerted by other factors. Similarly, Moran and Witter (1979) observed a solute-specific increase in arabinitol accumulation in Zygosacch. rouxii. The cells increased their content of arabinitol when grown in the presence of increased concentrations of glucose, but not with increased sucrose concentrations. Van Zyl and Prior (1990) followed polyol accumulation in Zygosacch. rouxii during growth in continuous culture at lowered water potential, and demonstrated that glycerol was the principal compatible solute in cells grown in media adjusted with sodium chloride. When sodium chloride was substituted by isotonic concentrations of polyethylene glycol 400, the intracellular glycerol concentration remained essentially similar to that reached in the saline medium, while the intracellular retention of arabinitol increased several-fold, so that the concentration of this polyol became, on a molar basis, similar to that of glycerol. Thus, arabinitol, which is normally an only slightly osmoresponsive polyol, may in response to specific stress solutes be used as an osmoregulatory compound. Evidence that polyols play an important role in water relations of filamentous fungi has accumulated during the last decade. Adler etal. (1982) examined the pol yo1 content in vegetative mycelium of Penicillium chrysogenum and Aspergillus niger grown in concentrations of sodium chloride ranging from 0 to 2 M. Both organisms contained glycerol, erythritol, arabinitol and mannitol and the total polyol contents increased strongly in response to raised salinity; glycerol and erythritol became increasingly predominant in the highly saline media (Fig. 7). Using published values for dry weight:fresh weight ratios at low water potential (Luard, 1982a; Beever and Laracy, 1986), the intracellular polyol concentrations can be estimated to counteract 40-60% of the external water potential in media containing 1.4 and 2.1 M sodium chloride. Luard (1982a) reported that glycerol was

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increasingly accumulated in P . chrysogenum and the osmotolerant species Chrysosporium fastidium as the water potential was decreased by adjustment with potassium chloride or glucose. The higher polyols identified (erythritol, arabinitol and mannitol) did not change consistently and were for this reason, and from behaviour in osmotic-shock experiments (Luard, 1982b), not considered to be of importance in osmotic adjustment. From analysed intracellular solute levels, glycerol accounted for 1540% of the calculated osmotic potential after growth on media adjusted with potassium chloride or glucose to -10 MPa. Similar observations that glycerol is the major osmoresponsive compatible solute within mycelia exposed to low water potential (generated by various stress solutes) have since been reported by Hocking and Norton (1983), who used natural-abundance I3C NMR to examine four osmotolerant and one non-tolerant species, by Gadd et al. (1984) for Penicillium ochro-chloron and by Beever and Laracy (1986) for Aspergillus nidulans. Although there appears to be a clear emphasis on glycerol production and accumulation under conditions of water stress in filamentous fungi, glycerol is not, as stressed by Jennings (1984, 1986), the solely osmoresponsive polyol. The type of polyol accumulated appears to be dependent on culture age, nutrient conditions, type of stress solute used and the organism studied.

1

I 1.4

1,

I 2.1

Concentration of sodium chloride ( M )

FIG. 7. Intracellular polyol contents of Aspergillus niger grown in media containing 0, 0.7, 1.4 or 2.1 M sodium chloride. For every salt concentration each set of open

columns describes, from left to right, contents of glycerol, erythritol, arabinitol and mannitol, respectively. The filled-in columns describe the total content of polyols. Redrawn from Adler et al. (1982).

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Hocking (1986a) followed the time-course of glycerol accumulation in five fungi grown at low water potential and demonstrated, as observed for yeasts (e.g. Adler and Gustafsson, 1980; Meikle et al., 1988), that culture age influences the glycerol content. The glycerol content remained high during vegetative growth, but decreased as the culture senesced and started spore formation. When the marine fungus Dendryphiella salina was grown in media in which the water potential was adjusted by addition of various electrolytes, the total soluble polyol concentration increased with decreased water potential (Wethered et al. ,1985). Within that pool, the proportions of the individual polyols differed depending upon the salt used. Highest glycerol values were found in media containing sodium chloride and the lowest when sodium sulphate was included in media. In the presence of 0.44 M sodium chloride (- 1.9 MPa), the polyols accounted for 30% of the calculated intracellular osmotic potential. The content of arabinitol and mannitol changed little with increasingly saline media but increased, on the other hand, when non-growing mycelium was exposed to saline media (Jennings, 1973). Although glycerol was the major osmoresponsive polyol in the species of Penicillium and Aspergillus examined, erythritol showed the behaviour of an osmoregulatory compound; it progressively increased with increasing external solute concentration (Adler et al., 1982; Gadd et al. , 1984; Beever and Laracy, 1986) and decreased after hypo-osmotic shock (Beever and Laracy, 1986). Furthermore, Da Costa and Niederpruem (1982) presented evidence for an osmoregulatory role for arabinitol in Geotrichum candidum, and Al-Hamdani and Cooke (1987) found that both mannitol and glycerol were the carbohydrates that increased most at decreased water potential in sclerotia of Sclerotinia sclerotiorum. Thus, though available evidence indicates that, in growing cells of yeasts and filamentous fungi, glycerol is the predominant osmoresponsive polyol it cannot, at least in filamentous fungi, be singled out as the exclusive osmolyte. It is probably significant, however, that in some instances where glycerol was found to give little contribution to cellular osmotic relations (Moran and Witter, 1979; Da Costa and Niederpruem, 1982; Al-Hamdami and Cooke, 1987) the organisms were washed before extraction under conditions which probably resulted in significant loss of this highly osmoresponsive solute. As pointed out by Pitt (1989), glycerol is the smallest polyhydroxy compound that can be used as a compatible solute. Therefore, from the point of carbon economy, glycerol production represents an effective way to osmoregulate. This may be the reason why glycerol is a preferred osmolyte among growing fungi while, as the organisms enter a non-growing stage, glycerol disappears and higher polyhydroxy compounds become the predominant intracellular organic compounds. These compounds, which are less permeable than glycerol and thus easier to retain,

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may not fully substitute for glycerol in terms of contribution to the osmotic potential. However, maintained turgor may be irrelevant to the resting cell, and the higher polyols may, nevertheless, serve to protect cellular structures against dessication stress. Many of these polyols, such as arabinitol in osmotolerant yeasts and mannitol in many filamentous fungi, are present in high concentrations irrespective of the external water potential, and could be regarded as constitutive compatible solutes in accordance with the terminology of Harris (1981). Hence, they give significant contribution to the internal osmotic potential, and thereby the turgor, also in growing cells under non-stress conditions, and their presence will tend to buffer such cells against dehydration effects by a fluctuating external water potential as discussed in Section 1V.C. Recovery of turgor-volume and growth after exposure to osmotic stress does not necessarily involve endogenous synthesis of compatible solutes. Uptake and accumulation of the external stress solute represent an alternative option, certainly when the external osmoticum has the characteristics of a compatible solute. Nobre and Da Costa (1985b) observed that, when Deb. hansenii was grown in medium containing 1 M sodium chloride in which glucose was substituted by erythritol as the carbon source, only slight intracellular accumulation of glycerol occurred and instead glycerol was replaced by erythritol as the internal compatible solute. Likewise, intracellular arabinitol was replaced by mannitol when mannitol served as the carbon source. When D.salina was furnished with the non-metabolizable sugar 3-O-methylglucose, it was intracellularly concentrated and the cell responded by converting intracellular mannitol into glycogen and preserved in this way a constant intracellular sugar concentration (Jennings and Austin, 1973). It is also apparent from numerous observations (e.g. Luard, 1982a; Gadd et al., 1984; Wethered et al., 1985; Meikle et al., 1988; Smith et al., 1990) that solutes used to change the water potential of a medium can to various extents be accumulated in yeast cells or in fungal hyphae. However, the contribution of the stress solute to the intracellular solute composition is usually difficult to determine experimentally, because a small amount within the cells has to be separated from a large amount in the medium. Beever and Laracy (1986) commented on these difficulties, and they observed in their study of A . nidulans that the external osmotica (sodium chloride, potassium chloride or glucose) played only a transient role in cellular osmoregulation after a hyperosmotic shock. There was an increased influx of the stress solute after such a shock, but the level decreased as endogenously produced glycerol and erythritol increased. When, on the other hand, glycerol was used as the stress solute, the glycerol content was maintained high and the intracellular rise of erythritol was depressed.

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2. Other Roles for Polyols

Apart from serving as compatible solutes, polyols may have other roles in fungi. Lewis and Smith (1967) suggested, in their review, that polyols, beside having a potential role in osmoregulation, could serve as carbohydrate reserves, translocatory compounds and a storage for reducing power. There is still not much information as to whether polyols are stored as carbon reserves, but mannitol is a most conspicuous candidate, since this compound is often found in mycelia, sclerotia, fruiting bodies and spores in large quantities (McCullough et al., 1986; Rast and Pfyffer, 1989). Such a role would not necessarily be in conflict with a function as an osmotic buffering agent as already discussed. Jennings (1987) discussed translocation of solutes in filamentous fungi. Available evidence points to trehalose as the main carbohydrate translocated (Hammond and Nichols, 1976; Brownlee and Jennings, 1981) and there is little to support a role for polyols in long-distance transport of carbon compounds. Turning to the role of a redox sink, a maintained cell metabolism is dependent upon a continuous supply of cytosolic NAD+ to sustain oxidation of substrates. Polyols are more reduced than sugars, and polyol formation represents a way of disposing of reducing equivalents to re-oxidize NADH. It is well established that glycerol formation in Sacch. cerevisiae is a means by which the cells oxidize NADH during alcoholic fermentation (Section V.A.4). Holligan and Jennings (1972) demonstrated that the type of nitrogen-containing compound included in media affects the relative amounts of arabinitol and mannitol in mycelia of Dendryphiella salina. It was suggested that the quest for NAD(P)H in reduction of the nitrogen source was responsible for the amount of the two polyols formed. Reduction of an aldose or ketose to the corresponding polyol has also been considered as a means of removing hydrogen ions from the cytoplasm (Jennings, 1984). Although the overall reaction for diversion of sugars to polyols is not proton-consuming, polyol production may be integrated in cellular p H regulation simply by constituting a neutral end-product. Experimental evidence for a role for polyols in pH control has yet to be produced.

