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Plant Physiol. (1989) 90, 955- 0032-0889/89/90/0955/07/$01 .00/
Received for (^) publication September 12, 1988 and in revised form (^) February 11, 1989
Lipid Composition of Plasma Membranes and
Endomembranes Prepared from Roots of
Barley (Hordeum vulgare L.4'
Effects of Salt
Dennis J. Brown*2 and Frances M. DuPont
U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center,
Albany, California 94710
ABSTRACT
Membrane fractions enriched in endoplasmic reticulum (ER), tonoplast and Golgi membranes (TG) and plasma membranes (PM) were prepared from barley (Hordeum vulgare L. cv CM 72) roots and the lipid compositions of the three fractions were analyzed and compared. Plants were grown in an aerated nutrient solution with or without (^100) millimolar NaCI. Each membrane fraction had a characteristic lipid composition. The mole per cent of the individual (^) phospholipids, glycolipids, and sterols in each fraction was not (^) altered when roots were grown in 100 millimolar NaCI. The ER had (^) the highest percentages of phosphatidylinositol and (^) phosphatidylcholine of the three fractions (7 and 45 mole per cent, respectively, of the total lipid). The TG contained the highest percentage of glycosylceramide (13 mole per cent). The PM had the (^) highest percentage of phosphatidylserine (3 mole per cent) and (^) nearly equal percentages of (^) phosphatidylethanolamine ( mole per cent and (^) phosphatidylcholine (18 mole per cent). The most abundant sterols in membranes (^) prepared from barley roots were stigmasterol (10 mole per (^) cent), sitosterol (^) (50 mole (^) per cent), and 24r-methylcholesterol (40 mole per cent of the total sterol). Salt-treated plants contained a slightly higher (^) percentage of stigmasterol than controls. The percentage of stigmasterol increased with age and a simple cause and effect relationship between salt treatment and sterol composition was not observed.
Membrane lipids form a physical barrier to the movement of the water soluble components of cells. They also provide a matrix for membrane transport proteins. At the PM,3 the combination of the (^) lipid barrier and the selective ion trans-
'Funded in part by a postdoctoral award to D. J. B. from the U. S. Department of Agriculture, Agricultural Research Service postdoc- toral research associates (^) program. 2 Present address: Laboratoire de (^) Physiologie Cellulaire Vegetale, Departement de Recherche (^) Fondamentale, Centre d'Etudes Nu-
cleaires de Grenoble, 85 x 38041 Grenoble-C6dex, France.
3Abbreviations: PM, plasma membrane; 2D, two-dimensional; CI- MS, chemical ionization mass spectrometry; DPG, diphosphatidyl- glycerol (cardiolipin); LSIMS, liquid secondary ion mass spectrome- try; lysoPC, lysophosphatidylcholine; lysoPE, lysophosphatidylethan- olamine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phos-
955
porters allows cells to accumulate essential ions while exclud-
ing ions which are toxic. The exclusion of Na+, for example,
is believed to be an important trait of salt-tolerant barley
cultivars (21). Selective transport also occurs at the tonoplast
and other endomembranes. The selectivity ofeach membrane varies with (^) the types of ion channels and pumps that are present, while the efficacy of each membrane as a barrier may
vary with the type and proportion of its lipid components (4,
5, 7). The major lipid components of plant membranes are
phospholipids. glycolipids, and sterols. The specific propor- tions of these lipids in the PM (24, 29, (^) 30) and the (^) tonoplast (25, 32) are known for a (^) variety of plant tissues. Only one
study (34) has examined the lipid composition of both the
PM and tonoplast prepared from a single source. There is some evidence that salt stress can induce (^) changes in plant (^) membrane lipids. A (^) survey of plant species of varying salt-tolerance reported an increase in the ratio of glycolipid to phospholipid in the roots of both a halophyte, Atriplex gme-
linia, and the salt-sensitive cucumber, Cucumis sativa L.,
when plants were grown in increasing concentrations of NaCl
(18). In Citrus roots, the sterol composition was altered in
salt-stressed plants and some of these changes occurred at the
PM (8, 9). It was not known ifsalt affects the lipid composition
of the PM or endomembranes of (^) barley.
