Carbohydrate Reserve and Dark Carbon Fixation in the Brown Macroalga, Laminaria hyperborea

Tissue discs originating from young, growing blade areas and from adult, mature frond regions of the brown macroalga Laminaria hyperborea (Fosl.) Gunn. (Phaeophyceae, Laminariales) were investigated with particular regard to photosynthesis, dark respiration, dark carbon fixation, and carbohydrate reserves. It was found that the mannitol/laminaran reserve of the young, developing blade meets the requirements of dark respiratory metabolism for only 7-10d at 10±2 °C under continuous darkness. A concomitant decrease in the potential for (β-carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) occurred along with the depletion of the stored carbohydrate. Restoring the intracellular pool of reserve carbohydrates by photosynthesis and by feeding of exogenously supplied mannitol resulted in a short term recovery of the rates of dark fixation. These findings support the view that (i) in the dark the substrate of (β-carboxylation is mainly derived from mannitol (along with glycolytic degradation of laminaran) and (ii) the young blade is not able to maintain its own carbon balance under the environmental conditions during midwinter and early spring, but relies on a carbon flow from the old blade.     

Effects of light and temperature on symptom development and virus content of tobacco leaves inoculated with potato mop-top virus

Necrotic spots or small rings develop after 3–4 days in leaves of Nicotiana tabacum cv. Xanthi-nc inoculated with potato mop-top virus and kept at 14 °C in continuous light (4320 lux); a series of concentric necrotic rings of increasing diameter then form at 2- to 3-day intervals around each initial lesion. Successive rings take longer to appear when either the light intensity or the photoperiod is decreased. Virus accumulation is much decreased and lesions rarely develop either at 14· in darkness or at 22° in light. Virus accumulates rapidly when plants are transferred from these conditions to 14° in light (4320 lux), and necrotic spots or rings develop whose size depends on the interval between inoculation and transfer, and on the conditions during this period. In such plants, necrosis seems to occur only when conditions become favourable for virus synthesis, it is confined to recently infected cells and it does not prevent virus spread to further healthy cells. From the sizes of the necrotic rings, the virus is estimated to invade tissue in light (4320 lux) at c. 38 μm/h at 22° and c. 16 μm/h at 14°. Invasion in darkness at either temperature is very slow. Necrotic rings develop, and the rate of virus accumulation increases when inoculated plants are transferred from 22° in light (4320 lux) to 14° in darkness, but no lesions appear when the order of the treatments is reversed. The process of lesion formation thus includes an early phase requiring light and a later phase requiring low temperature. The light-requiring phase takes about a day at 14° but less at 22°. The later phase takes about 2 days in light (4320 lux) or 3 days in darkness.     

FACTORS AFFECTING DARK SURVIVAL OF THE BROWN TIDE ALGA AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)1

Aureococcus anophagefferens Hargraves et Sieburth is a pelagophyte responsible for the harmful brown tides in New York, New Jersey, and Rhode Island, USA. Recent reports of blooms in new areas, Maryland, USA, and Saldanha Bay, South Africa, suggest that the alga may be expanding its range, possibly through anthropogenic transport. Experiments tested the ability of A. anophagefferens to survive dark conditions, such as those that might be encountered during transport in ballast tanks or recreational boats. Laboratory cultures were stored in complete darkness for various lengths of time under different conditions. After returning cultures to optimal light and growth conditions, we recorded the time until growth resumed. Cultured A. anophagefferens was able to survive for at least 30 days in the dark. Temperature played a major role in dark survival, because cultures stored at 6° C and 12° C recovered faster than those stored at 18° C or 24° C. Growth phase at the time of storage had a minor effect on recovery, with exponential phase cultures resuming growth more quickly than stationary phase cells. Although the alga is known to have heterotrophic capabilities, the addition of low levels (1–3 μM) of organic nutrients did not appear to increase dark survival. Salinities within normal estuarine and oceanic ranges also did not affect survival. The ability of A. anophagefferens to survive dark periods could allow it to be transported by anthropogenic means to new regions, and temperature and length of storage appear to be key factors determining cell viability during prolonged darkness.     

