Plant Physiol. (1980) 66, 316-3200032-0889/80/66/03 16/05/$00.00/0

Effect of Dehydration on Leakage and Membrane Structure inLotus corniculatus L. Seeds'

Received for publication January 10, 1980 and in revised form April 8, 1980

BRYAN D. MCKERSIE2 AND ROBERT H. STINSON32 Department of Crop Science and 3 Department of Physics, University of Guelph, Guelph, Ontario NJG 2 WICanada

ABSTRACT

Membrane damage as a result of dehydration was studied in Lotuscorniculatus L. cv. Carrol seeds which had been pregerminated for 0, 12,and 24 hours prior to dehydration. During reimbibition, desiccation-tolerant(0- and 12-hour) seeds leaked relatively low quantities of all solutes (totalelectrolytes, potassium, phosphate, sugar, amino acid, and protein). Desic-cation-sensitive (24-hour) seeds leaked higher levels, but evidence ofselective permeability remained. Membrane damage was not manifested asa complete removal of the diffusion barrier, although its permeabilityproperties were dramatically altered. Consequently, the plasmalemma wasnot ruptured or torn by the dehydration treatment, but a more subtlestructural alteration occurred.The possibility that seed membranes form a hexagonal rather than a

lamellar phase at moisture contents below 20% was investigated by x-raydiffraction. Phospholipids were extracted from desiccation-tolerant (0-hour) and desiccation-sensitive (24-hour) seeds and hydrated to 5, 10, 20,and 40% water. This phospholipid-water system was examined using low-and wide-angle x-ray diffraction and was found to be exclusively lamellar,even at 5% water. Consequently, membrane damage and the leakage ofcytoplasmic solutes from seeds cannot be explained by the formation of ahexagonal phase by membrane phospholipids.

lapse to form large discontinuities in the lipid bilayer. This possi-bility is supported by the observation that the transition fromdesiccation-tolerant to desiccation-sensitive occurs coincidentlywith the initiation of radicle elongation (9). Since elongation isfacilitated by the uptake of water into the vacuole (5), it isconceivable that the rapid loss of this water collapses the cell and,in so doing, breaks the continuity of the plasmalemma and/or thetonoplast. However, this model does not explain why dehydrationdamage is induced only when seeds are dehydrated below 20%water (9). An alternative model is that dehydration induces analteration in the hydrophobic-hydrophilic interaction within themembrane so that the structure of the cellular membranes below20% hydration is different than that above 20% hydration. Wateruptake by desiccation-tolerant seeds reinstates the original struc-ture of the cellular membranes, whereas the membranes of desic-cation-sensitive seeds are unable to reform completely. A struc-tural rearrangement of membrane phospholipids by dehydrationhas been proposed by Simon (13), who suggested that membranelipids in seeds form a hexagonal phase below 20% moisture.However, his proposal has not been experimentally verified usingseed phospholipids.

This study was intended to investigate these two models ofinduced damage. The results indicate that dehydration induces areorganization of the cellular membranes in dehydration-sensitiveseeds; however, this reorganization does not involve a lipid phasechange.

Dehydration treatments of seeds have also been shown toinfluence seed viability adversely and to increase electrolyte leak-age (1, 5, 9). Seeds which have been imbibed and dehydrated totheir original weight are able to maintain viability if the dehydra-tion treatment is imposed prior to radicle emergence (5). However,if the dehydration treatment is delayed until after radicle emer-gence, the subsequent germination and vigor of the seed is re-duced. For example, the seeds of birdsfoot trefoil (Lotus cornicu-latus L.) undergo a transition from a desiccation-tolerant to adesiccation-sensitive state coincidently with the initiation of radi-cle elongation (9). Seeds which were imbibed, dried back to theiroriginal weight, and reimbibed did not suffer impaired germina-tion or vigor, provided the dehydration was performed prior tothis transition. If the seeds had entered the desiccation-sensitivestate, dehydration prevented subsequent germination or greatlyreduced seedling vigor. The magnitude of the reduced viabilitycorrelated with an increase in electrolyte leakage during reimbi-bition, which indicated an alteration in cell permeability.

