Plant Cell, Tissue and Organ Culture 65: 149–158, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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

Stefania Egierszdorff & Alina Kacperska∗Institute of Experimental Plant Biology, Warsaw University, Pawinskiego 5A, Pl-02-907 Warsaw, Poland (∗requestsfor offprints; E-mail: alka@biol.uw.edu.pl)

Received 12 August 1999; accepted in revised form 2 February 2001

Key words: Brassica napus L. var. oleifera L., cold, cell morphology, extracellular freezing, microfilaments

Abstract

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

Abbreviations: CA – cold acclimated; 2,4-D – 2,4-dichlorophenoxyacetic acid; LS – Lindsmaier & Skoog;LT50 – temperature giving rise to 50% injuries; Mfs – microfilaments; NA – non acclimated; NAA – α-naphthaleneacetic acid; PBS – phosphate buffered salines; TRITC – tetramethylrhodamine isocyanate; TTC –2,3,5-triphenyltetrazolium chloride

Introduction

Low temperature affects plant cell functions and struc-ture in different ways. It may result in cell injury andcell or plant death or in the adjustment of cellularmetabolism to low temperature conditions and in theimprovement of plant resistance to freezing temper-atures (Kacperska, 1989). The effects depend on theduration and the intensity of the stress and on genetic-ally dependent properties of cell components (Sakaiand Larcher, 1987). In many biennial plants, theachievement of the maximum level of frost tolerance

requires a short exposure of cold-acclimated plants tofreezing at −2 to −3 ◦C (Sakai and Larcher, 1987;Kacperska, 1999). In contrast to the broad knowledgeof physiological and molecular mechanisms involvedin plant acclimation to cold (>0 ◦C ), information onthe nature of plant responses to the transient freezingis rather limited. It was proposed that cell dehydrationdue to extracellular ice formation results in a modific-ation of cell wall and plasma membrane interactionswhich, in turn, leads to changes in gene expressionand synthesis of products that protect cell constituentsagainst frost-induced dehydration (Kacperska, 1999).

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The cell wall/plasma membrane interactions maybe influenced by organisation of cytoskeleton (Tiwariet al., 1984; Derksen et al., 1990) which is modi-fied in response to internal and environmental stimuli,including low temperature. Temperatures approach-ing 4 ◦C were found to depolymerize microtubules inmany plant cell types (Wallin and Stromberg, 1995).Also, extracellular ice formation promoted micro-tubule depolymerization in several plants includingonion (Carter and Wick, 1984), rye (Kerr and Carter,1990) and spinach (Bartolo and Carter, 1991a). Inwinter rye root tips, the level of depolymerization wasrelated to the degree of freezing tolerance suggest-ing that microtubule depolymerization is importantfor achieving maximal freezing tolerance (Kerr andCarter, 1990). Stabilisation of microtubules with taxolactually increased frost-induced injuries in mesophyllcells of spinach (Bartolo and Carter, 1991b).

In plant cells, reorganisation of the actin cytoskel-eton affects several cellular processes such as cyto-plasmic streaming, cell division and differentiation,cell shape determination, organelle movement, cellwall deposition (Tiwari et al., 1984; Seagull et al.,1987; Traas et al., 1987; Staiger et al., 1997) aswell as binding and stabilisation of the microtubules(Shiboaka and Nagai, 1994). However, informationon the low temperature effects on the actin cytoskel-eton organisation is very scarce. Chilling temperatures(>0 ◦C) were found to be of no effect on the arrange-ments of actin-filament bundles in onion bulb cells(Quader et al., 1989). In the frost-affected rye root tips,stabilisation/depolymerization of microfilaments wasshown to depend on stabilisation/depolymerization ofmicrotubules (Chu et al., 1993). A discovery that coldtreatment results in a rapid up-regulation of a genewhich encodes an actin-binding protein may indicatethat the modulation of the intracellular actin structurecould be required for development of higher freezingtolerance (Danyluk et al., 1996).

Suspension cultured cells are a good model systemin the studies on actin microfilament organisation inhigher plants (Seagull et al., 1987; Traas et al., 1987).It is also known that cell suspensions are able to ac-climate in cold and to develop a higher frost tolerance(Chen and Gusta, 1982; Orr et al., 1985; Wallner et al.,1986; Robertson et al., 1987, Arora and Wisniewski,1995). Therefore, cell suspensions of winter oilseedrape were used in the present experiments.

