Plant Physiol. (1982) 70, 376-3800032-0889/82/70/0376/05/$00.50/0

Physical Properties of Mitochondrial Lipids from Lycopersiconhirsutuml

Received for publication June 8, 1981 and in revised form March 24, 1982

ADAM W. DALZIEL2 AND R. WILLIAM BREIDENBACHPlant Growth Laboratory/Department ofAgronomy and Range Science, University of California, Davis,California 95616

ABSTRACT

Mitochondrial lipids from Lycopersicon hirsutum undergo a broad ther-mal transition beginning well below 0°C and ending at approximately 25°C.Differential thermal analysis of mitochondrial lipids isolated from ecotypesof L. hirsutum that differ in chilling sensitivity indicates that these lipidpreparations have physically similar properties. This was confirmed byelectron-spin-resonance experiments, although this technique failed todetect the broad transition detected by differential thermal analysis. Noquantitative differences were observed between the percentages of individ-ual lipid classes (based on polar head group) or between the fatty acidcompositions of mitochondrial lipids from the two ecotypes investigated.These results suggest that the observed differences between the responsesof these ecotypes to prolonged exposure to 5°C may not be related todifferences between the physical properties of their mitochondrial Upids.

The cellular metabolism of ectothermic organisms is greatlyaffected by environmental temperature changes, because temper-ature influences the kinetic energy of reactive molecules (5).Temperature also influences the physical properties of cellularconstituents, ie. membranes and cytoskeletal elements. Some ec-tothermic organisms have adopted mechanisms that adjust therate of enzymic reactions to compensate for wide variations intemperature. Others are limited to narrower ranges of temperaturebecause they are unable to adjust their metabolic rates effectivelyor because of some other dysfunction, such as a loss of compart-mentation or alteration of cytoplasmic organization. Such a limi-tation is exhibited by many species of higher plants which, onexposure to low nonfreezing temperatures, suffer physiologicaldamage classified as chilling injury.

Lyons and Raison (9) and Raison et al. (16) proposed thatdifferences among physical properties of plant membranes are theprincipal factors influencing chilling sensitivity, and considerableevidence has accumulated in support of the hypothesis (see Ref.14 for review). However, this evidence is largely based on com-parisons between species that are chilling resistant and speciesthat are chilling sensitive. In the present study, we have comparedtwo ecotypes of the same species (Lycopersicon hirsutum) thatdiffer markedly in response to chilling temperatures. By studyingmetabolic processes in conjunction with membrane physical prop-

'Supported in part by Grant No. PCM 78-26254 from the NationalScience Foundation. Any opinions, findings, and conclusions or recom-mendations expressed in this publication are those of the authors and donot necessarily reflect the views of the National Science Foundation.

2 Present address: Department of Chemistry, Kline Chemistry Labora-tory, Yale University, P.O. Box 6666, New Haven, CT 0651 1.

erties, we hope to elucidate the biochemical characteristics thatallow ecotypes of L. hirsutum to live in habitats with diversetemperatures. Such ecotypes, collected along an altitudinal gra-dient in the Andes (19), have different rates of growth anddevelopment at low temperatures (12°C day, 5°C night) (C. E.Vallejos et al., unpublished data). Patterson et al. (13) have alsodemonstrated that the altitude of origin influences the sensitivityof seedlings to chilling injury using a similar collection of geneticlines of L. hirsutum.

In the present study, the chilling sensitivity of small rootedcuttings from L. hirsutum showed a similar dependency. From apreliminary study (2) we have reported the presence of a broadthermal transition in L. hirsutum membrane lipids extracted fromcrude mitochondrial preparations. Consequently, we studied thephysical and chemical properties of membrane lipids from altitu-dinal ecotypes of L. hirsutum. This study focused on the physicalproperties of mitochondrial membrane lipids, because previouscomparative studies of chilling-sensitive species and chilling-in-sensitive species indicated that the physical properties of mito-chondrial lipids correlated well with chilling sensitivity (9, 16). Inaddition, highly purified mitochondria can be obtained from plantroots, allowing study of a particular organelle membrane. Thephysical properties of the mitochondrial lipids were studied byboth DTA3 and ESR in an attempt to provide a rigorous interpre-tation of the observed physical properties.

