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Plant Physiol. (1998) 118: 591-598
Characterization of Transgenic Tobacco with an Increased
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Microsomal
-3 fatty acid desaturase catalyzes the conversion of 18:2 (linoleic
acid) to 18:3 (
-linolenic acid) in phospholipids, which are the main
constituents of extrachloroplast membranes. Transgenic tobacco
(Nicotiana tabacum) plants with increased 18:3 contents
(designated SIIn plants) were produced through the introduction of a
construct with the tobacco microsomal -3 fatty acid desaturase gene
under the control of the highly efficient promoter containing the
E12
sequence. 18:3 contents in the SIIn plants were increased by
about 40% in roots and by about 10% in leaves compared with the
control plants. With regard to growth at 15°C and 25°C and the
ability to tolerate chilling at 1°C and 5°C, there were no discernible differences between the SIIn and the control plants. Freezing tolerance in leaves and roots, which was assessed by electrolyte leakage, was almost the same between the SIIn and the
control plants. The fluidity of plasma membrane from the SIIn plants
was almost the same as that of the control plants. These results
indicate that an increase in the 18:3 level in phospholipids is not
directly involved in compensation for the diminishment in growth or
membrane properties observed under low temperatures.
Polyunsaturated fatty acids, especially 18:3 ( Although it remains unclear how important fatty acid polyunsaturation
is in determining membrane fluidity and low-temperature tolerance,
studies using Arabidopsis mutants with altered fatty acid composition
in glycerolipids have suggested that polyunsaturated fatty acids are
required for the growth and tolerance of higher plants at low,
nonfreezing temperatures. The Arabidopsis fad5 and
fad6 mutants, in which desaturation steps in the prokaryotic pathway are deficient and in which the contents of polyunsaturated fatty acids in the plastidic lipids are decreased, showed leaf chlorosis, reduced growth rates, and impaired chloroplast development at low temperatures (Hugly and Somerville, 1992 The Plasmid Construction
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-linolenic acid),
in membrane lipids have been found to increase in many plant species
when the plants are exposed to low temperatures (Smolenska and Kuiper,
1977
; Clarkson et al., 1980; Kodama et al., 1995). An increase in
polyunsaturated fatty acids in lipids has been thought to increase the
membrane fluidity by decreasing the scope for orderly packing of acyl
chains within the membrane interior (Chapman, 1975). Because a decrease
in membrane fluidity at low temperatures probably causes a loss of
membrane permeability (Kuiper, 1974) and diminishment of activities of
membrane-associated enzymes (Raison, 1973; Cronan and Gelmann,
1975), maintaining a constant fluidity of membranes has been considered
essential for survival at chilling and freezing temperatures (Wolfe,
1978). Although many attempts to find a causal relationship between an
increase in polyunsaturated fatty acid content and chilling or freezing acclimation have been made in higher plants, the results have been
controversial (Steponkus, 1984). The acclimation to low temperatures in
plants is associated with not only an increase in 18:3 content but also
with alterations in other biochemical metabolic processes: changes in
lipid composition and the synthesis of low-temperature-induced proteins
and cryoprotectants (Guy, 1990). Therefore, the precise role of
increases in the 18:3 content in membrane lipids is difficult to
ascertain from a comparison of low-temperature-acclimated and -nonacclimated plants.
). On the other hand,
the fad2 mutant defective in desaturation at the eukaryotic pathway, which exhibits a preferential decrease in polyunsaturated fatty acids in the microsomal lipids, showed increased chilling injuries such as inhibition of stem elongation at 12°C and loss of
viability at 6°C (Miquel et al., 1993).
