Journal of Experimental Botany, Vol. 47, No. 294, pp. 17-23, January 1996
Journal of
Experimental
Botany
Lipid changes in soybean root membranes in response to
salt treatment
A. Surjus1 and M. Durand
Laboratoire de Recherches sur les Substances Naturelles V6g6tales, Universite Montpellier II, Sciences et
Techniques du Languedoc, Place E. Bataillon, F-34095 Montpellier cedex 5, France
Received 10 March 1995; Accepted 27 September 1995
Abstract
Introduction
Soybean seedlings were grown hydroponically in a
nutrient medium with or without 25 mM NaCI. An analysis of proteins, phospholipids, total fatty acids, and
sterols was performed at the root level in the whole
tissue extract, in the crude microsomal fraction and
in the plasma membrane fraction purified by twophase partitioning. The free and conjugated forms of
sterols were analysed. Most of the sterols were found
in the free form, whereas steryl glycosides (SG) were
detected in small quantities and steryl esters (SE) did
not occur in the membrane fractions. The compositions in various moieties of sterols were quite similar
among the three materials, i.e. sitosterol was the major
sterol accompanied by campesterol, stigmasterol and
small quantities of cholesterol and isofucosterol. No
changes were detected in the whole root tissues after
salt treatment. Surprisingly, the main lipid changes
were observed in the microsomal fraction where the
phospholipid and sterol content decreased by 50%,
accompanied by an increasing saturation of the total
fatty acids. Only this last parameter was altered in the
plasma membrane fraction and to a lesser extent. The
saturated fatty acids (16:0) and (18:0) which both
accounted for 49% of the total fatty acids in the plasma
membrane increased 56% under salt stress. The
different responses of the lipid classes induced by
salt treatment and their possible significance are
discussed.
The response of plants to salt excess is complex and
involves changes in their morphology, physiology and
metabolism. It is now generally accepted that the first
deteriorating change during stress injury is an alteration
in the structure and function of cell membranes. In many
plants, alterations in lipids, particularly in phospholipids
and sterols were observed as a result of water stress
(Liljenberg, 1992) or salt treatment (Kuiper, 1984).
Changes in sterols, phospholipids and fatty acids may
contribute, in the membrane, to the control of fluidity
and the micro-environment surrounding proteins, which
in turn influences membrane functions such as bilayer
permeability (Schuler et al., 1991), carrier-mediated transport (Deuticke and Haest, 1987) and the activity of
membrane-bound enzymes including ATPase activity
(Cooke and Burden, 1990). Although there have been
many attempts to establish a causal relationship between
lipid alteration and salt stress in plants, the results are
often controversial. Therefore, in many of the studies the
lipid analysis was limited to the whole tissues or isolated
crude extract preparations.
The root cell is the key site of the plant's interaction
with salt in the surrounding medium. Previous works
have demonstrated the central role of roots in the salt
sensitivity of soybean (Lacan and Durand, 1995). The
aim of the present study was to characterize the changes
in root membrane composition in response to salt excess
in the growth medium of a soybean cultivar sensitive to
salinity. The lipid composition of the plasma membrane
fraction is presented and compared to the lipid composition of the microsomal fraction and to the whole root
extract. A moderate concentration of NaCI (25 mM) was
Key words: Glycine max, membrane lipids, root, salinity.
1
To whom correspondence should be addressed. Fax: +33 67 14 30 31.
Abbreviations: DW, dry weight; FS, free sterols; MF, microsomal fraction; PM, plasma membrane; PPL, phospholipids; SE, steryl esters; SG, steryl
glycosides plus acetylated steryl glycosides. Fatty acids are denoted by the number of C atoms and double bonds, respectively.
© Oxford University Press 1996
18 Surjus and Durand
chosen, as a higher concentration leads to a disturbance
of plant growth (Durand and Lacan, 1994).
Materials and methods
Plant material
Soybean (Glycine max L., Merr. cv. Hodgson) was germinated
as described by Durand and Lacan (1994) and the seedlings
were transplanted in a nutrient solution containing Knop's
macroelements, Berthelot's microelements and 0.02 M
FeKEDTA. The salinity treatment was imposed 1 d after
transplanting by adding 25 mM NaCl. The photoperiod of the
plant chamber was 16 h and the photon flux density was 230 pE
m ~ 2 s ~ ' . Temperatures in the light and dark were 25°C and
22 °C, respectively. Roots from 8-d-old plants were harvested,
rinsed with cold water and used immediately for plasma
membrane preparation or frozen in liquid N 2 and freeze-dried
for studies on root tissue lipids and analysis of protein content.
MES, p H 7 , 5mM IDP, 5 mM MgSO 4 , 0.7 mM Na 2 MoO 4 .
