Abstract. The ¯uidity of chloroplast thylakoid mem-branes of frost-tolerant and frost-sensitive needlesof three- to four-year-old Scots pine (Pinus sylvestrisL.) trees, of liposomes produced from the lipids of thethylakoids of these needles, and of liposomes containingvarying amounts of light-harvesting complex (LHC) IIprotein was investigated by means of electron paramag-netic resonance (EPR) measurements using spin-labelledfatty acids as probes. Broadening of the EPR-resonancesignals of 16-doxyl stearic acid in chloroplast mem-branes of frost-sensitive needles and changes in theamplitudes of the peaks were observed upon a decreasein temperature from +30 °C to )10 °C, indicating adrastic loss in rotational mobility. The lipid molecules ofthe thylakoid membranes of frost-tolerant needlesexhibited greater mobility. Moderate frost resistancecould be induced in Scots pine needles by short-daytreatment (Vogg et al., 1997, Planta, this issue), andgrowth of the trees under short-day illumination (9 h)resulted in a higher mobility of the chloroplast mem-brane lipids than did growth under long-day conditions(16 h). The EPR spectrum of thylakoids from frost-tolerant needles at )10 °C was typical of a spin label inhighly ¯uid surroundings. However, an additional peakin the low-®eld range appeared in the subzero temper-ature range for the chloroplast membranes of frost-sensitive needles, which represents spin-label moleculesin a motionally restricted surrounding. The EPR spectraof thylakoids and of liposomes of thylakoid lipids fromfrost-hardy needles were identical at +30 °C and)10 °C. The corresponding spectra from frost-sensitiveplants revealed an additional peak for the thylakoids,but not for the pure liposomes. Hence, the domains withrestricted mobility could be attributed to protein-lipidinteractions in the membranes. Broadening of the

spectrum and the appearance of an additional peakwas observed with liposomes of pure distearoylphosphatidyl glycerol modi®ed to contain increasingamounts of LHC II. These results are discussed withrespect to a loss of chlorophyll and chlorophyll-bindingproteins in thylakoids of Scots pine needles under winterconditions.

Key words: Electron paramagnetic resonance ± Frosthardening ± Membrane ¯uidity ± Photoperiod ± Scotspine ± Thylakoid

Introduction

Freezing of a plant tissue subjects the cells to a severedehydration stress. Assuming that loss of compartmen-tation within the cell leads to irreversible destruction ofthe cell, the cellular membranes are the primary sites ofpotential injury in this regard (Heber and Santarius1964; Steponkus et al. 1993). Cellular membranesundergo several changes upon adaption to subfreezingtemperatures during the frost-hardening period toensure that their functionality is sustained under coldconditions (for a review, see Sakai and Larcher 1987). Asigni®cant increase in the relative proportion of lipids aswell as in the degree of desaturation of the fatty acids ofthe lipids has been observed in the thylakoid membranesof conifer chloroplasts in this regard (OÈ quist 1982;Senser and Beck 1982a). Such changes indicate anincrease in membrane ¯uidity and, as a consequence, inmembrane stability at low temperatures in particular.However, direct evidence that the photosynthetic mem-branes of cold-adapted leaves are less viscous than thoseof frost-sensitive leaves is still lacking.

In this study we describe how changes in thecomposition of the photosynthetic apparatus in¯uencethe ¯uidity of the thylakoid membranes. It is demon-strated that a seasonal reduction in the relative contentof intrinsic membrane proteins, as described by Vogg

Planta (1998) 204: 201±206

Frost hardening and photosynthetic performanceof Scots pine (Pinus sylvestris L.). II. Seasonal changesin the ¯uidity of thylakoid membranes

