Content uploaded by Yuichiro Tsuchiya
Author content
All content in this area was uploaded by Yuichiro Tsuchiya
Content may be subject to copyright.
The embryonic leaf identity gene FUSCA3
regulates vegetative phase transitions by
negatively modulating ethylene-regulated gene
expression in Arabidopsis
Lumba et al.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8 (20 February 2012)
RESEARC H ARTIC L E Open Access
The embryonic leaf identity gene FUSCA3
regulates vegetative phase transitions by
negatively modulating ethylene-regulated gene
expression in Arabidopsis
Shelley Lumba
1†
, Yuichiro Tsuchiya
1†
, Frederic Delmas
1
, Jodi Hezky
1
, Nicholas J Provart
1,3
, Qing Shi Lu
1,2
,
Peter McCourt
1
and Sonia Gazzarrini
1,2*
Abstract
Background: The embryonic temporal regulator FUSCA3 (FUS3) plays major roles in the establishment of
embryonic leaf identity and the regulation of developmental timing. Loss-of-function mutations of this B3 domain
transcription factor result in replacement of cotyledons with leaves and precocious germination, whereas
constitutive misexpression causes the conversion of leaves into cotyledon-like organs and delays vegetative and
reproductive phase transitions.
Results: Herein we show that activation of FUS3 after germination dampens the expression of genes involved in
the biosynthesis and response to the plant hormone ethylene, whereas a loss-of-function fus3 mutant shows many
phenotypes consistent with increased ethylene signaling. This FUS3-dependent regulation of ethylene signaling
also impinges on timing functions outside embryogenesis. Loss of FUS3 function results in accelerated vegetative
phase change, and this is again partially dependent on functional ethylene signaling. This alteration in vegetative
phase transition is dependent on both embryonic and vegetative FUS3 function, suggesting that this important
transcriptional regulator controls both embryonic and vegetative developmental timing.
Conclusion: The results of this study indicate that the embryonic regulator FUS3 not only controls the embryonic-
to-vegetative phase transition through hormonal (ABA/GA) regulation but also functions postembryonically to
delay vegetative phase transitions by negatively modulating ethylene-regulated gene expression.
Keywords: Arabidopsis, embryonic development, phase transition, FUSCA3, hormones, ethylene
Background
Spatial patterning in most multicellular organisms
requires genes to both establish regions of cell differen-
tiation and specify cellular fate. In the early Drosophila
embryo, for example, cells are organized into boundaries
by the pair rule and segment polarity genes, then they
acquire distinct fates through homeotic gene expression
[1]. Homeotic genes are also required to establish bound-
aries during temporal patterning, whereas heterochronic
genes define the timing of the cell fate decisions within
those boundaries [2]. One challenge in developmental
biology is to identify and understand the overall develop-
mental role of genes involved in the temporal patterning
of genetic programs.
Higher plants are well-suited for identifying genes
involved in developmental timing because they continu-
ally produce easily distinguishable organs throughout the
life cycle, whose fates are dependent on the time of emer-
gence [3]. The types of leaves that emerge over time
often show distinctive developmental changes that allow
them to be classified into juvenile and adult leaves. Later,
when a plant enters reproductive development, the vege-
tative meristematic region switches to an inflorescence
* Correspondence: gazzarrini@utsc.utoronto.ca
†Contributed equally
1
Department of Cell and Systems Biology (CSB), University of Toronto, 25
Willcocks Street, Toronto, ON, M5S 3B2, Canada
Full list of author information is available at the end of the article
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
© 2012 Lumba et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, pro vided the original work is properly cited.
meristem that produces flower bracts with floral meris-
tems in their axils [4]. Genetic analysis in Arabidopsis
thaliana has identified a myriad of genes that converge
to control the juvenile to adult leaf transitions and the
switch of the vegetative meristem to reproductive devel-
opment [5].
Unlike flowers and leaves, which form from a shoot
apical meristem, the developmental relationship between
embryonic leaves (or cotyledons) and adult foliar organs
is complicated by cotyledon formation during embryonic
patterning. Furthermore, in many plants such as Arabi-
dopsis, cotyledons switch from a storage organ to a more
leaflike photosynthetic organ soon after germination.