The purpose of this study was to determine theeffects of cold (>0 ◦C) and freezing (<0 ◦C) temper-atures on the structure of actin cytoskeleton in cells

of winter oilseed rape plants. The plants are able toacclimate in cold and their freezing tolerance increases3 to 4 ◦C in response to a transient freezing treatmentat −4 ◦C (Kacperska and Kulesza, 1987). It is expec-ted that the freezing-induced changes in the structureof actin filaments may be an important element indevelopment of cell resistance to freezing.

Material and methods

Cell culture

Cell suspension cultures of winter oilseed rape(Brassica napus L., var. oleifera L.), initiated 8 yearsearlier from hypocotyl tissue, were maintained inliquid LS medium (Lindsmaier and Skoog, 1965), sup-plemented with 0.45 µM 2,4-dichlorophenoxyaceticacid (2,4-D) and 0.54 µM α-naphthalenacetic acid(NAA) and regularly subcultured at 6-day intervals (1ml of suspension culture to 50 ml of fresh medium).Cultures were grown at 25 ◦C in darkness and aer-ated at 100 revolutions per minute on rotary incubatorshaker (New Brunswick Scientific Co.). On the fourthday of the subculture, half of the cell suspensions weretransferred to the cold (2 ◦C) room for 7 or 24 days(cold-acclimated, CA cells). The other half continuedgrowth at 25 ◦C (non-acclimated, NA cells) for 8 days.Later on the viability of these cells, not transferred toa fresh medium, decreased markedly, as indicated bythe TTC reduction test.

Microscopic examinations

Suspension cells, collected at different days of the sub-culture, were fixed for 2 hours in 2% glutaraldehydein 0.1 M cocodylate buffer, pH 7.2 and postfixed in2% OsO4. Then, they were dehydrated using a seriesof graded washes of ethanol and embedded in Spurr’sresin (Spurr, 1969). Semithin sections were cut with anultramicrotome (Ultracut R Leica), stained with 0.1%toluidine blue in 1% borax solution and examined witha light microscope (NU, Carl Zeiss Jena). The im-ages were recorded on Black & White 100 ASA film(Foton, Poland).

Growth determinations

On selected days of subculture, 50 ml samples of NAor CA suspension cells (in three replicates) were col-lected on a Miracloth filter and their dry matters andmitotic activity were determined. Dry matter of each

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sample was estimated following oven drying at 95 ◦Cfor 2 h in aluminium cans. The rate of suspensiongrowth, calculated as daily dry matter increments, wasestimated. For determinations of mitotic indices (num-ber of cells undergoing division as a percentage ofall the cells), squashed cell preparations were used.Suspension samples were macerated and stained ina mixture of acetoorceine (2% acetoorceine in 45%acetic acid) and 1 N HCL, in proportion 1: 9 (v/v)(López-Sáez and Fernández-Gómez, 1965) for 0.5–1.0 h. On each sampling day, three readings weretaken, up to a total of one thousand cells.

Freezing treatments

NA or CA suspension cells were sampled on the dayof the experiment. They were collected on a Mir-acloth filter, washed with a fresh LS medium, driedwith a filter paper, and transferred to Eppendorf vials.For determination of freezing-induced modificationsin cytoskeleton structure, the suspensions (10 mg freshweight per a vial) were placed in a freezing bath at −3,−5, or −7 ◦C in cryostats (Hetofrig, CB7, Denmark).At each of these temperatures ice crystals were addedto the samples. In preliminary experiments no effectsof freezing temperatures on cytoskeleton organizationwere observed if freezing of extracellular water hadnot been provoked by inoculation of suspensions withice. After a 30-min incubation at the selected tem-perature, cells were allowed to thaw at 0 ◦C for 30min in the dark. Following the treatments, the actinmicrofilaments were visualised by staining.

For determination of freezing tolerance, the sus-pension samples (50 mg fresh weight per a vial)collected as indicated above, were cooled down incryostats to −3 ◦C. Extracellular freezing of waterwas initiated in vials by ice seeding and after 30-minequilibration the temperature of the freezing bath waslowered at the rate of 1 ◦C/10 min. Samples were re-moved at designated temperatures separated by 3 ◦Cintervals and thawed at 4 ◦C overnight. Cell viabil-ity was assayed by TTC reduction (Towill and Mazur,1975), the product of the reduction (formazan) beingextracted with 95% ethanol (5 ml per a vial) and de-termined spectrophotometrically at 485 nm. Freezingtolerance was expressed as the LT50, i.e. the temperat-ure which resulted in a 50% decrease in TTC reductionin comparison with TTC reduction by non-frozen con-trol suspension samples (maintained at 4 ◦C), taken as100%.