The interacting effect of prolonged darkness and temperature on photophysiological characteristics of three Antarctic phytoplankton species

Photophysiological characteristics of the Southern Ocean phytoplankton species Phaeocystis antarctica, Geminigera cryophila, and Chaetoceros simplex were assessed during 7 weeks of darkness and subsequent recovery after darkness at 4 and 7°C. Chlorophyll a fluorescence and maximum quantum efficiency of PSII decreased during long darkness in a species-specific manner, whereas chlorophyll a concentration remained mostly unchanged. Phaeocystis antarctica showed the strongest decline in photosynthetic fitness during darkness, which coincided with a reduced capacity to recover after darkness, suggesting a loss of functional photosystem II (PSII) reaction centers. The diatom C. simplex at 4°C showed the strongest capacity to resume photosynthesis and active growth during 7 weeks of darkness. In all species, the maintenance of photosynthetic fitness during darkness was clearly temperature dependent as shown by the stronger decline of photosynthetic fitness at 7°C compared to 4°C. Although we lack direct evidence for this, we suggest that temperature-enhanced respiration rates cause stronger depletion of energy reserves that subsequently interferes with the maintenance of photosynthetic fitness during long darkness. Therefore, the current low temperatures in the coastal Southern Ocean may aid the maintenance of photosynthetic fitness during the austral winter. Further experiments should examine to what extent the species-specific differences in dark survival are relevant for future temperature scenarios for the coastal Southern Ocean.     

Temperature and Time-course of the Carbon Balance of Vegetative Tomato Plants During Prolonged Darkness: Examination of a Method of Estimating Maintenance Respiration

In order to examine the suitability of estimating maintenancerespiration in prolonged darkness, the variation of structuraldry matter (SDM) was calculated on vegetative tomato plantsduring 48 h of darkness. For that purpose, the time-coursesof respiration rate and carbohydrate content were recorded inshoots and roots at temperatures of 10, 15, 20, and 25 °C Two exponential declines of respiration rate, separated by ashort resumption, were observed in shoots and roots, differentcarbohydrate pools might be involved. Respiration rate was alwayshigher in roots than in shoots: the part played by energy costsof mineral absorption has to be investigated. After 14 h ofdarkness, a fall in respiration rate was associated with a progressiveexhaustion of sucrose and starch - which was quicker at highertemperatures - and a decrease in shoot to root carbon translccation.After 24 h of darkness, respiration stabilized at all temperatures.However, structural growth persisted throughout the dark periodat 10 °C, stopped after about 14 h darkness at. 15 and 20°C, and became negative beyond 24 h at 25 °C The hypothesis of maintenance of SDM after a period of darknesscan thus be invalidated. The simple observation of the time-courseof respiration rate does not allow complete inferences to bemade concerning biomass maintenance Lycopersicon esculentum Mill., tomato, respiration, maintenance respiration, carbohydrate reserves, translocation, structural dry matter, temperature     

The Geotropic Curvature in Roots of Norway Spruce (Picea abies) Containing Anthocyanins

Seedlings of Norway spruce (Picea abies L.) have been found to synthesize anthocyanins in the root tips as well as in the hypocotyls upon irradiation with white light when kept at 4°C for 6–8 days. In addition, it has also been found that the elongation and the geotropic curvature of spruce roots are dependent on the light conditions. The course of the geotropic curvature in spruce roots containing anthocyanins has been followed during a period of 5 h, in which the seedlings were geotropically stimulated continuously in the horizontal position. When the stimulation was performed in white light and in darkness at 21°C, significantly larger curvatures were observed in the roots pretreated at 4°C in darkness than in the roots containing anthocyanins. The specific curvature (curvature in degrees per mm elongation), however, was approximately the same in both types of roots stimulated in white light. This was due to a retarded elongation of the roots pretreated with light at 4°C and containing anthocyanins. A smaller difference in elongation rate between roots with and without anthocyanins was observed in the dark than in the light, but even in the dark the anthocyanin-containing roots grew more slowly than roots without anthocyanins. In order to find out if it is the anthocyanin content or the illumination which affects the elongation and geotropic curvature in the roots, a series of similar experiments was performed using cress seedlings grown at 4°C in light or darkness. Roots of cress seedlings cultivated under conditions which would induce anthocyanin formation in spruce roots exhibited the highest geotropic responses both in light and darkness as compared to cress seedlings grown at 4°C in darkness. As in the case of spruce roots an increase in elongation was observed in cress roots illuminated during the geotropic stimulation. These similarities in the behaviour made it relevant to compare the development of the geotropic curvature in cress and spruce roots.     

