Plant PhysioL (1979) 64, 924-9290032-0889/79/64/0924/06/$00.50/0

Glycerolipid and Fatty Acid Changes in Eastern White PineChloroplast Lameliae during the Onset of Winterl

Received for publication June 30, 1978 and in revised form July 4, 1979

DAVID R. DEYOEForest Research Laboratory, School of Forestry, Oregon State University, Corvallis, Oregon 97331

GREGORY N. BROWNSchool of Forestry, University ofMinnesota, St. Paul, Minnesota 55455

ABSTRACT

Chloroplast lamellae of eastern white pine (Pinns m L) wereanalyzed to determine chnges In total glycerolpkids, component glycero-lipids, and glycerolipid fatty acids duing the onset of winter hardinss.Sampies were colected In September, November, and December when theaverage daily temperature varied between 23 and -10 C. Before November2, phosPhobPids decreased 40 to 85%, glycolpids only 30%. Analysis ofindivdual glycerolpids showed that glycerolipids containing 1&3 fatty acidwere retained at the expense of glyceroipids esterified with saturated (16:0 and 1&0) and monounsaturated (1&1) fatty acids.

Between mid-November and December, the total quantity of lamelarglycerolpib recovered to the September level. Increases in digalactosyldiglyceride and in 1&3 characterized the recovery period. High lamellarunsaturation achieved by mid-November appeared to be maintained duringrecovery thong preferentil incorporation of glycerolipids containg &3 (monogalactosyl d4gyceride, digalactosyl diglyceride, pbosphatidylglyc-erol, and phpatidyline).

These results suggest that eastern white pine chloroplasts maintainlaear vicosity by inceasing lamellar unsaturation and tolerate freezedesiccation by increasing the interfacial water-bhding capacity of thelamenae.

Recent attempts to explain plant or tissue tolerance to stressesimposed by subfreezing winter temperatures have focused onanalyzing membrane structural components (5, 18). The rationaleis that cell survival is a function of the integrity or fluidity of thelimiting membrane. Membrane fluidity, governed by the lipid andprotein complement of the membrane, is directly related to tem-perature (4). Temperature reduction initiates a physical change ofstate in the hydrophobic matrix of the membrane (temperature-specific for each glycerolipid-fatty acid type) and produces asemicrystalline to crystalline phase which increases the suscepti-bility of the membrane to stress (4, 16). Tissues of woody peren-nials can withstand winter freezing, although the same tissue isdamaged if exposed to freezing during the growing season (2).This suggests that adjustments in membrane composition accountfor maintenance ofmembrane integrity during the onset ofwinter.Previous investigators have used total tissue samples or crudemembrane preparations for examining hardening-inducedchanges in membrane lipids (5, 14, 18). Evaluation of a single,

I This study was completed at the Missouri Agricultural -ExperimentStation, Journal Series 8170. This is paper 1,340 from the Forestry Re-search Laboratory, School of Forestry, Oregon State University, Corvallis,Oregon 9733 1.

purified membrane system should make possible more reliableinterpretation of the role of composition adjustment in membraneintegrity.The most defined single membrane system in plants is the

chloroplast lamellae (1), and lamellar responses to winter harden-ing may be evaluated in coniferous needle tissue known to survivewinter stress (2). Although the plasma membrane is the logicaltarget, its purity in isolation has not been adequately demon-strated, and its functional characteristics are not well known. Weanalyzed chloroplast lamellae glycerolipids and their contingentfatty acids from tissue of eastern white pine needles collectedduring the onset of winter. The objective was to identify thecompositional adjustments believed to be critical to retention ofmembrane structure and function of conifers.

MATERIALS AND METHODS

We obtained needle tissue between September and December1976 from a 25-year-old eastern white pine (Pinus strobus L.)growing on the University of Missouri campus. All collectionswere made before sunrise. Maximum and minimum temperatureswere recorded daily during the examination period (Fig. 1).