-3 fatty acid desaturases are membrane-bound enzymes found in
microsomes and in plastid envelopes (for review, see Mazliak, 1994)
that catalyze the conversion of 18:2 (linoleic acid ) to 18:3 in
lipids. Three genes, FAD3, encoding the microsomal
desaturase, and FAD7 and FAD8, encoding the
plastid desaturases, were isolated from Arabidopsis (Iba et al., 1993;
Yadav et al., 1993; Gibson et al., 1994). Isolation of such desaturase
genes enabled us to modify the 18:3 content in both the plastidial and
extraplastidial membrane lipids. In fact, the transgenic tobacco
(Nicotiana tabacum) plants that contained increased trienoic
fatty acids, namely 16:3 (hexadecatrienoic acid) and 18:3, in plastid
membrane lipids were produced by an overexpression of the
FAD7 gene (Kodama et al., 1994). Because such
FAD7 transgenic tobacco leaves exhibited increased chilling
tolerance, the increase in trienoic fatty acids within the plastid
membrane would be one of the prerequisites for normal leaf growth at
low, nonfreezing temperatures (Kodama et al., 1995). We also reported
the successful production of transgenic tobacco plants that expressed
the transcripts of the tobacco microsomal -3 fatty acid desaturase
gene (NtFAD3) in sense and antisense orientations under the
control of the CaMV 35S promoter (Hamada et al., 1996). In this report
we describe engineering of the transgenic tobacco plants in which the
18:3 content in plasma membrane lipids increased drastically after the
introduction of NtFAD3 cDNA under the control of the El2
sequence, a highly efficient promoter for enhanced expression. Such
NtFAD3 transformants as those produced here allowed us to
determine the effects of an increase in 18:3 within plasma membrane
lipids on the function of higher plant cells without regard to other
biochemical changes associated with the acclimation to low
temperatures.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
) was subcloned into the BamHI unique site of pUC18 to
produce a plasmid, pF1-18S, which contains NtFAD3 cDNA in
antisense orientation relative to the lacZ region. Most of
the 5
noncoding region of the NtFAD3 cDNA was deleted by
digestion of pF1-18S with BbvII. The resulting fragment was
repaired with the Klenow fragment to create blunt ends, and then cut
with SacI to produce a truncated fragment. This cDNA
fragment was cloned into the SmaI-SacI site of
the binary plasmid pBE2113-GUS (Mitsuhara et al., 1996) to replace the
GUS gene. The resulting plasmid, pTF1SIIn, contained the
NtFAD3 cDNA fragment from nucleotides 52 to 1381 (see the NtFAD3 cDNA sequence, accession no. D26509) in sense
orientation relative to the promoter sequences.
Plant Transformation
Tobacco (Nicotiana tabacum cv SR1) was transformed using Agrobacterium tumefaciens LBA4404 containing the plasmid pTF1SIIn, as described previously (Hamada et al., 1996).
R1 seeds resulting from self-pollination were
aseptically germinated in continuous light (60 µmol
m
2 s1) at 26°C on
Murashige and Skoog medium (Murashige and Skoog, 1962)
supplemented with 100 µg/mL kanamycin. The kanamycin-resistant R1 and R2 seedlings were
subjected to further analyses. The seeds of the SIIn-24 plants
hemizygous for the T-DNA allele were produced by backcrossing the
homozygous R1 plant with the wild-type plant as a
pollen donor.
Lipid and Fatty Acid Analysis
Individual lipids were purified from leaves and roots as described previously (Kodama et al., 1995). The fatty acid compositions of whole
tissues and the individual lipids were determined by GC (GC-14B,
Shimadzu, Kyoto, Japan) as described by Kodama et al. (1994).
Measurement of Root Growth
The hemizygous SIIn-24 and wild-type seeds were germinated on plates with Murashige and Skoog medium containing 0.8% (w/v) gellan gum at 26°C. The Murashige and Skoog plates were arranged vertically to directly observe root elongation. One week after sowing, halves of these Murashige and Skoog plates with seedlings were transferred to 15°C, and then the positions of the root tips were plotted each day as a means of measuring root elongation.EL Test
In the leaf EL test, the third leaves (approximately 3-4 cm2) were excised from 4-week-old tobacco plants that had been cultured on soil under continuous light (3000 lux) at 26°C. Each detached leaf was cut in half and the halves were placed in a test tube. Deionized water (100 µL) was added to each test tube and leaf segments were fixed closely to the surface of the test tubes.
0.5°C for 30 min by using a program freezer
(model MPF-40, Tokyorika, Tokyo, Japan), and then ice formation
was achieved by introducing a small piece of ice into the test tubes.