Dehydration enhances the leakage of cytoplasmic electrolytesby inducing one of two possible types of membrane damage. Thefirst assumes that the membrane is ruptured during cellular col-

' Financial assistance was provided by Grant A6760 from the NaturalSciences and Engineering Research Council of Canada.

MATERIALS AND METHODS

Germination and Dehydration Treatments. Birdsfoot trefoil (L.corniculatus L.) cv. Carroll seeds were mechanically scarified and250 mg seed were germinated between two 9-cm filter discs inPetri plates, which had been moistened with 3.5 ml distilled H20.Seeds which had been allowed to germinate for 0, 12, and 24 hwere desiccated over silica gel for 24 h, which dehydrated theseeds to their original weight.Leakage Determination. The leakage from seeds during reim-

bibition was monitored by imbibing 250 mg seed in 20 ml distilledH20 in a 250-ml Erlenmeyer flask gently shaken at room temper-ature. In the first experiment, designed to compare the relativeleakage from 0-, 12-, and 24-h seeds, the seeds were allowed toleak for 2 h and the water was decanted for analysis. Each samplewas analyzed for conductivity, K, phosphate, amino acid, sugar,and protein. The initial content of each component within theseed was determined by homogenizing, with a glass homogenizer,250 mg of each seed group in 20 ml distilled H20. The resultanthomogenate was centrifuged at 10,000g for 15 min, and thesupernatant was analyzed for each component.

In the second experiment, 250 mg seed were imbibed andshaken in 20 ml distilled H20 as before. Duplicate samples wereremoved after 0.5, 1, 2, 4, and 6 h. The water was decanted fromthe seeds and analyzed as before. The seeds were blotted dry on

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MEMBRANE STRUCTURE IN SEEDS

filter paper and weighed to determine water uptake.Analysis. Conductivity of the leachate was measured using a

Barnstead conductivity bridge (9). K was quantified with an Orionspecific ion electrode. Phosphate was determined as Pi by themethod of Fiske and SubbaRow as outlined by Dittmer and Wells(3). Amino acids were quantified with ninhydrin using 2 mMleucine as a standard (10). Sugars were measured using the phen-olsulfuric acid method (4). Protein was quantified by the methodof Lowry et al. (6) using BSA as a standard.

Extraction of Phospholipids. Total lipids were extracted from25 g seeds which had been germinated for 0 and 24 h andsubsequently dehydrated. The dried seeds were ground to a finepowder in a grinding mill and extracted by homogenizing thepowder in 180 ml chloroform-methanol (2:1, v/v). The mixturewas centrifuged at 10,000g for 15 min and the supernatant wasdecanted. The extract was evaporated to dryness using a Rotava-pour and resuspended in 21 ml chloroform-methanol (2:1, v/v).To remove water-soluble materials, 4.2 ml 0.7% NaCl was addedand shaken (11). The lower chloroform layer was removed andevaporated to dryness.

Polar and neutral lipids were separated as previously detailedusing a silica gel column (8). The total lipid extract in chloroformwas added to a 2- x 20-cm glass column. The neutral lipids wereeluted with 300 ml chloroform and the polar lipids were elutedwith 300 ml methanol. The polar lipid fraction was evaporated todryness, weighed, and stored in chloroform solution at -20 Cunder N2. Separation of the lipid classes was confirmed for eachsample using TLC.

Hydration of Lipids for X-ray Diffraction. The polar lipidfraction from each seed group was split into four fractions ofapproximately 50 mg each. These were dried by a stream of N2and stored under vacuum for 24 h to ensure complete removal ofsolvent. Water was added to the four fractions from each group tobring water contents to 5, 10, 20, and 40%o water, relative to totalweight of hydrated sample. The samples were allowed to equili-brate for 24 h at room temperature under N2 before x-ray diffrac-tion analysis.