MATERIALS AND METHODS

Seeds from Lycopersicon hirsutum f typicum (LA 1777) werecollected by Vallejos (19) in Peru; L. hirsutum.f glabratum (LA1625) seeds from Ecuador were obtained from Professor C. Rick.The former were collected at an altitude of 3,200 m, and the latterwere collected at 100 m. Experiments aimed at determining thechilling sensitivity of the two ecotypes of L. hirsutum were con-ducted in constant-temperature growth rooms equipped with Gro-Lux lights operating on a 12-h light cycle. Cuttings were takenfrom greenhouse-grown plants and were rooted in sand. Theplants were watered with modified Johnson nutrient solution (3)as necessary. After 4 weeks at 20°C (RH 70%), the plants weretransferred to 5°C in similar lighting. Plants were transferred backto 20°C at 5-d intervals, and the number of plants surviving thechilling treatment was assessed.

Hydroponic culture was used to obtain more roots for mito-chondrial preparations. L. hirsutum cuttings were rooted in sandand transferred to a 50%Yo modified Johnson nutrient solution andgrown hydroponically in a greenhouse at the University of Cali-fornia at Davis. The temperature of the nutrient solution wasmonitored continuously. Average maximum and minimum tem-

3 Abbreviations: DTA, differential thermal analysis; ESR, electron spinresonance; 4N 14, (2,2-dimethyl-5-decyl-5-propyloxazolidine-N-oxide).

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PHYSICAL PROPERTIES OF MITOCHONDRIAL LIPIDS

peratures were 22.5°C and 18°C, respectively. Daily temperaturesranged from 16 to 29°C. After 40 d, the roots were harvested andwashed with distilled H20.Washed roots (250 g) were chopped with a razor into 0.5-cm

segments in 1 L of homogenizing buffer at 0°C (0.4 M sucrose, 50mm K-phosphate, 5 mm EDTA, 0.2% PVP-40, 0.1% fatty-acid-poor BSA, and 5 mm fl-mercaptoethanol at pH 7.2). The choppedroots were ground with a mortar and pestle in the presence of asmall amount of acid-washed sand. The homogenate was filteredthrough four layers of cheesecloth and centrifuged at 750g for 10min. The supernatant was passed through a single layer of cheese-cloth and centrifuged at 10,000g for 20 min. The mitochondrialpellet was suspended in 12 ml of homogenizing buffer, layeredonto six continuous (20-50%, w/w) sucrose gradients (50 mm Tes,I mM EDTA at pH 7.5), and centrifuged for 2 h at 100,000g. Eachgradient was fractionated into I -ml fractions, and the correspond-ing fractions were combined.Fumarase was assayed by the method of Racker (15) using 50

[lI of each gradient fraction in I ml of reaction mixture. NADHCyt c reductase was assayed by the method of Lord et al. (7). Thereaction was initiated by adding a 50-,ul sample from each gradientfraction.

Protein determinations were carried out using a modified pro-cedure of Lowry et al. (8). A 100-,ul sample from each fraction wasincubated overnight with 0.9 ml of 0.5 M NaOH. The coppertartrate reagent (1 ml) (minus NaOH) was then added. Theremainder of the procedure followed the original.The remaining portion of the mitochondrial fractions was di-

luted to a total volume of 250 ml with 50 mm Hepes (pH 7.5).Mitochondrial membranes were then pelleted by centrifuging at48,000g for I h. The pellet was suspended in 5 ml of buffer andadded to 50 ml of boiling isopropanol. Lipid extraction was carriedout by the procedure of Folch et al. (4), modified by the substi-tution of 0.2% NH40H for H20.

