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Lipid Composition in Barley Roots' Membranes: Salt Stress Effects, Study Guides, Projects, Research of Biochemistry

A research study on the lipid composition of plasma membranes, endoplasmic reticulum, and tonoplast membranes prepared from barley roots. The study investigates how salt stress affects the lipid composition of these membranes and identifies the most abundant sterols and phospholipids. The document also discusses the role of membrane lipids in ion transport and the effects of salt stress on membrane protein compositions.

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Plant
Physiol.
(1989)
90,
955-961
0032-0889/89/90/0955/07/$01
.00/0
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
(15
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
of
each
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
if
salt
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
of
injury
(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.
pf3
pf4
pf5

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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

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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
  1. Benveniste P (1986) Sterol biosynthesis. Annu^ Rev^ Plant^ Physiol 37: 275-
  2. Benz R, Cros D (1978) Influence of sterols on ion transport through lipid bilayer membranes. Biochim Biophys Acta 506: 265-
  3. Curatolo W (1987) The physical properties of glycolipids. Biochim Biophys^ Acta^ 906: 111-
  4. Curatolo W^ (1987) Glycolipid function.^ Biochim^ Biophys^ Acta 906: 137-
  5. Dekruijff B, Demel RA, Van Deenen LLM (1972) The effect of cholesterol and epicholesterol incorporation on the permeabil- ity and on the phase transition ofintact^ Acholeplasma^ laidlawii cell membranes and derived liposomes. Biochim Biophys Acta 225: 331-
  6. Douglas TJ (1985) NaCl effects^ on^ 4-desmethylsterol^ composi- tion of plasma membrane-enriched preparations from^ Citrus roots. Plant Cell Environ 8: 687-
  7. Douglas TJ, Sykes SR (1985) Phospholipid, galactolipid, and free sterol composition of fibrous roots from Citrus genotypes differing in^ chloride exclusion^ ability. Plant^ Cell^ Environ^ 8: 693-
  8. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F^ (1956) Colorimetric method for determination of sugars and related substances. Anal Chem^ 28:^ 350-
  9. DuPont FM (1987) Variable effects of nitrate on ATP-dependent proton transport by barley root membranes. Plant Physiol 84: 526-
  10. DuPont FM, Tanaka CK, Hurkman WJ (1988) Separation and immunological characterization of membrane fractions from barley roots.^ Plant^ Physiol^ 85:^ 717-
  11. Epstein E, Norlyn JD^ (1977) Seawater-based^ crop^ production:^ a feasibility study. Science 197: 249-
  12. Folch J, Lees M, Sloan-Stanley GH (1957) A simple method for the isolation and purification oftotal lipids from animal tissues. J Biol Chem 226: 497-
  13. Galliard T (1974) Techniques for^ overcoming^ problems^ of^ lipo- lytic enzymes and lipoxygenases in the preparation of^ plant organelles. Methods Enzymol 31: 520-
  14. Garbarino J, DuPont^ FM^ (1988)^ NaCl^ induces^ a^ Na+/H+^ antiport in tonoplast vesicles from barley roots. Plant Physiol 86: 231- 236
  15. Heupel RC, Sauvaire Y, Le^ PH, Parish^ EJ,^ Nes^ WD^ (1986) Sterol composition and biosynthesis in^ sorghum: importance to developmental regulation. Lipids 21: 69-
  16. Hirayama 0,^ Mihara^ M^ (1987) Characterization of^ membrane lipids of higher plants different^ in^ salt-tolerance.^ Agric^ Biol Chem 51: 3215-
  17. Hurkman WJ, Tanaka CK (1987) The effects of salt on^ the

pattern of protein^ synthesis^ in barley^ roots.^ Plant^ Physiol^ 83: 517-

  1. Hurkman WJ, Tanaka CK, DuPont FM (1988) The effect of salt stress on polypeptides in membrane fractions^ from^ barley roots. Plant^ Physiol^ 88:^ 1263-
  2. Jeschke WD (1984) Na+-K+ exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In RC Staples, GH Tonniessen,^ eds,^ Salinity^ Tolerance^ in Plants. Wiley and Sons, New York, pp 37-
  3. Kates M (1972) Techniques of lipidology isolation, analysis and identification of lipids. In TS Work, E Work, eds, Laboratory Techniques in Biochemistry and Molecular^ Biology. Elsevier, New York
  4. Lowry^ OH,^ Rosebrough^ NJ,^ Farr^ AL, Randall RJ^ (1951)^ Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-
  5. Lynch DV, Steponkus PL^ (1987) Plasma membrane^ lipid^ alter- ations associated with^ cold^ acclimation of winter^ rye^ seedlings (Secale cereale L. cv Puma). Plant Physiol 83: 761-
  6. Marty F, Branton D^ (1980) Analytical^ characterization^ of beetroot vacuole membrane. J Cell Biol 87: 72-
  7. Moore TS (1982) Phospholipid biosynthesis. Annu Rev Plant Physiol 33: 235-
  8. Mudd JB (1980) Phospholipid biosynthesis. In PK Stumpf, ed, The Biochemistry of Plants-A Comprehensive Treatise. Ac- ademic, New York, pp 250-
  9. Quarles RH, Dawson RMC (1969) The distribution of phospho- lipase D in developing and mature plants. Biochem J 112: 788-
  10. Rochester CP, Kjellbom P,^ Andersson^ B,^ Larsson^ C^ (1987)^ Lipid composition of plasma membranes isolated from light-grown barley (Hordeum vulgare) leaves: identification of cerebroside as a^ major component. Arch^ Biochem^ Biophys^ 255:^ 385-
  11. Rochester CP, Kjellbom P, Larsson C (1987) Lipid composition of plasma membranes from barley leaves^ and^ roots,^ spinach leaves and cauliflower inflorescences. Physiol Plant 71: 257- 263
  12. Thompson TE, Tillack^ TW^ (1985)^ Organization of glycosphin- golipids in^ bilayers^ and^ plasma^ membranes^ of^ mammalian cells. Annu Rev Biophys Chem 14: 361-
  13. Verhoek B, Hass^ R, Wrage K,^ Linscheid^ L, Heinz^ E^ (1983) Lipids and enzymatic activities in vacuolar membranes isolated via protoplasts from oat primary leaves. Z Naturforsch 38c: 770-
  14. Yang SF, Freer^ S,^ Benson^ AA^ (1967)^ Transphosphatidylation^ by phospholipase D. J Biol Chem 242: 477-
  15. Yoshida S, Uemura M (1986) Lipid composition of plasma membranes and^ tonoplasts^ isolated^ from^ etiolated^ seedlings^ of mung bean ( Vigna radiata L.). Plant Physiol 82: 807-
  16. Zlatkis A, Zak B (1969) Study of a new cholesterol reagent. Anal Biochem 29: 143-

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