Plant Science 161 (2001) 1117– 1123

www.elsevier.com/locate/plantsci

Characterization of Arabidopsis mutants that are associated with

altered C18 unsaturated fatty acid metabolism
Gorou Horiguchi 1, Hiroaki Kodama 2, Koh Iba *
Department of Biology, Faculty of Sciences, Kyushu Uni6ersity, Hakozaki, Higashi-ku, Fukuoka 812 -8581, Japan

Received 12 June 2001; accepted 3 August 2001

Abstract

Polyunsaturated fatty acids are a major component of glycerolipids in plant cellular membranes. We have isolated four novel
fatty acid mutants of Arabidopsis thaliana named regulator of fatty acid composition (rfc) 1 through 4. The rfc1, rfc2 and rfc3
mutants had an increased oleic acid (18:1) level at the expense of a-linolenic acid (18:3) level among total fatty acids especially
in root tissues. In these mutants, the transcripts of FAD2 and FAD3 gene for ER D12 and v-3 fatty acid desaturases that are
involved in the desaturation of 18:1 and linoleic acid (18:2), respectively, were accumulated to the wild-type levels. In the rfc4
mutant, over-accumulated 18:3 with a corresponding increase in the FAD3 mRNA over wild-type levels was observed. These
results suggest that C18 unsaturated fatty acid metabolism in extra-plastid membranes is controlled by multiple loci in addition
to the structural genes for fatty acid desaturases. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Arabidopsis; Fatty acid; Fatty acid desaturase; rfc mutants

1. Introduction The first double bond in C18 fatty acids is introduced

A unique feature of cellular membranes in higher
plants is the presence of significantly high levels of
polyunsaturated fatty acids. In addition, distinct fatty
acid compositions are observed in different intracellular
membrane systems. The predominant fatty acid found
in plastid membranes is a-linolenic acid (18:3) while
linoleic acid (18:2) is usually the most abundant fatty-
acid species in extra-plastid membranes [1,2].
In Arabidopsis thaliana, fatty acid desaturation path-
ways have been investigated in detail (reviewed in [3]).
Abbre6iations: CAPS, cleaved amplified polymorphic sequences;
dCAPS, directed CAPS; EMS, ethylmethane sulfonate; ER, endo-
plasmic reticulum; INDELs, insertions/deletions; PCR, polymerase
chain reaction; SNPs, single nucleotide polymorphisms; WT, wild
type; X:Y, fatty-acyl group of glycerolipid containing X carbons with
Y double bonds.
* Corresponding author. Tel./fax: +81-92-642-2621.
E-mail address: koibascb@mbox.nc.kyushu-u.ac.jp (K. Iba).
1
Present address: Plant Science Center, RIKEN, Hirosawa 2-1,
Wako, Saitama 351-0198, Japan.
2
Present address: Department of Bioresources Chemistry, Faculty
of Horticulture, Chiba University, Matsudo 648, Chiba 271-8510,
Japan.
by the activity of stearoyl-acyl-carrier-protein desat-
urase in the plastid stroma. The resultant oleic acid
(18:1) is utilized in the prokaryotic pathway in the
plastid and in the eukaryotic pathway in the endoplas-
mic reticulum (ER). In the prokaryotic pathway, 18:1 is
incorporated into phosphatidic acid to produce galac-
tolipids, sulfolipid and phosphatidylglycerol in plastids.
In the eukaryotic pathway, 18:1 is exported to ER as
18:1-CoA to produce phospholipids such as phos-
phatidylcholine and phosphatidylethanolamine. Subse-
quent desaturation reactions take place in both
pathways. The production of 18:2 and 18:3 is catalyzed
by D12 (v-6) and v-3 fatty acid desaturases, respec-
tively. In Arabidopsis, the ER D12, ER v-3, plastid v-6
and plastid v-3 desaturases are encoded by the FAD2,
FAD3, FAD6 and FAD7 genes, respectively [4–8].
Steady-state fatty acid composition in membrane
lipids is maintained by coordinating the processes for
the biosynthesis, desaturation, intra-membrane trans-
port and degradation of fatty acids. Although most of
the genes for the enzymes involved in glycerolipid
metabolism have been identified [9], the coordinated
regulation of these genes to establish a fatty acid com-