3. Other Candidates: Trehalose and Amino Acids Trehalose is a disaccharide containing glucose residues which is used as a compatible solute in bacteria (Giaever et al., 1988). It is widely distributed in fungi and is normally accumulated during conditions of decreased growth, particularly during periods of starvation and differentiation (Thevelein, 1984). Although stationary phase cells of Sacch. cerevisiae contain higher

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trehalose contents when grown in media of decreased water potential (Meikle et al., 1988), trehalose seems to have little role in osmoregulation during steady-state growth. In chemostat experiments, the trehalose content in Succh. cerevisiae grown in the salinity range of M . 8 6 M sodium chloride increased with elevated salinity ( 0 = 0 . 1 4 . 2 h-'), but the intracelM a r concentration never exceeded 50 mM (K. Larsson and R. Olz, personal communication). Likewise, Gadd et al. (1984) observed an insignificant contribution of trehalose to the intracellular solute composition of Penicillium ochro-chloron grown at concentrations of sodium chloride and copper sulphate up to 0.5 M. However, trehalose is implicated in osmotic-shock tolerance (see Section VI.3) and as a desiccation protectant of stationary phase cells exposed to harsh water-stress conditions such as during drying in air or on lyophilization (Van Laere, 1989; Wiemken, 1990). Many organisms use amino acids as compatible solutes (Yancey et al., 1982). In yeasts, the content of free amino acids does not appear to be osmoresponsive, as shown from 13C NMR measurements (P.-A Jovall and L. Adler, unpublished observations) and conventional analysis. In Deb. hansenii, the total amino-acid content was slightly lower in cells growing in medium containing 2.7 M sodium chloride than in dilute basal medium (Adler and Gustafsson, 1980) while Brown and Stanley (1972) reported that sodium chloride up to 0.5 M had no observable effect on the free amino-acid content and pool composition in Sacch. cerevisiae and Zygosacch. rouxii when grown in chemostat cultures. Malaney et ul. (1988) observed an increase in the proportion of basic amino acids and a strong relative increase in the content of citrulline in baker's yeast grown in medium containing 0.6 M sodium chloride. These changes were, however, insignificant in terms of their contribution to the osmotic potential of the cytoplasm. In sporangia of the fungi Thraustochytrium aureum and T. roseum, on the other hand, the content of the amino acid proline increased in a linear fashion as the salinity of the medium was adjusted to that of sea water (Wethered and Jennings, 1985). Although proline was the major organic solute in these sporangia, ions made by far the greatest contribution to the intracellular osmotic potential. It was not clear from the experiments whether proline was synthesized by the fungi or taken up from the medium. Luard (1982~) observed that proline accumulated in Phytophthora cinnarnomi to a concentration of 0.4-0.5 M as the external water potential was adjusted to -2 MPa with sucrose. It was suggested that proline serves as the main compatible solute in this organism which belongs to the Oomycetes, which are noted for their inability to produce polyols (Pfyffer et al., 1986). It is noteworthy, however, that Luard (1982~)observed the presence of low levels of arabinitol in P. cinnamomi and even higher amounts (arabinitol and mannitol) in Pythium debaryanum grown at -1.5 MPa.

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4. Yolyol Metabolism

Because of the importance of polyols as compatible solutes in fungi, a brief overview of polyol metabolism is presented in the following section. For more detailed information, the reader is referred to the thorough review by Jennings (1984). In Sacch. cerevisiae, biochemical evidence indicates that glycerol is formed from dihydroxyacetone phosphate which, as a first step, is reduced by an NAD+-linked glycerol-3-phosphate dehydrogenase (GPD, EC 1. l . 1.8; Gancedo et al., 1968). This reaction produces glycerol 3-phosphate which is subsequently dephosphorylated by a seemingly specific phosphatase (Tsuboi and Hudson, 1956) to yield glycerol. The same pathway is responsible for glycerol formation in Zygosacch. rouxii and Deb. hansenii (see Fig. 8), as indicated by enzyme studies (Edgley and Brown, 1983; Adler et al., 1985; Nilsson and Adler, 1990), although GPD in Zygosacch. rouxii is dependent on NADP+ rather than NAD+ (Verachtert and Dooms, 1969; Brown and Edgley, 1980). Glycerol is a mandatory end-product when yeasts ferment sugar to ethanol. Production of ethanol from glucose is namely a redox-neutral process, while biosynthesis of cell material is an overall oxidative process. To maintain redox balance, dihydroxyacetone phosphate Glucose -/+Gu l cose

c Fructose6- phosphote

DHAP-

14

A

16-phospate

+

7 6 -Phosphogluconate

Erythrose 4- phosphate

GAP Ribose5-phosphate

I

FIG. 8. Metabolic pathways for glycerol and arabinitol production in Debaryornyces hansenii. The scheme is based on information in Adler et al. (1985) and Jovall et al. (1990). 1, indicates the glycerol-transport system; 2, glycerol kinase; 3, mitochondria1 glycerol-3-phosphate dehydrogenase; 4, NAD+-specific glycerol-3phosphate dehydrogenase; 5, phosphatase. D H A P indicates dihydroxyacetone phosphate; GAP, glyceraldehyde phosphate.

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is reduced to glycerol (Lagunas and Gancedo, 1973; Nordstrom, 1966, 1968). Therefore, Sacch. cerevisiae which maintains a mainly fermenting metabolism under aerobic conditions, produces glycerol (about 5 mmol per gram of yeast (dry weight) formed) when cultured in a basal glucosecontaining medium, while the respiratory yeast Deb. hansenii does not. In filamentous fungi, this pathway for glycerol formation appears to occur in Neurospora crassa, as judged from the enzyme studies by Viswanath-Reddy et a f . (1977). Legisa and Mattey (1986) detected, on the other hand, high activity of a NADP+-specific glycerol dehydrogenase in crude extract of Aspergillus niger, and have suggested that glycerol formation in this organism is by reduction of dihydroxyacetone to glycerol. Growth on glycerol as the sole source of carbon was demonstrated for 89% of the 469 yeast strains described by Barnett et af. (1983). By isolating mutants defective in glycerol utilization, Sprague and Cronan (1977) provided genetic evidence that glycerol utilization in Sacch. cerevisiae involves phosphorylation by a glycerol kinase (EC 2.7.1.30) followed by oxidation by a mitochondria1 GPD (EC 1.1.99.5) to dihydroxyacetone phosphate. In Sacch. cerevisiae these enzymes are repressed by glucose. The structural gene (GUTZ) for glycerol kinase has been cloned by functional complementation of the corresponding gut mutant and Northern-blot analysis has confirmed the occurrence of glucose repression at the transcriptional level (B. Ronnow and M. Kielland-Brandt, personal communication). Enzyme and mutant studies have shown this so-called phosphorylative pathway for glycerol catabolism to be operative also in Deb. hansenii (Adler et a f . , 1985; Fig. 8) and it seems to be a common pathway for glycerol utilization in yeasts (De Koning et al., 1987). Biochemical and genetic evidence has been presented showing that the phosphorylative pathway is the main route for glycerol dissimilation in N. crussa (Courtright, 1975a,b; Denor and Courtright, 1982) and Aspergillus nidufans (Arst et a f . , 1990; Hondmann et al., 1991). The phosphorylative pathway is induced by glycerol in these fungi (Courtright, 1975b;Hondmann et al., 1991), as appears to be the situation also in Candida utilis (Gancedo et a f . , 1968). Based on enzyme-activity measurements, an additional route in which glycerol is utilized by an NADP+-dependent oxidation to glyceraldehyde has been suggested to operate in N. crassa (Tom et al., 1978), but the enzyme activities are considerably lower than those on the phosphorylative pathway (Denor and Courtright, 1982). A third alternative may occur in Aspergillus japonicus in which glycerol induces glycerol oxidase, an enzyme that oxidizes glycerol to glyceraldehyde and hydrogen peroxide in the presence of molecular oxygen (Uwajima et a f . , 1980). Glycerol catabolism through direct oxidation of glycerol was proposed for Schiz. pombe due to lack of a detectable glycerol kinase activity, the

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presence of an NAD+-linked glycerol dehydrogenase (EC 1.1.1.6) and a dihydroxyacetone kinase (May and Sloan, 1981; May et al., 1982). The operation of this so-called oxidative pathway in Schiz. pombe has received support from studies with mutants lacking the enzymes of the pathway (Gancedo et al. , 1986; Kong et al., 1987). Mannitol formation in fungi is believed to occur by an NAD+-dependent reduction of fructose 6-phosphate to mannitol 1-phosphate by mannitol-lphosphate dehydrogenase (EC 1.1.1.17) followed by dephosphorylation to mannitol by mannitol-l-phosphatase (EC 3.1.2.22) (Wang and Le Tourneau, 1972), while mannitol is considered to be dissimilated by an NADP+dependent oxidation to fructose by mannitol dehydrogenase followed by phosphorylation by hexokinase (McCullough et al., 1986). A cyclic pathway involving interconversion of fructose and mannitol for transfer of reducing equivalents from NADH to NADPH, has been proposed to occur in fungi (Hult and Gatenbeck, 1978). The presence of the enzymes constituting the mannitol cycle has been demonstrated in a number of fungi (Hult et al., 1980). However, doubts have been raised concerning the importance of the cycle for NADPH generation (McCullough et al., 1986; Singh et al., 1988). Spencer et al. (1956) determined the labelling pattern of arabinitol formed by an unidentified osmotolerant yeast from glucose labelled in position 1or 2. They concluded that the pentitol was formed by activities of the nonoxidative and oxidative branch of the pentose phosphate pathway. By using specifically labelled substrates, similar conclusions were reached for arabinito1 production in Dendryphiella salina (Lowe and Jennings, 1975) in which the final step in arabinitol synthesis involved reduction of xylulose (Holligan and Jennings, 1972). The situation is more unclear regarding the precursor for arabinitol in Zygosacch. rouxii. Blakely and Spencer (1962) suggested a direct conversion of xylulose to arabinitol according to the labelling pattern of arabinitol formed from xylulose labelled in position 5 , whereas Ingram and Wood (1965) found a labelling pattern from [6-'4C]glucose and enzymic activities which suggested formation through dephosphorylation of ribulose 5-phosphate and an NADP+-dependent reduction of ribulose to arabinitol. Using NMR spectroscopy, Jovall et al. (1990) examined the labelling pattern of arabinitol formed from [1-I3C]- and [6-'3C]glucose by Deb. hansenii. The almost exclusive labelling of C-5 of arabinitol from [6-'3C]glucose suggested formation via ribulose 5-phosphate (Fig. 8). Catabolism of arabinitol in yeasts appears to be initiated by NAD(P)+linked dehydrogenase to give the corresponding pentulose which may then be phosphorylated and catabolized by the pentose phosphate cycle (Barnett, 1976). In yeast, erythritol is believed to be formed by the action of transketolase on fructose 6-phosphate, which leaves a four-carbon fragment that is

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reduced and dephosphorylated to give erythritol (Spencer and Spencer, 1978). Utilization of erythritol was found to depend on synthesis of an NAD+-linked erythritol dehydrogenase in Schizophyllum commune (Braun and Niederpruem, 1969). A constitutive NADPf-dependent erythrose reductase activity was also present. Many polyol dehydrogenases have a low degree of specificity towards the carbohydrate substrate. Hence, a single enzyme may act on pentitols and pentoses as well as hexitols and hexoses. The NADP+-dependent alditol dehydrogenase (EC 1.1.1.21) which has been purified from Pichia querquum by Suzuki and Onishi (1975) is an example of a well-characterized dehydrogenase with a wide substrate specificity.