Despite these complexities, single loss-of-function muta-
tions in Arabidopsis have been identified in three genes,
LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2
(LEC2)andFUSCA3 (FUS3), whose mutations result in
the replacement of cotyledons with organs more similar
to vegetative leaves [6-9]. In lec1 and fus3 mutants, genes
that encode markers of late embryogenesis are reduced
or missing [9,10]. By contrast, germination markers that
normally proceed late embryogenesis are precociously
activated. These expression patterns suggest that LEC1
and FUS3 may establish temporal boundaries. Although
little is known about how these genes contribute to tem-
poral patterns, it is known that FUS3 regulates and is
regulated itself by the synthesis of two terpenoid hor-
mones, abscisic acid (ABA) and gibberellins (GA)
[10-12]. The ratio of these two hormones contributes to
proper cotyledon patterning by regulating the rates of
cell cycling [11].
Although extensive analyses of LEC1, LEC2 and FUS3
gene action have been carried out with respect to embryo-
genesis, the effects of these mutations on vegetative leaf
development have not been studied extensively [11,13,14].
It has been shown that after germination, the first juvenile
leaves of lec1 seedlings are shifted toward later leaf identi-
ties; however, this shift is not maintained, and successive
leaves and flowering time were corrected back to a wild-
type pattern [15]. This suggests that embryonic leaf devel-
opment can have a restricted impact on future vegetative
leaf identities. What remains unclear, however, is how
cotyledon development impinges on later vegetative devel-
opment, which is temporally and spatially distinct.
To address such questions, we decided to use a combi-
nation of controlled FUS3 activation during vegetative
development with whole-genome transcript profiling.
Using this approach, we discovered that FUS3 downregu-
lates a collection of genes involved in ethylene biosynth-
esis and signaling. Consistent with this finding, loss-of-
function fus3 mutants show ectopic ethylene responses at
both the developmental and molecular levels. The fus3
plants also show precocious vegetative phase change;
however, unlike the lec1 mutants, this change is not
corrected at later adult stages. More importantly, the
accelerated vegetative phase transition can be suppressed
by inhibiting ethylene action either genetically or phar-
macologically. Thus it appears that this previously
defined embryonic regulator also has roles in vegetative
development.OneroleofFUS3 during early seedling
growth is to dampen ethylene action, which in turn con-
tributes to a slowing of subsequent vegetative phase tran-
sitions. These results add ethylene to the list of
hormones that contribute to temporal patterning in
Arabidopsis.
Results
Downstream effectors of FUS3
The discovery that FUS3 misexpression outside embryo-
genesis can influence vegetative leaf identity suggests that
potential FUS3-dependent downstream effectors can be
identified through whole-genome microarray analysis
[11]. We constructed an inducible misexpression system
by transforming the fus3-3 (fus3)mutantwithaFUS3-
glucocorticoid receptor (GR) translational fusion under
the control of the epidermal specific AtML1 promoter
(fus3 AtML1:FUS3-GR) [11]. Transgenic seeds were ger-
minated and grown in minimal medium (Murashige and
Skoog, MS) for 5 days, then plantlets were transferred to
various concentrations of dexamethasone (DEX) to deter-
mine the minimal amount of activation needed to influ-
ence leaf identity. All concentrations of DEX tested had
an effect on vegetative leaf shape; increasing concentra-
tions of DEX resulted in a leaf with an increased paddle-
like shape and a progressively shorter petiole (Figure 1A).
At higher than 0.5 μM DEX concentrations, the appear-
ance of trichomes on the upper or adaxial surface of the
leaves was completely inhibited (data not shown). On the
basis of these conditions, 1.0 μMDEXwaschosenasthe
FUS3-activating condition.