Membrane fractions that are enriched in PM, tonoplast,
and ER are prepared by centrifuging a microsomal pellet
prepared from barley roots through a sucrose step gradient
(11, 12). When barley plants are grown in 100 mM NaCl the
distribution of marker enzymes on sucrose gradients does not
change, but the protein compositions of the PM, endomem-
brane, and cytoplasmic fractions are altered (19, 20), and a
Na+/H+ exchange is activated in the tonoplast membranes.
Shoot growth is reduced but the plants show no signs ofinjury
(19). In this study we identify and quantify the phospholipids,
glycolipids, and sterols contained in the three membrane
fractions. The effect of a high but noninjurious concentration
phatidylethanolamine; PI, (^) phosphatidylinositol; PS, phosphati- dylserine; TG, tonoplast and Golgi (^) membranes; 24v-methylcholes- terol, campesterol or dihydrobrassicasterol, orientation of (^) methyl group at carbon 24 not determined.
Plant Physiol. Vol.^ 90, 1989
of salt, 100 mM NaCl, on the lipid composition of each
fraction is also reported.
MATERIALS AND METHODS
Plant Materials
Seeds of barley (Hordeum vulgare L. cv CM 72) were sown
above aerated nutrient solutions (19). Control plants were
grown above a full nutrient solution (13), and salt-grown
plants were grown above a full nutrient solution plus 100 mm
NaCl. Solutions were adjusted daily to pH 5.6 with Ca(OH)2.
Seed germination and root growth was reduced by salt. In
order to obtain 60 g fresh weight of d 7 roots per treatment,
three containers of control plants and four containers of salt-
treated plants were required (400 seeds per container).
Membrane Preparation
Roots were homogenized and membranes were fractionated
by differential centrifugation and sucrose step gradients as
described (12). Membrane fractions (6 mL) were collected
from the sample/22%, 22/30%, and 34/40% interfaces of the
sucrose step gradients. The fractions were washed with a
buffered solution of 150 mm KCI and membranes were pel-
leted in a Beckman4 42.1 rotor at 100,000g. The membrane
pellets were resuspended in 2 mL of buffer consisting of 0.
M sucrose and 2 mm DTT in 5 mm Pipes-KOH (pH 7.2), and
stored frozen at -70°C. The identity and purity of the three
fractions, as defined by enzyme markers and immunoblots of
proteins on 2D gels, has been described (11, 12). The sample/
22% interface was enriched in ER, the 22/30% interface was
enriched in^ tonoplast and^ contained^ some^ Golgi membranes,
and the^ 34/40% interface^ was^ enriched^ in^ PM.
In some experiments, roots of different developmental ages
were prepared from control plants. Root tips are defined here
as the apical 2 cm of the roots and matured root tissue as that
portion of the roots 2 cm or more from the root apex.
The protein in the fractions was assayed by the method of
Lowry et al. (23) after precipitation with TCA.
Lipid Extraction
To inhibit the activity of endogenous lipases, lipids were
extracted from the membrane^ fractions^ with^ a^ mixture of
chloroform and^ isopropanol (22, 33). Isopropanol (2.12 mL)
and chloroform (0.6 mL) were mixed with 0.8 mL of mem-
brane fraction to form a monophasic solution. Insoluble
proteins were sedimented by centrifugation at 10OOg for 3
min and the supernatant was^ drawn^ off. Chloroform^ (3.
mL) and^ 0.1^ M^ KCI^ (0.8 mL) were^ added^ to^ the^ supernatant
to (^) produce a (^) biphasic solution. After (^) thorough mixing, the
phases were separated by centrifugation and the lower phase
was washed 3 times with 1.5 mL aliquots of 0.1 M^ KCI
saturated with chloroform. Proteins which collected at^ the
interface of the two phases were removed with the upper
phase and discarded. The lower phase was dried under a
(^4) Mention of a specific product name by the U.S. Department of Agriculture does not constitute an endorsement and does^ not^ imply a recommendation over^ other^ suitable^ products.
stream of N2 and the lipids were dissolved in 0.5 mL of
chloroform. The samples were stored at -20C until analyzed.
