Plant Physiol. (1981) 67, 4784830032-0889/81/67/0478/06/$00.50/0

Plasma Membrane Alterations in Callus Tissues of Tuber-bearingSolanum Species during Cold Acclimation'

Received for publication February 4, 1980 and in revised form October 27, 1980

MARIA A. TOIVIO-KINNUCAN, HWEI-HWANG CHEN, PAUL H. LI2, AND CECIL STUSHNOFFLaboratory ofPlant Hardiness, Department of Horticultural Science and Landscape Architecture, University ofMinnesota, St. Paul, Minnesota 55108

ABSTRACT

Plasma membrane alteration In two tuber-bearing potato species duringa 20-day cold acclimation period were investigated. Leaf-callu tssues ofthe frost-resistant Soeaum acaule Hawkes 'Oka 3878 and the frost-susceptible, comly grown SOa_w tuberom 'Red Pontiac,' were used.The former is a species that can be hardened after subjecting to the lowtemperature, and the latter does not harden. Samples for the electronmicroscopy were prepared from callus cultures after hardenin at 2 C Inthe dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acialincressed in frost hardiness from -6 to - 9 C (killing temperature),whereas frost hardiness of S. tubemsun remained unchanged (killed at -3C). Actually, after 15 days acclimation, a -9 C frost hardiness level In S.acaude callus cultures had been achieved.Membrane protein particle aggregation was monitored using freeze-

fracture electron microscopy. Protein particles were agegated In S.acauk up to 10 days after the initiation of acclimation treatment and thenredistributed abnost to the level of control after 15 days. No such changeswere observed for S. aabemsum under similar experimental conditions. Thechange in protein particle aggregation pattern In S. acadk Is interpreted as

Indicating the presence of an adaptive fludity control mechanism In thatspecies.

Studies in our laboratory have shown that the commonly grownpotato (Solanum tuberosum L.) possesses no frost tolerance (4),whereas a number of noncultivated species can survive at -4 C orcolder without frost injury. Some of these species, for example,Solanum acaule Hawkes and Solanum commersonii Dun., can alsobe cold acclimated to increase their frost hardiness up to -9 C orto -12 C, respectively (4). Also, similar increases in hardinesscould be achieved both for potted and leaf-callus cultures (4).Chen et al. (4) suggested that the callus, a homogeneous mass ofundifferentiated parenchyma cells, could serve for cold acclima-tion studies in place of intact potato plants. The stage of differ-entiation is easily controlled in callus cultures. They thus providea desirable material for the study of frost hardiness at cellularlevel.

Studies of ultrastructural alterations in relation to frost hardi-ness in plants are limited (3, 9, 20, 24). The observations ofchloroplasts (26) and mitochondria (3) during cold acclimationtreatments indicate some structural alterations in these organelleswhich may relate to plant adaptability to a given environment.

'Scientific Journal Series Paper 11,133 of the Minnesota AgriculturalExperiment Station. This research was supported in part by a researchcontract from the International Potato Center, Lima, Peru.'To whom reprint requests should be addressed.

Freezing injury often assumes as a result of rupture or loss ofsemipermeability of cell membranes (13, 27). During acclimation,plasma membrane alterations may occur which allow the cells tosurvive at lower temperatures.

Comparative studies have shown that freeze-fracturing electronmicroscopy can be used to visually observe the physical state ofmembranes subjected to phase-transition temperatures (1, 7, 8, 10,14, 25, 29). In these studies, the membrane lipid phase transitionhas been shown to be accompanied by a progressive aggregationof the IMP3 (7, 15, 16). In a complex lipid mixture, such as inbiological membranes, the protein particles tend to be associatedonly with the fluid phase (12). The particle-free areas in the freeze-fracture replicas represent areas of membrane lipid existing in thesolid state (10, 16). Therefore, we used freeze-fracture electronmicroscopy that could provide us with a direct visual probe fordetecting plasma membrane lipid phase transition in an intact cellsystem during cold acclimation.

MATERIALS AND METHODS

Leaf-callus cultures from two species of tuber-bearing potatoeswere used: (a) a noncultivated potato, S. acaule Hawkes 'Oka3878' that is frost-hardy (killed at -6 C) and can be acclimated to-9 C, and (b) the commonly grown potato, S. tuberosum L. 'RedPontiac' that is frost-sensitive (killed at -3 C) and does notacclimate to cold (4). The procedures of leaf-callus productionhave been established elsewhere (4).The cultures were grown in media for 8 to 10 weeks and then

subjected to an acclimation treatment. To compare the influenceof tissue age to the acclimation response, a second group of S.acaule 'Oka 3878' cultures were transferred to the acclimationtreatment at the age of 4 weeks. To acclimate the callus, flaskscontaining cultures were kept at 2 C in the dark for 0, 5, 10, 15,and 20 days. Frost hardiness of acclimated cultures at the end ofeach interval treatment was determined (4). Samples from eachculture were prepared for electron microscopy at the same chron-ological age.