G. Vogg3, R. Heim1, B. Gotschy2, E. Beck1, J. Hansen1

1Lehrstuhl fuÈ r P¯anzenphysiologie, UniversitaÈ t Bayreuth, UniversitaÈ tsstr. 30, D-95440 Bayreuth, Germany2Lehrstuhl fuÈ r Experimentalphysik II, UniversitaÈ t Bayreuth, UniversitaÈ tsstr. 30, D-95440 Bayreuth, Germany3GSF-Forschungszentrum fuÈ r Umwelt und Gesundheit, Institut fuÈ r Biochemische P¯anzenpathologie, D-85764 Oberschleiûheim, Germany

Received: 3 March 1997 /Accepted: 16 July 1997

Abbreviations: EPR=electron paramagnetic resonance; LHC =light-harvesting complex

Correspondence to: J. Hansen; E-mail: jens.hansen@uni-bay-teuth.de; Fax: 49 (921) 55 26 42

et al. (1997), is necessary to maintain the degree ofmembrane ¯uidity indispensable for survival at lowtemperatures. Membrane ¯uidity is a complex phenom-enon which is usually determined according to thedegree of mobility of a probe inserted into the lipidbilayer. In the experiments reported below we used aspin-labelled fatty acid as a probe, the mobility of whichin isolated thylakoid membranes could be investigatedby electron paramagnetic resonance (EPR) spectrosco-py. The EPR measurements with various spin labelsdi�ering in the position of the nitroxide probe along thefatty acyl chain have shown the lipid bilayer to consist ofa relatively mobility-restricted polar head region and amore mobile lipophilic region (Bigelow et al. 1986). Thenitroxide group of 16-doxyl stearic acid is situated closeto the terminal methyl group of the fatty acyl chain: theEPR spectra of membranes containing this labelledprobe thus illustrate the rotational mobility of thelipophilic acyl chains of the membrane lipids in thecenter of the bilayer. Such EPR spectroscopy wasapplied to examine seasonal changes in the ¯uidity ofthylakoid membranes of Scots pine needles with respectto membrane stability under winter conditions.

Materials and methods

Plant material. One-year-old needles of three- to four-year-oldScots pine trees (Pinus sylvestris L. ) were used in the experiments.The conditions of their growth under natural and arti®cial climateshave been described in detail by Vogg et al. (1997).

Isolation of thylakoids and extraction of membrane lipids. Thyla-koid membranes from Scots pine needles were isolated as describedin Vogg et al. (1997).

Membrane lipids were extracted from isolated thylakoidsaccording to Bligh and Dyer (1959) with the solvent chloro-form:methanol:water (1:2:0.8, by vol.). The extraction procedurewas repeated three times. The organic phases containing the lipidsand lipophilic pigments were combined and the solvent wasevaporated in a rotary evaporator. The samples were dissolved ina small volume of chloroform and used for liposome formation.

Preparation and spin labelling of liposomes. Liposomes wereproduced from distearoyl phosphatidyl glycerol (Sigma, Deisenho-fen, Germany) or from membrane lipids from isolated thylakoids.16-Doxyl stearic acid (Sigma) was added to spin-label the mem-brane lipids at a label:lipid-ratio of 1:100 (w/w). The solvent of theliposome preparation was removed under a stream of N2 andsubsequently by evaporation in a Speed Vac (RC 10.10; Jouan,Saint Nazaire, France) for 30 min. Aqueous bu�er (5 mM Hepes,pH 7.5; 50 mM MgCl2) was added to the residue and the resultantsamples were vortexed. The reaction tubes containing the sampleswere ¯ushed with argon and subsequently sonicated in a water bath(model 3200; Branson, Danbury, Conn., USA) for 30 min at 45 °C.