Preparation of Lameilae and Component Glycerolipids. Thirtyg of tissue were homogenized in buffer A containing 0.9 M sucrose,40 mm Tricine (pH 7.2), 1 mM Mg acetate, 10 mM KCL 10 mMGSH, 0.25% BSA (w/v), and 20% PEG4000 (w/v) (7, 20) with aBrinkmann polytron at 10,000 rpm for 20 s. A crude chloroplastpellet was prepared from needle tissue homogenates by the pro-cedure outlined in Figure 2. The chloroplast pellet was resus-pended in buffer B containing 10 mm Tricine (pH 7.2), 50 mmNaCL and 5 mm MgCl2 (8, 12), then incubated and layered on adiscontinuous sucrose gradient containing 5 mM Tricine (pH 7.2)and 3 mM MgCl2 (8). Centrifugation resulted in bands representingstroma and grana lamellae (29). The bands were collected, dilutedin hypotonic buffer, washed, and sonicated in distilled H20 toyield a final pellet of purified chloroplast lamellae (Fig. 2) (7).

Lipids were extracted from the lamellae pellet (Fig. 2) and Chlconcentrations were determined on this extract according to theformula presented by Sestak (26). The organic supernatant frac-tions were combined, concentrated under N2 to a volume of 2 ml,treated with 50 pg/ml butyl hydroxytoluene, and applied to aSephadex LH-20 column (1.8 x 50 cm) (12). Elution performedwith chloroform-methanol (2:1, v/v) yielded two major lipidfractions: F1, containing DGDG,2 MGDG, and PC; and F2,containing PG, PI, and SL. Each fraction was concentrated to a

2Abbreviations: DGDG: digalactsyl diglyceride; MGOD: monogalac-tosyl diglyceride; PC: phosphatidylcholine; PG: phosphatidylglycerol PI:phosphatidylinositol SL: sulfoquinovosyl diglyceride; PE: phosphatidyl-ethanolamine; PS: phosphatidylserine.

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Plant PhysioL VoL 64, 1979 WINTER LIPID CHANGE IN PINE CHLOROPLASTS

0

aid

0--

lmi

a6la

September October I November December

FIG. 1. Maximum and minimum daily temperatures attained September through Dember, 1976. Arrows indicate dates of collection of samples.

PINE NEEDLE TISSUE

NHO3GENIZATION(Buffer A)

FILTRATION(8 layers cheesecloth)

CENTRIFUGATION (6000 xg 10 sin)RESUSPENSION (Buffer B)

CENTRIFUGATION (2000 g 10 sLin)

RESUSPENSION / INCUBArION

(Rypotonic buffer / 20 sin)

SUCROSE DENSITY GRADIENT

x Sucrose ml / tube

Strom lasellae

Gra lealle

DILUTION(3 fold / hypotonic buffer)

RESUSPENSION(In distilled wter)(Polytron sonication:

30 8

42

60

9

8

CENTRIFUGATION (40,000 xg / 30 sin)

CENTRIFUGATION (40,000 xg / 30 sin)

PELLET

LIPID EXTRACTION

RESUSPENSION ( A / E (4:1)CENTRIFUGATION (11,000 xg 5 sin)

RESUSPENSION ( C M (2:1)

CENTRIPUGATION (13,000 / 5 &in)

LIPID ODNTAINING SUPERNATANT

GEL FILTRATION PROTEIN PELLET(Sephadex L.-20 (C/N (2:1)) (Saved for further analysis)

FRACTIONSF1 AND F2

FIG. 2. Flow diagram for isolation of chloroplast lamelle, separation

ofmembrane protein and lipid fractions, and preparation oflipid samples,F1 and F2. Solvent symbols: A, acetone; E, diethyl ether, C, chloroform;and M, methanoL

volume of0.5 ml under N2. Lipid fractions were stored in air-tightvials at -20 C in the presence of 50 pg/ml butyl hydroxytolueneand N2-

AnalYSIS of Glyceolp The analysis of glycerolipids in F1and F2 factions were performed on thin layer plates coated with

0.4 mm Silica Gel G. Resolution of individual glycerolipids withTLC was not complete until samples were separated on SephadexLH-20. Plates were activated at 100 C for 1 h and stored in adesiccator until analysis. Chloroform-methanol-NH4OH (65:33:4,v/v) or chloroform-methanol-acetic acid-water (170:75:25:6, v/v)was used for separation of lipids in the two fractions (7). Allsolvents were first distilled, and solvent mixtures were freshlyprpared for each separation. Lipid standards consisted ofMGDGand DGDG (Applied Science), and PC, PE, PG, PS, and PI(Sigma Chemical Co.). Membrane glycerolipids were quantita-tively determined according to methods described by DeYoe andBrown (7). Glycerolipid samples for quantitative glycerolipidanalysis or fatty acid analysis were first separated by TLC, theneluted from the absorbent by successive washes in chloroform,chloroform-methanol (2:1, v/v), and chloroform-methanol (1:1,v/v). Eluates were reduced to dryness under N2 before the anal-yses. We quantified glycerolipids spectrophotometrically usingstandard assays (7).