Microfilament (MF) staining

Rhodamine-phalloidin staining (Molecular Probes,Eugene OR) was used to visualise the structure ofactin cytoskeleton The cells, collected on a filter, werewashed once with phosphate buffered salines (PBS),pH 7.4, and drained off the buffer with a fine syringeneedle. Then, they were stained with 8.3 µM TRITC-phalloidin (Sigma) solution in the same buffer at 25◦C (NA cells) or 2 ◦C (CA cells) for about 4 hours.No fixation in formaldehyde was performed becausepreliminary experiments showed that such a treatmentresulted in fragmentation or disappearance of MFs andin increased non-specific background staining. Sim-ilar artifactual effects of formaldehyde fixation havebeen shown for other plant cells (Lehrer, 1981; Traaset al., 1987; Doris and Steer, 1996). Fluorescencefrom TRITC was observed with an epifluorescencemicroscope (Microphot-SA, Nikon) using 100 × CFFluor oil immersion objectives and G 2A filter block(single-pass excitation in 510–560 nm range and a 590nm barrier filter). Photographs were taken immedi-ately after staining using an automatic camera system(Nikon FX-35DX) and a black and white film (KodakT MAX p3200).

Results

Effects of temperature on growth and freezingtolerance of cell suspensions

The growth of cells was dramatically affected by thetemperature of the culture, as indicated by changes indry weight of the suspension samples (Figure 1A). At25 ◦C, dry matter of cells started to increase two daysafter subculture. The highest rate of dry matter pro-duction was observed between the fourth and the sixthday of the suspension growth. During further growth at25 ◦C the dry matter increases were much smaller andon the twelve day the rate of dry matter accumulationdecreased to the level observed at the beginning of thesubculture. The cold treatment, applied on the fourthday of the subculture, resulted in the marked inhibitionof cell growth (Figure 1A).

In NA cells, mitotic activity (Figure 1B) increasedmarkedly on the third day of the subculture. On thesixth day it decreased rapidly to the level noted atthe beginning of the subculture. No mitotic activitywas observed on day nine. Cold treatment delayed theloss of mitotic activity: in suspensions, grown in cold

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Figure 1. Dry matter accumulation (A), mitotic activity (B) andfreezing tolerance (C) in Brassica napus L., var. oleifera L. sus-pension cells grown at 25 ◦C (open symbols) or at 2 ◦C (closedsymbols). Bars indicate the standard deviation of the mean.

for 11 days, mitotic activity was still higher than thatnoted on the second day of the subculture.

Freezing tolerance of NA cells increased on thesixth day of subculture from −11 ◦C to −17 ◦C butlater on it decreased to −8 ◦C (Figure 1C). In CAsuspensions, a marked increase of freezing tolerancewas observed and twenty days after cell transfer to 2◦C the LT50 reached −25 ◦C.

Morphology of cell suspensions

On the 4th day of subculture, compact aggregates ofsmall cells with abundant cytoplasm and numeroussmall vacuoles were seen (Figure 2A). In nuclei, well-stained nucleoli could be observed. The developmentof new cross walls between the newly formed daughtercells could be also noted (Figure 2A, arrows). Afterfurther two days of the subculture at 25 ◦C, cells in-creased in size and fewer but larger vacuoles occurredin many cells (Figure 2B). Numerous starch grainswere also observed in some cells. The morphologyof the cold-treated cells, harvested 7 days after thebeginning of the cold treatment, did not differ muchwhen compared to that revealed by the four day-grownNA cells (Figure 2C, compare with Figure 2A). Newlyformed walls between the daughter cells could be seenand cytoplasm and nucleoli were heavily stained. Thenumber of small vacuoles increased in comparisonwith that found in the four-day-old cells. In subcul-tures grown in cold for 20 days (Figure 2D), cellsremained small but the volume of vacuoles increasedand a high number of starch grains in cytoplasm couldbe noted.

