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Regulatory Networks in Seeds Integrating Developmental,
Abscisic Acid, Sugar, and Light Signaling
1
Ine`s M. Brocard-Gifford, Tim J. Lynch, and Ruth R. Finkelstein*
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara,
California 93106
Progression through embryogenesis and the transition to germination is subject to regulation by many transcription factors,
including those encoded by the Arabidopsis LEC1 (LEAFY COTYLEDON1), FUS3 (FUSCA3), and abscisic acid-insensitive
(ABI) ABI3,ABI4, and ABI5 loci. To determine whether the ABI4,ABI5,LEC1, and FUS3 loci interact or act independently,
we analyzed abi fus3 and abi lec1 double mutants. Our results show that both ABI4 and ABI5 interact genetically with both
LEC1 and FUS3 in controlling pigment accumulation, suppression of vivipary, germination sensitivity to abscisic acid, gene
expression during mid- and late embryogenesis, sugar metabolism, sensitivity to sugar, and etiolated growth. However, the
relative strengths of the observed interactions vary among responses and may even be antagonistic. Furthermore, the
interactions reveal cryptic effects of individual loci that are not detectable by analyses of single mutants. Despite these strong
genetic interactions, but consistent with the disparities in peak expression of these loci, none of the ABI transcription factors
appear to interact directly with either FUS3 or LEC1 in a yeast (Saccharomyces cerevisiae) two-hybrid assay system.
Angiosperm embryo development can be divided
into three phases: morphogenesis, cell enlargement,
and desiccation (for review, see Rock and Quatrano,
1995). Cell division and histodifferentiation are com-
pleted during the morphogenesis phase, leading to
an embryo with all structures formed. This is fol-
lowed by a growth phase during which the embryo
fills the seed sac, accumulating storage reserves that
can be used later by the germinating seedling before
the onset of photosynthetic activity. During the final
phase, embryos develop desiccation tolerance, dehy-
drate, and enter developmental arrest, possibly be-
coming dormant.
Progression through embryo development to seed
maturity and the transition to germination is coordi-
nated by the interactions of stage-specific develop-
mental regulators and the competing effects of hor-
monal signals such as abscisic acid (ABA), GAs, and
ethylene (for review, see Finkelstein et al., 2002). In
addition, metabolites such as sugars may act as de-
velopmental signals regulating seed maturation (for
review, see Wobus and Weber, 1999). The most crit-
ical hormone promoting embryo maturation and
preventing germination is ABA, which reaches its
peak concentration midway through embryogenesis.
Severely ABA-deficient mutants of some species,
e.g. maize (Zea mays), produce viviparous seeds
(Robertson, 1955); this effect can be phenocopied by
transgene-driven production of antibodies directed
against ABA (Phillips et al., 1997). The Arabidopsis
ABI (ABA-INSENSITIVE) loci were initially identified
on the basis of the ABA-resistant germination of
mutants at these loci (Koornneef et al., 1984; Finkel-
stein, 1994). ABI3, which is an ortholog of the maize
VP1 (VIVIPAROUS1) locus, has the most pleiotropic
effects on seed maturation, regulating sensitivity to
ABA inhibition of germination, expression of some
seed-specific genes, acquisition of desiccation toler-
ance, and dormancy (Giraudat et al., 1992; Parcy et
al., 1994). However, severe abi3 mutants differ from
vp1 mutants in that they are not viviparous but pro-
duce desiccation-intolerant green seeds (Nambara et
al., 1992; Ooms et al., 1993). In addition to altering
ABA sensitivity of germination, the other ABI loci
regulate subsets of these responses: ABI1 and ABI2
regulate dormancy, but the monogenic mutants have
not been found to disrupt embryonic gene expression
(Koornneef et al., 1984; Finkelstein and Somerville,
1990; Parcy and Giraudat, 1997). In contrast, ABI4
and ABI5 do not regulate dormancy, but do control
some embryonic gene expression and also regulate
some seedling responses to ABA and sugars (Finkel-
stein, 1994; Finkelstein et al., 1998; Arenas-Huertero
et al., 2000; Finkelstein and Lynch, 2000a; Huijser et
al., 2000; Laby et al., 2000; So¨derman et al., 2000;
Lopez-Molina et al., 2001; Rook et al., 2001). ABI3,
ABI4, and ABI5 encode transcription factors and ap-
pear to act combinatorially to control embryonic gene
expression and seed sensitivity to ABA (Giraudat et
al., 1992; Finkelstein et al., 1998; Finkelstein and
Lynch, 2000a; So¨derman et al., 2000). Recent yeast
(Saccharomyces cerevisiae) two-hybrid studies have
shown that ABI3 and ABI5, and their rice (Oryza
sativa) homologs OsVP1 and TRAB1, can interact di-
1
This work was supported by the National Science Foundation
(grant nos. IBN–9728297 and IBN–9982779 to R.R.F.).
* Corresponding author; e-mail finkelst@lifesci.ucsb.edu; fax
805–893– 4724.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.011916.
78 Plant Physiology, January 2003, Vol. 131, pp. 78–92, www.plantphysiol.org © 2003 American Society of Plant Biologists
rectly (Hobo et al., 1999; Nakamura et al., 2001) and
presumably form part of a regulatory complex in
plants.
Unlike the ABI loci, the LEC1 (LEAFY COTYLE-
DON1) and FUS3 (FUSCA3) loci were identified on
the basis of developmental defects reflecting a failure
to temporally separate embryonic and vegetative dif-
ferentiation (Keith et al., 1994; Meinke et al., 1994;
West et al., 1994). In addition to producing cotyle-
dons with leaf-like characteristics such as trichomes,
starch accumulation, and anthocyanin accumulation,
the latter giving a purple color to the seed, both lec1
and fus3 are desiccation intolerant and occasionally
viviparous. Although both fus3 and lec1 embryos
have defects in the expression of some maturation-
specific genes (Nambara et al., 2000; Vicient et al.,
2000), only LEC1 affects ABA sensitivity of germina-
tion (Parcy et al., 1997).
ABI4, aside from its role in seed development and
germination, participates in a sugar signal transduc-
tion pathway. Additional abi4 alleles have been iso-
lated by screens including Suc insensitivity (sis5 and
sun6) (Huijser et al., 2000; Laby et al., 2000) or Glc
insensitivity (gin6) (Arenas-Huertero et al., 2000) in
early seedling growth because ABI4 is required for
Glc-induced developmental arrest at this stage. The
other abi mutants have been tested for their sugar
insensitivity and abi5 was shown to be mildly Glc
resistant (Arenas-Huertero et al., 2000; Huijser et al.,
2000; Laby et al., 2000), but abi5 mutants have never
been isolated by any screens for sugar insensitivity.
Although abi1-1,abi2-1, and abi3-1 display a Glc-
sensitive phenotype, overexpression of ABI3,ABI4,
or ABI5 confers sugar hypersensitivity (Finkelstein et
al., 2002). From these observations, it appears that the
ABA-mediated Glc signaling pathway belongs to a
branch in which ABI4 and, to a lesser extent, ABI5
and ABI3, participate as signaling molecules.
