全 文 ：Received 6 Jun. 2003 Accepted 1 Sept. 2003
Supported by the State Key Basic Research and Development Plan of China (G1998010100) and the National Natural Science Foundation
of China (0833193A).
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http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 211-215
Alternation in Lipid Composition of Wheat Leaves Induced by Phosphate
Deficiency Is Related to Both Lipid Biosynthesis and
Phosphatidylglycerol Degradation
YANG Wen, FENG Fu-Ying, HOU Hai-Tong, JIANG Gui-Zhen, XU Yi-Nong*, KUANG Ting-Yun
(Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: In this study, the causes of the changes in lipid composition induced by different phosphate
nutrient levels were investigated. Wheat plants were grown in phosphate-deficient and phosphate-
sufficient conditions, respectively, and lipid compositions in the leaves of 9-day-old and 16-day-old plants were
analyzed. We found that phosphate deficiency induced a dramatic change at the lipid levels in photosynthetic
membranes of wheat leaves and the extent of changes in lipid composition depended on the leaf ages.
Phosphate deficiency induced a gradual decrease in PG and MGDG and a concomitant increase in DGDG
and SQDG from the first leaf to the second and the third leaf on 16-day-old plants. In addition, as compared
to leaves grown under phosphate sufficient solution, PG content in the first leaf of 16-day-old plants was
significantly lower than that of 9-day-old leaf with 2.5 mol% versus 5.5 mol% when these plants were grown
under phosphate deficient condition. From these results, it is suggested that the alternation in lipid
composition in wheat leaves induced by phosphate deficiency is related to both lipid biosynthesis and PG
degradation. PG decrease in younger leaves is mainly due to insufficient phosphate supply for PG biosynthesis,
while PG degradation mainly resulted in the PG decrease in older leaves.
Key words: phosphate transport; phosphate deficiency; thylakoid membrane lipids; phosphatidylglycerol;
wheat leaf
The reaction process of photosynthesis depends on
highly organized pigment protein complexes that are em-
bedded in the polar glycerolipid matrix of thylakoid mem-
branes inside chloroplasts in the higher plants (Essigmann
et al., 1998). Therefore, glycerolipids play an important role
in maintaining the structure and function of protein
complexes. Thylakoid membranes consist of four
glycerolipids: monogalactosyl diacylglycerol (MGDG),
digalactosyl diacylglycerol (DGDG), phosphatidylglycerol
(PG) and sulfoquinovosyl diacylglycerol (SQDG)
(Siegenthaler and Murata, 1998a).
In general, lipid composition is highly conserved in
higher plant photosynthetic membranes (Benning, 1998). It
has been shown that phosphate deficiency strongly af-
fected lipid composition of the thylakoid membrane by de-
creasing the relative content of PG and increasing that of
DGDG and SQDG in both photosynthetic bacteria
(Benning et al., 1993; Güler et al., 1996) and Arabidopsis
thaliana (Essigmann et al., 1998). In addition, the culture
condition of phosphate deficiency resulted in an increase
of SQDG and a decrease of PG in Chlamydomonas
reinharditti (Sato et al., 2000a). These results corroborated
that plants were able to respond to phosphate deficiency
by a selective accumulation of SQDG and DGDG to a lesser
extent (Härtel et al., 1998).
Phosphate is a key compound in metabolic processes in
plants, including energy transfer, photosynthesis, respira-
tion and lipid biosynthesis (Raghothama, 2000). Higher
plants possessed two distinct pathways for the synthesis
of glycerolipids in photosynthetic membranes, the prokary-
otic pathway in inner envelope of the chloroplasts and the
eukaryotic pathway in endoplasmic reticulum (Ohlrogge
and Brows, 1995). PG in photosynthetic membranes is the
only product of the prokaryotic pathway, and the remain-
ing chloroplast lipids MGDG, DGDG and SQDG in many
plants including wheat, are synthesized entirely by the eu-
karyotic pathway. In addition to enzymes, some compo-
nents containing phosphate are used as substrates in
glycerolipid biosynthesis through both pathways. For
example, both pathways begin with the synthesis of phos-
phatidic acid (PA) and phosphatidylcholine (PC) are the
substrate for MGDG and SQDG synthesis in the eukaryotic
pathway (Siegenthaler and Murata, 1998b). Therefore,
changes in the lipid composition induced by phosphate
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004212
deficiency are involved in lipid biosynthesis.