0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 2 3 - 4
1118 G. Horiguchi et al. / Plant Science 161 (2001) 1117–1123
position observed in a particular membrane system is
undefined. However, at least in wheat, the expression of
plastid and ER v-3 desaturase genes is enhanced in
differentiating mesophyll cells and in apical meristem,
respectively, where active biogenesis of chloroplast or
ER membranes occurs [10,11].
In order to elucidate the regulatory mechanisms that
determine the degree of membrane unsaturation, a ge-
netic approach, as used for the identification of fatty
acid desaturase genes (reviewed in [3]), should be effec-
tive. In root tissues, the FAD2 and FAD3 desaturases
are responsible for most of polyunsaturated fatty acid
production [1,7], while their plastid isozymes (FAD6
and FAD7 desaturases) are not [12,13]. This simplicity
of the desaturation pathway in root tissues should be
suitable for the isolation of novel fatty acid mutants.
Here we report the characterization of four mutants of
Arabidopsis, named regulator of fatty acid composition
(rfc). These mutants show altered C18 unsaturated
fatty acid levels among the total fatty acid content.
2. Materials and methods
2.1. Plant materials
The wild-type (WT) plant used in this study is the
Landsberg erecta (Ler) ecotype. M2 seeds descended
from Ler were purchased from Lehle seeds. The seeds
of fad2 -1 [14] were obtained from the Arabidopsis
Biological Resource Center. Seedlings were grown on
vertical plates containing half-strength MS medium,
solidified with 0.4% gellun gum. For fatty acid, lipid
and mRNA analyses, shoot and root tissues were har-
vested from 14-day-old seedlings grown at 26 °C under
continuous illumination.
2.2. Lipid and fatty acid analyses
The extraction and separation of lipids and the deter-
mination of fatty acid composition were carried out as
previously described [15].
2.3. Screening of fatty acid mutants
Approximately 4100 each of fast neutron- and ethyl-
methane sulfonate (EMS)-mutagenized M2 seedlings
were subjected to a screening of mutants for an altered
root fatty acid composition. Four mutant lines, rfc1,
rfc2, rfc3 -1 and rfc4 were isolated using this strategy.
rfc3 -2 was isolated from 20,000 EMS-mutagenized M2
seedlings by the presence of an aberrant lateral root
phenotype. All mutants were backcrossed twice and
outcrossed twice with Ler prior to genetic and pheno-
typic analyses were carried out.
2.4. Genetic analyses
Each rfc mutant line was crossed with the Columbia
(Col-0) ecotype, and F2 seeds were used for genetic
mapping. Mapping of the chromosomal location of the
rfc loci was accomplished using simple-sequence-length
polymorphism (SSLP) and cleaved amplified polymor-
phic sequences (CAPS) markers (see The Arabidopsis
Information Resource (TAIR) web page, http://
www.arabidopsis.org/). In addition, insertions/deletions
(INDELs) and single nucleotide polymorphisms (SNPs)
markers developed by Cereon Genomics (http://
www.arabidopsis.org/Cereon/index.html) were used.
Several CAPS and directed CAPS (dCAPS) [16] mark-
ers were identified in this study. For K14A17-NdeI
(dCAPS), the polymerase chain reaction (PCR) primer
pair used was 5%-CGAGATCATGAGCATGAATAC-
3% and 5%-CAGTGAAATTTCTATGTTACATAT-3%.
The amplified DNA fragment was 116 bp in length, and
that from Col-0 was cleaved into 93 and 23 bp frag-
ments by NdeI. For MGD8M (CAPS), the PCR primer
pair used was 5%-AGAAGACGCGCAAAGACAGA-3%
and 5%-ACAAATCTGGCAAGTGACCA-3%. The am-
plified DNA fragment was 178 bp in length and that
from Ler was cleaved into 115 and 63 bp fragments by
HaeIII. For fine mapping of the RFC1 locus, the fast
isolation of recombinant (FIRE) strategy [17] was em-
ployed. The rfc3 /rfc3 individuals in the F2 mapping
population were selected based on the lateral root
phenotype rather than the fatty acid phenotype. For
mapping of the RFC4 locus, RFC4 /RFC4 plants were
selected from the F2 mapping population.
2.5. mRNA analysis
The isolation of total RNA and Northern analysis
was performed as previously described [15]. Hybridiza-
tion probes, labeled with [a-32P] dATP were prepared
using cDNA fragments amplified with following gene
specific primer pairs. Primer pair for FAD2 cDNA is
5%-GAGAAACCGCCTTTCTCGGT-3% and 5%-CAT-
AATGAGCAGCCAAAATG-3%, for FAD3 cDNA is
5%-GTATGTTGATAGCTGGTTCC-3% and 5%-ATGT-
CATGGTGGATGTTGTT-3%, and for FAD7 cDNA is
5%-CAATACTTTGGACAAGCCGA-3% and 5%-TCGT-
CGCTCACGTAATGATC-3%.
3. Results
3.1. Isolation of rfc mutants
By directly measuring the root fatty acid composition
of individual M2 seedlings, four rfc mutant lines were
isolated. rfc1, rfc3 -1 and rfc4 were isolated from an M2
population that was mutagenized using fast-neutron
 G. Horiguchi et al. / Plant Science 161 (2001) 1117–1123 1119