Freeze-fracture Electron Microscopy. Small segments of thefriable callus were fixed for 5 h in 2.5% v/v glutaraldehyde in 0.1M Na-phosphate buffer (pH 6.8) at 20 to 22 C, washed with threechanges of buffer, transferred to the same buffer containing 30%ov/v glycerol, and incubated for a further 18 to 20 h at 5 C priorto freezing. The glycerol concentration was selected based onpreliminary results showing ice-damaged cells and consistantlypoor replica yields at lower glycerol concentrations. However, acontrol without glycerol treatment was carried out, allowing crit-ical evaluation of the true nature of the particle aggregation

3Abbreviations: IMP, intramembranous particle; PM, plasma mem-brane; T, tonoplast; PF, protoplasmic fracture face; ES, exoplasmic fractureface.

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PLASMA MEMBRANE ALTERATIONS IN POTATO

3 | b | |

__

FIG. 1. Freeze-fractured plasma membranes of S. acauk leaf-calluscells showing various degrees of particle aggregation on the PF duringcold-acclimation treatment at 2 C. The photographs show gradually in-creasing aggregation and provide a set of standards with arbitrarily as-

signed aggregation (AGG) values of 0, 2, 4, 6, and 8. The experimentaltreatments of the S. acaule cultures used were: 5-day acclimation (at 2 C)at the age of 4 weeks (A and B); 5-day acclimation at the age of 8 weeks(C and D); 10-day acclimation at the age of8 weeks (E). In all micrographs,bar represents 0.2 pm. CE: cell wall.

patterns in the fractured specimens.The samples were frozen onto gold specimen mounts by im-

mersion in liquid nitrogen-cooled Freon 22 (-156 C) and thenquickly plunged into liquid nitrogen (-196 C). Frozen specimenswere stored in a liquid-nitrogen cryostat. Fracturing was donewithin a 2-month period from initial freezing using a Balzers 369M unit at -105 C and at 2 x 10- toff. After minimum etching,platinum/carbon shadowing and replicating materials were evap-orated on the fracture surfaces. The thickness ofplatinum shadow-ing was monitored to 1 to 2 am using a quartz crystal QSG 201.Replicas were cleaned using 2% w/v Cellulysin (lot 800591, Bgrade; Calbiochem) (1-2 days), followed by HNO3 (70%o v/v) (3-4 days), and 2.5% v/v NaOCl (diluted commercial bleach "Clo-rox") (1 h), with a thorough wash in glass-distilled H20 after eachtreatment. Replicas were picked up with 300-mesh copper speci-men support grids and examined with a Philips 300 transmissionelectron microscope. The membrane fracture face nomenclature

FIG. 2. Freeze-fractured cells of S. acaule (A) and S. tuberosum (B)grown at 27 C constant temperature in the dark for 8 weeks (controls).Cells were fixed with 2.5% glutaraldehyde at 20 to 22 C for 5 h andincubated in 30% glycerol at 5 C for overnight before freezing andfracturing. Neither PM nor T exhibits any particle aggregation on eitherthe PF or the EF. CW, cell wall. Direction of platinum shadowing isindicated (T). Bar equals 0.5 pm.

is that of Branton et al. (2).Membrane Particle Distribution. Membrane particle distribu-

tion was determined using techniques described by James andBranton (10). In the first method, electron micrographs werecompared to a predetermined set of standard pictures showingvarious degrees of particle aggregation on the PF (Fig. 1). Thescale of 0 to 8 was used to describe the degree of aggregation, sothat a low mean value would indicate little aggregation and a highvalue would indicate great aggregation. The second method in-volved the analysis of particle distribution on prints (total mag-nification, x 67,000) which were overlaid with a grid of 1-cmsquares. Particles in 25 adjacent 1-cm squares were counted foreach membrane. Counts were normalized as per cent of totalcount for each membrane to eliminate the effect of variationbetween total particle counts in different membranes. The countsare presented by plotting the number ofareas with varying particledensity (the mean density for each membrane is 4% = 100%odivided by the number of counts per membrane, 25) for eachtreatment. Each treatment was evaluated by these two techniquesusing 5 to 10 membrane photographs taken from different cells.Statistical analysis confirmed that both methods gave similarresults. Counts were made only in regions where no membranedistortions were evident.