Spin labelling of thylakoid membranes and EPR spectroscopy. The¯uidity of the isolated thylakoid membranes was investigated byEPR spectroscopy after insertion of the labelled probe (16-doxylstearic acid). The spin label was dried in a stream of N2 and theprepared suspension of thylakoid membranes was added, vortexedand subsequently stirred at room temperature for 30 min. The lipidcontent of the membrane preparation was estimated from itschlorophyll content (Senser and Beck 1982a; Chapman et al. 1983;Murphy and Woodrow 1983). After incorporation of the label themembranes were washed twice with aqueous bu�er (5 mM Hepes,

pH 7.5; 50 mM MgCl2). A spin label:lipid ratio of 1:100 (w/w)proved to be optimal with respect to the quality of the signals. Nospin-spin interactions were detected at a ratio 2:100 (w/v). The EPRspectra of the samples were obtained with an EPR-spectrometer(ER 200D-SRC; Bruker, Rheinstetten, Germany) at a constantfrequency of approx. 9.7 GHz. The rotational correlation time sRwas determined as a measure of spin label mobility (Gordon andCurtain 1988). The temperature of the sample holder was adjustedover a range from +30 °C down to )10 °C by the use of standardcryotechnique procedures.

Preparation of light-harvesting complex (LHC). The LHC II fromspinach was enriched according to Krupa at el. (1987). The purityof the samples was examined by recording chlorophyll a ¯u-orescence emission spectra at 77 K and by SDS-PAGE.

Results

Studies with isolated thylakoids. The EPR spectra wererecorded with photosynthetic membranes isolated fromfrost-sensitive or frost-tolerant Scots pine needles withina temperature range from+30 °C to )10 °C. A decreasein temperature from +30 °C to )10 °C resulted in abroadening of the EPR-resonance signals from 16-doxylstearic acid integrated within the chloroplast membranesfrom frost-sensitive needles, and changes in the ampli-tudes of the concomitantly observed peaks (Fig. 1A).These phenomena indicate a drastic loss in rotationalmobility of the spin label. An at least threefold increasein the rotational correlation time as a quantitativemeasure of spin-label mobility was calculated from thespectra for the temperature range from+30 °C to )5 °C(Fig. 1B). The use of 5-doxyl stearic acid as a spin label,

Fig. 1. A, Temperature dependence of EPR signals of 16-doxylstearic acid in thylakoid membranes from one-year-old Scots pineneedles. The needle sample was taken in September while still in thefrost-sensitive state. B, Temperature dependence of the rotationalcorrelation time sR calculated from the spectra shown in A

202 G. Vogg et al.: Frost hardening and photosynthetic performance of Scots pine

the nitroxide group of which is close to the hydrophilichead group of the fatty acid molecule, indicatedrestricted mobility throughout the whole temperaturerange investigated (data not shown). Hence, the e�ect oflow temperature on the rotational mobility of the spinlabel could be traced to the lipophilic center of the lipidbilayer.

The mobility of 16-doxyl stearic acid in chloroplastmembranes of Scots pine needles showed considerabledi�erences between chloroplasts isolated from frost-hardy and frost-sensitive needles (Fig. 2). The membranelipid molecules of thylakoids of frost-tolerant winterneedles exhibited a higher mobility than did those ofneedles in the frost-sensitive summer state. This ®ndingwas in line with the e�ect of temperature on the mobilityof the spin label at two temperatures +30 °C and 0 °C.Chloroplast membranes from frost-hardy plants showedonly a small di�erence in spin label mobility at +30 °Cand 0 °C, whereas the same temperature span had amuch greater e�ect on thylakoids from less frost-tolerantplants. Since some frost resistance could be induced byshort-day treatment of the plants (Vogg et al. 1997),growth under a daily illumination period of 9 h resultedin a higher mobility (lower sR) of the chloroplastmembrane lipids than growth under long-day conditions(Fig. 2). During spring the loss of frost tolerance of theneedles was accompanied by a substantial decrease in themobility of the membrane lipids. Thus, the EPR spectrarevealed an evident dependence of thylakoid membrane¯uidity on the degree of frost hardiness of the needles.The EPR spectrum of thylakoids of frost-hardy needlesrecorded at )10 °C was typical of a spin label in highly¯uid surroundings (Fig. 3, trace A), whereas the chlo-roplast membranes of frost-sensitive needles showed anadditional resonance peak in the low-®eld range (Fig. 3,trace B, arrow). As shown in Fig. 1A, this additionalpeak developed gradually in the subzero temperaturerange of the measurement. Spectra subtraction resulted