Some samples were^ extracted with mixtures^ of^ chloroform
and methanol. For these samples, the membrane fractions
were heated to 100°C for 2 min or were left untreated prior
to extraction.
Lipid Analyses
Lipids were separated by TLC. TLC plates (Silica gel 60,
20 x 20, 0.25 mm layer thickness, EM Merck) were prerun
in chloroform/methanol (2: 1) and then activated at 1 10C for
60 min.^ The plates were^ developed first^ in^ chloroform/meth-
anol/ammonium hydroxide/water (65:30:2:2) and air-dried.
Then the plate was rotated^900 and^ developed in^ chloroform/
methanol/acetic acid/water (170:25:25:6). Lipids were located
by exposing air-dried plates to l2 vapors. The most abundant
lipids were identified with specific stains for phosphorus,
sugar, primary amine, and sterol substituents (22) and by their
comigration with standards in the 2D solvent system. For
quantitative analysis, phospholipids were scraped from the
TLC plates and analyzed by the method of Bartlett (2). Lipids
which contained sugar substituents were scraped from the
TLC plates, hydrolyzed in 2 N H2SO4 at 100°C for 30 min,
and assayed for sugars (10). Sterols were scraped from the
TLC plates and eluted into chloroform/methanol (2:1). The
solvent was separated from the silica gel by centrifugation and
evaporated under a stream of N2. The sterols were quantified
by the method of Zlatkis and Zak (35).
Lipid dry weights were determined on 100 ,uL of the lipid
extract in chloroform. The samples were dried at 500C, stored
in a desiccator until cool, then weighed. The procedure was
repeated until the samples reached constant weights.
Identification of Sterols
Samples of total membrane lipids were loaded onto silica
gel plates (LK6F, Whatman) and developed in^ chloroform/
acetone/acetic acid (50:50:2). Sterols (RF =^ 0.71) were scraped
from the plate and eluted from the silica gel with chloroform/
acetone (1:1). The solvent was removed under a stream^ of^ N
and the samples were dissolved in 20^ to^40 ,uL chloroform.
Initially, samples were^ analyzed and the^ sterol^ components
were identified by GC-MS^ (17).^ For^ routine^ quantification,
the sterols were analyzed on a HP 5830A gas chromatograph
equipped with a flame ionization detector. The sterols were
separated on a DB 1701 capillary column (15 m; injection
temperature, 250°C; temperature program, 235-2750C^ for^10
min and held at^ 2750C^ for^ 20 min; carrier^ gas, He)^ with
cholesterol as an internal standard.
Identification of Glycosylceramides
As a first step in the^ purification of^ glycosylceramides,
membrane lipids were saponified in 2.0 mL methanolic KOH.
After the saponification step, a biphasic Folch solution (14)
was prepared by adding 1.5 mL 0.1 M^ KCl and 4 mL chloro-
form to the 2 mL^ of methanolic KOH.^ The^ nonsaponified compounds, including the^ glycosylceramides, partitioned into
the lower phase. The upper phase was discarded and the lower
956 BROWN AND DUPONT
Plant Physiol. Vol. 90, 1989
4(
2
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0 2
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LIPID CLASS Figure 1.^ Lipid composition of membrane fractions prepared from barley roots. Barley plants were grown in the presence (solid bar) or absence (open bar) of 100 mm NaCI. A, Composition of the ER fraction; B, composition of the TG fraction; C, composition of the PM fraction. Values are the averages from three replicate experiments with standard errors of approximately 10% of the values shown. Abbreviations: LPC, (^) lysophosphatidylcholine; LPE, lysophosphatidy- lethanolamine; PG, (^) phosphatidylglycerol; PX1,2, unidentified phos- pholipids; DGD, (^) digalactosyldiglyceride; MGD, monogalactosyldigly- ceride; GCM, (^) glycosylceramide; SG, steryl glycoside; ASG, acylated steryl (^) glycoside; ST, sterol.