The phenotypes of the monogenic mutants indicate
that these loci control overlapping responses, but
they do not show whether these loci interact or act
independently. Previous digenic mutant studies have
shown synergistic effects of mutations in ABI3,FUS3,
and LEC1, resulting in production of highly pig-
mented viviparous seeds (Keith et al., 1994; Meinke
et al., 1994; Parcy et al., 1997). Studies of the molec-
ular basis of this synergism have shown that ABI3
protein accumulation is reduced in the double mu-
tants (Parcy et al., 1997). Both LEC1 and FUS3 have
now been cloned and found to encode transcription
factors (Lotan et al., 1998; Luerssen et al., 1998; Reidt
et al., 2000). LEC1 encodes a CCAAT box-binding
factor HAP3 subunit and FUS3 encodes a transcrip-
tion factor with a conserved VP1/ABI3-like B3 do-
main. In this paper, we report the construction and
characterization of four digenic mutants combining
mutations in either ABI4 or ABI5 with those in FUS3
or LEC1. Seed of digenic mutants was compared with
that of wild-type and monogenic parents in terms of
pigment content (chlorophyll and anthocyanin), em-
bryonic gene expression, and sensitivity to ABA for
inhibition of germination. Because ABI4 and ABI5 ap-
pear to play a role in sugar response and the digenic
mutants exhibit some characteristics of wild-type
seedlings grown on high sugar, we also assayed sugar
sensitivity of germination and seedling growth, and
accumulation of soluble sugars and starch. All tested
combinations appear to reflect genetic interactions,
but the strength of the interaction varies with the loci
involved and the response. Although some strong
genetic interactions were observed between the ABIs
and both FUS3 and LEC1, none of these appeared to
reflect direct physical interactions detectable by a
yeast two-hybrid assay system, consistent with
previous observations that peak expression of these
loci occurs at disparate periods of embryogenesis
(Lotan et al., 1998; Luerssen et al., 1998; Finkelstein
and Lynch, 2000a; So¨derman et al., 2000; Brocard et
al., 2002).
RESULTS
Previous studies have shown that Arabidopsis seed
development is subject to control by the ABA-
insensitive loci ABI3,ABI4, and ABI5, as well as by
the developmental regulators FUS3 and LEC1.To
determine whether ABI4 or ABI5 interacts genetically
with either FUS3 or LEC1, we constructed double
mutants combining each abi mutation with either a
fus3 or lec1 mutation. The alleles used were abi4-1,
abi5-1,fus3-3, and lec1-1. The abi4-1 mutation is a
frame shift that results in production of a truncated
protein that includes the presumed DNA-binding
domain (BD; Finkelstein et al., 1998), but lacks any
transcription activation function and confers ABA
resistance similar to that of alleles that also lack the
DNA-BD (So¨derman et al., 2000). The abi5-1 allele
contains a “nonsense”mutation, resulting in produc-
tion of a truncated protein lacking the basic Leu
zipper (bZIP) domain required for DNA binding and
dimerization (Finkelstein and Lynch, 2000a); expres-
sion of this allele is also severely reduced, reflecting
autoregulation (Brocard et al., 2002). Thus, although
not genetic null alleles, both the abi4-1 and abi5-1
mutations are probably biochemical null alleles.
However, it is still possible that the limited amounts
of truncated products might interfere with the activ-
ity of other unidentified proteins. The lec1-1 mutation
is a deletion that removes the entire LEC1 gene and,
therefore, is a true null allele (Lotan et al., 1998). The
fus3-3 mutation produces a defective exon/intron
boundary within the region encoding the conserved
B3 domain, resulting in accumulation of aberrant
transcripts that are predicted to not encode a func-
tional FUSCA3 protein (Luerssen et al., 1998).
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 79
Germination of Digenic Mutant Seeds Is Highly
Resistant to ABA
Previous studies have shown that abi4-1 and abi5-1
mutants are 10- and 3-fold, respectively, less sensi-
tive to ABA inhibition of germination than the wild-
type (Finkelstein, 1994), whereas fus3 mutants have
normal ABA sensitivity (Keith et al., 1994; Parcy et
al., 1997), and lec1 mutants have been described as
having either normal (Meinke et al., 1994; West et al.,
1994) or roughly 10-fold reduced (Parcy et al., 1997)
ABA sensitivity for this response. The discrepancies
in results with the lec1 mutants could reflect differ-
ences in the alleles tested, assay media, and criteria
used for scoring ABA resistance. Resistance was re-
ported only for germination and cotyledon expan-
sion of lec1-1 incubated on media that included Suc
(Parcy et al., 1997), a condition subsequently shown
to reduce sensitivity to exogenous ABA (Garciarru-
bio et al., 1997; Finkelstein and Lynch, 2000b). To
determine whether the fus3 or lec1 mutations en-
hanced the ABA resistance of the abi mutants, early
desiccation stage seeds were excised and cultured on
media containing a range of ABA concentrations, but
no sugars. All genotypes were pre-incubated for 3 d
at 4°C to eliminate any effects of residual dormancy
on germination potential. Under these assay condi-
tions, the fus3 mutants have an essentially wild-type
sensitivity to ABA, lec1 seeds are resistant to only 3 to
10
mABA, and the monogenic abi mutants are
resistant to only 3 to 30
mABA. In contrast, the abi
fus3 double mutants and most of the abi4 lec1 mutant
seeds were capable of germinating on media contain-
ing up to 300
mABA (Fig. 1A), reflecting strong
synergistic effects. Although abi5 lec1 and its mono-
genic parents showed similar responses to low ABA
(reaching only 30%–40% germination after 1 week),
only the digenic mutant could germinate in the pres-
ence of 300
mABA, albeit at a lower frequency than
the other digenics, suggesting a weaker interaction.
Furthermore, although germination of the mono-
genic abi mutants on low concentrations of ABA is
usually first observed 2 to 4 d post-stratification, the
abi fus3 digenic mutants begin to germinate on 100
mABA within 30 min even before stratification (Fig.
1B). This germination behavior reflects a complete
loss of dormancy as well as enhanced resistance to
ABA. The abi4 fus3 digenic mutants were most resis-
tant to ABA, reaching 50% germination within5hon
100
mABA; the abi5 fus3 and abi4 lec1 mutants
required several days to reach this level of germina-
tion. The abi5 lec1 digenic mutants germinated even
more slowly, reaching less than 10% germination
after 1 week on 100
mABA (Fig. 1A). Surprisingly,
the observed degree of resistance did not correlate
with the frequency of vivipary in these lines; the
double mutants carrying the abi5 mutation were
more predisposed toward vivipary than those carry-
ing the abi4 mutation, despite being less resistant to
inhibition of germination by exogenous ABA (Finkel-
stein et al., 2002). The degrees of ABA resistance or
vivipary were also poor indicators of subsequent
seedling growth; many of the mono- or digenic lec1
lines did not grow well on minimal media, possibly
reflecting their poor root growth, but inclusion of Glc
Figure 1. Sensitivity of early desiccation stage mono- and digenic
abi,fus3, and lec1 mutants to inhibition of germination by ABA. A,
Germination was scored after7dofincubation in continuous light
on media with indicated concentrations of ABA. Top, Combinations
involving fus3 compared with monogenic parents; bottom, combi-
nations involving lec1 compared with monogenic parents. B, Kinet-
ics of digenic mutant germination on 100
MABA. Graphs show
mean values of at least two independent experiments for each
genotype.
Brocard-Gifford et al.
80 Plant Physiol. Vol. 131, 2003
or Suc greatly improved their growth (data not
shown).
Pigment Accumulation in Mutant Embryos
Arabidopsis embryos are completely green from
torpedo stage until the onset of desiccation, when
they lose color as a result of chlorophyll breakdown
(Meinke, 1994). However, mutants that fail to com-
plete the maturation process, such as fus3,lec1, and
the severe alleles of abi3, also fail to lose chlorophyll
at this stage (Nambara et al., 1992; Ooms et al., 1993;
Keith et al., 1994; Meinke et al., 1994). In contrast to
chlorophyll, anthocyanin does not accumulate to sig-
nificant levels in Arabidopsis embryonic tissues until
after germination. The precocious accumulation of
anthocyanin in the leafy cotyledon class of mutants
(e.g. fus3 and lec1) is part of the basis for describing
these as heterochronic mutants (Keith et al., 1994;
Meinke et al., 1994).