Actin cytoskeleton

Actin microfilaments were visualised in peripheralcells of small cell aggregates. In NA cells collected4 days after subculture (Figure 3), the actin cytoskel-eton consisted of a network of randomly arrayed shortand thin cables, seen in the cortical cytoplasm. A fewlong and relatively thick cables could be also observedin some cells. The 30 min freezing at −3 or at −5◦C in the presence of extracellular ice, resulted in de-polymerization of the fine cortical meshwork and infragmentation of thicker bundles. In some cells frozenat −7 ◦C, microfilaments of the nuclear basket be-came visible. In the six-day-old NA suspension cells(Figure 4), the actin filaments formed a network com-posed of thick cables which resisted depolymerizationat −3, −5 and −7 ◦C. In cells acclimated in cold forseven days (Figure 5), actin microfilaments formed adense network, resembling those seen in the four-day-old NA cells (Figure 5, compare with Figure 3). Inthese cells the actin filaments were sensitive to freez-ing since they had already undergone fragmentationat −3 ◦C, in the presence of extracellular ice. Lowerfreezing temperatures induced a higher degree of actindepolymerization. In cells acclimated in cold for 20days, the transvascular strands of actin microfilamentswere still observed (data not shown).

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Figure 2. Morphology of Brassica napus L., var. oleifera L. suspension cells, grown at 25 ◦C for 4 (A) and 6 (B) days or at 2◦C for 7 (C) and20 (D) days. N: nucleus, n: nucleolus, v: vacuole, s: starch grain. Black arrowheads indicate newly formed walls between the daughter cells.Bar is 20 µm in each photograph.

Discussion

Examination of time courses for dry matter produc-tion and mitotic activity in suspensions grown at 25◦C (Figure 1) indicates a relatively short lag phaseof growth of approximately two days after the sus-pension had been subcultured. This was followed bya sigmoidal increase in cell dry matter during thenext ten days. A high rate of dry matter accumula-tion observed between the fourth and the sixth day ofthe subculture (Figure 1A), preceded by a relativelyhigh mitotic activity (Figure 1B), indicate the phaseof a rapid cell growth. A dramatic decrease of mi-totic activity in the six-day-old suspensions, followedby a decrease in the rate of dry matter accumulation

and coincident with a development of larger vacuolesin a protoplasm (Figure 2B) indicate the beginningof a stationary phase of cell growth. Our results arein line with the observations of Bonner et al. (1988)who found that growth curves plotted on the basis ofcell dry weight changes could be successfully used fortiming of growth phases.

The cold treatment arrested growth of cells at thebeginning of a rapid growth phase (Figure 1), pre-vented exhaustion of carbohydrates, as indicated bystarch accumulation (Figure 2D) and led to a devel-opment of a high number of small vacuoles (Figure2C, D). It seems that a higher degree of cell vacu-olisation in winter oilseed rape suspension cells wasassociated with a higher freezing tolerance of cells

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Figure 3. Actin microfilaments in the four day-old suspension cells grown at 25 ◦C and exposed to extracellular freezing at temperaturesindicated in photographs. A network of fine cortical arrays (white arrow) surrounding small vacuoles (asterisk) is seen non frozen cells.Fragmentation of the network (white arrowheads) is observed in cells subjected to freezing at indicated temperatures. Nuclear basket cables arenot affected by freezing at −7◦C (black arrow). Bar is 5 µm in each photograph.

since the tolerance increased both in the six-day-oldNA cells and in CA cells (Figure 1C). A similarphenomenon was observed in phloem and cortical par-enchyma cells of cold-acclimated woody perennials(Pomeroy and Siminovitch, 1971; Niki and Sakai,1981) and in suspension cells of alfalfa, bromegrassand peach (Borochov et al., 1989; Tanino et al., 1991;Arora and Wisniewski, 1995).

Two different types of actin cytoskeleton structurehave been observed in winter oilseed rape suspensioncells. The first type, observed in cells rich in cyto-plasm (Figures 2A, C), sampled at the beginning ofthe phase of a rapid cell growth at 25 ◦C or after 7-

day-growth in cold, consisted of a fine, dense filamentnetwork (Figures 3 and 5). The second one, seen incells showing a higher degree of vacuolisation (Fig-ure 2B), consisted of the arrays of thick transvacuolarbundles (Figure 4). Similar types of actin filamentdistribution were described in carrot suspension cells(Seagull et al., 1987). In small isodiametric carrotcells, a fine network of randomly arrayed MFs wasseen in cortical cytoplasm, immediately adjacent to theplasma membrane. In larger vacuolized cells, showinga rapid cellular streaming, large and brilliantly stainedMFs bundles, localised in subcortical regions of cyto-plasm were noted. Our observation that distribution

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Figure 4. Actin microfilaments in the six-day-old suspension cells grown at 25 ◦C and exposed to extracellular freezing at temperaturesindicated in photographs. Thick, long actin transvacuolar strands (white arrows) do not undergo depolymerization in response to extracellularfreezing in the full range of tested temperatures. Vacuoles are indicated by asterisks. Bar is 5 µm in each photograph.