5 . Polyol Transport A transport system with high specificity for glycerol has been identified in Deb. hansenii (Adler et al., 1985; Lucas et al., 1990). The system is constitutive and the K , value remains relatively constant at about 0.5-1 mM at external concentrations of sodium chloride ranging from 0 to 3 M. Cells starved for potassium and sodium ions transported glycerol by passive diffusion while addition of sodium or potassium gave transport according to Michaelis-Menten kinetics (Lucas et al., 1990). Addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) induced collapse of the glycerol gradient. Since the levels to which the transport system could accumulate glycerol were correlated with the extracellular concentration of sodium chloride, it was suggested that glycerol was transported by a glycerol-sodium symporter and that the transmembrane sodium-ion gradient which drives the glycerol uptake is maintained by an Na+/H+ antiporter. Potassium ions were accepted as a cosubstrate when the sodium-ion concentration was low. This system is so far the only one characterized in fungi, but genetic evidence has been presented for a glycerol-transport system in Aspergillus nidulans (Visser et al., 1988). Less is known about the mechanism by which higher polyols are taken up. Observations by Canh et al. (1975) led them to suggest that erythritol and a number of pentitols and hexitols crossed the plasma membrane of Sacch. cerevisiae by passive diffusion. However, mannitol uptake in Sacch. cerevisiae was shown to have the characteristics of an energy-dependent carrier-mediated transport system (Maxwell and Spoerl ,1971). Kloppel and Hofer (1976) demonstrated that Rhodotorula gracilis possesses a constitutive transport system for mannitol, xylitol and arabinitol, and an inducible uptake system with lower polyol affinity by which the polyol uptake was associated with proton absorption. In Schizophyllum commune, an uptake

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system for mannitol and arabinitol was demonstrated which appeared to be both specific and inducible (Aitken and Niederpruem, 1972). 6. Membrane Permeability An intriguing fact that is difficult to reconcile with intracellular accumulation of glycerol is the reported high permeability of glycerol through lipid bilayers (e.g. Stein, 1986). However, membrane permeability to glycerol may vary greatly. Collander (1937) observed 1000-fold differences in permeability coefficients for glycerol with different types of cells, and more recent NMR studies of glycerol permeability by Brown et al. (1982) yielded a cm s-', while permeability coefficient for phospholipid vesicles of about the corresponding value for the osmotolerant algae Dunaliella salina was exceptionally low (lo-" cm s - l ) . Expressed in another way, egg phosphatidylcholine vesicles permitted glycerol leakage with half-lives of seconds, whereas the half-life for glycerol leakage through the cell membrane of D . salina was more than 400 hours. The structural and molecular background for such a low permeability to glycerol is not known. The general observations are, however, that factors which increase order in the hydrocarbon region decrease membrane permeability to polyols (Cullis et al., 1985). By introducing double bonds into phospholipid fatty-acyl groups, and by decreasing the fatty-acyl chain length, glycerol permeability through artificial membranes is increased (De Gier et al., 1968). There is little information as to whether plasma membranes of osmotolerant cells are intrinsically different from those of non-tolerant cells, or to the extent to which a decreased water potential induces changes in membrane-lipid composition. It is commonly found, however, that a decreased water potential causes a decrease of the polyenoic CI8acids (usually c 1 8 : 2 ) and an approximately corresponding increase in the content of oleic acid ( C 1 8 : 1 ) residues in fungal phospholipids (Adler and Liljenberg, 1981; Hocking 1986b; Tunblad-Johansson and Adler, 1987; Watanabe and Takakuwa, 1988). Saccharornyces cerevisiae is a notable exception to this rule; it lacks polyunsaturated acids and, in this yeast, increased salinity was observed to cause a slight decrease in the proportion of C l hacids and a corresponding small increase in the proportion of CI8acids (Tunblad-Johansson and Adler, 1987). The ability of sterols to induce a significantly more ordered acyl-chain region is reflected in decreased permeability properties to glycerol and erythritol across artificial lipid membranes (De Gier et al., 1968; D e Kruyff et al., 1973). A high level of free sterols may therefore contribute to the osmotolerance of fungi. Tunblad-Johansson et al. (1987) noted that the molar ratio of stero1:phospholipid decreased but was still maintained high

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(1:3) in the osmotolerant yeast Deb. hansenii when cultured in the presence of 2.7 M sodium chloride. Thus, there is meagre information to substantiate acclimations in fungal membrane-lipid compositions related to, and relevant to, membrane permeability and osmotic stress. The studies cited clearly suffer from the limitation of having analysed only the overall lipid composition of the cellular array of membranes and membrane organelles. Detailed studies on the composition of lipid molecular species performed on isolated plasmamembrane preparations are still awaited. In bacteria, the high apparent permeability for glycerol seems to be associated with facilitated diffusion, involving membrane proteins (Ingraham et a f . ,1983). The glycerol transport facilitator in Escherichia coli is described as a channel in the plasma membrane allowing passage of polyols but not charged molecules like glycerol 3-phosphate and dihydroxyacetone phosphate (Heller et al., 1980). The lipid composition of the membrane also affects the osmotic stability of the yeast plasma membrane. Alterthum and Rose (1973) observed that sphaeroplasts of Sacch. cerevisiae enriched in linoleic or y-linolenic acid residues, rather than oleic acid residues, were more sensitive to lysis in hypotonic solutions. It was also observed that sphaeroplasts enriched with phosphatidylethanolamine were more resistant to osmotic lysis than those enriched in phosphatidylcholine (Hossack et al., 1977). Likewise, sphaeroplasts enriched with ergosterol or stigmasterol were more stable than those enriched in cholesterol, campesterol or 7-dehydrocholesterol (Hossack and Rose, 1976). It was suggested that stigmasterol and ergosterol, the latter being a predominant sterol in yeasts, conferred better stability by more effectively restricting the mobility of the fatty-acyl chains, which increases as the membrane is stretched. Qualitative alterations of the endogenously produced sterols, as observed in nystatin-resistant mutants of Sacch. cerevisiae, did not, on the other hand, affect the resistance to osmotically induced membrane stretching (McLean-Bowen and Parks, 1982).

B . INORGANIC IONS

Inorganic ions play an important role in the osmotic responses of bacteria (Csonka, 1989), algae (Hellebust, 1976) and plants (Munnsetal., 1983). The role of these ions in the osmotic relations of fungi is less clear, although it is known that adjustments of the major intracellular cations do occur in response to osmotic stress. The following section considers the inorganic ion relations of fungi on exposure to low water potential.

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I . Intracellufar Levels The intracellular levels of sodium and potassium ions in Sacch. cerevisiae can vary depending on p H value, the availability of substrate and concentration of electrolytes in the medium. (Rothstein, 1963, 1974; Borst-Pauwels, 1981). In resting cells, levels of these ions are about equal while, on resumption of growth, sodium-ion level decreases and potassium-ion level inreases rapidly (Jones et al., 1965). In chemostat experiments, Watson (1970) observed that Sacch. cerevisiae grown in medium lacking or containing 1 M sodium chloride, contained the same amount of potassium ions (about 0.3 M) while the internal sodium-ion concentration was increased from about 0.05 M at low to about 0.13 M at high external salinity. Similar chemostat studies of Deb. hansenii grown in concentrations of sodium chloride ranging from 0.004 to 1.4 M demonstrated an increase in the internal sodium-ion potassium-ion level from about 0.4 M in basal medium to about 0.7 M at the highest salinity. Concomitantly, internal K+:Naf ratios decreased from >10 at low salinity to about 0.5-in the presence of high salinity (Larsson et al., 1990). Experiments in which the salinity was progressively increased by feeding the chemostat with strongly saline medium (Burke and Jennings, 1990) showed that Deb. hansenii adjusted on a short-term basis by a marked influx of sodium ions, which contrasted with the adjustment under steady-state growth, which involved decreased levels of sodium ions and increased glycerol content. It is also evident from the data of Hobot and Jennings (1981) and Burke and Jennings (1990) that Deb. hansenii grown in the presence of high concentrations of sodium chloride admits more sodium ions in alkaline media (concentrations above 1 M were reported) than at lower p H values, In the marine fungus Dendryphiella salina, ionic content is kept around 0.1 M potassium and 0.2 M sodium and chloride ions in organisms grown in basal medium (Jennings, 1983a). When salinity was adjusted to 0.8 Osm with sodium chloride or sulphate, sodium-24 efflux studies showed that the content of sodium ions changed slightly to 0.19 and 0.26 M, respectively, while the potassium-ion content reached a value of about 0.09 M (Wethered et al., 1985). While the organisms discussed so far admit sodium ions to a certain extent, it was observed that this ion is effectively excluded by Penicillium ochro-chloron (Gadd et al. , 1984) and Aspergillus nidulans (Beever and Laracy, 1986), and that potassium ions remain the predominant cations in these fungi also when cultured in media containing sodium chloride. In cases where the water potential of the growth medium has been amended with a non-ionic solute, only small adjustments of the internal concentration of potassium and sodium ions occur, as observed for P. chrysogenum,

+

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Chrysiporium fastidium (Luard, 1982a), D . salina (Wethered et al., 1985) and A . nidulans (Beever and Laracy, 1986). An osmotically induced increase in the cation content requires an equivalent increase of anions to maintain electroneutrality. Wethered et al. (1985) made an attempt to balance cations and anions in D . salina grown in media adjusted to low water potential with different osmotica. The authors did not achieve charge balance and discussed the difficulties in this task. It is generally observed that, in media amended with salts, an increased uptake of the cation is accompanied by an increased, although not equivalent, uptake of the anion. The ability to absorb the prevalent chloride anion appears to vary. While Sacch. cerevisiae seems to exclude this ion (Conway and Armstrong, 1961), other fungi admit it to varying extents (Shere and Jacobson, 1970; Luard, 1982a,c; Wethered and Jennings, 1985; Wethered et al., 1985).