Effects of thermoperiods on diapause induction in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae)

Abstract. The effects of thermoperiods on diapause induction in continuous darkness or under a 12 : 12 h LD photoperiod were investigated in the cabbage beetle, Colaphellus bowringi Baly, a typical short‐day species. The diapause response curves both at different constant temperatures and at the thermocycle of format CT x: (24 ? x) h (16 : 28 °C) under continuously dark rearing conditions showed that the incidence of diapause depended mainly on whether or not the mean temperature was ≤20 °C or >20 °C. If the mean temperature was ≤20 °C, all individuals entered diapause; if >20 °C, the incidence of diapause declined gradually with increasing mean temperatures. The thermocycle (CT 12 : 12 h) with a series of different cryophases (8–22 °C) and thermophases (24–32 °C) under continuous darkness demonstrated a cryophase response threshold temperature of approximately 19 °C and a thermophase response threshold temperature of approximately 31 °C. Thermoperiodic amplitude (temperature difference between cryophase and thermophase) was shown to have a significant influence on diapause induction at the mean temperatures of 22, 23 and 24 °C, but not at ≥25 °C. Thermoperiodic responses under LD 12 : 12 h clearly showed that the incidence of diapause was influenced strongly by the photophase temperature. The thermoperiod under LD 12 : 12 h induced a much lower incidence of diapause than the thermoperiod with the same temperature in continuous darkness. The ecological significance of thermoperiodic induction of diapause in this species is discussed.     

PHYSIOLOGICAL RESPONSES DURING DARK SURVIVAL AND RECOVERY IN AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)1

The harmful bloom alga Aureococcus anophagefferens Hargraves et Sieburth can survive prolonged darkness, which could facilitate overwinter survival and dispersal by anthropogenic vectors such as ballast water. Experiments were conducted to examine the biochemical and photosynthetic changes in cells during dark storage. Cells were stored in the dark for periods from 2 to 14 days and were sampled at days 0, 1, 2, 4, 7, 10, and 14. Samples from days 2, 7, and 14 were monitored during a recovery period of 4–5 days. Physiological and photosynthetic parameters were measured during the dark storage and recovery periods. Cultures resumed growth quickly when returned to light, and bacterial counts remained constant during the dark storage period but increased rapidly during recovery periods. Cellular protein, carbohydrate, and lipid concentrations declined slightly during the dark period. There were no changes in chl a per cell or in RUBISCO per cell during 14 days of darkness. The data therefore suggest that A. anophagefferens is able to maintain its photosynthetic apparatus during dark storage periods of at least 2 weeks and relies on cellular reserves until it is returned to light to resume photosynthesis. During the recovery period in the light, the cells are able to acclimate rapidly to current light levels and resume growth.     

Effect of anaerobic digestion temperature on odour, coliforms and chlortetracycline in swine manure or monensin in cattle manure

Aims: This study evaluated the effect of anaerobic digestion at 22, 38 and 55°C on odour, coliforms and chlortetracycline (CTC) in swine manure or monensin (MON) in cattle manure. Methods and Results: Swine or cattle were fed the respective growth promotant, manure was collected, and 2‐l laboratory methane digesters were established at the various temperatures and sampled over 25 or 28 days. After 21 days, the concentration of CTC in the 22, 38 and 55°C swine digester slurries decreased 7, 80 and 98%, respectively. Coliforms in the 22°C digester slurries were still viable after 25 days; however, they were not detectable in the 38 and 55°C slurries after 3 and 1 days, respectively. After 28 days, the concentration of MON in the 22, 38 and 55°C cattle digester slurries decreased 3, 8 and 27%, respectively. Coliforms in the 22°C cattle digester slurries were still viable after 28 days; however, they were not detectable in the 38 and 55°C slurries after 14 and 1 days, respectively. Conclusions: These studies indicate that anaerobic digestion at 38 or 55°C may be an effective treatment to reduce coliforms and CTC; however, it is not an effective treatment to reduce MON. Significance and Impact of the Study: More studies are needed to determine which pharmaceuticals are susceptible to degradation by a specific manure treatment to prevent negative environmental consequences.     

Prolonged survival of mouse epididymal spermatozoa stored at room temperature

Summary: The viability and fertility of isolated mouse epididymal spermatozoa kept for up to 7 days at various temperatures (4°C, 22°C, and 37°C) were determined. Spermatozoa kept for 3 days at 22°C were still active, while those kept at 37°C or 4°C exhibited great reduction in motility within 2 days after isolation. In vitro fertilizing abilities of spermatozoa left for 0, 1, 2, and 3 days at 22°C were 69.2, 32.5, 9.5, and 4.9%, respectively, when the cleavage rate to two‐cell stage was examined. Transfer of two‐cell embryos produced in vitro with spermatozoa left for 1, 2, and 3 days at 22°C resulted in production of fetuses with efficiencies of respectively 30.2, 11.5, and 16.7%, which were lower (63.3%) than that of embryos derived from in vitro fertilization with fresh spermatozoa. These findings indicate that spermatozoa kept for up to 3 days at 22°C can fertilize oocytes, although at relatively low efficiency. genesis 31:147–155, 2001. © 2001 Wiley‐Liss, Inc.     