Fig. 1. Root phenotype of rfc3 -1 mutant. (A) Eighteen-day-old

seedlings of WT and rfc3 -1 plants. Arrowheads indicate aberrant
lateral roots. Ar, adventitious root; Lr, lateral root; PR, primary
root. Bar = 1 cm. (B) A dome-shaped lateral root of the rfc3 -1
mutant is shown at a larger magnification. Bar = 0.5 mm.

bombardment. rfc2 was isolated from an EMS-treated

M2 population. Since rfc3 -1 showed an aberrant lateral
root phenotype (Fig. 1), an allelic mutant, rfc3 -2, was
also isolated based on the abnormal lateral root shape
from an independent pool of the EMS-mutagenized M2
population. A complementation assay between rfc3 -1
and rfc3 -2 showed that these two mutations are allelic
(data not shown). Thus, both the fatty-acid and the
lateral-root phenotypes resulted from genetic lesions at
the RFC3 gene.
The fatty acid compositions in the root tissues of rfc
mutants are shown in Table 1. The fatty acid composi-
tions of rfc1 and rfc2 were almost identical. In the root
tissues of these mutants, the 18:3 level was decreased,
the 18:1 level was increased and the 18:2 level was
slightly increased when compared to those in WT
plants. In the rfc1rfc2 double mutant, the 18:3 level was
further decreased and the 18:1 level was correspond-
ingly increased. The fatty acid compositions of rfc3 -1
and rfc3 -2 were similar to those of rfc1 and rfc2. In
contrast to rfc1, rfc2 and rfc3, rfc4 over-accumulated
18:3 at the expense of 18:2.
Fig. 2. Fatty acid composition of Fl plants between the rfc mutants
and the fad2 -1 mutant. The fad2 -1 plant was crossed with WT, rfc1,
rfc2 and rfc3 -1 plants. Fatty acid compositions in root tissues of Fl
progenies from each cross and those of parental lines were deter-
mined. The levels of 18:1, 18:2 and 18:3 are shown. Data are mean 9
SD (n =5).
The rfc1, rfc2 and rfc3 mutations were single reces-
sive nuclear traits (data not shown). Since fad2 is
deficient in the desaturation of 18:1 in ER [7], we
examined rfc1, rfc2 and rfc3 to see if they represent
weak alleles of the FAD2 locus. As shown in Fig. 2, the
F1 progenies from a cross between a WT plant and an
fad2 -1 plant showed intermediate root 18:1 and 18:3
levels compared to those found in each parent plant.
Therefore, the FAD2 /fad2 -1 heterozygote was co-domi-
nant in the root tissues. When each rfc1, rfc2 and rfc3 -1
plant was crossed with an fad2 -1 plant, the Fl progenies
showed almost the same fatty acid composition as that
of the heterozygous fad2 -1 plant. Since rfc1, rfc2 and

Table 1
Fatty acid composition of root tissues

Fatty acid (mol%)

16:0 18:0 18:1 18:2 18:3

rfc1 22.9 (1.1) 4.2 (0.3) 15.3 (0.7) 39.8 (1.7) 17.8 (1.2)
rfc2 24.4 (1.6) 3.6 (0.5) 18.7 (1.0) 36.4 (1.2) 16.9 (1.3)
rfc1rfc2 23.4 (0.5) 3.1 (0.2) 26.3 (0.8) 38.4 (0.4) 8.9 (0.4)
rfc3 -1 24.7 (0.8) 1.7 (0.2) 15.0 (1.6) 37.6 (0.2) 20.9 (2.1)
rfc3 -2 22.8 (0.4) 2.7 (0.2) 17.2 (1.0) 37.4 (1.1) 19.9 (0.3)
rfc4 20.4 (0.8) 3.1 (0.3) 9.3 (0.3) 27.5 (0.7) 39.6 (1.6)
WT 22.8 (2.8) 3.4 (0.4) 9.9 (1.4) 34.7 (1.6) 29.2 (2.7)