As previously described for the abi3 fus3 and abi3
lec1 digenic mutants (Keith et al., 1994; Parcy et al.,
1997), the abi4 and abi5 combinations with fus3 and
lec1 were first recognized as highly pigmented seeds
among the segregating F
2
progeny. To quantify the
effects on pigment accumulation before the onset of
vivipary, early desiccation stage seeds were excised
and used for extraction of chlorophyll or anthocyanin
(Fig. 2). The large sds reflect the fact that chlorophyll
decreases rapidly, whereas anthocyanin increases
rapidly, at this stage in seed development. As a con-
sequence, substantial variation can be observed
within a single silique.
The amount of chlorophyll in monogenic mutant
immature seeds was similar to that in the corre-
sponding wild-type lines (Fig. 2A). Despite the lack
of effect of the single mutations, chlorophyll accumu-
lation was significantly enhanced in all of the double
mutants except abi5 lec1. Although mean anthocyanin
levels in fus3 and lec1 increased 1.5- to 3-fold relative
to their corresponding wild-type lines (Fig. 2B), there
was no statistically significant difference in anthocy-
anin content between any of the monogenic mutants
and their wild-type progenitors at this stage. Al-
though the average anthocyanin content of abi5 lec1
seeds was slightly higher than that in lec1 seeds at
this stage, this was also not a statistically significant
difference. In contrast, the anthocyanin levels in the
double mutants revealed a strong synergistic inter-
action (from 10–16-fold higher than the highest of the
corresponding monogenic parents) between both abi
mutations and fus3 and between abi4 and lec1.
Embryonic Gene Expression
Mutations in ABI4 and ABI5 have been shown pre-
viously to have minor effects on gene expression
during embryogenesis, indicating that these loci are
required for only a subset of the ABI3-regulated
genes (Finkelstein and Lynch, 2000a; So¨derman et al.,
2000). In contrast, fus3 and lec1 mutations result in
severely reduced embryonic gene expression (Keith
et al., 1994; Parcy et al., 1997; Nambara et al., 2000;
Vicient et al., 2000). For example, genes for the stor-
age proteins At2S3 and CRC, and the lipid body
protein oleosin (PAP147), show essentially no change
in expression in the abi4 or abi5 mutants, whereas
their transcript accumulation in midembryogenesis is
severely reduced in the fus3 and lec1 mutants (Fig.
3A). In late embryogenesis, transcript levels for the
late embryogenesis-abundant (LEA) genes AtEm1
and AtEm6 are greatly reduced in abi5 mutants and
are even lower in lec1 mutants (Fig. 3B). However,
this has previously been shown to reflect delayed
expression in the lec1 mutants such that AtEm1 tran-
scripts reach at least wild-type levels in dry lec1 seeds
(Vicient et al., 2000), possibly because of a stress
response preceding death of these desiccation-
Figure 2. Pigment accumulation in early desiccation stage mono-
and digenic abi,fus3, and lec1 seeds. Chlorophyll and anthocyanin
content are expressed in arbitrary units and normalized to the num-
ber of seeds used in each sample. Values are the mean of two to 11
measurements with samples of 23 to 46 seeds each. Bars indicate
SDs. A, Chlorophyll content. B, Anthocyanin content.
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 81
intolerant seeds. In contrast, although AtEm6 tran-
scripts accumulate to at least wild-type levels in abi4
mutants at late embryogenesis (So¨derman et al.,
2000), they do not increase further by seed maturity,
resulting in slightly lower than wild-type levels in
dry seeds (Finkelstein, 1994). AtEm6 expression is
also reduced in fus3 mutants, but AtEm1 expression
increases in fus3 seeds (Fig. 3B), as previously docu-
mented for AtEm1 promoter activity (Vicient et al.,
2000). The expression of RAB18 and M17 is severely
reduced in fus3 and lec1 mutants, and near normal in
abi4 mutants, but M17 expression is slightly in-
creased in abi5 mutant seeds at desiccation stage.
These results also confirm previous observations
(Finkelstein and Lynch, 2000a; Nambara et al., 2000;
So¨derman et al., 2000; Vicient et al., 2000).
Comparison of transcript accumulation at mid- and
late embryogenesis in mono- and digenic mutant
seeds suggests that the abi mutations slightly enhance
the effects of the fus3 and lec1 mutations with respect
to expression of some storage reserve (At2S3,cruci-
ferin C, and oleosin; Fig. 3A) and LEA (AtEm1,AtEm6,
and RAB18; Fig. 3B) genes. With respect to M17 ex-
pression, the decreased expression due to fus3 or lec1
appears epistatic to the increase observed in the abi5
mutant. In contrast, the decreased expression of
AtEm1 because of the abi5 mutation is epistatic to the
increase observed in the fus3 mutant. These complex
double mutant phenotypes suggest that different tar-
get genes are regulated by varying combinations of
transcription factors.
Previous studies have demonstrated cross regula-
tion of ABI3,ABI4, and ABI5 transcript accumulation
and/or promoter activity (So¨derman et al., 2000), as
well as synergistic effects of LEC1 or FUS3 and ABI3
on ABI3 protein accumulation (Parcy et al., 1997). To
determine whether the observed genetic interactions
might reflect cross regulation of ABI4,ABI5,FUS3,
and LEC1 expression, we assayed their transcript
accumulation in the various mutant backgrounds
(Fig. 4; data not shown). We had shown previously
that ABI4 expression was near normal in abi5,fus3,
and lec1 seeds (So¨derman et al., 2000). Although ABI4
transcript levels vary slightly among seed lots, these
levels show no significant change in the digenic mu-
tants relative to the monogenic parents (data not
shown). In contrast, ABI5 expression is reduced in
abi5,lec1, and all digenic mutant seed. However, this
reduction is probably not a trivial reflection of the
failure of these seeds to reach maturity, when ABI5
transcript levels are highest, because monogenic fus3
seeds express ABI5 at or above wild-type levels de-
spite failing to complete maturation. FUS3 expres-
sion is significantly reduced only in the lec1 mutants,
and LEC1 expression is eliminated in all the lec1
mono- and digenic mutants, reflecting the fact that
the lec1-1 allele is a deletion. These results show cross
regulation of ABI5 and FUS3 by LEC1, and are con-
sistent with the previously described autoregulation
of ABI5 (Finkelstein and Lynch, 2000a; Brocard et al.,
2002). The major peak of LEC1 expression occurs
during the 1st week of embryogenesis (Lotan et al.,
1998), preceding the major peak of ABI5 transcript
accumulation by approximately 2 weeks (Finkelstein
and Lynch, 2000a; Brocard et al., 2002), suggesting
that LEC1 regulates ABI5 expression indirectly.
Regardless of whether cross regulation is observed,
the strong synergy among some of these mutations
cannot be explained simply by altered expression or
stability of the ABI gene products alone; the abi4,abi5,
and lec1 alleles used in this study are biochemical
Figure 3. Embryonic gene expression in mono- and digenic abi,
fus3,and lec1 mutants. RNA was extracted from immature siliques,
then analyzed by RNA gel blots hybridized to cloned probes for the
indicated transcripts. A, Maturation stage siliques (8–11 DPA). B,
Late embryogenesis stage siliques (17–21 DPA). Filters in A and B
contain 5 and 2.4
g of total RNA, respectively.
Brocard-Gifford et al.
82 Plant Physiol. Vol. 131, 2003
nulls and their intrinsic defects cannot be enhanced.