Differential thermal analysis was carried out using a MettlerTA 2000 (B) system. Samples were prepared by adding 5 to 10 mgof mitochondrial membrane lipid (in chloroform) to a preweighedaluminum sample pan. The chloroform was removed, and thesample was dried over P205 in vacuo for 24 h. After weighing, thesample buffer (20 ,Il 50%o ethylene glycol in 50%o 2 mm Hepes, 10mM NaCl, 0.1 mm EDTA, pH 7.2) was added and the sample panwas hermetically sealed in a N2 atmosphere. The lipid was allowedto hydrate overnight at room temperature and then was precon-ditioned by heating and cooling of the sample three times overthe temperature range to be studied. The sample buffer was usedas the reference material, and the heat capacity of the referenceclosely resembled the heat capacity of the sample.The thermograms shown are representative of results for lipid

extracts from two mitochondrial preparations for each ecotype.The onset temperatures for the cooling thermograms were meas-ured as that temperature at which the thermogram deviatedsharply from the base line. In some samples, there was a slightcurvature at temperatures above the onset temperature. The datapresented have been corrected for the thermal lag of the referencebehind the programmed furnace temperature. This was calculatedby measuring the onset temperatures for either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or distilled H20 at various scanningrates (I to 5C min-'). To eliminate the effect of the heat capacityof the reference on the constant for the lag time, the heat capacityof the sample closely matched the heat capacity of the referencesample used in the remainder of the experiments. The onsettemperature was plotted versus the scanning rate, and a straightline was obtained. The slope on this line was taken to be the celltime constant, and they intercept gave the true fusion temperature.Both materials used in this calibration gave a value of 0.76. Thus,at the scanning rate of 5°C min-' used, ATLAG was found to be3.80C ([ATLAG = TLAG dTp/dtj, in which ATLAG is the thermal lag,

TLAG is the cell time constant, and dTp/dt is the scanning rate).ESR experiments were carried out using a JEOL (JESME IX)

X band spectrometer equipped with a laboratory-constructed var-iable-temperature device. A thermistor probe was used to measurethe temperatures (+0.2°C) at which spectra were recorded.

Samples were prepared by drying a chloroform solution of thelipid extracts containing the nitroxide spin label, 4N14, in theapproximate molar ratio of 200:1. The remaining chloroform wasremoved in vacuo for 2 h, and buffer (2 mm Hepes, 1O mm NaCl,0.1 mim EDTA, pH 7.2) was added (molar ratio of lipid:buffer wasapproximately 1:100). Lipid dispersions were formed by brieflyheating the lipid sample in a steam bath followed by rapidvortexing. This procedure was repeated until the lipid was welldispersed. The sample was transferred to a glass capillary tubeand centrifuged in order to form a pellet.Some samples contained a component that reduced some of the

spin label. When this occurred, the lipid was reextracted withchloroform:methanol (2:1), and a solution of potassium ferricya-nide was added to cause phase separation. The chloroform phasewas dried and used as described above. In some cases, the samplewas also prepared with deuterated water to slow down the reduc-tion of the probe. Even with these precautions, some loss of signalwas detected.

Rotational correlation times were calculated using the equation= 6.5 X 1010 Wo [(h0/h )1/2 - 1] (11), in which TOr is the

empirical rotational correlation time, WO is the width (gauss) ofthe midfield line, ho is the height of the midfield line, and h-, isthe height of the highfield line. The ratio ofthe integrated intensityof the midfield: highfield peaks was calculated from IoII- =W02h0/W1_2h-1), in which Io and i-, are the integrated intensitiesof the midfield and highfield lines, respectively, and W-1 is thewidth of the highfield line. In some samples, this value increasedrapidly at low temperatures, indicating that there was loss ofintegrated intensity in the highfield line because of overlappingpeaks. This can lead to an erroneous discontinuity in an Arrheniusplot, so points for which 4/JIi was more than 10%9o greater thanthe average value at higher temperatures were not included in theresults.