After a 40-min equilibration period at 0.5°C, the bath temperature
was reduced automatically by 1°C increments every 30 min. The samples
were withdrawn from the bath at specified temperatures and placed on
ice. Samples were thawed overnight on ice and then incubated with 5 mL
of deionized water at 25°C for 1 h. The EL from the frozen
samples (ELfrozen) was measured by using a
conductivity meter. The EL from unfrozen samples
(ELunfrozen) and from samples frozen in liquid
N2 (ELLN) were taken as 0%
and 100% EL, respectively. The percentage of EL from frozen leaves and
roots at the specified temperatures was calculated by the following
equation:
Isolation of Root Plasma Membrane
Roots excised from 2.5-month-old tobacco plants were washed with distilled water and prechilled at 4°C before use. The microsomal fraction from the roots was obtained as described by Yoshida et al. (1986). Root plasma membranes were purified from this fraction by
aqueous two-phase partitioning and subsequent discontinuous Suc-density
gradient (Yoshida et al., 1986). In the alternative method, plasma
membranes were isolated from the microsome fraction by the aqueous
two-phase partitioning method as described by Larsson et al. (1994).
The fraction enriched with plasma membrane was diluted with Suc medium
(0.25 M Suc, 5 mM Mops-KOH [pH 7.3], and 1 mM DTT) and the plasma membrane was pelleted by
centrifugation at 156,000g for 20 min. The membrane pellets
were suspended in a sorbitol buffer containing 0.25 M
sorbitol, 5 mM Mops-KOH (pH 7.3), and 1 mM DTT.
When the plasma membrane was prepared for the ESR assay, the membrane
pellets were suspended in a sorbitol buffer without DTT, frozen in
liquid N2, and stored at 80°C until use.
ESR Spectroscopy
The spin-label molecule (5-SLS) was dissolved in dichloromethane to a final concentration of 0.104 mM. The solution containing 3.12 nmol of 5-SLS was placed into a sample tube and then dried under a vacuum for 1 h. The solution containing the plasma membrane equivalent to 600 to 900 nmol of fatty acids was placed into the sample tube and shaken vigorously for 2 min to stimulate the incorporation of 5-SLS into the membrane fragments. The ESR spectra of the samples were measured using a JES-RE-1X spectrometer (Jeol, Tokyo, Japan) equipped with a temperature controller.ATP Hydrolysis
The ATPase activity was determined by measuring the Pi released from ATP. The reaction mixture contained 3 mM Na-ATP, 3 mM MgSO4, 30 mM Tris-Mes (pH 7.0), and 50 mM KCl in a final volume of 150 µL, and was placed on ice until use. One minute before commencement of the reaction, the temperature of the reaction mixture was equilibrated at specified temperatures (0.5°C-40°C). The reaction was started by adding a plasma membrane solution containing 5 to 20 µg of proteins and was run for 30 min at specified temperatures. The reaction was stopped by adding 30 µL of 50% (v/v) ice-cold TCA, then centrifuged at 14,000g at 0°C for 10 min. The Pi concentration in the supernatant was determined by the method of Taussky and Shorr (1953).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Transgenic Tobacco Plants Harboring the NtFAD Construct
Previously, we showed that modulation of the NtFAD3 mRNA level is effective for modifying the 18:3 content in transgenic tobacco plants (Hamada et al., 1996). To produce
plants with extremely high 18:3 content, we introduced the
NtFAD3 cDNA under the control of the El2 sequence.
The El2 sequence contains two tandem repeats of a part of the
CaMV 35S promoter, which are followed by the sequence (Mitsuhara et
al., 1996). The El2 sequence can confer about 10-fold higher levels
of expression than the CaMV 35S promoter. The sequence has been
reported to improve the translation efficiency within plants when it is
located within the 5 untranslated region of the target gene. To ensure
the function of the sequence, most of the 5 untranslated region of
the NtFAD3 cDNA was deleted and then the cDNA was linked
behind the sequence to create a plasmid, pTF1SIIn (see ``Materials and Methods''). The tobacco transformants with pTF1SIIn were
designated SIIn lines. Of the 23 SIIn transgenic lines, two lines
(SIIn-20 and SIIn-24), which were segregated as a single T-DNA
insertion plant, were selected as representatives of those plants with
high 18:3 content and used in the following analyses.