However, this synergy might reflect additional cross
regulation of other factors that interact with the ABI
transcription factors. For example, several other
members of the ABI5 subfamily of bZIPs form het-
erodimers with ABI5 (Kim et al., 2002) and display
varying patterns of cross regulation by overexpres-
sion of several of the ABIs in vegetative tissue (Bro-
card et al., 2002). Most of these are expressed most
abundantly in early to midembryogenesis (Bensmi-
hen et al., 2002), so we compared their transcript
accumulation at midembryogenesis (Fig. 4). All
members of this bZIP family with detectable expres-
sion at this stage were expressed normally in the abi
monogenic mutants, but these transcript levels were
much lower in the lec1 monogenic and all digenic
mutants. Expression of these bZIP genes was more
variable in fus3 mutants, ranging from slightly to
strongly underexpressed (Fig. 4; data not shown). In
addition to the possible synergistic regulation of
these bZIPs by the ABI and FUS3 genes, the com-
bined loss of the ABI factors and these potentially
interacting bZIP factors could result in significantly
enhanced signaling defects in the digenic mutants.
Interactions Affecting Sugar Response and
Accumulation of Sugars
Stunted growth and increased anthocyanin accumu-
lation are characteristic of wild-type plants grown on
high concentrations of sugar (⬎250 mm). Therefore the
high anthocyanin content of abi4 lec1 or abi fus3 digenic
mutant embryos and seedlings is reminiscent of the
effects of sugar on seedling growth. Mutations in ABI4
and ABI5 result in strong and weak sugar-resistant
phenotypes, respectively (Arenas-Huertero et al., 2000;
Huijser et al., 2000; Laby et al., 2000), whereas over-
expression of either gene confers hypersensitivity to
sugar (Brocard et al., 2002; Finkelstein et al., 2002). To
determine whether the LEC1 or FUS3 loci affect sugar
metabolism and/or response, and whether they inter-
act with the ABI loci in this regard, we compared
sugar sensitivity and accumulation of soluble sugars
and starch in wild-type, monogenic, and digenic mu-
tant seeds.
Sugar sensitivity was assayed by scoring growth
(i.e. germination and production of true leaves) of
seedlings derived from early desiccation stage em-
bryos, after incubation on Glc concentrations ranging
from 0% to 6% (w/v; up to 333 mmGlc) under either
continuous light or dark conditions after stratifica-
tion in dim light. Although over one-half of the seeds
of all genotypes except Columbia (Col) and abi4 ger-
minated on up to 4% (w/v) Glc in light (Fig. 5A),
subsequent seedling growth was severely reduced by
4% (w/v) Glc in all of the wild-type and monogenic
lines such that true leaves were seldom seen until
after 1 week (Figs. 5B and 6, A and B). The monogenic
fus3 and lec1 mutants appear to display stage-specific
Figure 4. ABI5,FUS3,LEC1, and ABI5 homologs expression in
mono- and digenic abi,fus3, and lec1 mutants. RNA was extracted
from dry seeds or immature siliques, then analyzed by RNA gel blots
hybridized to cloned probes for the indicated transcripts. ABI5 ex-
pression was assayed in dry seeds (5
g of total RNA per lane), FUS3
and the ABI5 homologs were assayed at maturation stage (8–11 DPA;
10 or 7
g of total RNA per lane for FUS3 or the ABI5 homologs,
respectively), and LEC1 was assayed in 1- to 5-DPA siliques (20
g
of total RNA per lane). ABI5 homologs tested were AtDPBF2/
AtbZIP67,AtDPBF3/AREB3,AtDPBF4/EEL/AtbZIP12,AtDPBF5/ABF3,
and ABF4/AREB2;AtDPBF5/ABF3 and ABF4/AREB2 transcripts were
not detected in any genotype at this stage. The heterogeneity of
transcript sizes observed for FUS3 and AtbZIP67/AtDPBF2 have been
reported previously (Luerssen et al., 1998; Bensmihen et al., 2002).
Each hybridization was performed with a fresh blot; the rRNA control
depicted is from rehybridization of one representative blot.
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 83
defects in sugar response: They germinate at a higher
frequency than wild type on media containing high
Glc, but appear hypersensitive to Glc effects on seed-
ling growth, producing many stunted pink seedlings
on 4% (w/v) Glc (Fig. 5). Although stunted, the fus3
seedlings do not arrest growth, some producing
dark-pink plants with true leaves (Fig. 6C). In con-
trast, the abi fus3 and, to a lesser extent, abi lec1
digenic mutants, germinate rapidly and maintain
substantial growth on high Glc with some plants
remaining green and producing true leaves even on
6% (w/v) Glc (Fig. 5). Thus, the digenic mutants
appear resistant to germination inhibition, induction
of anthocyanin accumulation, and repression of
growth by high Glc even though the Glc response of
the monogenic abi mutants resembles that in wild-
type seedlings at this stage (Fig. 5). Similar to results
previously described for dry seeds (Laby et al., 2000),
sorbitol was less inhibitory of germination in early
desiccation stage wild-type seeds than an equimolar
concentration of Glc (⬎90% versus ⬍20% germina-
tion for seeds exposed to 222 mmsorbitol and Glc,
respectively), consistent with the view that the inhib-
itory effects of Glc are not simply because of osmotic
stress. However, germination of the fus3 mono- and
digenic mutant seeds was even more resistant to
sorbitol, reaching 100% even on 333 mmsorbitol,
indicating that the Glc-resistant germination of these
genotypes is accompanied by increased resistance to
osmotic stress. In contrast to their relative effects on
germination, sorbitol was slightly more inhibitory of
seedling growth (i.e. production of true leaves) than
Glc, for all genotypes except Col wild type and abi4
(data not shown). The inhibition of growth imposed
by sorbitol was especially pronounced in the fus3 and
lec1 mono- and digenic mutants, possibly because at
least some of the Glc serves a nutritional function
(compare growth on 0% [w/v] versus 1% [w/v] Glc;
Fig. 5B). Consistent with this possibility, supplement-
ing the sorbitol with 1% (w/v) Glc substantially im-
proved seedling growth of these genotypes (data not
shown). These results indicate that the highly Glc-
resistant seedling growth of the abi fus3 digenic mu-
tants might also be partially because of resistance to
osmotic stress. In contrast to Glc, no concentration of
sorbitol tested induced anthocyanin accumulation in
any genotype, indicating that the observed hypersen-
sitivity to Glc for this response in the fus3 and lec1
mutants was not because of osmotic stress.
Dark-grown plants germinated at lower frequen-
cies in all but the fus3 mono- and digenic lines (Fig.
7), possibly reflecting some residual photodormancy
in the other genotypes. The wild-type and monogenic
mutant seedlings are all etiolated in the dark, but the
length of Col and abi4 seedlings is quite variable,
probably reflecting delayed germination of some in-
dividuals. Among germinated dark-grown plants,
the anthocyanin accumulation characteristic of the
sugar sensitive phenotype was not observed. How-
ever, these plants still respond to high sugar by sup-
pressing the extreme hypocotyl elongation character-
istic of etiolated growth. In contrast, low sugar (1%
[w/v] Glc) slightly promotes elongation of most ge-
Figure 5. Glc effects on germination and growth in mono- and
digenic abi,fus3, and lec1 mutants. Seeds were excised at early
desiccation stage and cultured on minimal media supplemented with
0%, 1%, 4%, or 6% (w/v) Glc. Germination (A) and seedling color
and production of true leaves (B) were scored after7dofincubation
in continuous light. Graphs show mean values of at least two inde-
pendent experiments for each genotype. Bars indicate SD for germi-
nation data.
Brocard-Gifford et al.
84 Plant Physiol. Vol. 131, 2003
notypes, but the lec1 mono- and all digenic seedlings
are still significantly shorter than the majority of
wild-type and monogenic abi seedlings exposed to
1% (w/v) Glc (Table I). The fus3 seedlings are also
shorter than the majority of wild-type seedlings, but
the variability within the wild-type set prevents this
observation from being a statistically significant dif-
ference. Surprisingly, all of the digenic lines have
expanded leaves and cotyledons in the dark, resem-
bling de-etiolated mutants (Fig. 6, H, J, and K; data
not shown).