RESULTS

The leaf callus produced from S. tuberosum showed no increasein frost hardiness in response to low temperature. The frost-killingtemperature of 'Red Pontiac' calluses was -3 C, similar to that ofpotted grown plant leaves (4). The leaf callus of S. acaule can be

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TOIVIO-KINNUCAN ET AL. Plant Physiol. Vol. 67, 1981

Table L. Density ofProtein Particles ± SD on Fracture Faces and Mean Aggregation Values (AGG)for PlasmaMembranes of S. acaule and S. tuberosum Leaf-callus Culture Cells with Various Cold Treatment Periods (at 2 C)

Killing Plasma Membrane

Species Time Tem- PF EFat 2C pera -____________ _______ _____ture Density AGG Density AGG

days C particles!lIm2 particleksm2S. acaule 0 -6 2899 ± 683 1.2 ± 1.1 473 ± 154 1.6 ± 1.5

5 -7 2727 ±633 4.6 ±2.2 370± 178 1.5 ± 1.310 -8 1742 ± 230 6.4 ± 1.5 438 ± 257 5.3 ± 2.215 -9 2153 ±290 2.5 ± 1.8 530±401 1.0± 1.220 -9 1682 ± 363 2.7 ± 1.0 234 ± 72 0.6 ± 1.1

S. tuberosum 0 -3 2292 ± 618 0.4 ± 0.5 238 ± 117 0 ±05 -3 3435±485 0.5±0.5 419±47 0±010 -3 1735±943 0.8 ± 1.0 445 159 0.4±0.515 -3 2601 ± 435 0.5 ± 0.5 400 230 0.1 ± 0.420 -3 1729± 132 0.4±0.5 260±94 0±0en . ?M , -,- ,

W 100 5 DAYS Mean Agg.Volues4.6N=1250

80

60

40-0z

_ 20

40 10 DAYS Mean Agg.Volue64_N =125

2060 15 DAYS Mean Agg. Value*2.5100 2 3 250

Cl) 60

cr 40-w

20-

60 -20 DAYS Mean Agg.Value 2.7N =150

40-

20

FARICLE DENSITY [PERCENT SQUARE COUNT(lcm2)OF THE TOTAL MEMBRANE COUNT(25cm2)]

FIG. 3. Particle distribution on the PF of freeze-fractured membranesfrom S. acaule leaf-callus cells acclimated at 2 C for 0, 5, 10, 15, or 20days. The histogram shows the number of membrane areas (I cm2 pho-tograph = 2.23 X 10-2 Am2 membrane) of particular density (4% median)found at different stages of acclimation. The corresponding visual aggre-gation value and the total number of 1-cm squares counted (N) for eachacclimation period is indicated in the histograms.

hardened to -9 C after 15 days at 2 C, at 3 C increase in frosthardiness. Again, these results agree with observations of pottedplants (4).Throughout the experiment, mean particle counts on the mem-

FIG. 4. Fracture faces of plasma membranes after 5 days of cold

acclimation at 2 C. The PF of S. acaule (8-week-old cultures) plasmamembrane (A) showed high aggregation of protein particles. Similar

aggregation is not evident in the PF of the S. tuberosum (B) plasma

membrane (also 8-week-old cultures). Bar equals 0.5 ttm.

brane EF were sigiiatydifferent (P = 0.001) from those

observed on membrane PF whether the callus tissues of both

species were grown at 2 or 27 C (Fig. 2). On the average, EF had

about one-fifth of the particles observed on PF. Over-all, the two

species had similar trends in their total particle counts during the

acclimation treatment. After 20 days at 2 C, both species showed

significantly (P = 0.01) reduced (17%) particle counts. However,

after 5 days acclimation, S. tuberosum showed a significant (P =

0.05) initial increase in the total protein particle count (Table I).The main objective of the particle counting was to determine

the time course of particle aggregation after the beginigof

acclimation treatment. The histograms (Fig. 3) show that mem-

branes of S. acaule callus cultures treated at 2 C for 5 days or less,

or 15 days or more, had most of their particles in the mean densityclass (41%). However, as early as 5 days after transferring to 2 C,

the particles had begun to aggregate (Fig. 4). This is shown in the

histograms (Fig. 3) by a decrease in the median peak and the

appearance of some areas where the particle density was less or

greater than observed in the control. Maximum aggregation oc-

curred at the 10-day acclimation period for S. acaule (10-week-old callus), after which the particles started redistributing around

the mean 4% value. Maximum hardiness could be achieved in

about 15 days in S. acauke by acclimating at 2 C (4).