It is well known that under phosphate deficiency the
phosphate in older leaves can be transported to younger
leaves (Mimura, 1995). PG is the sole phospholipid in thyla-
koid membranes. A lot of studies have demonstrated that
PG plays an important role in the structure and function of
thylakoid membranes (Hagio et al., 2000; Kruse et al., 2000;
Sato et al., 2000b; Hagio et al., 2002; Xu et al., 2002a;
Gombos et al., 2002). So far, there is little knowledge about
whether phosphate in PG is able to remobilize from older
leaves to younger ones.
In the present study, the causes of the changes in lipid
composition of wheat leaves induced by phosphate defi-
ciency were investigated. We found that phosphate defi-
ciency induced drastic changes at level of lipids in wheat
leaves, which are not only involved in lipid biosynthesis
but also in PG degradation.
1 Materials and Methods
1.1 Materials
Seeds of winter wheat (Triticum aestivum var. Zhongyou
9507) were soaked for 5 h in distilled water and were germi-
nated at 25 ℃. Seedlings were cultured in Hoagland solu-
tions containing 0.01 mmol/L and 1.0 mmol/L KH2PO4,
respectively, in a controlled environment chamber under a
14 h photoperiod (400 mmol.m-2.s-1) at 25 ℃/20 ℃ (day/
night). Nutrient solutions in the hydroponic systems were
changed every two days. The first fully expanded leaves of
9-day-old plants and the first, the second and the third
expanded leaves of 16-day-old plants were used in this
investigation.
1.2 Lipid extraction and separation
Wheat leaves were ground into powder in liquid
nitrogen. Lipids were extracted according to the method of
Bligh and Dyer (Bligh and Dyer, 1959). Lipid extracts were
separated into individual lipid classes by two-dimensional
silica gel TLC (G model, 10 cm×10 cm, Qingdao Oceanic
Chemical Plant). TLC plates were developed with acetone/
toluene/water (91:30:8, by volume) in the first direction and
with chloroform/methanol/isopropylamine/concentrated
ammonia (65:35:0.5:5, by volume) in the second direction.
Spots were visualized by spraying the plates with 0.01%
primuline in acetone/water (60:40, by volume) under UV
(360 nm).
1.3 Fatty acid analysis
The fatty acid analysis was carried out according to the
method of Xu et al. (2002b). Individual lipids separated by
TLC were transesterified to the fatty acid methyl esters
with 5% H2SO4 in methanol at 85 ℃ for 1 h. The fatty acid
methyl esters were extracted with hexane and were sepa-
rated on a Hewlett-Packard 6890 gas chromatography sup-
plied with a hydrogen flame ionization detector and a cap-
illary column HP INNOWAX (30 m; i.d. 0.25 mm). The col-
umn was run at 170-210 ℃ (5 ℃/min). Heptacanoic acid
(17:0, from Sigma) was used as the internal standard. The
relative content of individual lipids was demonstrated by
molar percent (mol/%).
2 Results
The major polar glycerolipids detected in leaves of
higher plants were MGDG, DGDG, PG, phosphatidyletha-
nolamine (PE) and phosphatidylcholine (PC) (Siegenthaler
and Murata, 1998b). Phosphate deficiency induced a de-
crease in the relative content of all phospholipids and a
concomitant increase in the relative content of other lipids
(data not shown).