Putative FUS3 targets involved in vegetative phase
transitions were identified by examining the transcrip-
tome of seedlings that transiently activate FUS3 using
the AtML1:FUS3-GR DEX-inducible system [11]. To do
this, transgenic seeds from AtML1:FUS3-GR were ger-
minated and grown for 5 days in MS. After this time,
half of the seedlings were transferred to MS media sup-
plemented with 1.0 μM DEX (+DEX), and the other half
were transferred to MS media supplemented with
dimethyl sulfoxide (DMSO) (-DEX or control) for 2 or 4
days (Figure 1B). Putative FUS3 targets were chosen on
the basis of the following criteria. Genes that increased
(red bars) or decreased (blue bars) at least twofold in
expression after two days of FUS3 activation (Figure 1B;
+DEX) compared to the control (Figure 1B; -DEX) were
considered to represent candidate FUS3 targets involved
in phase transition. Utilizing this regime and the average
of two replicate experiments, 19 genes increased
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 2 of 15
A
[DEX] (μM)
0 0.1 0.5 1.0
B
C
Other molecular functions
Transcription factor
activity
Other binding (non-
nucleic acid)
DNA or RNA binding
Transferase activity
Other enzyme activity
Kinase activity
Hydrolase activity
Nucleotide binding
Transporter activity
Structural molecule
activity
Receptor binding or
activit
y
Repressed
Activated
+ DEX, downregulated
+ DEX, upregulated 2 days ± DEX
0
2
4
6
8
0
2
4
6
4 days ± DEX
– DEX
Ratio
Genes
+ –
Day 0 2 Days 4 Days
–
ACS6
ERF1
EDF4
EDF2
ACT7
D
DEX + –
Figure 1 Microarray analysis of seedlings ectopically expressing FUS3.(A) Images showing the development of leaf 4 in 5-day-old fus3
ML1:FUS3-GR seedlings transferred to different concentrations of dexamethasone (DEX). (B) Genes that change at least twofold in expression in
fus3 ML1:FUS3-GR seedlings treated with 1 μM DEX. The upper graph (2 days ± DEX) shows the ratios of fold changes in gene expression of 5-
day-old seedlings grown for 2 days in the absence of DEX (black bars) or in the presence of DEX (red bars represent upregulated genes, and
blue bars represent downregulated genes). The same order of genes is represented in the lower graph (4 days ± DEX), where 5-day-old
seedlings were grown for 4 days in the absence of DEX (black bars) or in the presence of DEX (red and blue bars). (C) The proportions of genes
associated with various molecular functions (gene ontology) are represented in the pie chart. (D) RT-PCR verification of ethylene-related genes
identified by microarray analysis downregulated by FUS3 activation. Expression of four ethylene-related genes and an ACTIN7 (ACT7) control in
the absence (-DEX) or presence (+DEX) of FUS3 activation.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 3 of 15
(Additional file 1, Table S1) and 34 genes decreased
(Additional file 2, Table S2) in expression (at least two-
fold) as the direct result of 2-day FUS3 activation
(2 days +DEX) compared to the control (2 days -DEX).
To independently verify the expression changes of the
2-day DEX induction experiment, a temporal replicate
experiment was performed in which FUS3 activation
was extended by transferring 5-day-old seedlings onto
DEXfor4days(4days+DEX).Allbut1ofthe53
genes selected by the 2-day experiment exhibited similar
expression ratios in the 4-day experiment, demonstrat-
ing that activation of FUS3 results in changes in expres-
sion levels of a relatively small but reproducible gene
set.
Gene ontology (GO) was utilized to categorize the acti-
vated and repressed genes by function (Figure 1C).
Thirty-eight percent of genes upregulated by FUS3 acti-
vation appear to be involved in enzymatic activity,
whereas a number of repressed genes are annotated as
having roles in hormone synthesis or action. For exam-
ple, the CYP707A3 gene that encodes the cytochrome
p450 monooxygenase that catabolizes ABA is repressed
more than twofold by FUS3 activation (Additional file 2,
Table S2) [16]. This observation, in conjunction with the
lack of FUS3-dependent induction of known ABA bio-
synthetic genes, suggests the increased ABA levels
observed previously in FUS3 misexpression lines are due
to decreased ABA catabolism [11]. A second class of hor-
mone-related genes that are repressed by FUS3 activation
at all time points and in both replicates are either
involved in ethylene biosynthesis (ACS6, ACC synthase 6)
or ethylene response (ERF1, ETHYLENE RESPONSE
FACTOR1;ERF104, ETHYLENE RESPONSE FAC-
TOR104;ESE3/EREBP, ETHYLENE AND SALT INDUCI-
BLE3;EDF4, ETHYLENE RESPONSE DNA BINDING
FACTOR4). ERFs and EREBPs are transcription factors
which contain an AP2 DNA binding domain, whereas
EDF (or RAV) transcription factors contain both an AP2
and a B3 DNA-binding domain. Indeed, transcription
factors constitute a dominant category (17%) of FUS3-
repressed genes (Additional file 2, Table S2).