X-ray Diffraction. The hydrated lipid samples were sealed inair-tight holders with thin mica windows. Sample thickness was 1mm. Samples were mounted in a small-angle x-ray diffractionsystem in which Cu K a radiation from a sealed source at 40 kvand 20 mamp was focused by a Franck's mirror through thesample and onto the film cassette. Sample to film distance was192.4 mm. All diffraction experiments were at 22 ± 1 C. Exposuretimes were typically 6 h. Kodak No-screen NS-5T film was used.The positions on the film of reflections was determined with anoptical comparator.Wide- and small-angle patterns were recorded with a toroidal

camera for the 5% water, 24-h samples as those were thought tobe the samples least likely to be in a lamellar phase.

Lipid Analysis. The phospholipid composition of the polar lipidfraction was determined by TLC as previously described (8). Analiquot of each polar lipid fraction was spotted onto an AnaltechTLC plate coated with a 1,000-,um thick layer of Silica Gel G, anddeveloped in one dimension using chloroform-methanol-aceticacid-water (25:15:2:1, v/v). Spots were detected with iodine vaporor with molybdenum blue reagent (8). The phospholipids wereidentified by co-chromatography with phospholipid standards(Sigma). Individual phospholipid spots were scraped from theTLC plate into centrifuge tubes and were extracted with 2 mldeveloping solvent, followed by 2 ml methanol. Extracts werepooled, evaporated to dryness, and quantified as Pi after digestionwith HC104 (3).

RESULTS AND DISCUSSIONTotal Leakage. Birdsfoot trefoil (L. corniculatus L. cv. Carroll)

seeds leaked a variety of cytoplasmic components into the imbib-

ing solution. Significant quantities of K, phosphate, sugars, aminoacids, and protein were detected in addition to total electrolytes asdetermined by conductivity (Fig. 1). When seeds were given adehydration treatment after 12- and 24-h germination, leakage ofall cytoplasmic components tested was increased during reimbi-bition. A slight increase was observed in the 12-h samples, butleakage was the most pronounced from the 24-h germinated seeds.Previous experiments have indicated that dehydration of trefoilseeds at 12 h does not impair either germination or seedling vigor,whereas dehydration at 24 h is detrimental to the survival of theseed (9).

Analysis of the K, phosphate, amino acid, sugar, protein, andtotal conductivity content of total seed homogenates indicated nosignificant change during the first 24 h of germination. Therefore,increased rates of leakage are not the result of increased cytoplas-mic concentrations.

Ungerminated seeds leaked over 13% of the total potassiumpresent in the seed during the first 2 h of imbibition (Fig. 1). Inaddition, almost 9% of the total soluble protein was leached out.On the other hand, during the same time period, the leakage ofsugars, phosphate, and amino acids was moderate with only 3.6,3. 1, and 1.4%, respectively, being leaked (Fig. 1).When seeds were germinated for 24 h, dehydrated to their

original weight, and reimbibed, over 32% of the total K content ofthe seed was lost after 2-h reimbibition. The leakage of phosphateand amino acid was markedly increased to the point whereapproximately 18% of the total cytoplasmic pool of each solutewas leached from the seed (Fig. 1). Only 14% of the soluble proteinand 8% of the sugar was present in the imbibing solution (Fig. 1).The increase in total leakage between 12 and 24 h suggests an

increase in membrane permeability. Although dehydration ofdesiccation-sensitive (24-h) seeds stimulated leakage, the efflux ofall solutes was not uniformly increased. Furthermore, the com-positions of the leachate and total homogenate differed, indicatingthat some aspects of membrane semipermeability remained afterdehydration damage.Water Uptake. Differential rates of leakage may have devel-

oped in response to different rates of water uptake and hydrationof the cellular compartments. However, examination of the rate ofwater uptake by 0-, 12-, and 24-h germinated seeds during thefirst 6 h of rehydration indicated that the differences in leakagebetween 12- and 24-h samples could not be attributed to differentrates of hydration (Fig. 2). The rate of water uptake by ungermi-

30

a,

10

K Pi SUGAR AA PROTEIN COND

FIG. 1. Per cent leakage of cytoplasmic solutes from birdsfoot trefoil(L. corniculatus L.) seeds dehydrated after 0-, 12-, and 24-h germination.Seeds were reimbibed in distilled H20 at 20 C for 2 h; AA, amino acids;Cond, conductivity. Values represent amount of solute leaked/amount ofsolute in dry seed x 100oand are the means of four to six determinations.