Comparison of soluble sugar accumulation in dry
seeds showed that Suc levels approximately doubled
in all of the fus3 or lec1 mono- or digenic seeds except
those of abi5 fus3 (Fig. 8A). In contrast, abi5 fus3 seeds
had significantly increased levels of Fru, which was
near the limit of detection in the wild-type and mo-
nogenic lines and only slightly increased in the other
digenic mutants. More dramatic differences were ob-
served in comparing starch accumulation; as previ-
ously described (Keith et al., 1994; Meinke et al.,
1994), fus3 or lec1 seeds accumulate starch, whereas
wild-type have almost no detectable starch. Al-
though monogenic abi mutant seeds also lack starch,
double mutant seeds contain 7- to 11-fold or 1.5- to
2-fold more starch than the corresponding mono-
genic fus3 or lec1 seeds, respectively (Fig. 8B). Be-
cause fus3 and lec1 mono- or digenic seeds fail to
complete the desiccation phase of embryo develop-
ment, we also assayed sugar and starch accumulation
in early desiccation stage seeds of all genotypes.
These seeds contain more starch than dry seeds, but
similar trends were observed in comparisons among
genotypes (data not shown). It is not clear whether
the increased starch accumulation reflects increased
synthesis, a failure to hydrolyze starch as normally
occurs during Arabidopsis seed maturation, or a
combination of these effects. However, it does not
result in decreased accumulation of Suc (Fig. 8A), the
other major storage form for fixed carbon.
Figure 7. Germination of mono- and digenic abi,fus3, and lec1
mutants in darkness. Seeds were excised at early desiccation stage
and cultured on minimal media supplemented with 0%, 1%, 4%, or
6% (w/v) Glc. Germination was scored after 7 d of incubation in the
dark. Graph shows mean values ⫾SD of at least two independent
experiments for each genotype.
Figure 6. Morphology of mono- and digenic
abi,fus3, and lec1 mutant seedlings. A through
D, Seedlings grown for8dincontinuous light
on medium with 4% (w/v) Glc. A, Wild-type
Wassilewskija (Ws); B, abi5;C,fus3;D,abi5
fus3. E through J, Seedlings grown for9dinthe
dark on medium with 1% (w/v) Glc. E, Wild-
type Ws; F, abi4;G,fus3;H,abi4 fus3;I,lec1;
J, abi4 lec1;K,abi5 lec1.
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 85
Test of Physical Interactions between ABI, FUS, and
LEC Gene Products
Our studies show that ABI4 and ABI5 interact ge-
netically with FUS3 and LEC1. Although the severity
of the digenic mutant phenotypes vary among re-
sponses, all appear to reflect interactions disrupting
some subset of the following processes: suppression
of vivipary, germination on ABA, pigment accumu-
lation, embryonic gene expression, and sugar sensing
and/or metabolism. Similar results have been ob-
tained previously for ABI3,FUS3, and LEC1 (Keith et
al., 1994; Meinke et al., 1994; Parcy et al., 1997),
although the earlier studies did not examine sugar
sensing or metabolism. All five of these loci encode
transcriptional regulators that might participate in a
regulatory complex. To determine if the observed
genetic interactions reflect direct physical interac-
tions, we used a yeast two-hybrid assay system with
GAL4-DNA-BD fusions to ABI3, ABI4, and ABI5 as
“bait”constructs. These constructs used truncations
of ABI3 and ABI4 because the full-length proteins are
strong transcriptional activators in yeast (So¨derman
et al., 2000; data not shown). The ABI4, FUS3, and
LEC1 proteins were fused to the transcription activa-
tion domain (AD) of GAL4. The AD vector and an
AD-ABI5 fusion were included as negative and pos-
itive controls, respectively. Physical interactions
would be reflected by trans-activation of the GAL4-
responsive lacZ reporter gene. Although these bait
constructs do produce functional products (Naka-
mura et al., 2001; data not shown), no strong inter-
actions (i.e. resulting in greater than 2-fold enhance-
ment of lacZ expression) were observed between
FUS3 or LEC1 and any of the ABIs (Fig. 9). The high
intrinsic activating function of the BD-ABI4 fusion
makes it difficult to determine whether the statisti-
cally significant, but less than 2-fold, enhancement of
lacZ activation by the AD-FUS3 construct is biologi-
cally significant. The even higher intrinsic activation
because of a BD-FUS3 fusion precluded attempting
the reciprocal experiment (data not shown). Al-
though AD-LEC1 produced a 2-fold increase in trans-
activation by BD-ABI5, no interaction was detected
between BD-LEC1 and AD-ABI5 (data not shown).
These results also show apparent homodimerization
of ABI3, consistent with the previously documented
cooperative DNA-binding activity of the B3 domain
(Suzuki et al., 1997), as well as reproducing the pre-
viously documented ABI5 homodimerization and
ABI5/ABI3 interaction (Nakamura et al., 2001). We
also tested for formation of a ternary complex by
combining AD-FUS3 and BD-LEC1 with each ABI,
but no interactions were observed in these combina-
tions (data not shown).
Figure 8. Soluble sugar and starch accumulation in mono- and
digenic abi,fus3, and lec1 mutant seeds. Suc, Glc, and Fru (A) and
starch (B) were assayed enzymatically in extracts from 50 dry seeds
of the indicated genotypes. Graph shows mean values ⫾SD of three
samples of 50 seeds each. Asterisks indicate starch contents signifi-
cantly different from wild type for the monogenic mutants or from the
corresponding monogenics for the digenic mutants (P ⬍⬍ 0.05, based
on Student’sttest, unequal variance assumed).
Table I. Elongation of dark-grown seedlings
Seeds of the indicated genotypes were excised at early desiccation
stage and placed on minimal media with or without 1% (w/v) Glc,
incubated for3dat4°C, then for9dat22°C in darkness before
measuring hypocotyl lengths. Values shown are mean ⫾SD of lengths
of five to 14 seedlings.
Genotype
Elongation on 1% (w/v) Glc
Hypocotyl
length
Length on
minimal media
mm %
Col 18.1 ⫾6.7 78.9
abi4 14.1 ⫾4.0 96.6
fus3 12.6 ⫾3.7 137
Ws 22.8 ⫾1.3 110
abi5 23.5 ⫾1.45 94.3
lec1 4.5 ⫾2.3
a
109
abi4, fus3 8.3 ⫾2.1
a
152
abi5,fus3 7.8 ⫾2.2
a
136
abi4, lec1 1.8 ⫾0.5
a
134
abi5, lec1 6.6 ⫾2.2
a
254
a
Significantly different from wild type (P⬍0.05, based on Stu-
dent’sttest, unequal variance assumed).
Brocard-Gifford et al.
86 Plant Physiol. Vol. 131, 2003
DISCUSSION
Regulatory Networks in Seed
Development and Germination
FUS3 and LEC1 expression peak during the mor-
phogenesis phase of embryo development, but these
genes affect processes throughout seed development,
regulating the establishment of body plan, suppres-
sion of vivipary, reserve accumulation, expression of
some LEA genes, and induction of desiccation toler-
ance (Lotan et al., 1998; Luerssen et al., 1998; Raz et
al., 2001). Mutations in these genes result in seed
lethality unless embryos are rescued before desicca-
tion. Although ABI3,ABI4, and ABI5 genes are also
expressed early in embryogenesis, expression of ABI4
and ABI5 peaks at seed maturity, consistent with a
greater role in controlling the transition from embry-
ogenesis to germination (Parcy et al., 1994; So¨derman
et al., 2000; Brocard et al., 2002). Comparison of se-
vere loss of function alleles for these ABI loci has
shown that ABI3 is most critical for seed maturation
and sensitivity to ABA inhibition of germination, but
that ABI4 and ABI5 may be more important in regu-
lating seedling establishment, particularly under
stress conditions. Unlike the abi and lec1 mutants, fus3
mutants do not display ABA-resistant germination.