Analysis of Fatty Acids. Fatty acids from individual phospho-lipids and glycolipids were analyzed on a Bendix gas chromato-graph equipped with a flame ionization detector. Individual glyc-erolipids recovered from TLC plates were saponified in 15%methanolic KOH (w/v) (10). The resulting free fatty acids were

selectively partitioned into acidified hexane and stored at -20 C.Before GLC analysis, the fatty acids were preferentially removedfrom hexane and were methylated with trimethyl (aaa-trifluoro-m-tolyl) ammonium hydroxide (10). Fatty acids were separatedon 15% HI-EFF-IBP, 100/200 mesh Chromsorb W (acid wash)column (2.5 m x 4 mm, id.). The operating parameters for GLCanalysis were: column temperature, 155 C; injector temperature,260 C; detector temperature, 245 C; and flow rate of N2 carriergas, 20 cm/s. Standards for qualitative identification of endoge-nous fatty acids were: palmitic (16:0), palmitoleic (16:1), stearic(18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), 1 l-eicosenoic

(20:1), 11,14-eicosadienoic (20.2), 3,1l,14-eicosatrienoic (20:3),and arachidonic (20:4). Fatty acid quantity was determined byequations given by DeYoe and Brown (7).

RESULTS

The glycerolipid composition of eastern white pine chloroplastlamellae is shown in Figure 3. Three collections were made in

925

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DEYOE AND BROWN

1507

z

.r_

0

I-J

250

200

150

100

50

0C 2 8 11 H

TOTAL

KI

KK

KKK

3011li 11 i. i m K

C 2 8 11 H C 2 8 11 H C 2 8 11 H C 2 8 11 H C 2 8 11 H C 2 8 11 H

MGDG DGDG SL PG PC PiFIG. 3. Quantitative (umol lipid/mg Chi) representation of total (inset) and component glycerolipids of eastern white pine chloroplast lamellae in

response to onset of winter. Collection date symbols: C, control (September); H, hardened (December); and 2, 8, and 11, dormant (November) samplescollected on the dates indicated. Lipid abbreviations coincide with those listed in text.

each of the months September, November, and December. Noquantitative difference in UM lipid/mg Chl of total or individualglycerolipids existed among the three September samples or thethree December samples. Because quantity varied considerablyamong samples taken on November 2, 8, and 11, we evaluatedNovember collections individually for correlation between a rapidadjustment in lamellar glycerolipids and a prolonged period (17days) of freeze-thaw stress (Figs. 1 and 3).Chl concentrations in mg Chl/g fresh weight of needle tissue

for the samplings (Fig. 1) are: September 14, 1.17; September 17,1.14; September 22, 1.12; November 2, 1.14; November 8, 1.04;November 11, 1.06; December 5, 1.15; December 7, 1.04; andDecember 12, 1.10. These values express glycerolipid levels interms of lamelar Chl concentrations. It is apparent that Chlconcentrations of eastern white pine do not fluctuate significantly(less than 7% from the mean) during the onset ofwinter. Therefore,rapid mid-November to December increases in lamellar glycero-lipids appear to be real and not a reflection of Chl loss. Chlconcentrations here represent "functional" Chl within the lamel-lae. Because previous studies include both lamellar and free Chlin their analyses, compansons are not practical, even thoughwinter losses appear to be slight (11).

Lipid Composition of White Pine Lamellae. Component glyc-erolipids were MGDG, DGDG, SL, PG, PC, and PI (Fig. 3).More than 75% of all glycerolipids in each of the collections wereassociated with the glycolipid group. The quantitative order ofglycerolipids (DGDG > MGDG > SL > PG > PI) was similarfor each collection, except that PC showed a substantial declineby November and minimal recovery by December (Figs. 3 and 4).The total fatty acid complement and the fatty acid contingent

of each glycerolipid are shown in Figures 5 and 6, respectively.Fatty acids indigenous to the chloroplast lamellae were: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:1, 20:2, and 20:4. However, 16:1, 20:1,20:2, and 20:4 appeared only in the November and Decembercoliections (Fig. 5). Linolenic acid, in all instances, waz more than40% of the fatty acid complement of MGDG and DGDG. The