When the standard picture scale was used to analyze the prints,similar results were obtained (Table I). The simple correlation

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PLASMA MEMBRANE ALTERATIONS IN POTATO

CLi

J

w0.(I)

-J

4a.z

fr-0

a.

IJ-J

4-i

z0I-4J

LI)

41

0 5 10 0 5 10 15 20

DAYS AT 2° C

FIG. 5. Effect of acclimation at 2 C on the number (bar graph) andaggregation (line graph) of IMP on membrane PF of S. acaule leaf-callustissues at the age of 4 and 10 weeks.

I20C

w

I--

4t

w

-Th;10d

-Th;15d-Tq

-TI ;10d

- TI;15d

FIG. 6. Schematic presentation of the influence on membrane phase-transition behavior of a shift to lower temperatures in S. acaule membranemelting curve after 15 days of acclimation, as indicated by intramembran-ous protein particle aggregation patterns. The high end of melting range

(Th) and the low end of the melting range (T,) indicate the hypotheticalhigh and low ends of the melting range for melting curves after 10 and 15days of acclimation. The quenching temperature (Tq) is constant 2 C.

coefficient (r) for the standard deviation ofthe normalized particlecounts on PF and the visual aggregation value for the same

membrane face was 0.85. However, the grid-counting method

FIG. 7. Plasma membrane (PM) PF of S. acaule callus tissue after 5days acclimation at 2 C. Note the linear structures on the membranefracture face. C, cytoplasma. Bar equals 0.5 ,um.

using a 1-cm square unit did not describe accurately the particleaggregation on membrane EF due to the low total particle countson that membrane face (r = 0.59). Thus, the visual aggregationvalue better described the aggregation situation on membrane EF.The major discovery of our experiment was the sudden drop in

the aggregation value of S. acaule membranes after 10 daysacclimation. IMP throughout the entire experiment showed verylittle or no aggregation on the membrane faces of S. tuberosum(Table I).The first 10 days acclimation were repeated for S. acaule

cultures at the age of 4 weeks. The two age groups respondeddifferently under similar acclimation conditions. Although theyounger cultures had an average of 2,636 IMP/tim2, the oldercultures showed 2,832 particles (7% more) (Fig. 5). Both agegroups showed a decreasing trend in total protein particle countsduring acclimation. However, in the younger cultures, the IMPaggregation peak occurred at an earlier stage of acclimation thanin the older cultures. Maximum aggregation was achieved in the4-week-old callus cultures after 5 days acclimation, whereas the10-week-old cultures showed more pronounced aggregation peakafter 10 days cold acclimation. Both membranes showed lessparticle aggregation beyond the peak, implying that membranefluidity was regained in both culture types.

DISCUSSION

The aggregation of IMP as the membrane is cooled towards itsphase transition temperature is well documented for several or-ganisms (1, 7, 10). However, no visual observations of particleaggregation exist during acclimation treatment in plants. Evidencefrom electron spin resonance (28) and fluorescent probe (21)studies indicates that cool climate herbaceous plants may havetemperature-dependent mechanisms which allow alteration of thetemperature at which membrane solidification begins. This maybe achieved either by accumulating polyunsaturated fatty acidswith lower phase transition temperatures (7) and/or by increasingthe plant sterol content that has an ordering influence on thephase properties of phospholipid bilayers (17).The drastic drop in the aggregation value of S. acaule mem-

branes that was observed after 15 days acclimation could possiblybe explained by a decrease in phase transition temperature ofthose membranes. Figure 6 is a diagrammatic presentation ofhypothetical melting curves for the membrane lipids from 10- and15-day-acclimated S. acaule callus. The high and low ends of themelting range for each curve are indicated by temperatures Th andTi. The quenching temperature (T) was the acclimation temper-ature (2 C) in our experiment and it was kept constant throughoutthe study. Because the phase transition occurs over a range intemperature, quenching the 10-day acclimated tissues from 2 C

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TOIVIO-KINNUCAN ET AL.

can produce fracture faces in which 80% of the membrane is inthe solid state, whereas, if the same membrane is quenched afterthe melting curve has been lowered, that is after 15 days ofacclimation, it is possible that only 25% of the membrane lipidsare in the solid state. Thus, a decrease in the IMP aggregation isproduced. The biochemical nature of such a decrease in theplasma membrane phase transition temperature for S. acaule isunder investigation.