Fatty Acid Composition of Leaf and Root Polar Lipids
Both the SIIn-20 and SIIn-24 lines had similar fatty acid compositions, and the 18:3 content in leaf tissues of the SIIn-20 plants increased by about 10%, whereas the 18:2 content decreased correspondingly compared with that of the control plants transformed with pBI121 (Table I). In contrast, the total 16:3 level of the SIIn-20 plants was almost the same as that of the control plants. In root tissues the 18:3 level of the SIIn-20 plants increased by about 40%, and was associated with a corresponding decrease in 18:2.
|
Plant Growth at Low and Normal Temperatures
-3 desaturation step does not significantly affect the synthesis
of each lipid class.
View this table:
Table II.
Fatty acid composition of individual leaf polar
lipids from the control (Cl) and SIIn-20 plants grown at 26°C
View this table:
Table III.
Fatty acid composition of individual root polar
lipids from the control (Cl) and SIIn-20 plants grown at 26°C
Freezing Tolerance in Vegetative Tissues
H+-ATPase Activity and Fluidity of Plasma Membrane
During low-temperature acclimation of higher plants, there is a
preferential synthesis of polyunsaturated fatty acids. In particular,
an increase in 18:3 is observed in root tissues of herbaceous plants
such as wheat and rye when they are exposed to low temperatures. For
example, in the roots of rye during acclimation to low temperatures,
the level of 18:3 in PC and PE increased from 20% to 40%, whereas the
level of 18:2 correspondingly decreased from 50% to 30% (Clarkson et
al., 1980 Influences of High 18:3 on Plasma Membrane Properties
Possible Role of Increased 18:3 Content on Low-Temperature
Acclimation
Received April 17, 1998;
accepted June 26, 1998.
Abbreviations:
CaMV, cauliflower mosaic virus.
EL, electrolyte
leakage.
ESR, electron spin resonance.
MGD, monogalactosyldiacylglycerol.
PC, phosphatidylcholine.
PE, phosphatidylethanolamine.
PG, phosphatidylglycerol.
PI, phosphatidylinositol.
5-SLS, 5-(4 We thank Dr. Yuko Ohashi (National Institute of Agrobiological
Resources, Tsukuba, Japan) for providing the pBE2113-GUS plasmid. We
also acknowledge Dr. Shizuo Yoshida (Hokkaido University), Dr.
Toshinori Kinoshita (Kyushu University), and Mr. Michiharu Hara for
their technical advice.
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Chilling injury in plants.
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Arabidopsis requires polyunsaturated lipids for low-temperature survival.
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View larger version (15K):
[in a new window]
Figure 1.
Effect of temperature on root growth. Seedlings of
the SIIn-24 ( 
) and wild-type (
) plants were grown at 26°C (A)
and 15°C (B). Vertical lines indicate SD
(n = 9).
2
s1) for 7 d. Then, these seedlings were
transferred to 26°C under continuous light. Two days later, leaf
chlorosis was observed in both the SIIn and control seedlings, and the
number of seedlings with chlorosis and the leaf growth were almost the
same. When 10-d-old SIIn and control seedlings grown at 26°C were
transferred to 5°C under continuous light (60 µmol
m2 s1), the growth of
both types of seedlings was inhibited. Two weeks later, the leaves of
both types of seedlings turned pale green.
0.5°C to 3°C). In the
SIIn-20 and control plants, the EL of leaves and roots began to
increase at 1.0°C and reached a maximum at 2.5°C. The
temperatures at which 50% EL occurred in leaves from the SIIn-20 and
control plants were approximately 1.6°C and 1.7°C,
respectively, whereas those for roots from these two plants were
approximately 1.5°C. Furthermore, in the transgenic plants (T-4 and
T-6) overexpressing the plastidial -3 desaturase gene
(FAD7), the same experiments were carried out. Although
these plants had increased levels of 18:3 in plastid membrane lipids,
similar results were observed (data not shown). These results indicated
that an increase in 18:3 content in both plastidial and extraplastidial
membrane lipid was not associated with any alteration to the
freezing-tolerance ability in the vegetative tissues of tobacco plants.