The protein content was determined taking aliquots of membrane suspensions or of an extract of root tissue prepared with
0.1 N NaOH. The analysis was performed according to Bradford
(1976) with bovine serum albumin (1 g I" 1 ) as a standard.
LJpid extraction
An appropriate amount of betulinol (1 g I" 1 ) used as an
internal standard was added to the freeze-dried tissues and
membrane suspension prior to extraction. The freeze-dried root
samples were reimbibed and extracted three times with chloroform :methanol (1:2, v/v). The lipid extract was washed with
0.88% (w/v) K G then evaporated and the residue redissolved
in 5ml of chloroform:methanol (1:2, v/v). The membrane
suspensions were extracted according to Hartmann and
Benveniste (1987). The lipid extracts were stored at 4°C in
teflon-capped glass test tubes and used after a few days for
lipid analysis.
Sterol analysis
Microsomal and plasma membrane preparation
Plasma membranes were prepared using the two-phase aqueous
polymer technique. Roots (50 g) were cut into pieces and
immediately ground using a mortar and pestle in two volumes
of the isolation medium containing 25 mM TRIS-MES, pH 7.8,
0.25 M sucrose, 0.2 mM PMSF, 2.5 mM DTT, and 3 mM
EDTA. The homogenate was filtered through two layers of
miracloth and centrifuged for 30 min at 80 000 g to yield a
microsomal pellet which was resuspended with 5 ml of 5 mM
K-phosphate buffer, pH 7.8, 0.25 M sucrose. The microsomal
suspension (1 g) was loaded on to 7 g of Dextran T-500PEG 3350 mixture in order to give an 8 g phase system
containing 6.4% (w/w) Dextran T-500, 6.4% (w/w) PEG 3350
[according to Sandelius et al. (1986)] in 5 mM K-phosphate
buffer, pH 7.8, 0.25 M sucrose. The mixture was vigorously
homogenized by shaking and centrifuged for 5 min at 2500 g.
The plasma membrane-rich upper phase was removed and
washed twice with a fresh lower phase. Finally, the upper phase
was diluted 4-fold in 5 mM TRIS-MES, pH 6.5, 0.25 M sucrose,
2.5 mM DTT, and then pelleted for l h at 100 000 g. The
resultant plasma membrane pellet was resuspended in 2.5 mM
TRIS-MES, pH 6.5, 0.25 M sucrose, 1 mM DTT, 20% (w/v)
glycerol and stored at - 2 0 ° C .
Enzyme activities and protein determination
Nucleoside phosphate hydrolysis was performed in a 0.5 ml
reaction volume and was started by the addition of 20 yX
membrane suspension (15-20 ^g protein). After 20 min at 37 °C
the reaction was stopped by the addition of 1 ml Ames's reagent
(1966) containing 0.1% (w/v) SDS. The colour was allowed to
develop for 30 min, and then was stopped by the addition of
10% (w/v) sodium citrate. The absorbance was noted at 820 nm.
The assay medium of the plasma membrane Mg-ATPase
activity containing 30 mM TRIS-MES, pH 6.5, 5 mM TRISATP, 5 mM MgSO 4 , 100 mM KC1, 0.7 mM Na 2 MoO 4 , 0.025%
(w/v) Triton X-100, with or without 0.3 mM Na 2 VO 4 .
Tonoplastic contamination was estimated by NOf-sensitive
Mg-ATPase whose activity was measured at pH 8 in the same
basal assay medium of plasma membrane Mg-ATPase with
KNO 3 in place of KC1. Mitochondrial contamination was
estimated by N^-sensitive Mg-ATPase measured at pH 8.5 with
1 mM NaN 3 . Golgi 'concentration' was estimated by latent
IDPase; the latency was tested by the addition of 0.025% (w/v)
Triton X-100 to the assay medium containing 30 mM TRIS
Free and conjugated sterols were separated from 4 ml of the
lipid extract by thin layer chromatography (TLC) on silica gel
plates using a solvent system of n-hexane: ethyl acetate (60:40,
v/v). The Rfi of the FS and SE were, respectively, 0.5 and 0.8
whereas the SG (including steryl glycosides plus acylated steryl
glycosides) stayed at the origin. The sterols were viewed under
a UV light after spraying the plates with 0.1% (w/v) berberine
in 95% ethanol. The free sterols (FS), steryl esters (SE) and
steryl glycosides (SG) were identified by co-chromatography
with known reference standards, scraped and eluted with
chloroform: methanol (1:2, v/v). An internal standard of
betulinol was added to the SE and SG extracts. The SE were
saponified with 10% (w/v) KOH in 95% ethanol by heating
them under reflux for 30 min. The SG were hydrolysed with
0.18 N H 2 SO 4 in 95% ethanol under reflux for 12 h. The sterols
were then extracted twice with diethyl ether, washed with water
and rechromatographed using TLC with chloroform: diethyl
ether ( 9 : 1 , v/v). The sterols were viewed and collected as
before. The 4-desmethylsterols from the three sterol fractions
were acetylated with pyridine: anhydride acetate (1:2, v/v) for
12 h at room temperature. The acetate derivatives were analysed
by gas chromatography (GC) on a DELSI DI700 gas
chromatograph using a flame ionization detector and a
30 m x 0.25 mm fused silica capillary column coated with
polydimethylsiloxane. The operating conditions were: column
temperature 270 °C, flash heater and detector 280 °C, H 2 was
the carrier gas. Compound identifications were made on the
basis of the retention time relative to known standards; they
were quantified using a CR4A Shimadzu reporting integrator.