70

60

50

-oX 40

G). 300

020

10

o

2 8 111rDG

MG

SLPG

PC,pi

C D HCombination

FIG. 4. Representation of percentage of glycerolipids in lamellae dur-ing onset of winter. Collection date symbols: C, control (September); H,hardened (December); and 2, 8, and 11, dormant (November) samplescollected on the dates indicated. Lipid abbreviations coincide with thoselisted in text.

glycerolipids SL, PG, PC, and PI were characterized by highquantities of 16:0, although 18:3 predominated in Novembersamples of PG and in December samples of PC and PG.Changes in Glycerolpids during the Onset of Winter. Chloro-

926 Plant Physiol. Vol. 64, 1979

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WINTER LIPID CHANGE IN PINE CHLOROPLASTS

C D H C D H C D H C D H C D H C D H C D H C D H C D H

16:0 16:1 18:0 18:1 18:2 18:3 20:1 20:2 20:4TOTAL FATTY ACIDS

FIG. 5. Total fatty acid complement during onset of winter. Samplescontaiiningl aliquots from F1 and F2 lipid fractions of chloroplast lamellacwere reduced to dryness under N2, saponified in 15% methanolic KOH,partitioned into acidified hexane, and derivatized with tri-methyl (aaa-trifluoro-m-tolyl) ammonium hydroxide in preparation for GLC analysis.

plast lamellae responded to the onset of winter with substantialqualitative and quantitative changes in component glycerolipids(Fig. 3). The quantity of lamellar glycerolipids decrased 65%between September and November 11 but recovered by mid-December (Fig. 3, inset). The period of decline can be dividedinto two parts based on the environmental factors to which thetree was subjected (Figs. 1 and 4). Before November 2, freezingstress was not severe. Minimum daily temperatures were at or

below 0 C only three times and for no more than 2 consecutivedays (Fig. 1). During this time the total quantity of lamellarglycerolipids decrased 37%. Decrease in individual glycerolipidswas quantitatively unique. Beginning November 3, minimum

daily temperatures were between -3 and -8 C for 17 consecutivedays (Fig. 1). Total glycerolipid quantity declined 26% betweenNovember 2 and 11 (Fig. 3, inset), with both quantity and qualityof individual glycerolipids changing markedly during this periodof freeze-thaw stress (Fig. 3). Chloroplast lamellae from November11 collections contained only the glycerolipids DGDG (63%),MGDG (28%), and SL (9%) and were devoid of all phospholipids,which emphasizes the importance ofDGDG, and to a lesser extentMGDG, in acclimation of the lamellae to winter stress (Fig. 4).Mid-November to December recovery was marked by a pro-

gressive decline in temperature (Fig. 1) and an increase in totalquality of lamellar glycerolipids (Fig. 3, inset). Minimum dailytemperatures, frequently below -5 C, many times dropped below-IOC (Fig. 1). Glycerolipid levels were consistent among thethree December samples, so it appears that the lamellae hadachieved a stable degree of hardiness to winter stress. This issupported by the lack of variation in lamellar composition amongDecember collections even though the temperature was -20 C onDecember 7 and extracellular ice was present. A preferentialinaease in DGDG, 34% above the September leveL characterizedrecovery of individual glycerolipids. The increase appears to haveoccurred at the expense of complete quantitative recovery of SLand the component phospholipids (Fig. 3), which were 35% belowthe September collective value.Changs In Fatty Acids durin the Onset ofWinter. Quantitative

and qualitative changes in lamellar fatty acids were a function ofpre-November decreases in component glycerolipids and subse-quent glycerolipid recovery by early December. Increase in la-mellar 18:2 (11%) and 18:3 (24%) and a concomitant decrease of16:0 (16%), 18:0 (15%), and 18:1 (35%) (Fig. 5) during totalglycerolipid decline (Fig. 3) reflect a preferential retention ofglycerolipids containing 18:2 and 18:3. The quantitative recovery

of glycerolipids between mid-November and December dictateda similar recovery in total lamellar fatty acids. Increases in 16:0,

18:0, 18:1, and 18:2 were small relative to the increase in 18:3,indicating a preference for 18:3 esterification to glycerolipids inwinter (Fig. 5). Besides these adjustments in the major fatty acids,several new fatty acids were observed during hardening. Measur-able quantities of 16:1 and 20:1 appeared in November andessentially maintained their proportions in the lamellac duringrecovery, 20:2 and 20:4 appeared only in December.