2. Transport The plasma membrane H+-ATPase from fungal and yeast cells has been thoroughly characterized (Slayman, 1987; Serrano, 1989). This enzyme generates a proton electrochemical gradient which provides the primary driving force for transport of nutrients and inorganic ions across the membrane. In the best documented example, that in Neurospora crassa, a high-affinity transport system that carries potassium ions inward in cotransport with protons has been identified (Rodriguez-Navarro et al., 1986; Blatt and Slayman, 1987). Although potassium-ion transport is mechanistically coupled to proton uptake, parallel operation of the proton pump yields net proton export (Kf/Hf exchange). There is evidence that potassium-ion uptake is driven by the electrogenic proton pump also in Sacch. cerevisiue (Pefia 1975; Eddy, 1982). The transport systems responsible for potassiumion uptake in N . crassa and Sacch. cerevisiae are known to have dual affinities (Rodriguez-Navarro and Ramos, 1984; Ramos and RodriguezNavarro, 1985) and a gene encoding a plasma-membrane protein (TRKI) necessary for high-affinity uptake of potassium ions in Sacch. cerevisiae was recently cloned and sequenced (Gaber et al., 1988). Sodium ions are believed to enter the yeast cell through the potassium-ion carriers and probably also by sodium-ion substrate symporters (Roomans et al., 1977; Borst-Pauwels, 1981). Mutations in the H O L l of Sacch. cerevisiue confer increased uptake of sodium ions and sensitivity to a number of cations (Gaber et al., 1990). The H O L l protein was genetically distinct from the potassium-ion transporters and it was suggested that H O L l might encode an endogenous sodium-ion transporter. Sodium-ion efflux from yeasts has been proposed to be mediated by an Na+/Hf antiporter, which transports

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sodium ions out of the cell in exchange for protons (Eddy, 1978). The presence of such a mechanism has received some experimental support (Rodriguez-Navarro and Sancho, 1979; Rodriguez-Navarro and Ortega, 1982). By using patch-clamp electrical recording technique on sphaeroplasts of Sacch. cerevisiae, voltage-gated potassium-ion channels were demonstrated in the plasma membrane (Gustin et u f . , 1986). These channels were outwardly conducting and favoured the passage of potassium over sodium ions by more than 20-fold. Later Gustin et af. (1988) identified mechanosensitive ion channels in the yeast plasma membrane. These channels differed from the potassium-ion channels in inhibitory sensitivity, conductance and were permeable to both cations and anions. The strain energy that regulated the channels showed a dependence on cell size that indicated regulation by membrane tension rather than membrane pressure. The mechanosensitive ion channels suggest a molecular mechanism by which stretching of the membrane elicits permeability changes. C. SOLUTE COMPARTMENTATION

Subcellular compartmentation is an important aspect of solute accumulation. The presence of vacuoles in yeasts and filamentous fungi offers the organisms a possibility of segregating ions into this rather inert organelle and, in this way, to lower the ion concentration in the cytoplasm. In higher plant halophytes (Flowers and Lauchli, 1983; Munns et u f . , 1983) and the halophilic alga Dunafieffusafina (Hajibagheri et af., 1986) sodium and chloride ions are kept low in concentration in the cytoplasm and accumulated in the vacuole under saline conditions. In yeast, the vacuolar solution is isotonic with that of the cytoplasm but has a different composition (Diirr et af., 1975); the vacuole is considered as a storage compartment for cationic solutes (calcium ions, arginine, lysine, etc.). Several transport systems have been identified in the vacuolar membrane of Succh. cerevisiue, including an H+-ATPase that generates a proton-motive force across the membrane (Ohsumi and Anraku, 1981) and solutelnH' antiporters that transport calcium ions (Ohsumi and Anraku, 1983) and amino acids (Sato etuf.,1984). Ion channels that displayed a broad specificity for cations showed voltagedependent gating and required calcium ions to be opened were reported by Wada et a f . (1987). There is little to sustain a selective confinement of sodium or potassium ions to the vacuole in fungi exposed to saline environments. Ortega (1988) provided some evidence for a preferential distribution of sodium ions to the vacuole after exposure of Sacch. cerevisiae to a sodium-rich medium. However, in growing cells of the marine fungus Dendryphieffasafina,X-ray

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micro-analysis demonstrated that the vacuole, which constitutes as much as 22% of the protoplasmic volume (Clipson et al., 1989), did not accumulate ions when cultured under saline conditions up to sea-water salinity (Clipson et al., 1990). Evidence for a selective distribution of polyols between the vacuolar and cytoplasmic compartments is to our knowledge entirely lacking. D. REGULATION OF POLYOL ACCUMULATION

As is clear from the foregoing, the generalized scheme for osmoregulation in fungi, when growth occurs in dilute media, involves ions (potassium ions in yeasts) as the main contributor to osmotic relations. Following a hyperosmotic shock, there is a rapid osmotic adjustment resulting in loss of turgor and volume, which is followed by a longer phase characterized by a compensatory and selective accumulation of polyols. The steady-state level of individual polyols and ions appears dependent on the stress solute used and the organism studied. Accumulation of polyols in response to an osmotic challenge can result from an increased production, a decreased dissimilation or efflux, or from uptake of the polyol. In the following sections the strategies for polyol accumulation are described for organisms in which the process has been studied, and regulation of the process is discussed.

I . Debaryomyces hansenii The osmotolerant marine yeast Deb. hansenii adjusts to low water potential by increasing its content glycerol (Section V.A.l; Fig. 6). Production of glycerol is osmotically induced, but is not achieved by any marked induction or repression of the glycerol-metabolizing enzymes. At most, two-fold changes in specific activities were observed for these enzymes when the salinity of the growth medium was increased to 1.4 M (Adler et al., 1985; Nilsson, 1988). As judged from the low activity of the glycerol kinase, there is little turnover of glycerol under saline conditions. Glycerol kinase might even be feed-back inhibited by fructose, 1,6-bisphosphate under physiological conditions (Adler et al., 1985), although such inhibition could not be confirmed with the enzyme in its purified state (Nilsson et al., 1989). Glycerol-3-phosphate dehydrogenase is sensitive to high concentrations of salt when assayed with chlorides or sulphates; the enzyme maintains less than 10% of its control activity when the ionic strength exceeds 0.6 M (Nilsson and Adler, 1990). However, when assayed in the presence of sodium glutamate, the enzyme maintains an about two-fold increased activity still at 0.9 M ionic strength. Glutamate is a major organic anion in

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Deb. hansenii (Adler and Gustafsson, 1980; Jovall et a f . , 1990) and the characteristics of GPD in the presence of glutamate are obviously a key feature in explaining glycerol production of osmotically dehydrated cells, although control of glycerol production must involve additional regulatory mechanisms. Following transfer of cells to a highly saline medium, there is an immediate admission of sodium ions followed by a period of sodium-ion release and glycerol accumulation. It is of interest that Commerford et af. (1985) detected an ATPase activity with an alkaline pH optimum in Deb. hansenii grown at high salinity, which was not present in cells cultured in basal medium. Induction of additional ATPase activity under saline conditions might facilitate expulsion of cytoplasmic sodium ions in this yeast. Since the glycerol-transport system is responsive to the sodium-ion gradient (Lucas et a f . ,1990), the magnitude of this gradient will also affect the capacity for glycerol accumulation. The importance of the glyceroltransport system in determining the magnitude of the glycerol gradient across the membrane is presently not known. Chemostat experiments show that a 500&10,000-fold glycerol gradient is maintained during steady-state growth in the presence of 0.68 and 1.35 M sodium chloride (Larsson et a f . , 1990). It is also reported that Deb. hansenii accumulates glycerol to appropriate levels when sugars are used as external osmotica (Nobre and daCosta, 1985b), under which conditions the glycerol transport would lack its driving sodium-ion gradient. These authors also made the interesting observation that erythritol takes over the role of glycerol as the main compatible solute in cells cultured in saline media containing erythritol. It is likely that erythritol is taken up by a transport system which is different from that of glycerol, since glycerol uptake was not inhibited by a 25-fold excess of erythritol over glycerol (Adler et a f . , 1985), and that erythritol either represses glycerol production or causes the glycerol to be released from the cell. The information required to distinguish between these two possibilities is lacking but would allow predictions on the mechanisms for osmotic regulation of glycerol production in Deb. hansenii. The second polyol produced by Deb. hansenii, arabinitol, is accumulated to relatively high intracellular concentrations (0.3-0.5 M), irrespective of external water potential, thereby contributing to the relatively high turgor of these cells (Larsson et af., 1990) under non-stress conditions.

2. Zygosaccharomyces rouxii This yeast produces glycerol and arabinitol by the same general scheme as outlined for Deb. hansenii in Fig. 8 (Spencer, 1968). However, in Zygosacch. rouxii, glycerol is not osmotically induced but is produced constitutively, and the cells regulate their intracellular glycerol content by altering the

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proportion retained within the cell (Brown, 1978; Van Zyl and Prior, 1990). In agreement with such a regulation, Brown and Edgley (1980) noted only slight changes in specific activities of GPD and a few other enzymes of carbohydrate metabolism as the growth-medium salinity was increased. It is not quite clear by which mechanisms the cells regulate their retention of glycerol. The glycerol concentration ratio across the membrane of cells grown in glucose-limited chemostat in the presence of 1.2 M sodium chloride (-5.5 MPa) ranged from 150- to 900-fold (Van Zyl and Prior, 1990). An active glycerol uptake was suggested by radiotracer experiments (Brown, 1974). Conceivably, such a system would not be driven by sodium ions in Zygosacch. rouxii since, in their chemostat studies, Van Zyl and Prior (1990) observed a high retention of glycerol irrespective of whether sodium chloride or polyethylene glycol 400 was used as external osmoticum. In the latter medium, the cells responded not only by increasing retention of glycerol but also of arabinitol. Although no measurements on electrolyte contents were performed, it is likely that the cells increased their retention of arabinitol to compensate for lower contents of sodium ions when sodium chloride was exchanged for a non-ionic external osmoticum. Although most workers have found that arabinitol production responds but slightly to osmotic stress, induction of enzymes on the arabinitol pathway may occur under certain conditions. Moran and Witter (1979) reported an enhanced intracellular arabinitol accumulation by cells grown in media containing a high concentration of glucose and noted that such cells showed an enhanced participation of the pentose phosphate pathway and an increased arabinitol dehydrogenase activity (about three-fold at 60% (w/v) glucose).