In higher plant leaves, MGDG, DGDG, SQDG and PG are
mainly distributed in thylakoid membranes (Siegenthaler
and Murata, 1998a). Therefore, it is generally considered
that the levels of these four kinds of glycerolipids in the
leaves may represent those in thylakoid membranes. In this
study, we focused on the effect of phosphate deficiency
on the composition of these four lipids. The relative amount
of PG in leaves of seedlings grown in phosphate-deficient
solution decreased approximately 45 mol% and those of
SQDG and DGDG increased about 23 mol% and 10 mol%,
respectively, as compared to those of PG, SQDG and DGDG
in plants grown in phosphate-sufficient solution (Fig.1).
The phosphate in older leaves may be transported to
younger leaves under phosphate deficiency conditions
(Mimura, 1995). To investigate whether the phosphate in
PG can be reutilized under the phosphate-deficient
condition, it is necessary to know the changes in lipid com-
position among different leaves during wheat plant growth.
Fig.1. Composition of four lipid classes in the leaves of the 9-
day-old wheat grown in Hoagland solutions containing 1.0 mmol/
L (solid bars) and 0.01 mmol/L (blank bars) phosphate. Values
represent the means of three independent measurements. Error
bars are indicated.
YANG Wen et al.: Alternation in Lipid Composition of Wheat Leaves Induced by Phosphate Deficiency Is Related to Both
Lipid Biosynthesis and Phosphatidylglycerol Degradation 213
Therefore, we analyzed the relative amounts of lipids in the
different leaves on 16-day-old wheat plants, among which
the third leaf was just fully expended and the second and
first leaves had been expended for 3 and 7 d, respectively.
When wheat plants were grown in phosphate-sufficient
solution, no difference in lipid composition was found
among different leaves (data not shown). In this culture
condition, the relative amounts of PG, MGDG, DGDG and
SQDG were about 10 mol%, 51 mol%, 33 mol%, and 6 mol%,
respectively (Fig.2). It was very possible that in phosphate-
sufficient solution there was phosphate available sufficing
for plant growth and development. When wheat plants were
grown in phosphate-deficient solution, the levels of indi-
vidual lipid classes were apparently different among leaves.
Phosphate deficiency induced a gradual decrease in PG
and MGDG and a concomitant increase in DGDG and SQDG
from the first leaf to the third leaf (Fig.2). Among these four
lipid classes, the most obvious change was the PG content
which decreased by approximately 19 mol%, 58 mol% and
75 mol% for the third, second and first leaf, respectively,
grown in phosphate-deficient solution as compared to that
grown in phosphate-sufficient solution.
Despite phosphate-deficient condition affected signifi-
cantly the composition of lipids among the leaves, the level
of individual lipids extracted from all leaves in 16-day-old
plants was almost the same as those from leaves on 9-day-
old plants (Fig.3). This means that the relative higher level
of PG in younger leaves on 16-day-old plants induced by
phosphate deficiency is the results of the PG re-distribu-
tion among leaves, i.e. the phosphate in PG can transport
from older leaves to younger ones.
3 Discussion
It has been reported that phosphate was able to be re-
mobilized from older leaves to younger leaves to ensure
the preferential growth of younger leaves (Mimura, 1995).
Our results suggest that in phosphate-deficient wheat
plants, phosphate in PG can be also remobilized from older
leaves to younger leaves. First, the relative content of PG
in the first leaf on 16-day-old plants was significantly lower
than that on 9-day-old plants with 2.5 mol% versus 5.5
mol% (Figs.1, 2). Second, the relative content of PG in the
third leaf (7.9 mol%) was higher than that in the second leaf
(4.1 mol%) and the first leaf (2.5 mol%) (Fig.2). Third, the
level of individual lipids from all leaves on 16-day-old plants
was almost the same as that from the leaves on 9-day-old
plants (Fig.3). These results suggest that phosphate in PG
can be transported from older leaves to younger leaves
when phosphate was a limiting factor for wheat plant
growth.