Examination of individual glycerolipid fatty acids revealed thatthe five major fatty acids, 16:0, 18:0, 18:1, 18:2, and 18:3, werepresent in each glycerolipid; the fatty acid complement of therepective glycerolipids was quantitatively unique; dunng harden-ing, 16:1, 20:1, 20:2, and 20:4 appeared in one or more, but neverall, of the component glycerolipids; and except for September PGand PC fatty acid samples, 16:0 or 18:3 was the most abundantfatty acid in the lamellar glycerolipids for all collection dates.Between Siptember and November 2, 18:3 content increased

substantially in DGDG, MGDG, and PG (Fig. 6). In DGDG, 18:3 increased from 42 to 63% and in MGDG from 43 to 58%. Thesetwo glycerolipids accounted for 68% of all lamellar glycerolipidsin November 2 samples. Although the largest increase occurred inPG, 12 to 39%, this phospholipid represented only about 10% oftotal lamellar glycerolipids (Fig. 3). The increase of 18:3 in theseglycerolipids appears to be a consequence of a selective decreaseof DGDG, MGDG, and PG molecules containing predominantly16:0, 18:0, 18:1, and 18:2 fatty acids.Recovery ofDGWD, MGDG, and PG between mid-November

and December was characterized by maintenance of high 18:3levels (Fig. 6). In addition, 18:3 content increased dramatically inDecember PC samples, rising to 40% from the 16% of Septemberand November 2 samples. This implies preferential increase andincorporation into chloroplast lamellae of 18:3-containingDGDG, MGDG, PG, and PC. These four glycerolipids, in which40 to 60% of the respective fatty acids is 18:3, comprised morethan 90% of lamellar glycerolipids in December samples. Of this,84% was represented by DGDG and MGDG, in which more than55% of the respective fatty acids was 18:3.

DISCUSSIONBecause this investigation shows changes in a single membrane

system, rather than a mix of multiple membrane types, composi-tional adjustments in the chloroplast lamellae of eastern whitepine can be attnbuted diectly to winter stresses. Specific adjust-ments involve glycerolipid derivatives, degree of unsaturation inglycerolipid fatty acids, and hydrocarbon chain length of glycer-olipid fatty acids. Before November 2, response to decreasingtemperature and photoperiod resulted in an increase in lamellar18:3 content. Beginning on November 3, a 17-day period of dailyfreeze-thaw stress appeared to be correlated with increasedDGDG in the lamellae. Between mid-November and December,high lamellar representation ofDGDG and 18:3-containing glyc-erolipids was maintained during quantitative glycerolipid in-creases. In addition to these adjustments, long chain (C18 and C20)fatty acids increased. Each of these parameters influences, tovarying degrees, the physical status (viscosity, interface stability,hydrophobicity, etc.) of the membrane (3, 4, 9). Sterol glycosidesand ,8-hydroxy sterols, also affecting membrane physical proper-ties at low temperatures (4, 13), were not detected in any lamellarpreparation (6).The degree of fatty acid unsaturation has a pronounced effect

on membrane viscosity and hence on membrane-related processes(4, 16, 27). This has been demonstrated repeatedly in tissuessensitive to low temperatures, which exhibit a lipid phase transi-tion in the 8 to 12 C range (16).

Maintenance of lamellar vsosity at low temperatures wasindicated here by retention of photosynthesis in chloroplasts iso-lated during the experiment (6). The temperature optimum forPSI and PSIIdecreased (2, 19), and the rates of electron transportat the resPective optima remained between 70 and 85% ofsummer

927Plant.Physiol. Vol. 64, 1979

n L

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Plant Physiol. Vol. 64, 1979

PG

18:3

16:0

18:220:418:118:0

MG

18:3

18:2 16:0

18:0-18:1

C D H

Pi

16:0

16:1

18:318:2

18:018:1

c D H

PC

SL

6:0

18:318:118:2118:0\8:0 20:10 . 1~~~~~~~:

C D H

Glycerolipid Fatty Acid StatusFIG. 6. Fatty acid contingents of component glycerolipids during onset of winter. Glycerolipids were separated by TLC and eluted from the silica

gel with varying ratios of chloroform and methanol. They were reduced to dryness under N2, saponified in 15% methanolic KOH, partitioned intoacidified hexane, and derivitized with tri-methyl (aaa-trifluoro-m-tolyl) ammonium hydroxide in preparation for GLC analysis.

controls (6). This suggests that the increase in 18:3 representationduring temperature decline was, in part, responsible for mainte-nance of an optimal winter range of lamellar viscosity for struc-tural and functional stability of lamellae (4, 21, 22). Similarchanges in unsaturation of fatty acids assocated with the mem-brane have been shown in plants at different growth temperatures,the degree of unsaturation responding inversely to low (5, 18) or

high (21) temperature stress. In no instance have these tempera-ture-related fatty acid adjustments been shown to occur within a

single membrane of a mature evergreen growing under naturalconditions.