Phospholipid and fatty acid analysis
The phospholipid content was quantified from the phosphorus
content as described by Ames (1966) using L-a-phosphatidylcholinedilauroyl (0.51 jimol 1"') as a standard. The total fatty
acids analysis was performed after saponification of 0.5 ml of
the lipid extract with 0.5 N NaOH in methanol for 10 min and
methylation with BF 3 in methanol (Merck) for 2 min by heating
under reflux. The fatty acid methyl esters were extracted with
/j-hexane, and analysed by GC with a 30 m x 0.25 mm column
composed of polyethylene glycol. The analysis was programmed
from 180°C to 240 °C with a temperature rise of 4°C min" 1 ,
flash heater and detector were, respectively, 240 °C and 250 °C.
Statistical analysis was performed using the arialysis of
variance.
Lipids in salt-stressed soybean
Results
Table 1 summarizes the soluble protein, phospholipid and
sterol content expressed asmgg" 1 dry weight (DW) of
root tissue. The sterols were present as FS, SE and SG
including acylated steryl glycosides plus steryl glycosides,
but the FS class was the main sterol class (96% of total
sterols). A mild salt treatment of 25 mM NaCl induced
no change in these root characteristics. Additionally, this
table gives the molar ratio of FS to PPL which also
appeared to be significantly unchanged.
The 4-desmethylsterols identified in the roots were
(in the order of their relative abundance) sitosterol>
stigmasterol > campesterol > isofucosterol > cholesterol
(Table 4). The relative abundance of these primary components was almost the same among sterol classes (data
not shown). The salt treatment induced some significant
changes in the proportion of these compounds: the proportion of campesterol and stigmasterol decreased,
respectively, by 13% and 6%, whereas the rate of sitosterol
increased by 5% (Table 4). Consequently, the
(cholesterol + campesterol) to (sitosterol + stigmasterol)
molar ratio showed significant decrease between 0.154
and 0.132, whereas the sitosterol to stigmasterol showed
significant increase between 2.149 and 2.372.
Table 2 shows the distribution of various membrane
markers assayed on the microsomal fraction (MF) of the
root or on the plasma membrane (PM)-enriched fraction
after phase partitioning. The plasma membrane fraction
was enriched in vanadate-sensitive Mg-ATPase by greater
19
than 2.6 in the control and 3.7 in the salt-treated as
compared with the microsomal fraction. Moreover, low
activities were found for the NO^"-sensitive Mg-ATPase
and N^~ -sensitive Mg-ATPase in the plasma membrane
from both control and salt-stressed roots, indicating few
contaminations with tonoplastic and mitochondrial fractions. Only the latent IDPase activity was still unchanged
after two-phase partitioning in the microsomal and
plasma membrane fractions.
The phospholipid and sterol content of both fractions
was examined (Table 3). The PM fraction was shown to
contain a higher level (expressed as ^gmg" 1 protein) of
phospholipids and sterols compared to the MF. The
sterols remained mainly in free form in both fractions
with percentages of 92% and 85% of total sterols, respectively, in the microsomal and plasmalemma fractions.
However, the rate of SG was increased in the membrane
fractions compared to the tissue whereas the SE were not
detected. Saline treatment induced no significant change
in the phospholipid and sterol contents of the plasma
membrane fraction, whereas these parameters decreased
by about 2-fold in the microsomal membrane fraction.
On the contrary, the molar ratio of FS to PPL appeared
to be significantly unchanged by salt treatment in the two
membrane fractions. The decrease of the sterol content
in the microsomal fraction concerned the FS as well as
the SG leading to stable relative proportions of these
forms. Additionally, the protein content of membrane
fractions (expressed asmgg" 1 root DW) was unaffected
by salt treatment.