1 0

012247 0124 [7I 1224 [1124p Lu245

. . . . I . 1- I

y-

z

-A-

-

I

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McKERSIE AND STINSON

2.01

*- 24 h

A 2h/

0 h

1 2 3TIME (h)

FIG. 2. Rate of water uptake during reimb(L. corniculatus L.) seeds dehydrated at 0-, 1Values represent the increase in fresh weight cafter imbibing seeds in distilled H20 and blotti

200

1801

1 2 3TIME (h)

1T 1 ; _g.1 -I *-*-I 1]--,1n

are two processes contributing to the total leakage of a specificsolute. The initial leakage during the first minutes of imbibitionmay reflect the release of surface deposits or solutes not passingthrough a semipermeable membrane. The subsequent constantrate of leakage presumably indicates the release of solutes from

£ cytoplasmic sites which must pass through the plasmalemmabefore dissolving in the external solution. Both ofthese parameterscan be calculated from a regression analysis of the time profile(Fig. 3). The initial leakage is represented by the y intercept, andthe rate of cytoplasmic leakage is represented by the slope of theregression line between 30 and 360 min.

0 The initial leakage from ungerminated seeds is low and, in mostinstances, negative (Table I). The negative value may simplyreflect a 30-min lag period in water uptake (9). Seeds which hadbeen dehydrated after 12- or 24-h germination had higher rates ofinitial leakage. The initial leakage of K, sugars, and especiallyprotein had increased in the 12-h germinated seeds. This correlateswith the increased rate of water uptake between 0- and 12-hsamples which may contribute to an apparent increase in initialleakage; the leakage of phosphate and amino acids remained low

4 5 6 (Table I). The initial leakage from 24-h germinated seeds con-tained only a slight increase in K, sugar, and protein beyond the

ibition of birdsfoot trefoil 12-h values, whereas 6- and 20-fold increases in leakage of phos-2-, and 24-h germination. phate and amino acid occurred, respectively. These latter changes)f 250 mg seed determined occurred quite independently of large changes in water uptakeing dry. (Fig. 3) and, therefore, indicate an increased availability of freely

diffusable phosphate and amino acid in dehydration-damagedseeds.The constant rate of leakage between 30- and 360-min imbibi-

tion is a better indicator of membrane permeability than totalleakage after a specific time because the complicating factor ofinitial leakage has been removed. The rate of leakage was slightlydepressed in 12-h samples, as compared to ungerminated seeds,but was greatly increased between 12- and 24-h samples (Table I).The slight depression between ungerminated and 12-h germinatedseeds may be related to the differential rate of water uptake (Fig.2). If it is assumed that the measurement of initial leakage fromungerminated seeds is depressed, relative to 12-h seeds, because ofthe lag period in water uptake, then it must also be assumed that

Se the constant rate of leakage will be proportionately increasedbecause solutes leak from both intracellular and extracellular sites.Therefore, in ungerminated seeds with a slower rate of wateruptake, the estimates of membrane permeability are slight over-estimates. Consequently, the values of 12-h germinated seedswould be expected to be better estimates of membrane permea-bility in undamaged tissue.The rate of water uptake is similar in 12- and 24-h germinated

4 5 6 samples, and consequently differences in the rate of leakagebetween these samples reflect actual differences in permeability.