Potential downstream targets of FUS3 would be
expected to contain the RY promoter element motif
CATGCA, to which the B3 domain of FUS3 binds
[17,18]. In fact, the RY sequence was found to be statisti-
cally enriched in both the up- and downregulated gene
set compared to a randomized sample (p-value of 1.77 ×
10
-4
and 6.06 × 10
-3
for 1, 000 bp upregulated and down-
regulated genes, respectively; Additional files 1 and 2,
Tables S1 and S2). Together, these data strongly suggest
that our experimental conditions identified a small gene
setthatisresponsivetoFUS3. Interestingly, this set
includes only a few genes that typically mark seed
maturation and late embryogenesis, such as seed storage
proteins and late embryogenesis abundant proteins
(LEA).
FUS3 negatively regulates a subset of genes that are
responsive to ethylene
A closer inspection of our microarray data yielded addi-
tional ethylene-induced transcription factors (ERF2, EDF2
and EDF1) whose expression levels were repressed by
FUS3 (Additional file 2, Table S2). These genes did not
quitemeetthestringentcriteria of exhibiting a twofold
decrease following both 2- and 4-day periods of FUS3 acti-
vation. Of the 14 AP2/EREBP/ERF and RAV/EDF genes
that are known to be induced by ethylene exposure, six
genes (ERF1, ERF2, ERF104, EDF2, EDF4 and EDF1) were
dampened by FUS3 activation, suggesting that FUS3 plays
aroleinreducingtheexpression of genes involved in
ethylene action [19]. This was verified by RT-PCR per-
formed on ACS6, ERF1, EDF4 and EDF2 at 2 and 4 days
of DEX induction (Figure 1D).
FUS3-mediated repression of genes involved in ethy-
lene synthesis and action predict that fus3 loss-of-func-
tion mutants would exhibit an inverse expression
pattern of these genes. Indeed, expression of ACS6,
EDF2 and EDF4 genes were consistently higher in ger-
minating fus3 versus wild type at all time points sur-
veyed after imbibition, whereas ERF1 expression
increased after 24 hours (Figure 2A). These results again
supportanegativeroleforFUS3 in regulating the
expression of these ethylene responsive genes during
germination. The presence of the RY element in the
promoters of this gene subset may mean FUS3 directly
downregulates their expression. Alternatively, FUS3 may
act through ethylene synthesis or signaling to modulate
their expression. To test these possibilities, we repeated
the RT-PCR analysis of ACS6, ERF1, EDF4 and EDF2
on 2-day old wild-type and fus3 seedlings germinated in
the presence of either an ethylene biosynthesis inhibitor,
aminoethoxyvinylglycine (AVG), or an ethylene signaling
inhibitor, silver ions (AgNO
3
). In both cases, the inhibi-
tors partially suppressed the increased levels of the gene
transcripts in fus3 seedlings compared to untreated con-
trols (Figure 2B). Furthermore, a key transcriptional reg-
ulator of ethylene signaling, EIN3 [20,21], showed
increased protein stability in the emerging root cells of
fus3 compared to wild type, and this stability was elimi-
nated by the addition of silver ions (Figure 2C).
Although these results do not exclude a direct role for
FUS3 on ethylene-responsive gene transcription, it does
suggest that at least part of the aberrant gene expression
observed in fus3 mutants does require functional ethy-
lene synthesis or signaling.
To further probe the connection of FUS3 with ethy-
lene action during germination, we studied the growth
of dark-grown fus3 loss-of-function seedlings. When
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 4 of 15
wild-type seedlings are germinated in the dark in the
presence of ethylene, they show an exaggerated apical
hook, a shortening of the hypocotyl and reduced root
growth [22]. Termed the “triple response, “this develop-
mental assay has been extremely useful in genetically
dissecting the role of various genes involved in ethylene
synthesis or signaling in Arabidopsis [23]. Dark-germi-
nated fus3 plantlets grown in the absence of ethylene
exhibited shorter hypocotyl growth and hooked cotyle-
don development (Figure 2D), which is consistent with
their ectopic ethylene gene expression. Moreover, these
phenotypes were alleviated by the introduction of a
mutation (ein2-1) that reduces ethylene signaling into
the fus3 genetic background (Figure 2D). To further dif-
ferentiate ethylene synthesisandresponse,werepeated
the experiment in the presence of the ethylene biosynth-
esis inhibitor AVG. In contrast to the ein2 mutation,
AVG did not restore the hypocotyl length of fus3, which
suggests that the ectopic ethylene responses observed in
dark-grown fus3 seedlings were not due to increased
ethylene synthesis (Figure 2E).