Table 1. Soluble protein, phospholipid, free and conjugated sterols content (mg g ' DW) and molar ratio of FS to PPL in soybean
roots subjected or not to 25 mM NaCl
Values in brackets are proportion (weight %) with respect to total sterols. The molar ratio of FS to PPL was calculated using, respectively, the
molecular weight of sitosterol and phosphatidylchohne. Results are means for three independent experiments. Means of salt-stressed plants are not
significantly different from control at P<0.05.
Proteins
FS/PPL
Sterols
PPL
Control
30.4 ±1.2
17.6 + 3.1
Treated
28.8 ±0.7
20 3 ± 1.3
FS
SG
SE
4.11 ±0 10
(96)
3 46 + 0.08
(95)
0.12±0.02
(3)
0.10±0.01
(3)
0.05±0.01
0)
O.O7±OO3
(2)
O.36±O.ll
0.25 ±0.01
Table 2. Specific activities [fjjnol mg ' protein min ' / of marker enzymes in the microsomal and plasma membrane fractions of roots
from control and salt-stressed soybean plants
Vanadate inhibition is given as % inhibition for 0.3 mM vanadate. ND not detectable. Results are means±SD for three independent experiments
Marker enzymes
Vanadate-sensitive Mg-ATPase
Vanadate inhibition
Latent IDPase
No 3 " -sensitive Mg-ATPase
N3"-sensitive Mg-ATPase
Control
Treated
MF
PM
MF
PM
0.188±0.017
89±3.7
0 120 ±0.007
0.017 ±0.008
0.038 ±0.011
0.4% ±0.074
95.6±6.2
0.128 ±0.037
0.011 ±0.012
0.003 ±0.003
0 182 ±0.025
81.1 ±3.7
0.101 ±0.045
0 026 ±0.008
0 029 ±0.002
0.683 ±0.064
93.1 ±0.7
0.108 ±0.055
ND
0.013 ±0.008
22
Surjus and Durand
increased to 85% for the microsomes and 56% for the
plasmalemma. It is well known that phospholipid fatty
acids affect membrane properties in many ways, for
instance, in changing fluidity (Cooke and Burden, 1990),
membrane permeability (Spychalla and Desborough,
1990) or enzyme activity (Kasamo, 1990). It has also
been suggested that the degree of saturation of the acyl
chain and its length are important parameters in the
regulation of the activity of membrane proteins, in particular, ATPase activity (Palmgren et ai, 1988; Kasamo,
1990). Traditionally, the degree of unsaturation of the
fatty acids has been interpreted in terms of fluidity,
however, it is unlikely that the fluidity of the bulk lipid
phase has any important effect on the function of the
membrane proteins (Lee et ai, 1989).
The FS and SG level decreased in the MF, but stayed
unchanged in the PM in response to salt stress (Table 3).
In the literature, the FS either increased or decreased in
concentration depending on the duration and severity of
salt stress as well as the sensitivity of the plant. However,
a decrease seemed to be a characteristic of the saltsensitive plant (Kuiper, 1985). The molecular structural
feature of FS allows them to interact with the phospholipid core to regulate the membrane properties.
Phytosterols affect the lipid bilayer, increasing the fluidity
of membranes which were below their phase transition
temperature and decreasing the fluidity of membranes
which were above their phase transition temperature
(Henessey, 1992, and references herein). Hence, the effect
of FS decrease in the microsomal fraction was hardly
noticeable without knowing the phase transition
temperature.
In spite of the changes induced by salt treatment in the
microsomal fraction concerning the PPL and FS content,
the molar ratio of FS to PPL appeared significantly
constant. The same result was observed in the plasma
membrane fraction owing to an unchanging phospholipids and free sterols content. This could suggest that the
physical state of the membranes remained unchanged and
that the lipid composition was strongly regulated.
The 4-desmethylsterols composition of the two types
of membranes was similar to the composition in the crude
root extract, but in the root extract, salt stress did not
induce major changes in the proportion of these sterols
at the membrane level leading to the stability of the
(cholesterol +campesterol) to (sitosterol + stigmasterol)
as well as the sitosterol to stigmasterol ratio. A decrease
in the sitosterol to stigmasterol ratio is an index of
metabolic disorder (Guye, 1988). The stability of the
sterol composition in membranes can help to overcome
the injuries to the lipid bilayer caused by salinity, so the
plant has, at least in part, the capacity to adjust to
adverse conditions.
In conclusion, the main lipid change induced by a
moderate salt treatment in the plasma membrane fraction
was a change in the fatty acids composition and a
concomitant increase in the degree of saturation. This
change must have profound implications for membrane
functioning under salt stress. On the contrary, various
changes occurred at the microsomal level indicating that
an endomembrane was responding to the stress, as
plasmalemma only forms a small percentage of the root
microsomal membrane.
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