in a di�erential spectrum typical of spin-label moleculesof strongly restricted mobility (Fig. 3, trace B)A). Thus,the low-temperature spectrum of chloroplast membranesfrom frost-sensitive needles may be composed of at leasttwo components representing spin-label molecules withdi�erent mobilities. This leads to the interpretation thatthe membrane is not homogeneous at subzero temper-atures but contains areas of high viscosity.

Studies with liposomes. Liposomes were produced fromthe lipids extracted from the chloroplast membranes offrost-sensitive and frost-tolerant needles (Fig. 4). Where-as the EPR spectra of the thylakoids and of theliposomes from frost-hardy plants were identical at+30 °C and )10 °C, the corresponding spectra fromfrost-sensitive plants obtained at )10 °C showed asubstantial di�erence from those obtained at +30 °C:the additional peak in the low-®eld range mentionedabove was only seen with the thylakoids and not withthe liposomes. Since the spectrum of the liposomes at)10 °C was identical to the spectra obtained withmembranes and liposomes of the frost-tolerant plants,the additional peak in the membranes of frost-sensitiveplants must be due to an e�ect of the membraneproteins. Hence, the domains with restricted mobilityshould be attributable to protein-lipid interactionswithin the membranes.

The idea of protein-triggered formation of lipiddomains with restricted mobility was con®rmed by EPRmeasurements with liposomes consisting of distearoyl

Fig. 2. Seasonal course of rotational correlation time sR of 16-doxylstearic acid in thylakoid membranes of one-year-old needles of Scotspine. The trees were grown under di�erent photoperiods [short days(9 h light/day), h, 0 °C; s, +30 °C; long days (16 h light/day):n, 0 °C; ·, +30 °C]. The two vertical lines indicate the period of timeof extreme frost hardiness of the needles Fig. 3. Electron paramagnetic resonance spectra of 16-doxyl stearic

acid in chloroplast membranes of one-year-old needles of Scots pinetrees recorded at )10 °C. Trace A, chloroplast membranes from frost-tolerant needles [lethal temperature for 50% of cells (LT50)<)42 °C].Trace B, chloroplast membranes from frost-sensitive needles (LT50 �)10 °C). Trace B±A, di�erential spectrum ofB and A (solid line) and acalculated EPR spectrum of a spin label with completely restrictedmobility (dotted line) from Gri�th and Jost (1976). Total scan widthwas 100 G

G. Vogg et al.: Frost hardening and photosynthetic performance of Scots pine 203

phosphatidyl glycerol as the lipid and LHC II protein asthe protein component. The EPR spectra produced withsuch liposomes were observed at various lipid/proteinratios. The EPR spectrum of 16-doxyl stearic acid inpure phospholipid liposomes revealed three distinctresonance peaks, as is typical of spin-labelled moleculesin isotropic surroundings (Fig. 5). An additional reso-nance signal in the low-®eld range could not be seen.Broadening of the spectrum at )10 °C occurred whenLHC II protein (from spinach thylakoids) had beenincorporated into the liposomes at a lipid/protein ratioof 1:5. The additional peak was still recognizable even at+30 °C when liposomes with a lipid/protein ratio of1:10 were investigated, and this peak became verypronounced at )10 °C. The EPR spectrum of theliposome preparation was very similar to that of

chloroplast membranes from frost-sensitive needles(Figs. 4, 5).