Previous experiments showed that ABI3,FUS3, and
LEC1 act synergistically to control multiple processes
during seed development, including promotion of
chlorophyll breakdown, suppression of anthocyanin
accumulation, and control of sensitivity to ABA for
germination inhibition (Parcy et al., 1997). Similarly,
ABI1 and, to a lesser extent, ABI2 appear to act syn-
ergistically with FUS3;abi1 fus3 and abi2 fus3 double
mutant seeds have increased vivipary and are redder
than their respective monogenic parents, but the pig-
ment levels were not quantified (Keith et al., 1994).
We report herein the digenic analyses with FUS3 or
LEC1 and ABI4 or ABI5.
Synergistic effects on ABA-resistant germination
and pigment levels were observed for the abi4 fus3,
abi5 fus3, and abi4 lec1 mutants, but the defects of the
monogenic parents were at most slightly enhanced in
abi5 lec1 mutants. All double mutants tested had
synergistic effects on starch accumulation. Compari-
son of embryonic gene expression showed that most
of the transcripts assayed were greatly reduced in
fus3 and lec1 mutants such that it was difficult to
determine whether any further suppression occurred
in the double mutants. However, the observation that
the monogenic abi mutants have little or no effect on
storage protein gene expression, yet further decrease
storage protein gene expression in the digenic mu-
tants, is consistent with a synergistic interaction. The
observations that fus3 and lec1 appear epistatic to abi5
with respect to effects on M17 expression, yet abi5
appears epistatic to fus3 with respect to AtEm1 ex-
pression, are not readily explained by simple hierar-
chical genetic interactions. Surprisingly, although
sugar induction of anthocyanin accumulation in pre-
cociously germinating desiccation stage seeds was
not affected in abi mutants and was enhanced in lec1
and fus3 mutants, the digenic mutants were resistant
to this sugar-induced response: The fraction of seed-
lings with anthocyanin accumulation was consis-
tently lower in the digenics than in the monogenic
lines grown on any given Glc concentration. One
possible explanation for this is that in the digenic
lines the heterochronic lec1 or fus3 mutations change
the developmental context such that the abi muta-
tions function as they would during normal germi-
nation, when they confer sugar-resistant growth
(Arenas-Huertero et al., 2000; Huijser et al., 2000;
Laby et al., 2000). In this regard, the abi mutations
appear to have an epistatic effect on sugar induction
of anthocyanin accumulation. These results are all
suggestive of genetic interactions among these genes,
but the nature of the interaction varies depending on
the affected response.
To date, all of the ABI transcription factors have
been shown to interact genetically with FUS3 and
LEC1. Possible explanations for these results include
cross regulation of expression and/or direct physical
interactions. Although all the ABI and LEC class tran-
scription factors analyzed in these studies are ex-
pressed to varying degrees throughout embryo devel-
opment, they do not display the same developmental
profile of accumulation. LEC1 expression is most
abundant early in embryogenesis (Lotan et al., 1998)
and FUS3 transcripts peak at midembryogenesis (Lu-
erssen et al., 1998), whereas ABI4 and ABI5 tran-
scripts peak at seed maturity (Finkelstein and Lynch,
2000a; So¨derman et al., 2000). The recently cloned
LEC2 gene also encodes a B3 domain transcription
factor, is expressed from early to midembryogenesis
Figure 9. Yeast two-hybrid assays of interactions between ABI tran-
scription factors and LEC1 or FUS3.

-galactosidase activity of yeast
harboring plasmids encoding the GAL4-AD (AD) or the indicated
GAL4-AD fusions in combination with either GAL4-BD (BD) or the
indicated GAL4-BD fusions. The BD-ABI3 and BD-ABI4 fusions en-
code truncated forms of these ABI proteins to reduce their intrinsic
activation function (see “Materials and Methods”for details). Values
are the means ⫾SD of assays on at least three independent
transformants.
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 87
(Stone et al., 2001), and might also interact genetically
with these ABI loci, but this hypothesis has not yet
been tested. The major functions of the LEC class
genes appear to be maintenance of an embryonic
state, whereas the ABIs promote embryo maturation.
The observed effects of the mutations on transcript
accumulation are consistent with sequential or hier-
archical LEC1-dependent activation of FUS3 and
ABI5. Consistent with an indirect effect of LEC1 on
ABI5 expression, a LEC1-GAL4AD fusion failed to
significantly enhance expression of ABI5 promoter-
lacZ fusions in yeast (data not shown). An alternate
explanation for LEC1-dependent FUS3 and ABI5 ex-
pression is that the lec1 mutants might either reduce
or lose the stages when these genes are normally
expressed. Consistent with this hypothesis, the ob-
served restoration of FUS3 expression in the abi lec1
mutants at midembryogenesis (Fig. 4) might reflect
an abi-dependent failure to accelerate toward matu-
ration phase, when FUS3 transcript levels normally
decrease. Expression of several of the ABI5-
homologous bZIPs is also dependent on LEC1, and to
a lesser extent FUS3 and the ABIs, but the lack of
characterized mutants for most of these loci makes
their role less clear. Surprisingly, one of these (At-
bZIP12/EEL/AtDPBF4) has recently been shown to
act antagonistically to ABI5 with respect to AtEm
expression, possibly by competing for binding to the
same sites within the promoter (Bensmihen et al.,
2002). Similar to the reported phenotype of abi5 eel
digenic mutants, the fus3 digenics and all the lec1
mutant lines have reduced AtEm expression, consis-
tent with their decrease in both ABI5 and AtbZIP12/
EEL/AtDPBF4 expression. In contrast, the slight de-
crease in AtbZIP12/EEL/AtDPBF4 expression in fus3
mutants may be sufficient for the observed increase
in AtEm1 expression, yet does not enhance AtEm6
expression. Unlike ABI5 and its homologs, ABI4 ex-
pression appears to be unaffected by the LEAFY
COTYLEDON class mutants. Thus, of all the regula-
tory loci tested, expression of ABI5 and/or its ho-
mologs are most closely correlated with changes in
marker gene expression.
In addition to the observed cross regulation of
regulatory gene expression, levels of active protein
for these regulators might be subject to posttranscrip-
tional controls, as have been described for ABI5 in
seedlings (Lopez-Molina et al., 2001). ABI3 transcript
levels in abi3,fus3, and lec1 mono- and digenic mu-
tants did not correlate with severity of the mutant
phenotype, yet comparison of ABI3 protein levels in
these lines demonstrated that FUS3 and LEC1 act
synergistically with ABI3 to control ABI3 accumula-
tion (Parcy et al., 1997). Surprisingly, fus3 and abi3-4
monogenic mutants show the opposite effect: en-
hanced ABI3 accumulation. This complexity of regu-
lation is similar to our observations of FUS3 expres-
sion in mono- versus digenic mutants. One possible
explanation for the synergistic effects on ABI3 pro-
tein accumulation is that ABI3 might be stabilized by
direct interaction with FUS3 or LEC1, both of which
also encode transcription factors. However, on the
basis of yeast two-hybrid assays with the ABI and
LEAFY COTYLEDON class gene products, ABI3 ap-
pears to interact directly only with ABI5 and itself
(Nakamura et al., 2001; Fig. 9). Perhaps it is not
surprising that FUS3 and ABI5 do not appear to
interact directly because the interaction between
ABI3 and ABI5 requires the B1 domain of ABI3 (Na-
kamura et al., 2001) and FUS3 lacks a B1 domain,
even though both ABI3 and FUS3 are members of the
B3 domain family of transcription factors (Luerssen
et al., 1998). The failure to detect interactions among
the remaining transcription factors might reflect a
simple lack of physical interactions, a requirement
for modification (e.g. phosphorylation) that does not
occur in yeast, deletion of a domain required for
interaction (to reduce intrinsic activation by the BD
fusion), or a requirement for additional factors. As a
consequence, the lack of interaction in yeast does not
exclude the possibility that these factors participate
in a regulatory complex in plants. However, the dis-
crepancies in developmental timing of peak expres-
sion for most of these regulators argue against direct
interactions as a major mechanism of the observed
genetic interactions.