F-IG. J. l'me protiie o tOtaI 1eCtrOl1Yte leaKage aurung reFmwbDtioln OXbirdsfoot trefoil (L. corniculatus L.) seeds dehydrated after 0-, 12-, and 24-h germination. Values represent the conductivity of 20 ml water duringthe imbibition of 250 mg seed.

nated seeds, which is regulated by the permeability of the seedcoat, was linear for the first 6 h. The rate of uptake by 12- and 24-h germinated seeds was biphasic. Initially, the rate of water uptakewas rapid and the fresh weight of the seed had more than doubledduring the first 30 min of imbibition. Subsequently, the rate ofwater uptake slowed to the point that both groups of seeds were

almost fully hydrated at 6 h.Time Course of Leakage. The leakage of all solutes from the

seeds followed a biphasic pattern with time. As shown in Figure3 for conductivity, the pattern was similar for ungerminated seedsand for 12- and 24-h germinated seeds. Initially there was a rapidefflux of solutes from the seed but, after 30 min, this was replacedby a constant rate of leakage up to 6 h (Fig. 3). Similar patternshave been observed previously (2). This pattern suggests that there

Table I. Initial Leakage of Cytoplasmic Solutesfrom L. corniculatus L.Seeds

The times (0, 12, and 24 h) refer to the length of the pregerminationperiod prior to dehydration and reimbibition. Leakage was measured from250 mg seed imbibed in 20 ml distilled H20 at 20 C. Values represent they intercept (initial leakage) and the slope (rate of leakage) of the regressionline and are expressed per mg seed. K, phosphate, and amino acid areexpressed in nmol; sugar and protein, in ,ug; rates, per h.

Initial Leakage at Rate of Leakage atSolute

Oh 12h 24h Oh 12h 24h

K -0.25 2.2 3.6 0.09 0.06 0.09Phosphate -0.20 0.91 5.2 0.7 0.6 2.04Amino acid -1.3 0.5 10.4 1.4 0.5 9.4Sugar 0.19 1.62 1.73 0.7 0.5 1.2Protein -2.3 11.8 18.6 4.1 0.2 0.1

itz°1.5

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. I

318 Plant Physiol. Vol. 66, 1980

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MEMBRANE STRUCTURE IN SEEDS

The rate of K, phosphate, amino acid, and sugar leakage were allincreased. As observed in the measurements of total leakage, therates of leakage of the individual solutes were not uniformlyincreased. K increased by only 50% and sugar increased by 140%o.The rate of phosphate and amino acid leakage increased approx-imately 4- and 18-fold, respectively (Table I).The leakage pattern of protein does not follow the same general

pattern as the other solutes (Table I). The 12- and 24-h germinatedseeds leaked a large amount of protein initially and subsequentlyvery little (Table I). Ungerminated seeds leaked protein at arelatively high, constant rate (Table I). This leakage pattern withtime is very closely related to water uptake (Fig. 2). These datasuggest that most of the protein leaked from extracellular sites andthat very little is cytoplasmic in origin. Alternatively, cytoplasmicprotein may leak from seeds only when the cellular membranesare below a specific level of hydration. There was no significantdifference in the rate of protein leakage between dehydration-damaged and nondamaged tissues (Table I), suggesting that de-hydration-damaged membranes are not highly permeable to sol-uble proteins.The dehydration-damaged seeds (24 h) still displayed evidence

of selective permeability during rehydration. If cellular collapsehad induced a rupturing of the plasmalemma in such a manner asto destroy its continuity, it would be anticipated that the quantityof a solute in the leachate would reflect its cytoplasmic concentra-tion within the seed. The fact that some aspects of selectivepermeability have been maintained suggests that a general rup-turing of the cellular membranes has not occurred. A reorganiza-tion of the membrane at low levels of hydration may have inducedan increase in membrane permeability. However, these data donot preclude the possibility that the relative concentration ofsolutes in different cellular compartments was altered by dehydra-tion and that the increase in solute leakage was in response to arelative increase in the concentration gradient across the plasma-lemma. In addition, dehydration may not have affected all cellswithin the embryo uniformly which could make leakage measure-ments misleading.