About 85% of PG in higher plant leaves is mainly present
in chloroplast and the remains are located in extraplastidic
membranes (Browse et al., 1986). Someone may argue that
phosphate remobilization in PG may be not involved in the
photosynthetic membrane PG. However, under phosphate-
deficient condition PG in the first leaf on 16-day-old plants
decreased by 54.9 mol%, as compared to that in the same
leaf on 9-day-old plants (Figs.1, 2), which was far much
higher than the proportion of extraplastidic PG. This means
that the photosynthetic membrane PG mainly contributes
to the loss of PG in older leaves
Phosphate deficiency induced a decrease in the amounts
of both PG and MGDG, with a concomitant increase in those
of DGDG and SQDG (Figs.1, 2). The similar phenomena
were also observed in photosynthetic bacteria (Benning et
al., 1993; Güler et al., 1996), Chlamydomonas reinharditti
(Sato et al., 2000b) and A. thaliana (Essigmann et al., 1998).
Fig.2. Composition of four lipid classes extracted from leaves in
16-day-old wheat plants grown in different solutions. Lipid con-
tent was the same among leaves when plants were grown in phos-
phate-sufficient solutions and the means of lipid composition in
three leaves are shown here (solid bars). The individual lipid con-
tents in the third leaf (cross-hatched bars), the second leaf (hatched
bars) and the first leaf (dot bars) were shown under phosphate-
deficient conditions. Values represent the means of three indepen-
dent measurements. Error bars are indicated. DGDG, digalactosyl
diacylglycerol; MGDG, monogalactosyldiacylglycerol; PG,
phosphatidylglycerol; SQDG, sulfoquinovosyl diacyglycerol.
Fig.3. The levels of individual lipids extracted from all leaves in
16-day-old (solid bars) and 9-day-old plants (blank bars) grown
in phosphate-deficient culturing solution. Values represent the
means of three independent measurements. Error bars are indicated.
Abbreviations are the same as in Fig.1. The abbreviations are the
same as in Fig.2.
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004214
Our results suggest that the changes in lipid composition
induced by phosphate deficiency are related to both lipid
biosynthesis and PG degradation. There was only one leaf
on 9-day-old plants, and in this case, little phosphate of PG
could be transported from this leaf to the new leaves. In
addition, phosphate deficiency resulted in 45 mol% of de-
crease in PG, as compared to phosphate-sufficient condi-
tion (Fig.1). These results indicate that phosphate was in-
sufficient for PG biosynthesis under phosphate-deficient
condition. On 16-day-old wheat plants, there were three
fully expended leaves, among which the first leaf was the
oldest one and its PG level was the lowest. The third leaf
was the youngest one and has just expended. When plants
were grown in 0.01 mmol/L phosphate solution for 16 d, PG
level in the third leaves was 48 mol% and 69 mol% higher
than that in the second and first leaves, respectively, and
only 19% lower than that in leaves on plants grown in 1.0
mmol/L phosphate solution (Fig.2). These results suggest
that PG decrease induced by phosphate deficiency in dif-
ferent leaves results from different mechanism. PG decrease
in the younger leaves is mainly due to insufficient phos-
phate available for PG biosynthesis, while PG decrease in
the older leaves mainly results from PG degradation.
If phosphate levels in culture solutions only affected
PG biosynthesis and degradation, PG decrease induced by
phosphate deficiency should be accompanied with an in-
crease in other three lipids. However, phosphate-deficient
condition only induced the increase in DGDG and SQDG,
in contrast, the level of MGDG decreased (Figs.1, 2). These
results suggest that phosphate levels also affect at least
the biosynthesis process of MGDG and DGDG. Some com-
pounds containing phosphate, such as PA and PC, are in-
volved in the biosynthesis of MGDG and SQDG (Ohlrogge
and Brows, 1995). SQDG is the final product of photosyn-
thetic lipid biosynthesis, whilst some molecules of MGDG
are used to synthesize DGDG (Siegenthaler and Murata,
1998b). This latter process does not need any compound
containing phosphate. Therefore, if phosphate is only a
limiting factor for MGDG synthesis but not for DGDG syn-
thesis from MGDG, as a result, MGDG should decrease
whilst DGDG increase.
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(Managing editor: HE Ping)