The ABI/LEC Class Network Integrates Responses to
Developmental, Chemical, and Abiotic Signals
Physiological and genetic studies have demon-
strated both antagonistic and similar effects of ABA
and sugar in embryogenesis and germination (for
review, see Finkelstein and Gibson, 2002). The
midembryogenesis transition from growth by cell
division to growth by enlargement is correlated with
a decrease in Glc and an increase in endogenous
ABA. This transition suppresses vivipary and is also
dependent on FUS3 and LEC1 function (Raz et al.,
2001), but not ABI4 or ABI5. Exogenous ABA inhibits
germination at seed maturity, but this effect can be
suppressed by low levels of Glc or Suc, demonstrat-
ing another antagonistic interaction between sugar
and ABA (Finkelstein and Lynch, 2000b). However,
the developmental arrest and intense anthocyanin
accumulation induced by exposure to high concen-
trations of sugar during germination of mature seeds
is partially dependent on ABA and the ABI transcrip-
tion factors (Arenas-Huertero et al., 2000; Huijser et
al., 2000; Laby et al., 2000; Rook et al., 2001). Al-
though only ABI4 has been identified genetically by
sugar-sensing screens to date, under- and/or overex-
pression of ABI5 and ABI3 also modify sugar sensi-
tivity (Finkelstein et al., 2002).
Comparison of sugar metabolism and response to
sugar or osmotic stress demonstrated that all were
disrupted in fus3,lec1, and digenic mutant seeds.
Furthermore, sugar sensitivity appears to be regu-
Brocard-Gifford et al.
88 Plant Physiol. Vol. 131, 2003
lated differently before and after seeds reach matu-
rity. For example, although highly resistant to Glc
after seed maturity, abi4 mutants are only weakly
resistant to Glc inhibition of germination and growth
before seed maturity. In contrast, the fus3 and lec1
mutants confer sugar/osmotic stress-resistant germi-
nation, but hypersensitivity to sugar-induced antho-
cyanin synthesis at this stage; their lack of desiccation
tolerance precludes testing their sensitivity at the dry
seed stage. The growth defects of the fus3 and lec1
mutant embryos might be enhanced by the combina-
tion of hypersensitivity to sugar and doubled endog-
enous levels of Suc. However, the digenic mutants
are highly resistant to sugar/osmotic effects on ger-
mination and growth, as well as to sugar-specific
induction of anthocyanin synthesis, despite having
increased endogenous levels of Suc (or Fru, in the
case of abi5 fus3) and exhibiting intense pigmentation
as developing embryos. The degree of sugar/osmotic
resistance in the digenic mutants correlates with their
ABA resistance. One possible explanation for this
result is that the digenic mutants have undergone a
phase transition that permits extremely rapid germi-
nation and escape from the brief developmental win-
dow (up to approximately 36 h post-stratification) of
sensitivity to high concentrations of sugar (Gibson et
al., 2001), whereas the monogenic fus3 and lec1 mu-
tants precociously enter this sugar-sensitive phase
but do not escape it.
Our results also show that the extreme anthocyanin
accumulation characteristic of seedlings grown on
high concentrations of sugar is a synergistic effect of
sugar and light signaling. Anthocyanin accumulation
is a well-characterized response to UV or high-
intensity light stress (for review, see Mol et al., 1996)
and moderate anthocyanin accumulation in response
to low sugar (1% [w/v] Suc) has been demonstrated
to be phytochrome dependent (Montgomery et al.,
1999). However, neither moderate light nor a high
sugar concentration alone is sufficient to induce ex-
treme anthocyanin accumulation. The fus3 and lec1
mutants are characterized by the red color of their
embryos and their hypersensitivity to sugar-induced
anthocyanin accumulation, both consistent with a
role for these loci in repressing this light- and sugar-
induced response. Although fus3 differs from the
other FUSCA loci in that it has not yet appeared in a
screen for “de-etiolated”or “constitutively photo-
morphogenic”growth, we found that the abi fus3
digenic mutants had a mild de-etiolated phenotype
on low concentrations of sugar. Similarly, although
lec1 mutant seedlings tend to be stunted under all
conditions, cotyledon and true leaf expansion in the
dark is observed only in the abi lec1 digenic mutants.
These results suggest that the ABI and LEC class loci
interact in some aspects of light and sugar response
as well as ABA and seed developmental responses.
Consistent with this hypothesis, attempts to combine
lec1 with a constitutively photomorphogenic mutant,
cop1, resulted in embryo lethality at torpedo stage
(Meinke et al., 1994). This early lethality might reflect
a genetic interaction between lec1 and cop1 or an
additive effect resulting in heterochronic onset of
seedling lethality, as previously suggested (Meinke
et al., 1994).
Recent studies of ABI3 have also demonstrated a
role in some aspects of light response such as plastid
differentiation (Rohde et al., 2000). Furthermore,
ABI3 has been shown to interact genetically with
DET1 in regulating germination, plastid differentia-
tion, anthocyanin accumulation, floral determination,
and internode elongation (Kurup et al., 2000; Rohde et
al., 2000). However, as described herein for ABI4,
ABI5,FUS3,orLEC1, the interactions are complex,
ranging from synergistic effects on germination to
antagonistic effects on plastid differentiation (Rohde
et al., 2000).
Summary
The ABA INSENSITIVE and LEAFY COTYLEDON
class transcription factors regulate overlapping
events in seed development. Comparison of digenic
mutants shows that, for most responses in late em-
bryogenesis, the most severe defects are observed in
abi4 fus3, with progressively less severe defects in
abi5 fus3,abi4 lec1, and abi5 lec1. Genetic interactions
ranging from synergistic to antagonistic have been
documented for each combination of mutations, and
have revealed a variety of cryptic effects of these
mutations. Although FUS3 and LEC1 appear to inter-
act with all of the ABI transcription factors, previous
analyses of fus3 lec1 digenic mutants led to the con-
clusion that these loci participate in distinct regula-
tory pathways (West et al., 1994). The complexity of
these interactions is more consistent with combina-
torial controls than a hierarchical signaling pathway,
but some cross regulation of transcript or protein
accumulation has also been described. However, few
of these regulatory proteins appear to physically in-
teract in yeast two-hybrid assays. Finally, although
initially identified as regulators of ABA response
and/or seed development, all of these loci also ap-
pear to function to varying degrees in mediating
response to light, sugar, and osmotic stress. In fact,
the characteristic reddish color of the leafy cotyledon
class mutant embryos and seedlings may be ex-
plained by their combination of increased endoge-
nous soluble sugars and hypersensitivity to light-
dependent, sugar-induced anthocyanin synthesis.
MATERIALS AND METHODS
Plant Material
The abi4-1 and abi5-1 mutant lines were isolated from the Col and Ws
backgrounds, respectively, as described by Finkelstein (1994). The fus3-3
and lec1-1 mutant lines were isolated from the Col and Ws backgrounds,
respectively, as described by Keith et al. (1994) and West et al. (1994).
Seed Regulatory Networks
Plant Physiol. Vol. 131, 2003 89
For RNA isolation from siliques, plants were grown in soil in continuous
light at 22°C. Siliques were harvested in pools corresponding to four devel-
opmental stages: early embryogenesis (1–5 DPA), maturation (8–11 DPA),
late embryogenesis (17–21 DPA), and dry seed (⬎21 DPA). The siliques
were weighed, flash frozen in liquid nitrogen, and stored at ⫺70°C until
extraction. Dry seed were stored at room temperature.