3. Saccharomyces cerevisiae This yeast produces no polyol other than glycerol, and responds to water stress by increasing its production of this polyol and by losing increasing amounts to the surrounding medium. During growth on glucose, there appears to be no reutilization of glycerol since the glycerol-dissimilating enzymes are effectively repressed (Section V.A.4). In cells cultured at high salinity there was an about two-fold increased specific activity of phosphofructokinase (Brown and Edgley, 1980) and an up to 30-fold increased activity of GPD (Edgley and Brown, 1983). Estimations of the flux-control coefficient suggested a high controlling power for GPD in glycerol production, indicating a key role for this enzyme in formation of this polyol (Blomberg and Adler, 1989). Since the osmotically induced increase in specific activity was prevented by cycloheximide, de novo synthesis of the

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enzyme seemed likely. This was recently confirmed by Western-blot analysis (AndrC et a f . ,1991) using antibodies raised against GPD as purified by Chen et al. (1987). Using a probe that was deduced from the N-terminal aminoacid sequencing of GPD, clones were selected from a plasmid library. Limited DNA analysis of one of the clones showed about 54% identity of the predicted amino-acid residues with residues 1-80 of the corresponding mouse enzyme. This cloned DNA hybridized to an mRNA transcript that was salt induced (AndrC, 1990). Although further confirmation is required, the results strongly suggest that osmoregulation in Sacch. cerevisiae involves control at the gene level. In glycerol formation, dihydroxyacetone phosphate provided by glycolysis is not the only essential substrate; cytoplasmic NADH is required in equimolar amounts. During adaptive osmoregulation, the enhanced requirement for NADH is met by a decreased reduction of acetaldehyde to ethanol and an increased oxidation of this intermediate to acetate. This metabolic shift is reflected in an about two-fold decrease in the specific activity of alcohol dehydrogenase (EC 1.1.1.1) and an about equally large increase in that of aldehyde dehydrogenase (EC 1.2.1.5). The observed increase in the rate of acetate production did, however, not fully compensate for the increased quest for reducing equivalents in glycerol formation (Blomberg and Adler, 1989). The kinetics for glycerol uptake in Sacch. cerevisiae has been interpreted to indicate transport by passive diffusion (Gancedo et a f . , 1968; Brown, 1974). However, glycerol efflux from the cell does not appear to be uncontrolled. The glycerol that is produced under non-stress conditions (Section V.A.4) is quantitatively released to the surroundings, whereas cells which, by treatment with cycloheximide, are forced to maintain the same glycerol production rate under osmotic stress start accumulating glycerol (Blomberg and Adler, 1989). This observation indicates a degree of control at the level of the membrane. Membrane channels which are activated by membrane stretching are described in Sacch. cerevisiae (Section V.B .2). Since these channels readily pass both cations and anions, they could serve as general down-regulators of osmotic stress by permitting solute efflux from the cell. Specific membrane-stretching channels which selectively release glycerol when optimum turgor is adjusted would explain why glycerol is quantitatively released in the absence of osmotic stress but accumulated when cells are subjected to dehydration stress. In enterobacteria, the intracellular concentration of potassium ions acts as a signal which controls induction of a transport system for uptake of the compatible solute betaine (Sutherland et al., 1986). There is no evidence for a corresponding potassium-ion dependence of glycerol production in Sacch. cerevisiae. By manipulation of the composition of the growth

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medium, it was established that glycerol accumulation occurs on exposure to osmotic stress independent of the potassium-ion concentration in the cell (Meikle et al., 1991; I. Tunblad-Johansson and L. Adler, unpublished observations). Meikle et al. (1988) demonstrated that protoplasts of Sacch. cerevisiae increased the intracellular level of glycerol when subjected to osmotic stress (high-glucose media). The protoplasts produced more glycerol than did whole cells under corresponding conditions and, on continued incubation, the protoplasts increased in size and developed large vacuoles. These observations seem to indicate that turgor (rather than cell volume) mediates some degree of control of the osmotic adjustment. 4. Phycomyces blakesleeanus

Little is known of the control of polyol formation in response to osmotic stress in filamentous fungi. From the number of participating polyols, regulation appears more complex than in yeasts. However, it was suggested by Jennings (1984) that the salinity-induced increase in synthesis of both glycerol and erythritol in Aspergillus niger and Penicillium chrysogenum (Adler et al., 1982) might be due to the involvement of an unspecified dehydrogenase. An NADP+-linked polyol dehydrogenase with wide substrate specificity has been described in P. chrysogenum (Chiang and Knight, 1959). The most relevant studies on the water relation of polyol regulation have been conducted on germinating spores of Phycomyces blakesleeanus (Van Schaftingen and Van Laere, 1985; Van Laere and Hulsmans, 1987). During germination, the spore has to generate a low intracellular osmotic potential to mediate turgor and swelling. Interestingly, spores of P. blakesleeanus transiently produce large amounts of glycerol after induction of germination (Van Schaftingen and Van Laere, 1985). This glycerol is largely formed from trehalose by activation of trehalase and glycerol-3-phosphatase. The activity of glycerol-3-phosphatase was rapidly but transiently about 10-fold activated during early germination. Since this activation was mimicked in vifro by addition of CAMP,protein phosphorylation is probably involved in glycerol formation. These results are particularly interesting, indicating a CAMP-dependent phosphorylation cascade and a transient covalent modification might be involved in regulation of glycerol synthesis in fungi. VI. Osmotic Hypersensitivity Only a minor proportion of exponentially growing cells of Sacch. cerevisiae taken from cultures in basal medium and plated onto low water-potential

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media had the capacity to form colonies, even when the water potential of the plating medium was well above ymin. This phenomenon was qualitatively described by MacKenzie et al. (1986) and given the name “water stress plating hypersensitivity”. Here we will use the term “osmotic hypersensitivity” for the phenomenon. Either sodium chloride or glucose included as an osmotic agent in the plating media manifested the osmotic hypersensitivity of the cells. However, the water potential of the medium had to be below a critical threshold, as shown in Fig. 9, for the phenomenon to be observed. Saltcontaining media will give a somewhat higher value for the critical threshold compared with glucose-adjusted media (yh,,(NaCI) = -4 MPa; A. Blomberg, unpublished result). By culturing cells in salt, their subsequent degree of osmotic hypersensitivity was drastically decreased. At salt concentrations above 0.35 M , no hypersensitivity to 48% (wh) glucose was observed. Similarly, short-time conditioning (60 minutes) in media

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FIG. 9 Colony-forming capacity of a culture of Saccharomyces cerevisiae at indicated water potentials of glucose-adjusted media, expressed as the percentage of colonies formed relative to that on high water-potential medium (w = 0.5 MPa). The water potential was calculated from data given by Norrish (1966) at 25°C. For an explanation of v h y p , see the text. Redrawn from Mackenzie et a[. (1986).

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containing 0.7 M sodium chloride transformed the whole population into a resistant state (Blomberg and Adler, 1989). Different species of yeast were tested for the osmotic hypersensitivity response, of which only Sacch. cerevisiae and Candida krusei exhibited the phenomenon at 48% (w/v) glucose (MacKenzie et al., 1986). It should be noted, however, that the existence of a osmotic hypersensitivity threshold (Whyp) instigates a conditional character to the phenomenon, implying that those species-strains that did not display the osmotic hypersensitive phenotype at a particular y-value might do so at a lower y-value. This has been shown to be true for Deb. hansenii, which did not display the phenomenon in the presence of 48% (wh) glucose but did so with 2.8 M sodium chloride, with less than 1% of the mid-exponential culture being viable at this salt concentration. The v h y p value for Deb. hansenii seems to be about -10 MPa, since no plating discrepency was observed at 2.1 M sodium chloride (Larson, 1990). The importance of the physiological state of the culture in determining the degree of osmotic hypersensitivity was substantiated by a study in which growth, on either glucose or ethanol as the carbon source, was monitored by the use of a microcalorimeter (Blomberg et al., 1988). Exponentially growing cells in either medium were hypersensitive to an osmotic shock as only 0.01% of the culture formed colonies on 1.5 M sodium chloridecontaining medium, while cells from the stationary phase or the transition phase between respirofermentative and respiratory growth were more tolerant. Furthermore, the phenomenon was not restricted to plating, since the osmotic hypersensitivity of exponentially growing cells was also reflected in its long lag phase (more than 100hours) in media containing 1.5 M sodium chloride compared to 15 hours for cells from the transition phase. The physiological state of the culture has also been shown to be of the utmost importance for the resistance of Sacch. cerevisiae to a number of environmental factors such as heat (Schenberg-Frascino and Moustacchi, 1972) and chemical mutagens (Parry et al. , 1976). The phenomenon of osmotic hypersensitivity is probably a reflection of lethal effects on the cells during the initial dehydration, since a culture of Sacch. cerevisiae rapidly lost roughly 50% of its viability as a response to a sudden exposure to lower water potentials (Morris et al., 1986). Viability measurements were performed after 15 minutes of incubation in low water-potential media, and it was reported that prolonged incubation for up to about two hours did not significantly alter viability, i.e. the viability decrease was a rapid process. The response was shown to be quantitatively similar for both sodium chloride and glycerol at equal water potentials, while methanol at concentrations up to 6 Osm (about -15 MPa) did not lower cell viability. Since methanol even at high concentrations did



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not induce any changes in cell volume, while both sodium chloride and glycerol did, it was concluded that the decreased viability was a result of irreversible damage caused by osmotically induced cell shrinkage. The viability decrease of yeast during exposure to low water potential is not a new phenomenon, and was reported by Onishi (1959) to occur for the osmotolerant yeast Zygosacch. rouxii. An important observation by Onishi (1959) was that Zygosacch. rouxii, when exposed to 3 M sodium chloride (-14.9 MPa) in a phosphate buffer at pH 4.8 without an energy source, died off quite rapidly, and a 10,000-fold decrease in viability was observed after two days of incubation. However, if 5 % glucose was added, no major change in viability occurred over five days incubation in the salt solution. This implies that not only the magnitude of the osmotic stress but also the additional nutritional constituents will be of importance in determining the initial cellular response and the subsequent viability of the cells. A. OSMOTIC HYPERSENSITIVITY DETERMINANTS