Recently, a fluidity maintenance mechanism for the root tissueofwinter wheat was suggested by Willemot (30). The total increasein tissue 18:3, coinciding with a decrease in 18:2, was envisionedas 18:2 desaturation catalyzed by a desaturase (30), a conceptconflicting with that suggested by our data. Since glycerolipidcontent was decreasing before mid-November, the increase in 18:3 appears to represent preferential loss of glycerolipids containingpredominantly saturated and monounsaturated fatty acids. Main-tenance of high 18:3 content during recovery appears to showpreferential incorporation of glycerolipids containing 18:3 intopreexisting or newly assembled lamellae, suggesting adaptive re-organization of lamellar fatty acids during winter onset to main-tain a critical viscosity range for activity (4, 27).

Fatty acid chain length can also influence the physical status ofthe membrane (4, 28). Increased length of the n-alkyl chain causes

increased membrane hydrophobicity (28) which implies greaterover-all bilayer stability in an aqueous system (15, 28). Lamellar

stability also has been linked to interfacial H-bonding resulting

from enhanced insulation of the hydrophobic region (3, 9). Be-cause ofthe increased ratio ofC18 to C16 fatty acids, the appearanceof highly unsaturated C20 fatty acids, and the increased interfacialH-bonding capacity of glycolipids compared to phospholipids, itappears that greater hydrophobicity also may contribute to mem-brane integrity during hardening.

Winter cells are susceptible to dehydration injury from extra-cellular ice formation when cell volume falls below a criticalminimum or when the concentration of ions or solutes becometoxic (15, 17). Cell resistance to dehydration injury has beencorrelated with an increase in soluble sugars (15, 25) and theappearance, during acclimation, of soluble proteins associatedwith hardening (23). The protective ability is attributed to bindingof intracellular water by these components (9, 17). The lamellarinterface binds significant amounts of intracellular water in glyc-erolipids and protruding integral proteins (9). Lamellar polypep-tides do not appear qualitatively or quantitatively different innonhardened and hardened tissues of wheat (23) or eastern whitepine (6), but a general increase in tissue glycolipid to phospholipiddemonstrated for spinach (14), potato (24), and poplar cortical cell(31) tissues exposed to low (14, 24), or subfreezing (24, 31)temperatures is consistent with our data. Not only do lamellarglycolipids increase at the expense ofphospholipids, but the majorincrease occurs in DGDG, which, with two galactose residues atthe interface, is the lamellar glycerolipid with the greatest capacityfor binding water (9, 15).Changes observed in eastem white pine chloroplast lamellae

during the onset of winter lead us to expect that acclimatedlamellae will show increased capacity for binding intracellularwater at the lamellar interface, enhanced hydrophobic stability,maintenance of lamellar permeability characteristics, and main-

tenance of a critical range in lamellar viscosity that provides for

retention of structural and functional integrity of the system.

Acknowledgmentf-We extend thacns to Dr. Klaus Gerhardt and Dr. Charles Gehrke of the

Auklurl Experiment Station for permitting use of their GLC sytem

LITERATURE CrrED

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2. BAut H,W LARCHER, RB WALKE 1975 Influence oftemperature stres on COt gs exchange.in JP Cooper, ed, Photosynthesis and Productivity in Different Environments. Cambridge

60

4-

.)

20.

Q

%.O

CO)

4-cn

D 4060

0

20-

0

DG18:3

16:0

18:218:118:0 = _ 20:1

928 DEYOE AND BROWN

I

I

I

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WINTER LIPID CHANGE IN PINE CHLOROPLASTS

Univeraity Press, Cambridge, pp 557-5863. BROCKm{oFF H 1974 Model ofinteraction ofpolar lipids, cholesterol and proteins in biological

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Plant Physiol. Vol. 64. 1979

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