Double Mutant Construction
To construct double mutants, abi plants were crossed with either fus3 or
lec1 plants. The fus3 and lec1 mutations result in desiccation-intolerant
seeds, so individuals homozygous for either of these mutations must be
rescued by excision and culture of early desiccation stage seeds. For each
cross, the F
2
progeny clearly segregated a novel phenotypic class of highly
pigmented seeds (approximately 1/16 in this generation). These individuals
were rescued and found to display phenotypic markers of the fus3 and lec1
lines (i.e. a linked gl mutation and trichomes on the cotyledons, respective-
ly). The abi lines used for the crosses with fus3 carried the linked er and py
markers, such that abi individuals displayed the erecta growth habit and
thiamine auxotrophy. In addition, DNA polymorphisms corresponding to
each abi mutation could be scored by cleaved-amplified polymorphic se-
quence reactions, permitting direct confirmation of the abi genotype. For
abi4-1, an 895-bp fragment with an NlaIV polymorphism was amplified with
the following primers: 5⬘-CCCATAATAATCCTCAATCC-3⬘and 5⬘-
AAATCCCAAATACTCCCC-3⬘. For abi5-1, an 826-bp fragment with an
AvaII polymorphism was amplified with the following primers: 5⬘-
CAATCAACAAGCAGCAG-3⬘and 5⬘-TCTCTCCACTACTTTCTCCAC-3⬘.
Amplification conditions followed standard protocols (Konieczny and Aus-
ubel, 1993), using 50°C annealing for ABI4 and 60°C annealing for ABI5.
Additional double mutant lines were obtained by selecting abi F
2
segregants
by requiring germination on 3
mABA (mixed isomers, Sigma, St. Louis),
then screening the F
3
progeny for highly pigmented seeds (approximately
25% in this generation). Double mutant lines were maintained by excising
and culturing early desiccation stage seeds in each succeeding generation.
Germination Assays
Germination assays were performed with early desiccation stage seeds
(20–70 seeds per treatment). Siliques were harvested and surface sterilized
in 70% (v/v) ethanol, then seeds were excised and placed on minimal
medium (Haughn and Somerville, 1986) containing 0.7% (w/v) agar sup-
plemented with different concentrations of ABA, Glc, and/or sorbitol. The
dishes were incubated for3dat4°C to break any residual dormancy, then
transferred to 22°C in continuous light (50–70
Em
⫺2
s
⫺1
); germination was
scored after 7 d.
Quantification of Chlorophyll and
Anthocyanin Pigments
Early desiccation stage seeds were excised from the siliques, counted,
and stored at ⫺70°C. Immature seeds were ground at 4°Cin400
L of either
80% (v/v) aqueous acetone (chlorophyll) or 1% (v/v) HCl in 60% (v/v)
methanol (anthocyanin) as described by Parcy et al. (1997). The absorption
spectrum was recorded between either 500 and 700 nm (chlorophyll) or 400
and 650 nm (anthocyanin). The quantity of pigments was measured as the
value above baseline at the absorption maximum (663.5 nm for chlorophyll
and 533.5 nm for anthocyanin), then normalized to the number of seeds
used in each sample.
RNA Gel-Blot Analysis
RNA was isolated from immature siliques as previously described (So¨-
derman et al., 2000). Dry seed RNA preps were based on a modified
procedure of Vicient and Delseny (1999) as previously described (So¨derman
et al., 2000). RNA concentrations were estimated based on A
260
and 280 nm.
Total RNA (2.4–20
g per lane) was size fractionated on MOPS-
formaldehyde gels (Sambrook et al., 1989), then transferred to nylon mem-
branes (Osmonics Inc., Westborough, MA) using 20⫻sodium chloride/
sodium phosphate/EDTA as blotting buffer. RNA was bound to the filters
by UV cross-linking (120 mJ cm
⫺2
at 254 nm). Uniformity of loading and
transfer were assayed qualitatively by hybridization to an rDNA probe. The
ABI5 probe was a PCR-amplified genomic fragment excluding most of the
conserved bZIP domain. The ABI4 probe was an EcoRI fragment from a
cDNA clone encompassing all but the first two and final codons of the
coding sequence. Transcripts from FUS3 and LEC1 were detected by hy-
bridization to cDNA clones. Hybridization probes for the AtDPBF tran-
scripts were full-length cDNA clones described by Kim et al. (2002). Tran-
scripts from CRC,PAP147 (oleosin), At2S,M17,AtEm1,AtEm6, and RAB18
were detected by hybridization to cDNA clones labeled by random priming
to a specific activity of 10
8
cpm
g
⫺1
, as described by So¨derman et al. (2000).
At least two independent RNA samples were analyzed for each genotype,
stage, and probe tested.
Two-Hybrid Assays
Translational fusions between ABI3,ABI4,ABI5,LEC1, and FUS3 genes
and the GAL4 activation and DNA-BDs were constructed in the pGAD-C(x)
and pGBD-C(x) vectors, respectively (James et al., 1996). The GAL4-BD-
ABI4 construct encoded a slightly truncated form of ABI4 (amino acids
3–287) because a full-length ABI4 fusion provides very strong transcription
activation function in the absence of any AD fusion (So¨derman et al., 2000).
Similarly, the ABI3 fusion contains only the C-terminal basic domains of
ABI3 (amino acids 216–670) because the N-terminal acidic domains provide
a strong transcription activation function. The BD-ABI5 construct encoded
all but the first eight amino acids of ABI5, thus including all conserved
domains. The different fragments for the BD and AD fusions involving ABI
genes were cloned as previously described by Nakamura et al. (2001). The
newly constructed AD fusions encoded full-length LEC1 or all but the first
22 amino acids of FUS3, thereby including all conserved domains. All gene
fusions were transformed into yeast (Saccharomyces cerevisiae) as previously
described by Nakamura et al. (2001).
Quantification of Soluble Sugars and Starch
Starch and soluble sugar (Suc, Fru, and Glc) levels in samples of 50 dry
seeds were determined as previously described by Chia et al. (2000). After
extraction of soluble sugars, the extract was divided into three fractions for
parallel determinations of Suc, Fru, and Glc levels. Suc was digested with
400 units of invertase and 1 unit of phosphoglucoisomerase, followed by
measurement of released Glc by the infinity Glc reagent (Sigma). Fru and
Glc levels were determined by digestion of extracted soluble sugars with,
respectively, 1 unit phosphoglucoisomerase or no enzyme, followed by
measurement of released Glc by the infinity Glc reagent. Control experi-
ments indicated that the Glc reagent was not contaminated with phospho-
glucoisomerase, such that the measured Glc levels did not include any
contribution from endogenous Fru levels (data not shown).
Distribution of Materials
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes.
No restrictions or conditions will be placed on the use of any materials
described in this paper that would limit their use in noncommercial research
purposes.
ACKNOWLEDGMENTS
We thank Dr. J. John Harada for providing FUS3 and LEC1 cDNAs and
the lec1-1 mutant, Dr. Peter McCourt for providing the fus3-3 mutant, Drs.
Jerome Giraudat and Francois Parcy for providing the ABI3 cDNA, Dr.
Terry Thomas for providing the AtDPBF cDNAs, and Dr. Michel Delseny
and the Arabidopsis Biological Resource Center (Ohio State University,
Columbus) for providing the expressed sequence tag clones encoding em-
bryonically expressed transcripts. We thank Dr. James Cooper for use of his
SMZ-U stereoscopic microscope (Nikon, Tokyo).
Received July 30, 2002; returned for revision August 24, 2002; accepted
September 20, 2002.
Brocard-Gifford et al.
90 Plant Physiol. Vol. 131, 2003
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