FUS3 functions to repress ethylene action during
germination
The heightened ethylene responses observed in fus3
after germination suggest that loss of this gene function
WT
ACT7
EDF4
EDF2
ERF1
ACS6
A
fus3
0 h
C
+ Ag + Ag
MS MS
+ ACC
WT fus3
B
WT fus3 WT fus3 WT fus3
48 h
12 h 24 h
D
MS AVG Ag MS AVG Ag MS AVG Ag MS AVG
Ag
EDF4 EDF2 ERF1 ACS6
Fold changes normalized by ACT7
Hypocotyl length (cm)
Hypocotyl length (cm)
fus3
fus3 ein2
0.0
0.4
0.8
1.2
1.6
2.0
WT
fus3
fus3 ein2
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
fus3
WT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0.0
0.4
0.8
1.2
1.6
- AVG
+ AVG
WT
fus3
eto1
E
Figure 2 Increased expression of ethylene signaling and biosynthetic genes in fus3 mutant, and ethylene-related fus3 phenotypes.(A)
RT-PCR of ethylene-related genes in wild-type (WT) and fus3 seeds germinated for 12, 24 and 48 hours in minimal medium (MS). ACTIN7 (ACT7)
served as a control. (B) RT-PCR of ethylene-related genes in wild-type and fus3 seeds germinated for 48 hours in MS media or MS media
containing 10 μM aminoethoxyvinylglycine (AVG) or 100 μM AgNO
3
(Ag). Fold change in gene expression was normalized to ACTIN7, which
served as a control. Similar trends were seen in two independent experiments. (C) GFP-EIN3 fluorescence in wild-type and fus3 roots incubated
for 48 hours in minimal medium with (+) or without (-) 100 μM AgN0
3
(Ag). The inset shows the GFP-EIN3 fluorescence in wild-type roots
exposed to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Scale bar = 15.2 μm. (D) Images (right panels) and
quantification (left panel) of hypocotyls length of 5-day-old fus3 and fus3 ein2-1 seedlings grown in the dark in the air. Scale bar = 0.2 cm.
Averages from triplicate experiments ± SD are shown. fus3, n = 24; fus3 ein2-1, n = 21. (E) Quantification of hypocotyls length of 5-day-old wild-
type, fus3 and eto1-1 seedlings grown in the dark in MS in the absence (-) or in the presence (+) of the ethylene synthesis inhibitor AVG. The
eto1-1 seed, which overaccumulates ethylene gas, was used as a positive control to demonstrate rescue by AVG addition. Averages from
triplicate experiments ± SD are shown. Wild type, n= 25; fus3, n = 25; eto1-1, n = 24.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 5 of 15
might have other, uncharacterized effects on vegetative
development. Because FUS3 is a regulator of embryonic
leaf identity, we decided to examine the vegetative leaf
identity and phase transitions of the fus3 loss-of-func-
tion mutant more closely. From the emergence of the
first leaf after germination through to flowering, each
wild-type leaf adopts a unique identity based on size
and shape, and this graded growth variation is often
referred to as the “leaf heteroblastic series”[23].
Although rosette leaves always produce trichomes on
the adaxial side of the wild-type leaf, trichomes on the
abaxial side begin to appear on leaves only at later
nodes (leaf 5 in wild type). Leaves that produce only
adaxial trichomes are considered juvenile, whereas later
leaves that show trichomes on both sides are regarded
as adult [24]. Also, the ratio of leaf blade-to-petiole
lengths is generally lower in early juvenile leaves and
becomes higher as the plant transitions to adult leaves
and flowering [25].
Comparisons of leaf profiles between the fus3 mutant
andwild-typeplantsshowedthatthefirsttwoleaves
from the mutant plant were more similar in shape and
size to the third and fourth leaves of the wild type, and
that this shift continued throughout the heteroblastic ser-
ies (Figure 3A). This precocious shift was reflected in the
blade-to-petiole length ratio of each individual leaf
(Figure 3B) and in the leaf trichome distribution (Figure
3C). Abaxial trichomes frequently (63%) appeared on leaf
3infus3 mutant plants, approximately two leaves earlier
than the wild type.