View larger version (13K):
[in a new window]
Figure 2.
Freezing tolerance of leaves (A) and roots (B) of
the SIIn-20 ( ) and control () plants. Survival was determined by
the EL measurement from leaves and roots after a freeze/thaw treatment.
Vertical lines indicate SD (n = 4 in
leaves, and n = 3 in roots).
View this table:
Table IV.
Effect of inhibitors on the ATPase activity
of plasma membranes isolated from the roots of SIIn-20 and control (Cl)
plants
ATP activity was assayed at 30°C for 30 min in the presence of
nitrate (50 mM KNO3), azide (1 mM
NaN3), molybdate (100 µM
Na2MoO4), or vanadate (100 µM
Na3VO4).
). High 18:3 content was evident in the plasma membrane fraction purified from the SIIn-20 plants (data not shown). For elucidation of the effects of such a high 18:3 content on the
membrane-bound enzyme activity, the H+-ATPase
activity in the plasma membranes, which were isolated from the roots of
26°C-grown SIIn-20 and control plants, was assayed at temperatures
ranging from 0.5°C to 40°C (Fig. 3).
In Arrhenius plots of the vanadate-sensitive ATPase activity, breaks
were observed around 10°C, and observed slopes were indistinguishable
in the two membrane fractions prepared from the SIIn-20 and control
plants. These results indicate that plasma membrane
H+-ATPase activity is only slightly affected by
an increase in 18:3 content within the surrounding phospholipids.
View larger version (17K):
[in a new window]
Figure 3.
Arrhenius plots of ATPase activity found in the
root plasma membrane. Total ATPase activity ( and ) and
vanadate-sensitive ATPase activity (
and 
) were determined in
root plasma membranes prepared from SIIn-20 ( and ) and control
( and ) plants. Vanadate-sensitive ATPase activity was determined
in the presence of 100 µM vanadate. Each value is the
mean of two independent experiments.
; Utsumi et al., 1978), this ESR parameter can be used as an
indicator of membrane fluidity. Lower values of overall splitting
indicate that the membrane has become more fluid. The overall splitting values of the plasma membrane from SIIn-20 plants were almost the same
as those from control plants at temperatures from 3°C to 30°C
(Fig. 4). At temperatures from 3°C to
30°C, the plasma membrane with a high 18:3 content exhibited
thermotropic properties similar to those of the control plasma
membrane.
View larger version (20K):
[in a new window]
Figure 4.
Effect of temperature on the overall splitting
value of the spin-labeled membrane vesicles. The spin-label molecule
5-SLS was incorporated into the root plasma membranes prepared from the
SIIn-20 ( ) and control () plants. mT, Millitesla.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). In the root the major membrane lipids are PC and PE, which
together account for 70% to 75% of root polar lipids, and the 18:3
level would be regulated by the conversion of 18:2 to 18:3 in these
lipids, in which the conversion is catalyzed by the microsomal -3
fatty acid desaturase in the eukaryotic pathway (Browse et al., 1993).
For investigation of the role played by an increase in 18:3 in membrane
lipids, we thus introduced a construct with the NtFAD3 gene
under the control of the El2 sequence to produce transgenic tobacco
plants with an extremely high 18:3 content. Overexpression of the
NtFAD3 gene was effective at increasing the 18:3 content
in phospholipids in leaf and root tissues of transgenic tobacco (Tables
II and III). Because lipid composition and the ratio of fatty acid to protein content in the SIIn plants were almost the same as those in the
control plants (data not shown), it seems that the precise role of a
high 18:3 phenotype could be evaluated by using the SIIn plants
produced here.
). Hypothetically, a decrease in temperature leads to a
decrease in membrane fluidity, although an increase in polyunsaturated
fatty acids might compensate for this decrease in membrane fluidity.