1. Osmoregulation and Osmotic Hypersensitivity

The resistance to ultraviolet radiation of exponentially growing cells of Sacch. cerevisiae is dependent on active DNA repair mechanisms (Parry et al., 1976). By analogy, the most osmotolerant cells to respond to a sudden exposure to water potentials below v h y p could be those with a functional and active osmoregulatory system. The main features of this osmoregulatory system in yeast seem to be glycerol production and accumulation. The rate of glycerol production in Sacch. cerevisiae appears to be controlled by the amount of the enzyme GPD (Blomberg and Adler, 1989). If a high rate of glycerol production would promote osmotic-shock tolerance, induced levels of GPD should be a favourable osmotolerance feature for cells. The activity of GPD seems at first glance to be a good candidate for an osmotolerance factor, since both transition-phase, stationary phase (Blomberg et al., 1988) as well as osmotically conditioned cells (Blomberg and Adler, 1989) exhibit enhanced specific activities. The absolute specific activity of GPD, however, cannot be the determining factor per se, since ethanol-grown cells have high specific activities of GPD but no increased resistance to osmotic shock (Blomberg et al., 1988). It has been proposed that compatible solutes that accumulate intracellularly during growth in basal medium act as an osmotic buffer to minimize water loss from cells subsequent to osmotic shock (Harris, 1981). Debaryomyces hansenii, displaying a lower v h y p value than Sacch. cerevisiae, has been shown to produce constitutively and accumulate arabinitol (Adler and Gustafsson, 1980). In a study on Deb. hansenii, only minor variations in

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the intracellular pools of polyols were reported during growth, exhibiting no correlation with fluctuations in osmotic hypersensitivity (Larsson, 1990). It was furthermore shown by Blomberg and Adler (1989) that osmotically conditioned cells, washed free of glycerol, only partly increased their osmotic hypersensitivity. This precludes glycerol (polyols) as the general factor determining the degree of osmotic hypersensitivity, while leaving polyols as a candidate in setting the absolute value of V h y p in different species. 2. Proteins

In the study by Blomberg and Adler (1989), it was shown that the transition of Sacch. cerevisiae into an osmotolerant state (not displaying osmotic hypersensitivity) was dependent on protein synthesis. Addition of cycloheximide, which blocked protein synthesis, almost completely inhibited acquisition of osmotolerance, in spite of the fact that the cells accumulated glycerol during the conditioning. These findings indicate that the state of resistance to a sudden osmotic shock is protein mediated. The specific activity of some enzymes has been reported to be modulated during adaptation and growth to water stress. Brown and Edgley (1980) reported enhanced activities of phosphofructokinase, as well as of some key enzymes in the pentose phosphate cycle during growth of Sacch. cerevisiae at low water potentials. Similarly, the increased rate of acetate production during osmotic conditioning was reflected in the decreased specific activity of alcohol dehydrogenase and an increased specific activity of acetaldehyde dehydrogenase (Blomberg and Adler, 1989). In order to get a view of the overall shift in protein synthesis during osmotic conditioning, proteins were labelled with [35S]methionine, and subsequently separated by two-dimensional gel electrophoresis. It was found that strain Y41 of Sacch. cerevisiae increased expression of about 20 proteins more than 10-fold during osmotic conditioning, differential synthesis of the majority of proteins not being significantly altered. Interestingly, another strain (SKQ2n), which was found to exhibit a lower Vhyp compared with strain Y41 and thus was more resistant to a sudden osmotic shock, displayed a constitutive high expression at high water potentials of many of these osmoresponsive proteins found in strain Y41 (A. Blomberg, unpublished result). 3. Trehalose The maintenance of a sufficient quantity of intracellular trehalose has been attributed a key role in cellular resistance towards dehydration at water i.e. for cells not displaying osmotic hypersensitivity potentials above ymin,

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(MacKenzie et al. ,1988). These authors claimed that the threshold value for a single cell of Sacch. cerevisiae to be protected from the deleterious effects was 0.15 pmol (mg dry wt)-', which roughly corresponds to 75 mM (if cell water is taken as 2 pl (mg dry wt)-'). A correlation was found between the level of trehalose and the viability of the culture on low water-potential medium, trehalose approaching the threshold value concomitant to ethanol depletion and onset of the stationary phase. Additional support for the trehalose-threshold hypothesis comes from studies on a mutant lacking the ability to dephosphorylate trehalase, keeping the enzyme in its trehalosedegrading form. This mutant produces and accumulates trehalose to a lesser extent than the wild type (three-fold less) and exhibits no recovery from osmotic hypersensitivity in the stationary phase of growth (MacKenzie et al., 1988). Furthermore, dinitrophenol induced breakdown of trehalose in the wild type, eventually rendering the culture more sensitive to osmotic dehydration. Additional information on factors involved in cellular survival subsequent to dehydration comes from work on cellular desiccation. Desiccation could either be effected by freeze drying (Gadd et al., 1987) or incubation in a desiccator of filter-spread cells (Hottiger et al. ,1987a). The physicochemical principles underlying desiccation are qualitatively not different from osmotic dehydration in a concentrated solution, but will usually differ in the degree of stress applied and the kinetics of the dehydration process. For example, osmotic dehydration in a saturated solution of sodium chloride (about 35% (w/v), -30 MPa) is regarded as a major stress and found generally to be rapid. Cellular desiccation, on the other hand, usually proceeds in an atmosphere of not more than 10% relative humidity (a, = 0.10, w = - 315 MPa) which is a 10-fold decrease compared with saturated sodium chloride, and y-equilibrium, especially in the desiccator, will be slowly achieved. Desiccation should thus by all means be regarded as an extreme stress situation for the cell, which in nature can be properly dealt with only by a small number of organisms. Among these resistant forms are some fungal spores (Sussman and Lingappa, 1959), macrocysts of Dictyosreliurn spp. (Clegg, 1965) and dry baker's yeast (Payen, 1949). In all of these organisms and structures capable of surviving complete dehydration (the so-called anhydrobiotic organisms), trehalose is accumulated to as much as 20% of the dry weight. The function of trehalose in protection of cells to desiccation seems to be its unique capacity to substitute for water by hydrogen bonding of the hydroxyl groups of carbohydrates and the polar head groups of phospholipids (Crowe et al., 1984). This interaction is believed to protect the integrity of dry membranes during cellular desiccation. The work by Gadd et al. (1987) and that of Hottiger et al. (1987a) supports the role of trehalose as

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an inducer of desiccation tolerance in Sacch. cerevisiae, demonstrating increased resistance for cells in the stationary phase of growth which are reported to contain a high concentration of trehalose. Externally supplied trehalose (500 mM) during freeze drying dramatically increased resistance for both stationary phase and exponential-phase cells (Gadd ef al., 1987). These authors also applied a threshold hypothesis for intracellular trehalose to be effective, in this case being 120 mM trehalose. Furthermore, trehalose accumulation seems to be induced by a number of treatments besides glucose exhaustion, such as exposure to ethanol, copper sulphate or hydrogen peroxide (Attfield, 1987) and a rise in temperature (Hottiger et al., 1987a), all treatments which inhibit growth. Heat-induced trehalose accumulation was reported to increase the desiccation tolerance of Sacch. cerevisiae (Hottiger et al., 1987a). Heat conditioning of heat-sensitive exponential-phase cells has been shown to transform the culture into a state of acquired thermotolerance (Hottiger et al., 1987a; Trollmo et al., 1988), the state of tolerance being proposed either as being mediated by proteins (McAlister and Finkelstein, 1980) or trehalose (Hottiger et al., 1987a). Surprisingly, not only was one of the enzymes involved in trehalose synthesis, trehalose-6-phosphate synthase, rapidly induced by heat treatment and correlated to trehalose accumulation, but the degrading enzyme, trehalase, was also induced. This is believed to reflect not just an increased need for trehalose as such, but more importantly that the trehalose path might be a futile cycle acting as a sink for ATP over-production (Hottiger et al., 1987b). It was reported by Trollmo et al. (1988) that exponential cells of Sacch. cerevisiae submitted to heat conditioning did not improve their osmotic hypersensitivity, even though the cells became thermotolerant, concluding that the supposed heat-induced trehalose accumulation (unfortunately not measured) did not confer a higher degree of resistance towards osmotic This conclusion is further substantiated dehydration above the value for vmin. by trehalose analysis of osmoresistant cells exponentially growing in medium containing 0.86 M sodium chloride, with trehalose levels well below 50 mM (K. Larsson and R. Olz, personal communication). In summary, the role of trehalose in protecting cells from the lethal effects of desiccation at wvalues far below vmin seems to rest on a solid basis, while the involvement of the carbohydrate as a factor governing the osmotic hypersensitivity at water has to be further substantiated. potentials above vmin B . PHYSIOLOGICAL OVERLAP

The physiological overlap in Sacch. cerevisiae between tolerance to heat and osmotic stress was shown to be unidirectional; osmotically conditioned cells

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(0.7 M sodium chloride for 60 minutes) became thermotolerant while heatconditioned cells (37°C for 60 minutes) did not improve their osmotic hypersensitive state (Trollmo et al. , 1988). It was furthermore shown that the thermotolerance acquired by osmotic conditioning was not a response of the cellular dehydration per se, since thermal resistance was not imposed by suspending non-conditioned cells in 0.7 M sodium chloride during heat treatment in accordance with results earlier reported (Beuchat, 1981). A decreased water potential during heat treatment has been reported to generally alter thermotolerance (Van Uden, 1984), but microbial species and solute considerations have to be taken into account (Beuchat, 1981), sucrose being more generally effective as a thermoprotector than sodium chloride. In comparison with sucrose or sorbitol, glycerol exhibited a low protective effect on heat denaturation of pure enzyme solutions (Back et al., 1979). In accordance with this, only a slight decrease in thermotolerance resulted from a two-fold decrease in the glycerol content of osmotically conditioned cells (brought about by washing; C. Trollmo, unpublished result). The thermotolerant state is generally believed to rely on production of so-called heat-shock proteins. By use of two-dimensional gel electrophoresis, it was shown that some heat-shock proteins also display increased synthesis during the osmotic conditioning (A. Blomberg, unpublished result). VII. Cellular Factors Involved in Determining yminValues Superficially, it might seem a trivial pursuit to search for cellular factors values since, at extremes of water potentials, any limiting growth at ymin factor might be the cellular “Achilles heel” for a particular organism. At high concentrations of sodium chloride, for example, some membranebound proteins could suffer from ionic strength-mediated conformation changes inhibiting their activity and thus precluding growth. Thus, according to this line of thought, allocation of the limiting factor at yminin one species would not reveal any of the general cellular mechanisms of osmotolerance, but merely be a consequence of evolutionary chance. However, the physiological consensus between organisms in their response to the water potential of the environment, namely production of a compatible solute, mode of osmoregulation, growth response and osmotic hypersensitivity, indicates that the principles determining ymin values in one species might well be universally applicable and not species-specific. The simplest model for cellular osmotolerance is, of course, that there is only one cellular factor involved. Plant-breeding efforts over the years indicate, however, that there will be no single gene product which

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determines salt tolerance (Cheeseman, 1988). Even if no single gene might confer tolerance, a mutation in one gene might confer intolerance. Two loci involved in sensitivity to osmotic stress in Sacch. cerevisiae have been identified and named O S M l and O S M 2 (Singh and Sherman, 1978). The locus O S M 2 mapped close to the A R 0 7 locus, and it was later shown that AR07and O S M 2 are allelic (Ball e t a f . ,1986). LocusAR07is thestructural gene for chorismate mutase and the enzyme catalyses the first step from chorismate to prephenate on the phenylalanine and tyrosine pathway. The molecular basis behind the osmotic sensitivity of the aro7 mutant is not known, and furthermore, the transcriptional level of the AR07gene was not affected by alterations in the water potential of growth media. In the following discussion, factors involved in determining ymin will be treated individually. Information is scanty on the relative importance that these factors have in determining ymin values for a fungal species, and it might well be that the sum of two or more of them will co-operate in defining the ultimate limit for growth in relation to the water potential. A.