FUS3 has functions outside embryogenesis
There are two explanations for the vegetative phenotypes
of the fus3 mutant. Possibly the altered cotyledon devel-
opment during embryogenesis in the mutant advances
the progression of vegetative leaf identities after germina-
tion. Alternatively, FUS3 may function in the developing
vegetative meristem. We tested these possibilities directly
by measuring FUS3 transcript levels using quantitative
RT-PCR (qRT-PCR). In wild-type seeds, FUS3 transcript
is detected 6 hours postimbibition and declines over
time, as previously shown [26]; it is still detectable 5 days
after germination (Figure 4A). To further clarify the
source tissue of postembryonic FUS3 expression, a trans-
genic line containing a sensitive FUS3:GUS transcrip-
tional fusion was germinated and sampled over time for
b-glucuronidase (GUS)-dependent blue histochemical
staining [27]. The FUS3:GUS line used in these experi-
ments was previously confirmed to reliably report
embryonic FUS3 expression patterns based on FUS3 in
situ hybridization [27]. Whole-mount preparations of
seedlings showed blue staining in emerging leaf primor-
dia from 2- to 5-day-old seedlings (Figure 4B). A similar
expression pattern was found using a FUS3:GFP reporter
previously described [26] (data not shown).
To functionally determine the effect of FUS3 from
embryonic and vegetative tissues on the precocious vege-
tative phase transition, we constructed transgenic plants
in which FUS3 could be activated either in the embryo or
in vegetative tissue. fus3 was transformed with a FUS3-
GR translational fusion construct that was under the con-
trol of the native FUS3 promoter (fus3 FUS3:FUS3-GR).
Seeds produced from these lines in the absence of DEX
application were phenotypically indistinguishable from
fus3 loss-of-function mutants, with approximately 95% of
the seed being desiccation-intolerant after 6 weeks of sto-
rage (data not shown). Of the small number of seeds that
did survive desiccation, all produced ectopic trichomes
on their cotyledons, as expected. Thus, in the absence of
DEX application, the FUS3-GR fusion protein cannot res-
cue any of the known embryonic fus3 phenotypes. By
contrast, in two independent experiments, approximately
50% of the seed produced by transgenic plants sprayed
with 30 μM DEX during flowering showed desiccation
tolerance after 6 weeks of storage. Of these, approxi-
mately 85% lacked ectopic trichomes on the cotyledons.
The large reduction of fus3 seed phenotypes indicates
that DEX application during flowering is sufficient to res-
cue mutant embryos.
From the transgenic seeds that were rescued, seven
independent plants were randomly selected and grown
to maturity in the absence of DEX, and leaf profiles
were performed. Leaf profiles of all these lines still dis-
played accelerated heteroblastic development similar to
that observed in fus3 leaf profiles (Figure 3A). Further-
more, the lines showed the same advanced vegetative
phase transition as fus3 with respect to both individual
leaf blade-to-petiole ratios (Figure 3B). The abaxial tri-
chomes, however, did show a partial reversion to a wild-
type profile (Figure 3C). Together, these results suggest
that specifically removing embryonic FUS3 has a limited
influence on the development of the first two juvenile
leaves of Arabidopsis, but not on later leaves.
The inability of embryonic FUS3 to fully rescue the
precocious vegetative phase transitions of fus3 suggests
that this gene may function outside seed development.
We tested this possibility by using the fus3 FUS3:FUS3-
GR transgenic lines. Unlike in the previous experiment,
however, we allowed transgenic lines to produce seed in
the absence of DEX application and then germinated
the seed in the presence of 10 μM DEX for 2 days. This
short DEX application did not influence the heteroblas-
tic phase change in wild-type plants as measured by
either the blade-to-petiole transition or the appearance
of abaxial trichomes in comparison to untreated plants
(Additional file 3, Figure S1). By contrast, exposure of
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 6 of 15
Wild type
fus3
fus3 FUS3:FUS3
-GR
(seed + DEX)
A
B
C
1 2 3 4 5 6 7
0 0 0 0 87
100
0 0 63 100
100
0 0 0 100
100
fus3 FUS3:FUS3-GR
Genotype
wild type (n=23)
fus3 (n=24)
(seed + DEX; n=7)
Leaf position
Leaf position
0
2
4
6
1 2 3 4 5 6 7 8 9 10 11
Blade/petiole length
wild type (n=10)
fus3 (n=10)
fus3 FUS3:FUS3-GR
(seed + DEX; n=7)
8
88
60
52
100
100
Figure 3 Contributions of embryonic FUS3 to leaf identity and phase transitions.(A) Rosette leaf morphology of wild-type (top row), fus3
(middle row) and fus3 plants transformed with the FUS3:FUS3-GR construct (bottom row). fus3 FUS3:FUS3-GR parent plants were sprayed with 30
μM dexamethasone (DEX) during seed production. Leaves from ten plants were dissected, and representative profiles are shown (scale bar = 1
cm). (B) Ratios of blade-to-petiole lengths of individual rosette leaves in wild-type and fus3 and embryonically rescued fus3 FUS3:FUS3-GR plants.