Two-dimensional chromatography of the mitochondrial lipidsand fatty acid analysis of the fractionated phospholipids werecarried out using the method of Roughan et al. (17). Fatty acidmethyl esters were analyzed by GLC using a column of 15%ethylene glycol succinate on Gas Chrom P. Phospholipids weremeasured using the method of Rouser et al. (18).The results shown in the present paper are representative of the

results obtained using two replicate sets of plants for each ecotype.The observed DTA transition temperatures and the lipid analysis

100

80

High altitude* 60 _ ecotype

-J Low altitude> 40 _ \ ecotype

U) 20

00 5 10 15 20 25 30 35

TIME (DAYS)FIG. 1. Chilling sensitivity of L. hirsutum fJ glabratum (low altitude)

(O-O) and L. hirsutum f typicum (high altitude) (@-- ). Afterexposure to 5°C for number of days shown, plants were returned to a20'C environment. Percentage of survival was calculated from number ofplants able to grow after chilling treatment.

377

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DALZIEL AND BREIDENBACHI Plant Physiol. Vol. 70, 1982

0.8 -\

0,0.6CZE

-

0.4-

0.2 _

oLX

E 06050

E40

z! 30c 20

0

175

'S 150-(I 125

Z) ! 75-c 50

,25100 2 4 6 8 10 12 1416 1820

FRACTION NUMBER

FIG. 2. Distribution of protein and marker enzymes on 20o to 500 (w/w) sucrose gradient. Fraction 2 is top of sucrose gradient. Shaded areaindicates fractions collected for lipid analysis (densities between 1.16 and1.19 g cm-3).

Heating

-40 -20 0 20 40 60 80TEMPERATURE (*C)

FIG. 3. Heating thermograms for mitochondrial lipids from L. hirsu-tum. A, High-altitude ecotype (9.56 mg); B, low-altitude ecotype (7.09 mg);C, dynamic base line.

data are the average values obtained for lipid extracts from tworeplicate mitochondrial lipid preparations for each ecotype.

RESULTS

The responses of L. hirsutum f typicum and L. hirsutum fglabratum to continual exposure to 5C were quite different (Fig.1). Although visual differences were apparent within 2 d (notshown), both ecotypes were able to recover from a 5-d exposureto 5°C. However, after 10 to 25 d at 5°C, L. hirsutumf glabratum

r.-

LUJ

O-U

U

LUJ

I

0

LUJ

NZ-U

CoolingI I I I I I I I

60 40 20 0 -20 -40 -60 -80 -100 -120TEMPERATURE (C)

FIG. 4. Cooling thermograms for mitochondrial lipids from L. hirsu-tum. A, High-altitude ecotype (9.56 mg); B, Low-altitude ecotype (9.92mg); C, dynamic base line.

5040

30

u0

x

0

0

'/T eK x 103FiG. 5. Arrhenius plot of T., versus IIT (OK) for 4N14 in aqueous

dispersions of total mitochondrial lipids from A, L. hirsutumf typicum(high altitude) and B, L. hirsutumrf glabratum (low altitude).

was more seriously injured than L. hirsutum f typicum. Whenexposure to 5C was longer, both ecotypes were irreparably in-jured.

Because the responses of the two ecotypes of L. hirsutum tocontinuous exposure to 5°C differed considerably, mitochondriawere prepared from these ecotypes in order to compare the

378

I )-H igh altitude

L~ow altitudeecotype

I I I I I I I I I:t

Q

IjJ

I 0.15mW

II I I I I I I

I -I

A

High altitudeecotype

B

ecotype

I0.15mW

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PHYSICAL PROPERTIES OF MITOCHONDRIAL LIPIDS

Table 1. Distribution of Phospholipid Classes in Mitochondrial LipidsfromLycopersicon hirsutum

L. hirsutum L. hirsutumf typicum f glabratum

(High Altitude) (Low Altitude)