Microscopy was also carried out with a control that was frozenwithout the cryoprotectant glycerol to evaluate the real nature ofthe particle aggregation patterns during the acclimation period.Glycerol applied before tissue fixation has been known to induceartificial particle aggregation (5). In our experiment, the mem-branes fractured without glycerol pretreatment turned out to bevery rough (icy) and it was hard to count particles on them.However, similar aggregation patterns could be distinguished insamples treated with and without glycerol and, thus, the reportedparticle aggregation patterns should be real.

Often the S. acaule cells that showed the most particle aggre-gation were somewhat plasmolyzed. A space, filled with ice, wasobserved to separate the plasma membrane from the cell wall.Such a plasmolysis could result as a consequence of an increasedwater permeability in the plasma membrane during phase transi-tion. [There have been several studies published showing largeincreases in passive permeability in membranes within their phasetransition; see, for example, the article by Murata and Fork (19)and references cited therein.] Similar plasmolysis was not observedin the replicas of S. tuberosum which did not show any particleaggregation either.

Partial lipid crystallization during the early stage of acclimationprocess may turn out to be a necessary step of a successfulacclimation. The excess water needs to be removed from the cellsvia the plasma membrane during freezing in order to avoid lethalintracellular freezing. Some water may escape through the inter-facial regions between the solid and liquid areas of the membraneat low temperature (2 C). However, if the lipid phase transition isallowed to proceed too far, physiologically significant portions ofthe plasma membrane may become solidified and nonfunctional(14).The acclimation temperature used in our experiment is appar-

ently above the lower end of the melting range for S. acaule. ForS. tuberosum the phase transition temperature has been deter-mined to be 3 C by electron-spin resonance (6). However, studiesconducted by Fey et al. (6) and Pike and Berry (21) indicate thatphase transition temperatures for cool-climate herbaceous plantsmay well lie below 0 C. It is possible that at the temperaturesemployed in our experiment, acclimation may still proceed nor-

mally in S. acaule and the cells adapt to cooling temperatures byaltering their lipid composition. Evidence from studies with a

thermotolerant clone of Tetrahymenapyryformis has indicated thatmembrane fluidity is self-regulating. Therefore, the action of themembrane-bound phospholipid desaturases (22) can be modu-lated by the physical state of their membrane environment (15).The partially solidified membranes in S. acaule may signal themembrane proteins to modify their lipid environment, that is, thefluidity of the membrane which they inhabit. This happens as a

response to the cool acclimation temperature. How the initialsignal is modulated into a membrane fluidity change is not known,although it has been suggested that the action ofgrowth hormonesmay also be mediated through membrane fluidity. For example,IAA decreases the thickness of membranes, which has been takenas an indication of an increased membrane fluidity (18). Thiscould explain why the younger S. acaule callus, which still hasmost of its residue auxin reserve left in the medium, is able to

adjust its membrane fluidity to acclimation temperature fasterthan older cultures. Also, lipid composition (11) may vary accord-ing to the physiological age of the tissue affecting their membrane

fluidity characteristics.Organized rows of unknown material were observed on the PF

of the plasma membrane during the early stages of S. acauleacclimation (Fig. 7). The orientation parallels that of the wallmicrofibrils and a microfibril synthetic function by this materialis possible. They might represent a linear enzyme complex formicrofibril biosynthesis and orientation as proposed by Robeneckand Peveling (23). These linear structures could be associated witha membrane response to early stages of acclimation and beidentified as stress fibers. However, there is no evidence availableof their existence in plants.We have shown that, in the tuber-bearing potato species S.

acaule, cold acclimation at 2 C for 10 days causes a partial phasetransition as indicated by IMP aggregation. This is believed tofacilitate the movement of cellular water into the extracellularspace and to trigger operation of a self-regulating membranefluidity restoration mechanism. The protecting role of such mem-brane alterations before or during the actual freeze-thaw stressremains to be discovered.

Acknowledgments-We wish to thank Henry W. Kinnucan for his statistical help.The freeze-fracture electron microscopy was carried out at the Electron MicroscopeLaboratory of Biology, University of Minnesota, St. Paul. We are grateful to SharonRobinson and Prof. W. Cunningham for making these facilities available and wishto thank them for their interest and help during the study.

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