Data are mean of five independent analysis. SD values are shown in parentheses.
1120 G. Horiguchi et al. / Plant Science 161 (2001) 1117–1123

rfc3 -1 were recessive mutations, these Fl phenotypes

should result from the co-dominant feature of the
FAD2 /fad2 -1 heterozygote. Thus, we conclude that the
RFC1, RFC2 and RFC3 loci are independent from the
FAD2 locus.
The root fatty acid compositions of 29 WT plants
and 235 F2 progenies from a cross between rfc4 and
WT plants were examined (Fig. 3). The 18:2/18:3 ratios
of WT plants ranged from 1.1 to 1.4. In the F2 proge-
nies, 24.7% of seedlings showed an 18:2/18:3 ratio that
was equal to or higher than the WT range. Since the
high 18:2/18:3 (] 1.1) population versus the low 18:2/
18:3 (B 1.1) population in these F2 population segre-
gated to an approximately 1:3 ratio, the rfc4 allele may
be interpreted as dominant over the RFC4 allele. How-
ever, the broad distribution of the 18:2/18:3 ratio with-
out a sharp boundary between the RFC4 /RFC4 and the
RFC4 /rfc4 plus rfc4 /rfc4 population suggested a weak
(or incomplete) dominance of the rfc4 allele. Other 18:3
over-accumulating mutants, ela1 and ife, have been
isolated [14,18]. The ela1 mutation is recessive and its
chromosomal location is unknown. The ife mutation is Fig. 4. The chromosomal location of the RFC loci. Names of P1,
thought to be localized in the promoter region of the bacterial artificial chromosome (BAC) and transformation competent
artificial chromosome (TAC) clones on the physical map (http://
FAD3 gene on chromosome 2. Since the rfc4 mutant www.arabidopsis.org/servlets/mapper) around the RFC1 and RFC3
exhibited a weak dominance and mapped on chromo- loci are indicated by open boxes. Numerals in the parentheses indi-
some 3 (see below), this mutation defined a novel locus cate the number of recombinants between each marker and each
involved in the negative regulation of the 18:3 level. mutation site/the total number of chromosomes examined.
These genetic analyses demonstrate that all of the rfc
mutations represent novel loci related to unsaturated RFC2 and RFC3 loci were mapped on chromosome 3.
fatty acid metabolism. The RFC2 locus was located between ngal26 and At-
DMC1. The RFC3 locus was mapped between
K14A17-Nde and MGD8M. The RFC4 locus was
3.2. The chromosomal location of the RFC loci mapped between a Cereon INDEL marker, 456162,
and GL1 on chromosome 3.
The chromosomal location of the RFC loci is sum-
marized in Fig. 4. The RFC1 locus was mapped be- 3.3. Effects of rfc mutations on the eukaryotic pathway
tween Cereon SNPs markers, 432598 and 427646. The
In all of the rfc mutants, alterations of the fatty acid
compositions in seeds were similar to that in root
tissues of respective mutants (data not shown). The
fatty acid compositions in leaf tissues of rfc1, rfc2 and
rfc4 mutant were altered in a similar, but very limited
fashion as that seen in root fatty acid composition of
each mutant. The fatty acid compositions in leaf tissues
of rfc3 -1 and rfc3 -2 were indistinguishable from that of
WT plant (data not shown). In both leaves and seeds,
the effects of rfc mutations were evident in the levels of
C18 unsaturated fatty acids but not in the levels of C16,
C20 and C22 fatty acids (data not shown). The fatty
acid composition of major root lipid species examined
was altered in accordance with the fatty acid composi-
tions of the whole root in rfc1, rfc4 (Table 2), rfc2 and
rfc3 -1 (data not shown). The pronounced effect of the
Fig. 3. The frequency distribution of the 18:2/18:3 ratio determined in
mutations on the non-photosynthetic organs and the
WT plants and in F2 population from a cross between WT and rfc4 alteration of fatty acid composition of both phospho-
plants. lipids and galactolipids are also the characteristics of
 G. Horiguchi et al. / Plant Science 161 (2001) 1117–1123 1121

Table 2
Fatty acid compositions of individual lipid classes in root tissues

Lipid class Fatty acid (mol%)