% by wt of lipidOrigin 7 83-sn-Phosphatidyl inositol 13 163-sn-Phosphatidyl choline 40 353-sn-Phosphatidyl ethanolamine 29 27Unknown 6 83-sn-Phosphatidic acid 4 5

physical and chemical properties of their membrane lipids.The purity of the mitochondrial preparations was analyzed by

assay of mitochondrial and microsomal marker enzymes (Fig. 2).Most of the fumarase activity was associated with a single band,indicating that mitochondria were essentially intact. The fumaraseactivity in the top fractions of the gradient indicate that some ofthe mitochondria were disrupted during the suspension of themitochondrial pellet. A peak of NADH Cyt c reductase activitycoincided with the mitochondrial peak, which is consistent withthe results of Moore and Beevers (12) and Lord et aL (7). Noseparate peak in activity corresponding to microsomal membraneswas detected, but low activity was observed in the upper fractionsof the gradient.The thermotropic properties of the total lipid fraction from

purified mitochondrial membranes were analyzed by DTA (Fig.3, heating and Fig. 4, cooling thermograms). The results for thehigh-altitude ecotype (Fig. 3A) indicated that an endothermicprocess had already begun at -39°C and that it extended over abroad temperature range. A similar transition was observed forthe low-altitude ecotype (Fig. 3B). The cooling thermogramsdemonstrated that the physical properties of mitochondrial lipidsfrom these two ecotypes were similar: the onset temperature was23.2 ± L.0°C for the high-altitude ecotype (Fig. 4A) and 24.9 ±1.0°C for the low-altitude ecotype (Fig. 4B). Similar results wereobtained for successive scans and, therefore, the observed ther-motropic properties were completely reversible.The enthalpy of the transition was estimated by extrapolating

the base line at high temperatures until it intersected the thermo-gram at a low temperature. This was useful in comparing the twoectoypes, but there is likely to be considerable error in the absolutevalue. The estimated enthalpy was found to be 15.5 ± 1.5 J g-1 forthe high-altitude ecotype and 16.8 ± 0.5 J g-1 for the low-altitudeecotype. Thus, the two ecotypes do not appear to differ signifi-cantly in any of the parameters measured.To investigate the temperature-dependent changes in molecular

motion that occur in mitochondrial lipids of L. hirsutum, westudied the same lipid preparations using ESR spectroscopy.Figure 5 shows Arrhenius plots for the rotational correlation timeof the spin label probe 4N 14 in aqueous dispersions of these lipids.The linear plots indicate that the viscosity of these lipid prepara-tions decreases exponentially as the temperature increases. Essen-tially no difference was detected between the ecotypes althoughminor variations in slope between replicates prevented a detailedcomparison of this parameter; however, lower temperatures couldnot be investigated because the high-field spectral line becameasymmetric and invalidated the calculations of T.

Tables I and II show the lipid composition of the mitochondriallipids extracted from the high- and low-altitude ecotypes of L.hirsutum. The percentages of the phospholipid classes of the twoecotypes are similar (Table I). The presence of phosphatidic acidmay indicate some lipid degradation during the mitochondrialisolation, but phosphatidic acid has also been observed in steam-killed L. hirsutum leaves (R. W. Breidenbach and P. G. Roughan,

Table I1. Fatty Acid Composition of 3-sn-Phosphatidyl Choline (PC), 3-sn-Phosphatidyl Ethanolamine (PE), and 3-sn-Phosphatidyl Inositol (PI)from L. hirsutum Mitochondria (L.hirsutumf typicumfrom High Altitude

[HA] and L. hirsutumf glabratumfrom Low Altitude [LA])16:0 16:1 18:0 18:1 18:2 18:3

% wt of totalfatty acidsPCHA 15 6 2 2 62 12LA 14 7 2 2 63 9

PEHA 12 5 2 1 68 9LA 13 6 2 1 70 7

PIHA 24 10 3 2 47 13LA 22 11 3 2 52 9

m.7G

PA-

PE- A

-3r

'1

-4

PC

FIG. 6. Representative two-dimensional chromatogram of mitochon-drial lipids from L. hirsutum. Spots shown are 3-sn-phosphatidyl choline(PC), 3-sn-phosphatidyl ethanolamine (PE), 3-sn-phosphatidic acid (PA),and unidentified lipids (0-10).