Discussion

Electron paramagnetic resonance spectroscopy has beenwidely used for the quantitative assessment of therotational mobility of lipid molecules as a measure for¯uidity of cellular membranes (see Schreier et al. 1978;Devaux 1983; Campbell and Dwek 1984 for reviews). Inthe present paper we utilised 16-doxyl stearic acid forspin labelling, a fatty acid which possesses the spin labelat a position near the center of the lipophilic layer ofthe chloroplastic membranes. The EPR spectra of thephotosynthetic membranes of frost-sensitive Scots pine

Fig. 4. Comparison of the EPRspectra of 16-doxyl stearic acid inchloroplast membranes and inpure liposomes of the lipids fromone-year-old needles of frost-sen-sitive and frost-tolerant Scotspine plants. After extraction ofthe lipids from the chloroplastmembranes, liposomes were pro-duced with 5 mM Hepes bu�er(pH 7.5, containing 50 mMMgCl2). Spectra were recorded at+30 °C and )10 °C. An addi-tional peak is indicated by thearrow

Fig. 5. The EPR-spectra of16-doxyl stearic acid doped intophosphatidyl glycerol liposomescontaining di�erent amounts ofthe LHC II-complex from spin-ach as measured at +30 °C and)10 °C. The appearance of anadditional line in the EPR spectrais indicated by an arrow

204 G. Vogg et al.: Frost hardening and photosynthetic performance of Scots pine

needles exhibit a gradual broadening of the signalconcomitant with an increase in rotational correlationtime upon cooling of the membranes, thus indicatingdecreasing membrane ¯uidity (Fig. 1). The observedgradual e�ects do not imply an abrupt thermotropicchange in the lipid hydrocarbon phase. Since fatty acylchains and polar head groups are quite heterogeneous innative membranes, a sharp liquid-crystalline to gel phasetransition cannot be expected (Quinn 1981).

Chloroplastic membranes of needles in the frost-hardened state yield a homogeneous EPR spectrumindicative of a highly ¯uid membrane throughout thewhole of the examined temperature range from +30 °Cto )10 °C (Fig. 3, trace A). The EPR spectrum of spin-labelled chloroplastic membranes from frost-sensitiveneedles measured at )10 °C shows a hybrid spectrumresulting from spin-label incorporated into lipid envi-ronments of di�erent mobilities (Fig. 3, trace B). Onecomponent in the central range of the spectrum corre-sponds to spin-labelled molecule of high mobility. Asecond minor component represents spin label caught ina domain of restricted mobility (Fig. 3, trace B)A). Theoccurrence of bimodal EPR spectra has been attributedto interactions between integral membrane proteins andthe lipid molecules (Li et al. 1989, 1990). The spectrallybroadened EPR signal is presumed to re¯ect a fractionof motionally hindered lipid molecules directly adjacentto the protein, the so-called ``boundary layer lipids''(Jost et al. 1973). The boundary lipids are understood tobe molecules ®xed around an intrinsic protein. Theyparticipate, however, at a reduced and strongly temper-ature-dependent rate in the exchange movements of thebulk lipid molecules (Ryba et al. 1987; Horva th et al.1988). Our experiments with protein-free and LHC II-containing liposomes corroborate the idea of proteinsforming nuclei for domains of restricted mobility. Ourresults indicate that intrinsic protein-lipid interactionsmay be of crucial importance for obtaining a certaindegree of frost hardiness. The proportion of immobilelipid domains in the photosynthetic membranes of frost-tolerant plants ± if at all present ± was smaller than thethreshold for detection by EPR. Thus, the lipid boun-dary around the proteins of these membranes should notbe very wide and therefore should not appreciably a�ectthe ¯uidity of the membranes.