X-ray Diffraction. Simon (13) proposed that the lamellar struc-ture of cellular membranes was altered below moisture contentsof 20%, which has particular significance to the leakage of solutesfrom seeds. Based on the low-angle x-ray diffraction studies ofLuzzati and Husson (7), he suggested that, above 20%o hydration,the phospholipid molecules were packed side by side in a bilayerwith the hydrophilic head groups oriented outwards toward theaqueous layer and the hydrophobic fatty acid tails oriented in-wards. Below 20% hydration, the x-ray diffraction studies indi-cated that the bilayer was rearranged into a hexagonal phasewhich is predominantly hydrophobic but in which a hexagonalarray of "pores," lined with the hydrophilic head groups and filledwith water, would be normal to the membrane surface. Accordingto Simon's model (13), leakage from seeds would occur throughthe water-lined cavities of the membrane until the membrane was

sufficiently hydrated to reform a lamellar structure, which thenwould terminate free diffusion. However, Luzzati and Husson's(7) experiments were performed using lipids from an ether extractof human brain. The phospholipid and fatty acid composition ofthis extract might be markedly different from that found in seeds,and, therefore, the two phospholipid bilayers might be expectedto differ in their hydration requirements. Moreover, hexagonalphases as detailed by Luzzati and Husson (7) have been observedonly in bulk phospholipid extracts and have not been reported tooccur in phospholipid bilayers as thin as a biological membrane.In the bulk extracts, all of the water was contained within thehexagonal array of pores which, on a molecular scale, were ofgreat length. Formation of a thin membrane pierced by shortpores with some water on both sides of the membrane would seem

entropically unlikely since a thin slice of the hexagonal model

would indicate that both hydrophilic head groups and hydropho-bic tails would be exposed to the surrounding water layer.The possibility that cellular membranes from a hexagonal phase

as a result of dehydration was investigated by repeating Luzzatiand Husson's experiments using phospholipid extracted frombirdsfoot trefoil seeds. Phospholipids were extracted from unger-minated and 24-h germinated seeds and were hydrated to differentlevels. Analysis of the phospholipid fraction used in the x-raydiffraction experiments indicated the presence of four major phos-pholipids and trace amounts of phosphatidic acid. Phosphatidyl-choline was the predominant phospholipid in both extracts, ac-counting for 50 to 55% of the total phospholipid. Phosphatidyli-nositol and phosphatidylethanolamine accounted for 25 to 30%oand 15% of the total phospholipid, respectively. Approximately 2to 3% of the total phospholipid was phosphatidylglycerol, and 2%was phosphatidic acid.

Hydrated phospholipids were analyzed by low- and wide-anglex-ray diffraction to determine the repeat spacing, d, and to deter-mine whether the phospholipids were packed into a lamellar or ahexagonal phase. The d spacings in A which represent the repeatdistance within the phospholipid-water preparation are shown inTable II for the ungerminated and 24-h germinated samples at 5,10, 20, and 40% (w/w) water. Each sample produced two firstorder bands, an intense primary band and a weaker secondaryband at a slightly higher d spacing. Higher orders were alsoobserved. As the hydration of the phospholipid increased, the dspacing also increased. Lamellar systems ofone pure phospholipidand water usually increase slightly in thickness (d spacing) aswater content increases (14). When cell membrane extracts areused, there are larger increases in d spacing as water contentincreases (12). The presence of two bands indicates the presenceof two distinct regions or phases of different thickness within agiven sample, presumably due to segregation of different compo-nents into separate lamellar regions. The identity or compositionof these two phases is not known, nor is it possible to predictwhether these phase separations occur in the intact seed mem-brane. However, phase separations on the basis of differences infatty acid saturation or phospholipid head group have been re-ported ( 1 5).

Lamellar packing can be distinguished from hexagonal packingon the basis of the ratio between the first, second, third, and fourthorder Bragg spacings. In a lamellar phase, the ratio is l:1/2:1/3:¼/4,whereas in a hexagonal phase the ratio is 1: 1//3: 4i/'/7 .All phospholipid-water preparations analyzed had a ratio of theBragg spacings characteristic of a lamellar structure. Even at 5%hydration, the ratio of the Bragg spacings was l:1/2:/1:1/4 for bothlipid extracts. There was no evidence for the presence of hexago-nally packed phospholipid at any moisture content examined ineither lipid extract.The diffraction pattern obtained with the toroidal camera for

the 5% water, 24-h sample showed d spacings consistent with

Table II. Bragg Spacings (d) from Low-angle X-ray Diffraction AnalysisLiposomes were prepared from polar lipid extracts of 0- and 24-h

germinated birdsfoot trefoil (L. corniculatus L.) seeds. Values represent thefirst order spacings of the main and secondary bands.