16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3

Monogalactosyldiacylglycerol
WT 6.2 0.4 0.4 4.9 1.6 2.6 12.3 71.6
Digalactosyldiacylglycerol 19.4 0.3 0.2 0.2 3.8 3.7 20.7 51.7
Phosphatidylglycerol 41.0 1.7 0.0 0.0 3.9 4.0 29.4 19.9
Phosphatidylethanolamine 22.0 0.6 0.0 0.0 1.9 6.6 42.6 26.3
Phosphatidylinositol 35.6 0.3 0.0 0.0 2.6 5.2 33.9 22.4
Phosphatidylcholine 20.9 1.1 0.0 0.0 1.7 8.9 38.5 28.7
Monogalactosyldiacylglycerol
rfc1 8.5 1.2 1.9 6.4 1.2 10.4 29.1 41.3
Digalactosyldiacylglycerol 20.6 0.8 0.0 0.4 2.9 9.7 30.0 35.7
Phosphatidylglycerol 41.2 1.6 0.0 0.0 2.6 13.7 33.4 7.6
Phosphatidylethanolamine 23.5 0.6 0.0 0.0 1.5 12.4 51.5 10.5
Phosphatidylinositol 31.4 0.5 0.0 0.0 2.2 13.8 43.1 9.0
Phosphatidylcholine 24.4 0.7 0.0 0.1 2.3 23.1 41.4 7.8
Monogalactosyldiacylglycerol
rfc4 7.8 1.1 0.3 5.3 2.3 4.0 10.9 68.3
Digalactosyldiacylglycerol 19.6 0.9 0.0 0.0 4.0 4.6 12.2 58.6
Phosphatidylglycerol 41.7 3.4 0.0 0.0 4.3 3.7 21.7 25.2
Phosphatidylethanolamine 22.0 0.7 0.0 0.0 1.8 6.2 31.2 38.0
Phosphatidylinositol 35.1 0.6 0.0 0.0 2.7 5.1 24.8 31.6
Phosphatidylcholine 21.2 1.0 0.0 0.0 2.9 9.0 28.0 37.9

Fatty acid composition of each lipid class was determined. Values are mean of three independent analyses.

the fad3 mutation [1]. Therefore, these results suggest
that the RFC genes are involved in C18 unsaturated
fatty acid metabolism in the eukaryotic pathway.
3.4. Expression of FAD2 and FAD3 genes in the rfc
mutants
We examined the expression of FAD2 and FAD3
genes in the rfc mutants (Fig. 5). Unexpectedly, the
levels of the FAD2 and FAD3 mRNAs in shoot and
root tissues of the rfc1, rfc2 and rfc3 -1 plants were
similar to those in the WT plant. In contrast, the level
of FAD3 mRNA in root tissues of the rfc4 plant was
l.6390.06 (n=3) times higher than that in the WT
plants, while the FAD2 mRNA accumulated at a simi-
lar level to that in the WT plants (1.0790.08, n =3).
The FAD7 mRNA in four rfc mutants accumulated to
a similar extent to that in the WT plant.
fatty acid phenotype we consider that the FAD3 desat-
urase activity is also reduced in rfc1 (and rfc2 and rfc3 ).
In this scenario, the reduced production of 18:2 do not
lead to the reduced 18:2 level since the amount of 18:2
that is converted into 18:3 by the FAD3 desaturase is
simultaneously reduced.
Recently, we found that the temperature-dependent
change in the 18:3 level in wheat root tips occurs
without changing the TaFAD3 mRNA level [19]. In this
case, a key regulatory step is at a translational level.
Therefore, our results that the levels of desaturase
mRNAs did not correlate with the content of polyun-
saturated fatty acids suggests the presence of a common
regulatory step for the FAD2 and FAD3 gene expres-