and organisation of MFs bundles was similar in four-day-old non-acclimated cells, entering the phase of arapid growth and in 11-day-old CA in which the ex-pansion growth was inhibited (compare Figure 2A andC) supports the view that distribution of microfilamentarrays depends on the phase of the cell cycle, as pro-posed by Staiger and Schliva (1987) and not on thecell chronological age or cold acclimation status. It isinteresting that differences in organisation of actin mi-crofilaments in suspension cells, observed in our work,resemble those observed during the development ofcells in Equisetum hyemale root tips (Derksen et al.,1986).

The two types of actin cytoskeleton differed intheir reaction to the transient freezing treatment. The

fine filament network was very labile and disas-sembled at −3 ◦C, both in non- acclimated and cold-acclimated cells, independently of the level of cellfreezing resistance. The stability of thicker filamentbundles of vacuolized cells was higher since they didnot undergo the freezing-induced depolymerization aseasily as fine cortical network. Therefore, it appearsthat the stage of cell cycle rather than cell cold ac-climation status modifies the sensitivity of MFs tofreezing temperature.

The possible reason for a high freezing sensitiv-ity of a fine network of cortical actin arrays adjacentto plasma membrane is rather unclear. The reorgan-isation of the actin cytoskeleton is mediated by actinbinding proteins that serve to anchor, crosslink or

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Figure 5. Actin microfilaments in the 11-day-old suspension cells acclimated in cold for 7 days (see material and methods). A network of finecortical arrays (white arrow) surrounding small vacuoles (asterisk) is seen non frozen cells. Fragmentation of the network (white arrowheads)is observed in cells subjected to freezing at indicated temperatures. Bar is 5 µm in each photo.

regulate the network within the cell (Meagher et al.,1999). Many of actin binding proteins are regulated bycalcium either directly or by calcium/calmodulin com-plex (Janmey, 1994). It appears that proteins whichlead to disruption of actin network by filament frag-mentation or end-blocking, are activated by Ca2+;whereas, filament crosslinking proteins are inhibitedby Ca2+ (Janmey, 1994). The cumulative effect of in-creased Ca2+ appears to be a solubilization of the actincytoskeleton which would allow (among others) forpreparing the cytoskeleton for remodelling in responseto a subsequent signal (Janmey, 1994). The transientincrease in the cytosolic calcium level was actuallyreported for the cold-affected plant cells (Knight et al.,

1991, 1996). However, the reasons of lower sensitiv-ity of cable-like filaments than of fine cortical arraysto freezing temperatures cannot be explained by thecalcium effects, unless different intracellular locationof these filaments and the existence of intracellularcalcium gradients will be taken into consideration.

On the other hand, numerous actin-binding pro-teins are regulated by phosphoinositides (Janmey,1994; Staiger et al., 1997). The phosphoinositide-regulated actin binding proteins generally promoteactin polymerisation and strengthen the cortical actingel, especially near the plasma membrane (Janmey,1994). It appears that the actin-depolymerising func-tion of one of the cofilins is inhibited by its binding

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to phosphatidylinositol 4,5-bisphophosphate (PIP2) inthe plasma membrane (Gungabissoon et al., 1998).Low temperature was shown to affect the phos-phatidylinositol metabolism in winter oilseed rapeleaves (Smoleñska-Sym and Kacperska, 1994). Tran-sient sublethal freezing was found to induce hydrolysisof phosphatidylinositol 4,5-bisphosphate (Smoleñskaand Kacperska 1996). This in turn, may result in liber-ation of actin-depolymerising factor(s) and in depoly-merization of the actin cortical network. Decreasedphosphatidylinositol bisphosphate levels along withincreased Ca2+ favour depolymerization of filamentsand solation of the cell cortex in response to mech-anical forces generated by a change in hydrostaticpressure (Janmey, 1994).

It seems that solation of the cell cortex may bean important factor in protection of rich-in-cytoplasmcells against effects of mechanical strains evoked at thecell wall/cytoplasm border line by the frost-inducedcell dehydration and contraction. The microfilamentbundles, localised in the subcortical region of cyto-plasm of enlarged, vacuolised cells, seem to be of lessimportance for this type of interactions.

Acknowledgements

This work was financially supported by grant 6 P204009 07 from the State Committee for Scientific Re-search of Poland.

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