The grinding mix contained EDTA and had a high (^) pH, two conditions known to limit (^) phospholipase D as well as (^) many other lipolytic enzymes (15). In addition, preliminary (^) experi- ments showed that membrane fractions prepared from barley roots contain^ little phospholipase D^ activity. When methanol, which stimulates (^) phospholipase D (^) activity (33), was (^) added to a microsomal (^) pellet before the (^) lipids were extracted there was no increase in PA above the levels in a control and there was only a small amount (less than 1% of the total phospholipid) of (^) phosphatidylmethanol, another (^) artifact sometimes pro- duced (^) by phospholipase D (^) activity (data not (^) shown). Additional phospholipases or lipolytic acyl hydrolases were present in the membrane fractions. Lipolytic activity was observed in^ experiments when the membrane fractions^ were extracted in (^) chloroform/methanol (2:1) without (^) prior treat-
ment of the membranes at 100°C (Table II). The (^) chloroform/
methanol lipid extracts contained 3 to 5 times more lysoPC,
a product of the hydrolysis, than fractions which were ex-
tracted in chloroform/isopropanol or which were heated to
100°C before extraction with chloroform/methanol. The
greatest content^ of^ lysoPC (5% ofthe total phospholipids) was
in the PM fraction. All other data in this paper are from
extractions and analyses ofheat-treated or chloroform/isopro-
panol-extracted membrane fractions. Small amounts of
lysoPE and lysoPC were still observed on some 2D TLC plates
and the lysophospholipids may have been produced during
the preparation and fractionation of the membranes despite
the precautions which were taken to inhibit lipid-degrading enzymes.
Glycosylceramides
On 2D TLC plates, two spots were identified as glycosyl-
ceramides. These lipids were stable to alkaline hydrolysis and
contained a sugar substituent but no phosphorus, primary
amine, or sterol substituents. LSIMS spectra ofthe glycolipids
contained prominent MNa+ ions at m/z 864.8 and 866.8.
The calculated mol wt for the compounds, 841 and 843, were
similar to those of the monoglycosylceramides of PM and
tonoplast prepared from plant leaves and hypocotyls (24, 29,
34). The glycosylceramides were the most abundant glycolipid
in all three fractions (Fig. 1). The TG fraction contained the
highest percentage of glycosylceramides, which accounted for
15% of the total lipid in this fraction. This amount surpassed
all phospholipids except PC and approximately equaled the
amount of PE and sterol. The glycosylceramides in the ER
fraction accounted for about 10% of the total lipid. In the
PM fraction glycosylceramides were present in nearly equi-
molar amounts with the steryl glycosides and the acylated
steryl glycosides, each accounting for 7% of the total lipid.
Sterols
Five sterols were observed by GC-MS analysis (Fig. 2).
Sitosterol predominated in all three membrane fractions and
accounted for about 50% of the sterol in each. The next most
abundant sterol was 24v-methylcholesterol which accounted
for about 40% of the total sterols. The 24D-methylcholesterol
may be a mixture of campesterol and dihydrobrassicasterol,
as has been determined for other members of the Poaceae
( 17). These sterol epimers vary only in the orientation of the
methyl group on^ carbon 24 and were difficult to separate
chromatographically. Stigmasterol was^ also^ present in^ all^ three
membrane fractions and accounted for as little as 5% of the
sterol in the ER fraction and up to 12% of the sterol in the
PM fraction. Isofucosterol and cycloeucalenol, both interme-
diates of sitosterol biosynthesis (3, 17), were the only other
sterols which were (^) present in (^) large enough quantities to be
detected. This is the first report of cylcoeucalenol in barley
roots. Both were present in the ER and TG fractions. A trace
amount of isofucosterol, but no cycloeucalenol, was present
in the PM fraction.
Small differences in sterol composition were observed be-
tween control plants and plants grown in 100 mm NaCl (Fig.