4. Discussion

When it is assumed that the FAD2 desaturase activ-

ity is reduced by the rfc1 mutation, for example, the
level of 18:1 should be increased and the level of 18:2 is
correspondingly decreased as is seen in the fad2 mutant
[7]. However, the small increase of 18:2 does not ac-
count for the excess 18:1 in the rfc1 mutant. Instead,
the 18:3 level is significantly decreased. To explain this
Fig. 5. The expression of desaturase genes in the rfc mutant back-
grounds. Steady state mRNA levels for FAD2, FAD3 and FAD7
genes were determined by Northern analysis. Total RNAs were
isolated from shoot and root tissues of WT, rfc1, rfc2, rfc3 -1 and rfc4
plants. Each lane contains 20 mg of total RNAs.
1122 G. Horiguchi et al. / Plant Science 161 (2001) 1117–1123
sion downstream of transcription. In addition, the
RFC1 and RFC2 genes may be components of indepen-
dent pathways since the rfc1rfc2 double mutant exhibits
a more severe alteration of fatty acid composition
compared to that of either single parental mutant.
Alternatively, the degradation of polyunsaturated
fatty acids may influence the fatty acid composition. In
the case of the Arabidopsis aim1, a defect in b-oxidation
results in a slight increase in the 18:1 and 18:2 levels at
the expense of 18:3 level in leaves, when compared to
those in the WT plant [20]. Arabidopsis ped1, ped2 and
ped3 also have a defect in b-oxidation and these mu-
tants require sucrose for post-germinative growth [21].
The lack of b-oxidation allows aim1 and the three ped
mutants to germinate and grow on media containing
2,4-dichlorophenoxybutyric acid, which is otherwise
metabolized into synthetic auxin, 2,4-dichlorophenoxy-
acetic acid through b-oxidation [20,21]. However, from
the following observations, we consider that b-oxida-
tion functions normally in the rfc1, rfc2 and rfc3 : (i) the
RFC1, RFC2 and RFC3 loci are mapped at the posi-
tions different from those of AIM1, PED1, PED2 and
PED3 ; (ii) these rfc mutants are as sensitive as WT
plants to 2,4-dichlorophenoxybutyric acid (data not
shown); and (iii) these rfc mutants can germinate and
grow normally in medium containing no sucrose (data
not shown).
A third possibility for the role of RFC1, RFC2 and
RFC3 genes is in electron transfer that is essential for
the desaturation reaction. In ER membranes, the re-
duced form of cytochrome b5 is considered to act as an
electron donor for desaturation. The oxidized form of
cytochrome b5, itself, can be reduced by cytochrome b5
reductase and also by cytochrome P450 reductase in
vitro [22]. However, genes for these electron transfer
systems (including putative ones annotated by the
Arabidopsis Genome Initiative: see http://www.
arabidopsis.org/info/agi.html) were not found around
near any of the rfc loci.
In leaf tissues, a part of the 18:3 produced by the
FAD7 desaturase could be transferred into extra-
chloroplast membranes [23]. On the other hand, in the
fad7 root tissues, the 18:3 level is hardly affected [13].
This observation suggests that 18:3 production in root
tissues is almost dependent on the FAD3 desaturase
and that the flow of 18:3 from plastid to extra-plastid
membranes is very small if present. Thus, it is unlikely
that RFC gene products are involved in the inter-mem-
brane transport of fatty acids. Thus, among the four
possible roles of RFC1, RFC2 and RFC3 genes men-
tioned above, the most plausible one is the positive
regulation of the FAD2 and FAD3 genes at the down-
stream of transcriptional level.
Among the rfc mutants, rfc3 is associated with the
aberrant lateral root phenotype. Since two rfc3 alleles
with similar phenotypes were isolated from different
M2 pools, both lateral root and fatty acid phenotypes
resulted from the same genetic lesion. However, the
altered fatty acid composition would not be a direct
cause of the aberrant lateral root phenotype of rfc3
because the primary roots of fad2 through fad8 mutants
normally develop lateral roots (data not shown). The
alf 3 mutation, which is mapped on the different posi-
tion from the RFC3 locus, also results in the aberrant
lateral root formation [24]. Further characterization of
these mutants and cloning of corresponding genes will
provide a cue for understanding the mechanism of
lateral root development and the role of polyunsatu-
rated fatty acids during such a developmental process.
Over-accumulation of 18:3 in rfc4 resulted from an
approximately 1.6 times higher FAD3 mRNA level
than that in WT plant. This situation is very similar to
the ife mutant which is believed to have a mutation in
the promoter region of the FAD3 gene [18]. Therefore,
the product of the RFC4 gene may be involved in the
negative transcriptional regulation of the FAD3 gene in
combination with a cis element that may be mutated in
the ife mutant.
In summary, the metabolism of C18 unsaturated
fatty acids produced by the eukaryotic pathway is
controlled by multiple RFC loci in addition to the
FAD2 and FAD3 desaturase. Further investigation of
these rfc mutants will aid in understanding the regula-
tion of C18 unsaturated fatty acid metabolism.
Acknowledgements
G. Horiguchi is a recipient of a scholarship from the
Japanese Society for Promotion of Science. We thank
the Arabidopsis Biological Resource Center for mutant
seeds and Cereon Genomics for accessing polymor-
phism information. Part of this study was supported by
grant RFTF96L00602 from the Japanese Society for
Promotion of Science.
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