unpublished data). No major differences were detected betweenthe fatty acid compositions of the phospholipid classes examined(Table II), but minor quantitative differences between these eco-types cannot be ruled out.A typical two-dimensional chromatogram (Fig. 6) shows that

although phospholipids appear to be the major resolvable constit-uents of these lipid preparations, other lipid classes are present.Spots 6, 7, and 8 developed a color characteristic of sterols whensprayed with 501% H2SO4 and are probably sterol glycosides,esterified sterol glycosides, and sterol esters plus sterols, respec-tively. Spot 10 may be free fatty acids created by the action of aphospholipase, but it appeared to be a minor constituent and wasnot positively identified. Some of the material at the solvent frontis probably neutral lipid derived from mitochondrial membranes.No qualitative difference between the ecotypes in any of thesecomponents was detected.

DISCUSSION

The results of the chilling-injury experiment verify that underthe conditions used the low-altitude ecotype of L. hirsutum is more

379

5-

2-

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DALZIEL AND BREIDENBACH

susceptible to continual exposure to 5°C than is the high-altitudeecotype. However, the high-altitude ecotype was killed by longexposure to 5°C, suggesting that it too is sensitive although to alesser degree. The DTA results indicated that under the conditionsused, lipids extracted from highly purified mitochondrial mem-branes underwent a broad transition, beginning at approximately25°C and continuing well below 0°C. It is not entirely expectedthat mitochondrial lipids from L. hirsutum would undergo athermal transition above 0°C, because lipid analysis indicates thatthe phospholipids in these preparations contain a predominanceof highly unsaturated fatty acids. Soybean phosphatidylcholine,phosphatidylethanolamine, and phosphatidylinositol, which all

contain a predominance of highly unsaturated fatty acids, undergotransitions at temperatures considerably less than 0°C (1). It ispossible that phospholipid polar head group interactions, theeffect of other lipid components, or fatty acid positional isomerspresent in the phospholipids strongly influence the physical prop-

erties of mitochondrial lipid extracts.The slopes of the cooling thermograms from L. hirsutum mito-

chondrial lipids were difficult to interpret. In contrast with resultsobtained with phospholipids containing one or less unsaturateddouble bond per esterified fatty acid (10), the base line with theseheterogeneous mitochondrial lipids was not continuous before andafter the transition. The observed decrease in the apparent heatcapacity of the sample at low temperatures may be due to theformation of amorphous regions in the bilayer caused by irregu-larities in molecular packing. The decrease in apparent heatcapacity at low temperatures may be caused partly by the inter-action of thelipids with the buffer, but only a slight slope wasdetected when 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was

investigated under similar conditions (not shown).The ESR results with mitochondriallipids from both ecotypes

of L. hirsutum did not detect the onset of the transition detectedby DTA. Linden et al. (6) have reported that, in Escherichia colisupplemented with unsaturated fatty acids, spin-labeled probessimilar to those used in the present study may detect only thecompletion of the phase transition. This is consistent with theDTA results, because the transition does not appear to be completeuntil well below0°C. In the future, we plan to use other spectro-scopic probes in order to determine whether they are more sensi-tive to the transition detected by DTA.The results of the lipid analysis indicated that theselipid

preparations are similar. Without a quantitative analysis of thepositional distribution of fatty acids within each phospholipidclass, and the identification of the other lipid components presentin these preparations, we cannot say that they are identical.However, the lipid analysis, coupled with the evidence from DTAand ESR, strongly suggests that any differences that might existdo not contribute significantly to the physical properties of thelipid preparations.

It will be important to use more sensitive techniques to studythe physical properties of isolated mitochondria, mitoplasts, and

other membrane systems to determine whether the physical prop-erties of the intact membrane reflect the physical properties oftheir lipids. Because of the results presented in this paper, it isquite possible that mitochondrial lipids from these two ecotypesof L. hirsutum do not differ significantly, so other mechanismsmay have to be invoked to account for the differential suscepti-bility of these ecotypes to chilling injury.