A small e�ect of the boundary layer on membrane¯uidity could, in principle, be due to a higher motility ofthe fatty acid residues created by a higher degree ofdesaturation (OÈ quist 1982; Senser and Beck 1982a), or toa signi®cantly decreased protein content of the mem-branes. Analyses of the fatty acid composition of thephotosynthetic membranes of Scots pine showed a slightincrease in the proportion of unsaturated fatty acids inthe frost-tolerant plants (data not shown), but atwofold-higher total chloroplast membrane lipid contentin comparison with frost-sensitive needles (3.4 mg fattyacids per g FW as compared to 1.7 mg). Accordingly, anincrease in the lipid/protein ratio has been found inmicrosomal fractions of frost-hardened Scots pine nee-dles (data not shown) and in thylakoid membranes ofcold-grown pea plants (Chapman et al. 1983). The

motility of the membrane lipids and therefore the ¯uidityof the membrane is increased with increasing ratios oflipids to proteins. This is consistent with the idea that thestoichiometry of motionally restricted lipid moleculesper molecule of protein is constant over a wide range oflipid/protein ratios (Ryba et al. 1987). Hence, a selectivedecrease in the protein content of membranes results in adecrease in the proportion of motionally hindered lipidmolecules.

However, as indicated above, protein-lipid interac-tions are considerably a�ected by temperature, as alowering of the temperature intensi®es the in¯uence ofintegral membrane proteins on the boundary lipid layer.In other words, decreasing temperature increases thediameter of the boundary lipid layer. However, acomplete immobilisation of boundary lipids by pro-tein-lipid interactions has not been found even in veryfrost-sensitive plants (Li et al. 1989).

The idea of intrinsic proteins a�ecting the biophysicalproperties of membranes at low temperatures is sum-marised in Fig. 6. An e�ect of intrinsic proteins on theboundary lipids exists even at moderate temperatures,and is enhanced at low temperatures when largedomains of rigid membrane lipids develop. Thesemembrane areas have been identi®ed as the primarysites of membrane injury, since an elastic response of themembrane to mechanical and osmotic stress is therebyconsiderably diminished (Steponkus et al. 1993).

Vogg et al. (1997) have shown the in¯uence of thephotoperiod on the induction of the frost hardening ordehardening process. In the present paper the length ofthe photoperiod was also identi®ed as one majorexogenous factor a�ecting the ¯uidity of the lipid phase:chloroplasts of Scots pine trees under short-day condi-tions revealed a higher membrane ¯uidity than thosefrom trees under long days (Fig. 2). Since the observed

Fig. 6A,B. Schematic illustration of membrane alterations duringfrost hardening. Top view of membrane surfaces of frost-sensitive (A)and frost-resistant (B) cells. Membrane lipids ( ) surroundingintegral membrane proteins (P) are motionally restricted by protein-lipid interactions. Cooling enhances the protein-lipid interactions dueto a delayed di�usional exchange of boundary lipid molecules withbulk lipid molecules ( ). A, Upon cooling of non-frost-hardenedmembranes, lipids are restricted in their mobility due to protein-lipidinteractions constituting rigid domains ( ). These appear to be theprimary site of membrane injury at low temperatures. B, Large areasof bulk lipids with high mobility prevail due to a decreased proteincontent resulting in a low protein-lipid ratio of frost-hardenedmembranes

G. Vogg et al.: Frost hardening and photosynthetic performance of Scots pine 205

increase in membrane ¯uidity corresponds to a decreasein chlorophyll-binding protein under short-day treat-ment (Vogg et al. 1997), frost hardening should beaccompanied or even caused by a signi®cant increase inthe lipid/protein ratio of the photosynthetic membranes.This interpretation is in accordance with observations onfreeze-fracture electron micrographs of spinach thy-lakoids, whereby a decrease in the number of proteinparticles relative to the membrane area upon frostacclimation was noted (Garber and Steponkus 1976). Anaugmentation of the fraction of phospholipids of chlo-roplasts from spruce needles has also been observedunder short days (Senser and Beck 1982b): this similarlyindicates the attainment of frost resistance resultingfrom an increase in the lipid/protein ratio of thethylakoids.

This study was supported by EUROSILVA-EUREKA No. 447(grant No. BEO 51 OEF 1201 8). The linguistic help of Dr. PaulZiegler (UniversitaÈ t Bayreuth, Germany) is gratefully acknowl-edged.

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