Responses of the rice shoot apex to irradiation with red and far-red light

Summary Determinations of cell-doubling times using the technique of colchicine-induced metaphase accumulation showed that after 40 h exposure to red or far-red light the rates of cell division in young rice (Oryza sativa L. cv. Ballila) shoot apices were faster than in dark controls. In red light, the increase was already taking place after 16 h of irradiation but in far-red the rates at this time were slower than in the dark controls. Seedlings became more responsive to far-red light as they continued to grow in darkness between 2 1/2 and 4 1/2 days. Mitotic activity at the apex increased in the leaf primordium and decreased in the sub-summit corpus between the 4th and 5th days of growth in darkness at 30° C.     

Survival in the dark: strategies adopted by Tetraselmis indica (Chlorodendrophyceae,Chlorophyta)

The adaptive strategy of Tetraselmis indica to survive darkness was evaluated over a period of 182 days. Resting cells formation and reduction in catabolic activity facilitated dark survival and their rejuvenation. The occurrence of thick-walled cells (type 1) and thin-walled cells with the condensed chloroplast (type 2) in light-dark and dark conditions respectively indicated differential resting cell formation. The dissolved organic carbon remained nearly constant throughout the dark period signifying T. indica did not favour heterotrophy. Upon re-illumination of dark-adapted cells, the lag phase lengthened from 2 to 28 days when darkness period increased from 8 to 182 days, respectively. However, this negative influence on growth was compensated by an increase in growth rate from 0.35 to 0.95 d^?1. This resurgence capability of T. indica after six months of dark exposure and distinctive resting cell formation indicate a differential population sustenance mechanism. Further elucidation of dark adaptability and survival strategies in T. indica may have significance in ecological and evolutionary processes.     

N‐ASSIMILATION IN THE NOXIOUS FLAGELLATE HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE): DEPENDENCE ON LIGHT,N‐SOURCE,AND PHYSIOLOGICAL STATE1

The capabilities of the diel vertically migrating flagellate Heterosigma carterae Hulburt for assimilating ammonium and nitrate into cell‐N in light and in darkness were studied using cells of different N‐status. Ammonium utilization in darkness, except by N‐replete cells, attained>50% of use in the light. However, the capacity to use nitrate was poor in darkness, and less than 20% of nitrate‐N that was taken up in darkness was then actually incorporated into cell‐N. The ability to assimilate N in darkness improved as N‐status (N:C) declined, concurrent with an increasing content of water‐soluble carbohydrate. This carbohydrate was used to support dark N‐assimilation. Cells held in darkness for over a day and that had halted nitrate‐uptake were still capable of taking up ammonium. Furthermore, the act of taking up ammonium appeared to make available a source of C to support nitrate uptake that was previously unavailable. The implications of these results for the ecophysiology of this organism and for the construction of mathematical models of algal growth are considered.     

Effects of temperature and light on germination and early seedling development of the pine pink orchid (Bletia purpurea)

Orchid seed physiology is a poorly understood phenomenon owing to an emphasis on production and the challenges associated with propagating orchids from minute seed. We investigated the role of simulated south Florida temperatures and illumination (dark and 12 h photoperiod) in regulating germination and seedling development using asymbiotic seed germination assays of Bletia purpurea. Our objectives were to determine whether in situ germination is limited by seasonal temperatures and to determine whether temperature alters responses to illumination. Bletia purpurea seeds were able to germinate to > 90% under all treatments. The greatest germination after 3 weeks was observed at 29/19°C under continual darkness and at 25°C under dark and illuminated conditions. The slowest germination was observed at simulated winter temperatures (22/11°C). Illumination initially inhibited germination and development, but resulted in equal or greater development by week six. Germination under 22/11°C was strongly inhibited by illumination, indicating an interaction between temperature and light sensing systems.     

Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype

A temperature-sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts-H6-15) is a derivative of SV-3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3-0-methyl-D-glucose (3-0-MeG) transport is observed as cell population density increases. At all cell population densities tested, 3-0-MeG transport rates (at a common assay temperature) were greater in H6-15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum-arrest on H6-15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum-arrest the rate of 3-0-MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2-deoxyglucose uptake rates were growth state-dependent at 39°C, indicating perhaps metabolic as well as membrane-level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.     