Acknowledgments-We thank Mettler Instrument Corporation for allowing us touse the TA 2000 B system and Dr. A. D. Keith for his cooperation in carrying out theESR experiments. We greatly appreciate the technical assistance of Mimi Thomasand Karen Yamamoto. The advice of Dr. G. Roughan, Dr. B. Mudd, EduardoVallejos, and Fargo Rousseau is also appreciated.

LITERATURE CITED

1. DALZIEL AW 1980 The physical properties of tomato (genus Lycopersicon)membranelipids. Ph.D. dissertation, University of California, Davis

2. DALZIEL AW, RW BREIDENBACH 1979 Differential thermal analysis of tomatomitochondriallipids. In JM Lyons, D Graham, JK Raison, eds, Low Temper-ature Stress in Crop Plants: The Role of the Membrane. Academic Press, NewYork

3. EPSTEIN E 1972 Mineral Nutrition of Plants: Principles and Perspectives. J Wiley& Sons, Inc, New York, pp 38-39

4. FOLCH J, M LEES, GH SLOANE-STANLEY 1957 A simple method for the isolationand purification of total lipids from animal tissues. J Biol Chem 226: 497-509

5. HOCHACHKA PW,GN SOMERO 1973 Strategies of Biochemical Adaptation. WBSaunders Co, Philadelphia, pp 179-271

6. LINDEN, CD, AD KEITH, CF Fox 1973 Correlations between fatty acid distri-bution in phospholipids and the temperature dependence of membrane phys-ical state. J Supramol Struct 1: 523-534

7. LoRD JM, T KAGAWA, TS MooRE, H BEEVERS 1973 Endoplasmic reticulum asthe site of lecithin formation in castor bean endosperm. J Cell Biol 57:659-667

8. LowRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-ment with the Folin-phenol reagent. J Biol Chem 193: 265-275

9. LYONS JM, JK RAISON 1970 Oxidative activity of mitochondria isolated fromplant tissues sensitive and resistant to chilling injury. Plant Physiol 45: 386-389

10. MELCHIOR DL, JM STEIM 1976 Thermotropic transition in biomembranes. AnnuRev Biophys Bioeng 5: 205-238

11. MEHLHORN RJ, AD KEITH 1972 Spin labeling of biological membranes. In CFFox, AD Keith, eds, Membrane Molecular Biology. Sinauer Associates, Inc.,Stamford, CT, pp 192-227

12. MOORE TS, H BEEVERS 1974 Isolation and characterization of organelles fromsoybean suspension cultures. Plant Physiol 53: 261-265

13. PATTERSON BD, R PAULL, RM SMILLIE 1978 ChilLing resistance in Lycopersiconhirsutum Humb & Bonpl, a wild tomato with a wide altitudinal distribution.Aust J Plant Physiol 5: 609-617

14. QUINN PJ, WP WILLIAMS 1978 Plantlipids and their role in membrane function.Prog Biophys Molec Biol 34: 109-123

15. RACKER E 1950 Spectrophotometric measurements of the enzymatic formationof fumaric and cis-aconitic acid. Biochim Biophys Acta 4: 211-214

16. RAISON JK, JM LYONS, RJ MEHLHORN, AD KEITH 1971 Temperature-inducedphase changes in mitochondrial membranes detected by spin labeling. J BiolChem 246: 4036-4040

17. ROUGHANPG, CR SLACK, R HOLLAND 1978 Generation of phospholipid artifactsduring extraction of developing soybean seeds with methanolic solvents. Lipids13: 497-503

18. ROUSER G, AN SIAKOTOS, S FLEISCHER 1966 Quantitative analysis of phospho-lipids by thin-layer chromatography and phosphorus analysis of spots. Lipids1: 85-86

19. VALLEJOS CE 1979 Genetic diversity of plants for response to low temperatureand its potential use in crop plants. In JM Lyons, D Graham,JK Raison, eds,Low Temperature Stress in Crop Plants: The Role of the Membrane. AcademicPress, New York pp 473-489

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