Because fatty acid desaturation at low temperatures in plants is
observed mainly from 18:2 to 18:3, we investigated the effects of
increased 18:3 content on membrane fluidity. As shown in Figure 4, a
decrease in temperature caused an increase in the viscosity of plasma
membranes, although increased 18:3 content was not associated with the
discernible changes in the ESR parameter showing membrane fluidity.
This observation suggests that the role of desaturation from 18:2 to
18:3 in plasma membranes during acclimation to low temperatures is very
limited in its compensation for a decrease in membrane fluidity at low
temperatures. By using the Arabidopsis fad7 mutant,
thylakoid membranes with a drastically reduced content of trienoic
fatty acids (16:3 and 18:3) were assessed for their membrane fluidity,
and these membranes were found to have only a slightly rigid nature at
the temperatures tested compared with the corresponding membranes
prepared from wild-type plants (McCourt et al., 1987). These results
show that conversion from 18:2 to 18:3 in both plasma membrane lipids
and thylakoid membrane lipids only slightly affects membrane fluidity.
; Steponkus, 1984). The
proportion of polyunsaturated species of PC and PE in the plasma
membrane increases naturally during the 1st week of cold acclimation in
both rye and oat (Uemura and Steponkus, 1994). Previously, the
correlation between an increase in the 18:3 content in wheat seedlings
during cold acclimation and the establishment of enhanced freezing
tolerance was investigated by use of the inhibitors of 18:3 synthesis
(Willemot, 1977; de la Roche, 1979). However, the results obtained were
controversial. The freeze-induced lesions can be classified into two
different destabilization mechanisms of the plasma membrane (Uemura et
al., 1995). Over the range of 2°C to 4°C, the predominant
freezing injury is caused by expansion-induced lysis, which is a
consequence of the osmotic contraction by endocytotic vesiculation of
plasma membranes during a freeze-thaw cycle. Over the range of 4°C
to 8°C, the predominant phase of injury is freeze-induced
lamellar-to-hexagonal II phase transitions of plasma membranes and
other cellular membranes. The data shown in Figure 2 indicate that high
18:3 content in the plasma membrane did not directly affect freezing
tolerance, such as avoiding leakage of ions from cytosol at
temperatures of 0.5°C to 3°C in tobacco cells. Therefore, the
increased 18:3 content was not effective at preventing the probable
expansion-induced lysis.
), did not show any detectable phenotypic
alteration in growth at either low or high temperatures (Browse et al.,
1995). These results indicate that high or low 18:3 content in
microsomal membrane lipids had almost no effect on cell division or
expansion.
, 1995). Such different behavior
regarding chilling tolerance may be explained by the fact that the
target membrane system is quite different between transgenic tobacco
plants introduced with the plastidial and microsomal -3 fatty acid
desaturases. The chilling tolerance observed in the FAD7
transformants could be mediated by enhancement of the chloroplast
functions required at low temperatures. During acclimation the
expression of a number of low-temperature-responsive genes is induced.
Among them, the Cor15a gene product is reported to interact
with chloroplast membranes as a cryoprotectant (Artus et al., 1996).
It is possible that during acclimation, molecular mechanisms
conferring low-temperature tolerance are developed based on some
interaction between cellular membranes modified at low temperatures and
newly expressed proteins that are protective against chilling and
freezing temperatures.
1
This work was supported in part by a
grant-in-aid (Biotechnology no. 1317) from the Ministry of
Agriculture, Forestry, and Fishery, Japan, and by a grant from the
Japan Society for the Promotion of Science (no. JSPS-RFTF96L00602).
FOOTNOTES
2
Present address: The Ishikawa Agricultural
College, Nonoichimachi, Ishikawa 921-8836, Japan.
3
Present address: Department of Biochemistry,
Faculty of Horticulture, Chiba University, Matsudo City, Chiba
271-8510, Japan.
*
Corresponding author; e-mail koibascb{at}mbox.nc.kyushu-u.ac.jp; fax
81-92-642-2621.
ABBREVIATIONS
,4-dimethyloxazolidine-N-oxy) stearic acid.
X:Y, a
fatty acyl group containing X carbon atoms and Y cis
double bonds.
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
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[Abstract]
Copyright Clearance Center: 0032-0889/98/118//08
© 1998 American Society of Plant Physiologists
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