DIEL CHANGES IN PHASED-DIVIDING CULTURES OF CERATIUM FURCA (DINOPHYCEAE): NUCLEOTIDE TRIPHOSPHATES,ADENYLATE ENERGY CHARGE,CELL CARBON,AND PATTERNS OF VERTICAL MIGRATION1

The diel pattern of cell division, cell carbon, adenine nucleotides and vertical migration was determined for laboratory cultures of the photosynthetic marine dinoflagellate, Ceratium furca (Ehr.) Clap. & Lachm., entrained on an alternating 12:12 LD schedule at 20 C. Cell division was initiated during the latter portion of the dark period with ca. 30% of the population undergoing division. Cell C increased during the light period and exhibited a linear decrease with a loss of 33% during the dark period. ATP · cell^?1 increased during the light period and decreased by ca. 40–50% during the dark period. The diel patterns of cell C and ATP tended to “buffer” the magnitude of the change in C:ATP ratios around an overall mean value of 89. There was no obvious trend in the concentration of [GTP + UTP] · cell^?1 over the cell cycle. The cellular adenylate energy charge was maintained at values between 0.8 to 0.9 throughout the 24 h LD cycle, despite a ca. 40% decrease in total adenylates (A_T= ATP + ADP + AMP) during the dark period on 12:12 LD, and over a 68% decrease in ATP during 42 h of continuous darkness. These data lend experimental support to the theory of cellular metabolic control by the adenine nucleotides. With lateral illumination on 12:12 LD cycles, the cells began to concentrate at the surface of the experimental tubes shortly before the lights were turned on, and at the bottom of the tubes shortly before the lights were extinguished. This pattern continued for 6 days in continuous darkness, suggesting that the vertical migration pattern is independent of a phototactic response and may be under the control of an endogenous rhythm.     

Investigation of morphological change of Aulacoseira baicalensis using a small desktop incubator controlling light and temperature

A desktop incubator with temperature control over the range 1–20°C (±0.5°C) was designed to hold two microtitre plates. Illumination of individual wells in the plate was by a matrix of 96 light-emitting diodes, whose intensity, period and pulsation could be controlled individually in each of 12 rows of eight chambers. The incubator was used to test how the length of light period affected cell length of the planktonic diatom Aulacoseira baicalensis, which only grows below 4°C. Short cells (mean length 26 µm) were formed under a 8-h : 16-h light–dark cycle, intermediate cells (mean length 35 µm) under a 4-h : 20-h light–dark cycle and longer cells (mean 38 µm) under a 2-h : 22-h light–dark cycle. These were similar to changes in mean cell length from 28 to 49 µm found in Lake Baikal during a decrease in light period caused by increased convective mixing from 15 m under-ice to 94 m after ice break-up. The laboratory experiments confirmed that decreasing light period was the environmental cue that initiated the production of long cells that then developed into resting stages.     

Fickean and Non-Fickean Water Desorption During Vacuum Drying of <Emphasis Type="Italic">L. bulgaricus</Emphasis>

The recovery of Lactobacillus bulgaricus was studied in correlation to the kinetics of cell drying. When bacteria were dehydrated at 30 °C, either in the presence or the absence of sucrose, the drying kinetics corresponds to a Fickean diffusion in correspondence with a short lag time. In contrast, when the bacteria were dehydrated at 70 °C in the absence of sugar, the kinetics corresponds to an anomalous diffusion, and the lag time is four to five times higher than that at 30 °C. However, when drying at 70 °C was carried out in the presence of sucrose, drying kinetics turned into a Fickean process parallel to a substantial decrease in the lag time. The pattern of water desorption was correlated with the critical water activity. When the drying kinetics corresponds to a Fickean diffusion, the lag time started to increase at 0.7 water activity, but when the cells were dried at 70 °C, the damage started at 0.5 water activity. This observation indicates that the drying rate affects the pattern of water desorption, and it can change the value of critical water activity. These results put into relevance that the cell recovery is due to the drying history and that the recovery increase produced by sucrose can be related to the maintenance of kinetic barriers for water desorption.     

Low temperature effects on growth and actin cytoskeleton organisation in suspension cells of winter oilseed rape

Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of cells and in the increased freezing tolerance of cells as indicated by a decrease of LT₅₀ from −11 °C to −17.5^o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6. Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization of actin microfilaments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.