2). In each membrane (^) fraction, the (^) percentage of (^) stigmasterol
(^958) BROWN AND DUPONT
MEMBRANE LIPIDS FROM BARLEY ROOTS
Table II. Phospholipid Composition of Membrane Fractions Prepared from Barley Roots Membrane fractions were heated to 1 00°C for 2 min (heat) or remained untreated (no heat) prior to extraction of lipids in a mixture of chloroform and methanol. Membrane Fraction Treatmenta ER TG PM Heat No Heat Heat No Heat Heat No Heat mol % LPC 0.7^ ±^ 0.3b 2.2 ±^ 0.8 0.6 ±^ 0.2 3.2 ±^ 1.7 0.9 ± 0.2 5.0 ± 2. PI + LPEC 9.8 ± 2.6 (^) 10.0 ± 2.1 7.4 ± (^) 2.0 8.7 ± (^) 2.5 5.1 ± (^) 0.7 7.6 ± 2. PS 1.5 ± 0.4 1.4 ± 0.4 2.5 ± 0.6 (^) 2.9 ± 0.7 (^) 6.6 ± 1.1 7.1 ± (^) 1. PA 1.0 ± 0.3 1.0 ± 0.3 1.8 ± 0.5 1.9 ± (^) 0.2 5.6 ± 0.7 6.3 ± 0. PC 59.5 ± 13.3 58.0 ± 11.6 57.6 ± 13.1 53.6 ± 12.4 42.7 ± (^) 6.1 36.9 ± (^) 3. PE 20.6 ± 4.9 20.9 ± 3.4 23.2 ± 5.1 23.0 ± 4.5 33.8 ± 5.2 32.1 ± 2. PG 6.9 ± 1.7 6.4 ± 1.2 6.3 ± 1.5 6.0 ± 1.3 3.6 ± 0.7 3.0 ± 0. DPG 0.1 ± 0.1 0.0 ± 0.0 0.6 ± 0.3 0.6 ± 0.3 1.7 ± (^) 0.4 2.0 ± (^) 0. a (^) LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine. bAll (^) values are ± (^) SD. Data are averaged from three replicate experiments. c PI (^) and LPE (^) were not separated by 2D (^) TLC in (^) all experiments and data for these lipids were combined.
LL] w U)
-j 0 LFJ 0
LL zC) bi
a-- 0 wi
tYJIO~ tYL^ - LLJO tYLiI-^ LJI- 1 2 3 4 5 STEROLS Figure 2. Sterol (^) composition of membrane fractions (^) prepared from barley roots.^ Barley plants were^ grown in^ the^ presence (solid bar) or absence (^) (open bar) of 100 mm NaCI. Values are the (^) averages from three replicate experiments with standard errors less than 5% of the values shown. 1, 24v-Methyl cholesterol; 2, stigmasterol; 3, sitosterol; 4, isofucosterol; 5, cycloeucalenol.
was greater in salt-treated plants than in controls. The increase
in the percentage of stigmasterol for the PM fraction was
typical. The percentage of stigmasterol was 1 1% for the con-
trols and 14% for the plants grown in 100 mm NaCl. There
also was a small decrease in sitosterol in salt-treated plants.
No change in the percentage of 24v-methylcholesterol was
detected.
When barley plants were grown in full nutrients and their
roots were apportioned into root tips and matured root tissue,
unique sterol compositions were found for the two tissues
(Fig. 3). There were higher percentages of both sitosterol and
0
w
LLI F--
F-
0
F-L z LLJw^ C)
F-- 0
WHFD L I-Dt L WH(.D^ L WL LJHDL 1 2 3 4 5 STEROLS
Figure 3. Sterol (^) composition of membrane (^) fractions prepared from barley roots^ of^ differing age. Barley roots^ were divided into root tips (solid bar), the^ apical 2 cm^ of^ the roots, and matured^ root tissue (open (^) bar), that (^) portion of the roots 2 cm or more from the root (^) apex. Values are the averages from duplicate experiments. 1, 24-v-Methyl cholesterol; 2, stigmasterol; 3, sitosterol; 4, isofucosterol; 5, (^) cycloeu- calenol.
stigmasterol in^ matured^ root^ tissue^ than^ in^ root^ tips. As^ a
percent of^ total sterols, stigmasterol content was three to five
percentage points higher in matured root tissue than in root
tips and sitosterol content was one to two percentage points
higher. The percentages of 24t-methylcholesterol and isofu-
costerol were highest in root tips. Cycloeucalenol was detected
only in^ matured^ root^ tissue.
DISCUSSION
The plasma membrane and the endomembranes had dis-
tinct lipid compositions, which were maintained despite ex-
959
MEMBRANE LIPIDS FROM BARLEY ROOTS
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