GENERATION OF ENERGY (ATP) AT LOW WATER POTENTIALS

Norkrans (1968) investigated the effect of a decreased water potential on respiration and fermentation for yeasts isolated from the sea. The main species under study were Deb. hansenii and Sacch. cerevisiae, their water relations being described in an earlier work (Norkrans, 1966). She found respiration and fermentation of both strains almost unaffected by a minor increase in sodium chloride from 0 to 0.68 M . A further decrease in water potential, however, considerably lowered the respiratory and fermentative value for Sacch. cerevisiae (about 1.9 M sodium chloride values. At the ymin both fermentation and respiration rates decreased to about 20% of the high water-potential values. The initial inhibition on these ATP-generating reactions was similar in magnitude for both yeasts, and thus not related to their degree of osmotolerance. These declining values were recorded for cell suspensions with externally supplied glucose, which contrasted with the almost constant values for endogenous respiration-fermentation (no glucose supplied). The unaffected endogenous rates suggest a minor impact of lowered water potential on central metabolic paths involved in fermentation as well as respiration. Rather, the sharply decreased activity at concentrations above 0.68 M sodium chloride in the case of externally supplied glucose indicates a major detrimental impact on the transport system for glucose. This hypothesis was later investigated in studies on the effect of sodium chloride on the K , and V,,, values for the glucose-transport system, by use of D-glucosamine as substrate (Lindman, 1981). Although Sacch. cerevisiae

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displayed a much higher V,,, value than Deb. hansenii, it was more strongly affected by an increased concentration of sodium chloride. Debaryomyces hansenii exhibited a two-component system for glucose transport, with two different affinities for D-glucosamine. The K , value for the high-affinity system was increased from about 1 to 24 mM when cells were grown in the presence of 2.7 M sodium chloride compared to non-saline medium, while the V,,, value was just slightly decreased. It was concluded that the transport system for glucose in Deb. hansenii was affected but still functional at this high concentration of sodium chloride. The transport system was further characterized and found to demonstrate an energy-dependent uptake. Since uptake was neither affected by p H value nor by alkalization upon sugar addition, a proton gradient was rejected as the driving force (Lindman, 1981). This is in contrast to the situation in the marine fungus Dendryphiella salina for which a proton symport of glucose has been reported (Davies et al., 1990). The glucose-transport system in both Deb. hansenii and Sacch. cerevisiae displayed increased K , values at lower water potentials, a feature which will considerably diminish the scavenging capacity for an energy source in an energy-poor environment. In summary, studies on respiration-fermentation and transport indicate diminished rates of ATP generation by decreased water potentials. B . COST OF MAINTENANCE AT LOW WATER POTENTIALS

A cellular complication to growth at low water potentials is the reported increased cost of maintenance, as demonstrated in chemostat experiments with a respiration-deficient mutant (petite) of Sacch. cerevisiae (Watson, 1970). The uptake rate of an energy-yielding substrate specifically used for maintenance (me)was shown to increase by a factor of 10, from 0.2 to 2 pmol glucose (mg dry wt)-' h-', when cells were cultured in medium containing 1.0 M sodium chloride compared with growth in the absence of salt. By studying the carbon dioxide production, it was shown that an increased fraction of the glucose used for maintenance was, in sodium chloridecontaining medium, diverted into non-ATP-generating activities. This was clearly demonstrated to be a function of the increased maintenance production of glycerol, the me value in salt being 1 pmol (mg dry wt)-' h-', i.e. 50% of the glucose maintenance value. Even in medium lacking salt, glycerol was formed but production was in this case independent of growth rate and strictly related to biomass production (see Section V.A.2). Thus, under growth at low water potentials, glycerol production was included in the cost of maintenance. The use of respiratory deficient mutants enabled calculations of the amount of ATP generated per unit of glucose utilized, care being taken of

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the glycerol effect. It was accordingly shown that the ATP yield, regardless of culture media, was 11 mg dry wt (mol ATP)-’, indicating a constant cost of ATP in biosynthetic reactions irrespective of the water potential of the media. However, the me value for ATP increased four-fold from 0.52 to 2.2 pmol (mg dry wt)-’ h-’ in media lacking or containing 1.0 M sodium chloride, respectively. Taken together, the result by Watson (1970) clearly states that, during water-potential stress, Sacch. cerevisiae deviates an increased amount of energy source into maintenance reactions, partly as glycerol production and partly as an increased expenditure of ATP. The degree of increase in ATP utilized for maintenance seems to match the energy dispensed on enhanced ion pumping (Norkrans and Kylin, 1969; Watson, 1970). It has also been found that the increased catabolic energy expenditures during growth in medium containing 0.68 M sodium chloride, was about four times larger for Sacch. cerevisiae compared with that of Deb. hansenii. This value was calculated from measurements on heat dissipation from growing cultures, care being taken to subtract the “glycerol effect” (Gustafsson and Larsson, 1990). Increased maintenance cost has also been reported for growth at low pH values for Sacch. cerevisiae, the rate of maintenance being proportional to the proton concentration (Verduyn et al., 1990). Similarly, Gustafsson and Larsson (1990) found an increased maintenance expenditure for Deb. hansenii at low pH values, and it was concluded that the maintenance requirement at p H 3 was of the same order of magnitude as for growth in the presence of 1.0 M sodium chloride. This might explain the earlier discussed ymindependence on pH value (see Section III.A.3), additional environmental factors besides low water potentials adding an extra load onto the total maintenance cost of the cells. Maybe more significant from an ecological point of view than the saltinduced initial decrease in energy generation (Norkrans, 1968) is the fact that many natural environments, e.g. the sea, are nutrient limited, so that energy generation per cell and hour is determined mainly by supply from the environment, provided efficient cellular nutrient-transport systems exist. This implies that the report by Watson (1970) indicating an increased cost of maintenance in the presence of salt probably has long-ranging implications on the ecology and evolution of organisms. A simple model for this is depicted in Fig. 10, where the rate of maintenance cost is assumed to increase, and the energy-generating capacity of an organism predicted to decrease, in response to a decreased water potential of the environment. Note that maintenance will be a function of the physicochemical environment and not related to energy supply. A decreased water potential will both decrease the rate of ATP generation and increase maintenance, the two

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1

1

1

I

Rich J

al 0 C

C

0 .-a-

m C

h

al

a-

a, C

: 5 C

1-

0)

cn

a I-

-I

Lc

0

-4-

0 al

al

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m

w

m U

a:

Water potential (MPa)

FIG. 10. A simple model showing how the rate of maintenance (. . .) and rate of energy generation (-) will influence the value of ymin.Indicated are two environments, energy-rich and energy-poor, and arrows indicate the different ymin values obtained.

processes eventually being equal at ymin and growth no longer possible. An appropriate example of the rich habitat might be the conical flask in the laboratory, supplemented with a high concentration of easily accessible nutrients and energy sources. In the other situation, when energy supply is will be reached at a much higher water-potential value, since the poor, ymin energy supply is not sufficient for the increased cost of maintenance at lower y-values. This might represent the situation sea-living organisms have to face and which will be important components of their evolutionary pressure: both genetic adaptation to effective energy scavenging and to low-cost maintenance at -2.6 MPa (0.5 M salt). The high osmotolerance of, for example, Deb. hansenii as measured in the laboratory (growth in the presence of 4.1 M sodium chloride) (Norkrans, 1966) might thus not be an indication of adaptation to these extremes of water potentials, but merely a consequence of a high degree of genetic adaptation in minimizing its cost spent on osmoregulation. Thus, at the water potential of the sea, minimizing the cost of maintenance spent on osmoregulation might be an important selection pressure.