d Spacings

Moisture 0-h Germination 24-h GerminationContent

Main Secondary Main Secondary% X45 46.8 51.6 45.7 49.210 48.7 53.9 48.3 53.120 51.0 56.0 49.0 54.440 55.9 6 1.9 56.7 63.1

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McKERSIE AND STINSON

patterns from the small-angle camera plus a broad wide-anglediffraction ring corresponding to a d spacing of approximately4.6A. This is the typical spacing observed.from the "tails" ofphospholipids when they are in a lamellar phase above the tran-sition temperature, ie. in a lamellar liquid crystal phase.As previously mentioned, the hexagonal phase has been ob-

served only in bulk phospholipid extracts and it would seementropically unlikely that this phase as detailed by Luzzati andHusson (7) could exist in a thin biological membrane. The low-angle x-ray diffraction data reported in this study indicate that ahexagonal phase does not form in bulk phospholipid extracts fromseeds. Since the original experiments of Luzzati and Husson (7)were performed with lipid extracts from human brain, it must beassumed that the observed difference in response to hydration bythe two systems is a result of differences in lipid composition.Consequently, dehydration damage in seeds does not appear tobe related to a hydration-induced lipid phase change in the bulkphospholipid.

Acknowledgments-The authors gratefully acknowledge the technical assistanceof Ms. A. Moore. Facilities provided by the Ontario Ministry of Agriculture andFood are also gratefully acknowledged.

LITERATURE CITED

1. BEWLEY JD 1979 Physiological aspects of desiccation tolerance. Annu Rev PlantPhysiol 30: 195-238

2. BRAMLAGE WJ, AC LEOPOLD, DJ PARRISH 1978 Chilling stress to soybeans

during imbibition. Plant Physiol 61: 525-5293. DITTMER JD, MA WELLS 1969 Quantitative and qualitative analysis of lipids and

lipid components. Methods Enzymol 14: 482-5304. DuBois M. KA GILES, JK HAMILTON, PA REBERS, F SMITH 1956 Colorimetric

method for determination of sugars and related substances. Anal. Chem. 28:350-356

5. HEGARTY TW 1978 The physiology of seed hydration and dehydration, and therelation between water stress and the control of germination: a review. plantCell Environ 1: 101-1 19

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

7. LUZZATI V, F HuSSON. 1962 The structure of the liquid-crystalline phases of thelipid-water systems. J Cell Biol 12: 207-219

8. McKERSIE BD, JR LEPOCK, J KRUUV. JE THOMPSON 1978 The effects ofcotyledon senescence on the composition and physical properties of membranelipid. Biochim Biophys Acta 508: 197-212

9. McKERSIE BD, DT TOMES 1980 Effects of dehydration treatments on germina-tion, seedling vigour. and cytoplasmic leakage in wild oats and birdsfoot trefoil.Can J Bot 58: 471-476.

10. MOORE S. WH STUEIN 1948 Photometric ninhydrin method for use in thechromatography of amino acids. J Biol Chem 176: 367-388

11. NICHOLs BW 1964 Separation of plant phospholipids and glycolipids. In ATJames, ed, New Biochemical Separations. D Van Nostrand. Toronto, pp 321-337

12. RAND RP, V LUZLATI 1968 X-ray diffraction study in water of lipids extractedfrom human erythrocytes. Biophys J 8: 125-137

13. SIMON EW 1974 Phospholipids and plant membrane permeability. New Phytol73: 377-420

14. TORBET J, MHF WILKINS 1976 X-ray diffraction studies of lecithin bilayers. JTheor Biol 62: 447-458

15. WUNDERLICH F, W KREUTZ. P MAHLER. A RONAI, G HEPPELER 1978 Thermo-tropic fluid-ordered "Discontinuous" phase separation in microsomal lipids ofTetrahymena. An x-ray diffraction study. Biochemistry 17: 2005-2010

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