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C. ION TRANSPORT AND ACCUMULATION

In order to maintain appropriate activities of cellular enzymes, fairly low internal levels of sodium ions are accepted. It was shown for both Sacch. cerevisiae and Deb. hansenii that the rate of sodium influx was proportional to the external sodium-ion concentration (Norkrans and Kylin, 1969). Both yeasts selectively accumulated potassium ions in preference to sodium ions, which was reflected in the high intracellular K+:Na+ratio of around 100-300 during growth in basal medium, Deb. hansenii consistently displaying higher ratio values than Sacch. cerevisiae. The competition for the same ion carrier (see Section V.B.2) might explain the decreased ratio of about unity during growth in the presence of 2.7 M sodium chloride (-13.4 MPa), the competition theory being supported by an almost constant proportion between the intracellular K+:Na+ ratio and the extracellular K+:Na+ ratio, irrespective of medium salinity. The salt-tolerant yeast Deb. hansenii exhibited a higher capacity for sodium-ion efflux than Sacch. cerevisiae (Norkrans and Kylin, 1969). The sodium-ion extrusion is reported to be coupled to proton import (RodriguezNavarro and Ortega, 1982). The driving force, the proton gradient, is produced by the plasma-membrane ATPase which is an electrogenic proton pump. The ATPase gene has been cloned (Serrano et al., 1986), and was by site-specific deletion shown to be essential for growth. By modifying the promoter region of the ATPase gene, Vallejo and Serrano (1989) were able to construct transformants with altered expression of the ATPase gene. One of these constructs with a low ATPase content was shown to be unaffected in its osmotolerance as demonstrated at 1.15 M sodium chloride (-5.7 MPa, pH 6.0), under which conditions the mutant grew at the same rate as the wild type. No results were reported for growth of the mutant in media closer to value of Sacch. cerevisiae. Whether active and efficient sodium-ion the ymin extrusion can explain the differences in yminmerits further investigation. Among the ATPase mutants of Sacch. cerevisiae obtained by McCusker et al. (1987) some, but not all, were reported to display an osmosensitive phenotype. It was later shown that, in one of the osmosensitive strains, the mutated ATPase interacted with a voltage-gated potassium-ion channel (Ramirez et al. , 1989). This might indicate a central role for potassium-ion transport in osmotolerance, which is supported by studies of potassium iontransport mutants (isolated by Gaber et al. 1988,1990) which displayed high ymin values (A. Blomberg, unpublished result). D. PRODUCTION AND ACCUMULATION OF A COMPATIBLE SOLUTE

Substantial indirect evidence for the importance of accumulation of a compatible solute for growth to occur at low water potentials comes from the

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vast literature on polyol accumulation (see Section V.A.la). A more direct proof of the prerequisite for a compatible solute came from studies on a mutant of Deb. hanseriii with impared glycerol production, the rate of glycerol production being about four-fold lower than for the wild type in the presence of 1.4 M sodium chloride (Andre etul., 1988). The requirement for glycerol accumulation at low water potentials was demonstrated by an experiment in which addition of 0.5 mM glycerol completely restored the colony-forming capacity of the mutant in the presence of 2.4 M sodium chloride, a concentration that is normally inhibitory for the mutant but not for the wild type. No colonies appeared o n plates lacking glycerol o r on media where glycerol was substituted by 1 mM concentrations of other wellknown osmoprotectants (Yancey et al., 1982) like mannitol, arabinitol, proline, trimethylaminoxide and betaine. It was furthermore shown that the mutant at high salt concentrations rapidly accumulated externally supplied glycerol to wild-type intracellular levels. This clearly indicates that accumulation of sufficient intracellular levels of a compatible solute was required to permit growth at low water potentials. Further demonstration of the importance of appropriate regulation of production and accumulation of a compatible solute during growth at low water potentials comes from the isolation of osmosensitive mutants of Zygosacch. rouxii (Yagi and Tada, 1988). Twenty mutants unable to grow in the presence of 2 M sodium chloride were isolated and classified into physiological groups by investigating their glycerol production and accumulation at permissive water potentials. For most of the mutants, the glycerol criteria explained their growth response, glycerol being produced but not retained or production per se being meagre. For some mutants, however, additional unknown factors seemed to be involved in their osmosensitivity. A possible explanation why the ymin value of Sacch. cerevisiae is much higher than for Zygosacch. rouxii has been substantiated by work by Brown and his coworkers (Brown, 1978). It was found that Sacch. cerevisiue responded much more vigorously in its total glycerol production to increased concentrations of sodium chloride compared to Zygosacch. rouxii with roughly 30 mol% of the consumed glucose being converted into the polyol at 1.4 M sodium chloride compared to 5 mol% in basal medium. Zygosaccharomyces rouxii, on the other hand, showed a much higher “unstressed” production of around 15 mol% but, in contrast to Sacch. cerevisiae, the production was kept almost constant over the range of 0-3 M sodium chloride. However, as salinity increased, Zygosucch. rouxii retained an increased proportion of the glycerol produced (see Section V.D.3). The explanation for the high cost of glycerol maintenance in Sacch. cerevisiue is an apparent lack of a transport system for glycerol, glycerol retention thus being set by the balance between rate of production and

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leakage. The theoretical maximum of glycerol produced is 1 mol of glycerol from each mole of consumed glucose, since the cell cannot divert all triose phosphates produced in the early glycolytic steps to glycerol, but has to channel half of them into pyruvate production to generate NADH, which is needed as the reducing moiety in conversion of DHAP into glycerol 3phosphate (see Section V.A.4). A figure close to this theoretical optimum was reported for well aerated cultures containing 1.35 M sodium chloride (Larsson and Gustafsson, 1987), for which it was found that 68 mol% of the consumed glucose ended up as glycerol. A more glucose-conserving mode of glycerol production would be NADPH-dependent reduction of DHAP into glycerol 3-phosphate, resulting in a theoretical value of two units of glycerol formed per unit of glucose consumed. This is the mode of glycerol production proposed for Zygosacch. rouxii (Brown and Edgley, 1980). The of Sacch. cerevisiae is mainly conclusion is that the absolute value of ymin dictated by its poor retention and wasteful mode of production of its compatible solute glycerol. Presumably, the existence of a transport system for the compatible solute in possibly both Deb. hansenii and Zygosacch. rouxii (see Section V.A.3) allows the glycerol maintenance of these species to be significantly smaller. The part of the total ATP maintenance involved in energy expenditure on the uphill transport of glycerol during growth at low water potentials, however, will in these species probably be relatively high. VIII. Conclusion

The response of a fungus to osmotic stress involves the integrated function of many components of cell metabolism (Fig. 11). An important mechanism by which the dehydration stress is countered entails accumulation of polyols, primarily glycerol, to achieve an internal environment that is conducive for enzyme function and growth under water stress. The changes in the composition of the cytoplasm are controlled by systems for biosynthesis of polyols and for transport of inorganic ions. The detailed regulation of these systems is little understood, but evidence points to regulation at gene as well as protein level. The intracellular retention of glycerol is controlled at the level of glycerol efflux and by systems for glycerol uptake. The osmoregulatory processes require energy to drive transport and carbon supply for polyol formation. The capacity for substrate uptake under osmotic stress and the efficiency by which the osmoregulatory processes are operated are important in setting the limits for growth at low water potentials. The fungal response to changes in the external water potential must involve sensing as well as transduction of the received signal. Are

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Hf

FIG. 11. A schematic representation of cellular functions involved in osmoregulation in fungi. 1 indicates glucose uptake; 2, glycerol production;3, glycerol uptake; 4, glycerol efflux; 5 , energy supply; 6, efflux of sodium ions; 7 , influx of potassium ions.

mechanosensation (e.g. turgor sensing) and phosphorylation cascades involved in this response? To what extent and in what way does the signal affect gene expression? It appears that combined genetic and physiological analysis is required for a deeper understanding of fungus-water relations. Analysis at this level has revealed sequential induction of osmotically controlled genes in enteric bacteria and given exciting insights in signal transduction and regulation of the process (see the review by Csonca, 1989). The experimental tractability and developed genetics of many fungal species makes these organisms equally attractive eukaryotic experimental systems for exploration of fundamental mechanisms in osmoregulation and osmotolerance.

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IX. Acknowledgements The compilation of data and the writing of this review were made possible by a grant from NFR (B-Bu4645-302). We greatly appreciate the efficiency of the staff at the Botanical Library, University of Goteborg, with a special thanks due to Helena G r a v b for her technical help with the computer search in the BIOSIS database. The everlasting patience and support from our families during the period of writing are deeply acknowledged. REFERENCES Adebayo, A. A. and Harris, R. F. (1971). Proceedings of the Soil Science Society of America, 35, 465. Adebayo, A. A., Harris, R. F. and Gardner, W. R. (1971). Transactions of the British Mycological Society 57, 145. Adler, L. and Gustafsson, L. (1980). Archives of Microbiology 124, 123. Adler, L. and Liljenberg, C. (1981). Physiologia Plantarum 53, 368. Adler, L., Pedersen, A. and Tunblad-Johansson, 1. (1982). Physiologia Plantarum 56, 139. Adler, L., Blomberg, A. and Nilsson, A. (1985). Journal of Bacteriology 162, 300. Aitken, W. B. and Niederpruem, D. J. (1972). Archiv fur Microbiologie 82, 173. Al-Hamdani, A. M. and Cooke, R. C. (1987). Transactions of the British Mycological Society 89, 51. Altherthum, F. and Rose, A. H. (1973). Journal of General Microbiology 77, 371. Anand, J. C. and Brown, A. D. (1968). Journal of General Microbiology 52, 205. Andrews, S . and Pitt, J. I. (1987). Journal of General Microbiology 133, 233. Andre, L. (1990). Ph.D. Thesis: University of Goteborg, Sweden. AndrC, L., Nilsson, A. and Adler, L. (1988). Journal of General Microbiology 134, 669. AndrC, L., Hemming, A. and Adler, L. (1991). FEBS Letters 286, 13. Arst, H . N., Hondmann, D. H. A. and Visser, J. (1990). Molecular and General Genetics223, 134. Attfield, P. V. (1987). FEBS Letters 225, 259. Ayerst, G. (1969). Journal of Stored Products Research 5 , 127. Bachmann, B. J. and Bonner, D. M. (1959). Journal of Bacteriology 78, 550. Back, J . F., Oakenfull, D. and Smith, M. B. (1979). Biochemistry 18, 5191. Ball, S . G., Wickner, R. B., Cottarel, G., Schaus, M. and Tirtiaux, C. (1986). Molecular and General Genetics 205, 326. Bank, H. (1973). Cryobiology 10, 157. Bank, H. and Mazur, P. (1973). Journal of Cell Biology 57, 729. Barnett, J . A. (1976). Advances in Carbohydrate Chemistry and Biochemistry 32, 125. Barnett, J . A., Payne, R. W. and Yarrow, D. Y. (1983). “Yeasts: Characteristics and Identification.” Cambridge University Press, Cambridge. Beever, R. E. and Laracy, E. P. (1986). Journal of Bacteriology 168, 1358. Bellinger, Y . and Larher, F. (1988). Canadian Journal of Microbiology 34, 605. Beuchat, L. R. (1981). Journal of Food Protection 44,765. Blakely, E. R. and Spencer, J. F. T. (1962). Canadian Journal of Biochemistry and Physiology 40, 1737. Blatt, M. and Slayman, C. L. (1987). Proceedings of the National Academy of Sciences ofthe United States of America 84, 2737. Blomberg, A. and Adler, L. (1989). Journal of Bacteriology 171, 1087. Blomberg, A,, Larsson, C. and Gustafsson, L. (1988). Journal of Bacteriology 170, 4562. Borst-Pauwels, G. W. F. H. (1981). Biochimica et Biophysica Acta 650, 88. Braun, M. L. and Niederpruem, D. J. (1969). Journal of Bacteriology 100, 625.

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