(C) Percentage of wild-type, fus3 and embryonically rescued fus3 FUS3:FUS3-GR (seed + DEX) rosettes that developed an abaxial trichome at each
leaf position.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 7 of 15
B
A
Wild type
fus3 FUS3:FUS3-GR
– DEX
fus3 FUS3:FUS3-GR
+ DEX
Full rescue
Partial rescue
C
D
0
5
10
15
20
25
Time after germination
Relative mRNA levels
Day 5
Day 3
Day 1 Day 2
1 Day 2 Days 5 Days 6 h
1 2 3 4 5 6 7
0 0 0 0 80 100 100
0 0 29 92 100 100 100
0 0 71 100 100 100 100
Genotype
Leaf position
Wild type (n=5)
fus3 FUS3:FUS3-GR + DEX (n=24)
fus3 FUS3:FUS3-GR – DEX (n=21)
Figure 4 FUS3 is expressed and functions postembryonically.(A) Relative expression of the FUS3 gene at various time points after
germination. Quantitative RT-PCR was performed on germinating wild-type seeds imbibed for 6 hours, 1 day, 2 days and 5 days in minimal
medium (MS). Transcript levels were normalized using ACTIN7 as an internal control. Results from triplicate samples are shown with error bars
(SD). Experiments were repeated twice with similar results. Data from one of these replicates are shown. (B) Histochemical staining of FUS3:GUS
seedlings at 1, 2, 3 and 5 days after germination in MS. Arrows indicate emerging leaf primordia showing b-glucuronidase (GUS) staining. Scale
bars = 0.2 mm (day 1) and 0.4 mm (days 2, 3 and 5). (C) Wild-type and fus3 FUS3:FUS3-GR seeds germinated in MS without dexamethasone
(DEX) (-DEX) or on 10 μM DEX (+DEX) for 2 days, then transferred to soil until bolting. Of the plants analyzed, 8.3% showed a full rescue of the
rosette leaf morphology, and an example is shown (full rescue). A partial rescue was obtained for 62.5% of the plants, and an example is shown
(partial rescue). The morphologies of the first six to eight fus3 FUS3:FUS3-GR leaves were rescued, but those of subsequent leaves were variable.
Twenty-one to twenty-four plants were analyzed, constituting a representative profile. Scale bar = 1.0 cm. (D) Percentage of abaxial trichomes in
wild-type rosettes and in fus3 FUS3:FUS3-GR rosettes of plants grown with (+) or without (-) DEX.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 8 of 15
germinating fus3 FUS3:FUS3-GR transgenic seeds to
DEX for 2 days resulted in a partial (62.5%) or full
(8.3%) shift of leaf heteroblasty back toward a wild-type
profile (Figure 4C). This rescue was partially reflected in
trichome appearance (Figure 4D) and in full-leaf mea-
surements; in wild type, the largest leaf was leaf 7,
reachingasizeof3.9±0.2cm(n= 24). Similarly, the
largest leaf in fus3 FUS3:FUS3-GR plants treated with
DEX was leaf 7, which reached a size of 3.6 ± 0.3 cm
(n= 23). In contrast, leaf 6 was the largest leaf in fus3
FUS3:FUS3-GR untreated plants (3.0 ± 0.3 cm; n= 23).
In conclusion, a short pulse of FUS3 during the first 2
days of postembryonic growth can influence leaf identity
in juvenile leaves.
FUS3-dependent vegetative phase variation requires
functional ethylene signaling
Loss-of-function fus3 mutants show advanced vegetative
phase transition, increased expression of ethylene-regu-
lated genes and phenotypes that are characteristic of
increased ethylene signaling. To test if these phenotypes
are related, a mutation conferring ethylene insensitivity
was introduced into the fus3 background. The introduc-
tion of the ein2 mutation did suppress defects in vegeta-
tive phase change compared to the fus3 single mutant as
measured by leaf profiles, blade-to-petiole ratios and
abaxial trichome appearance (Figures 5A to 5C). These
results suggest that increased ethylene signaling does
contribute to the advanced vegetative phase transition
phenotype observed in fus3.
To further refine the ethylene contribution, we germi-
nated fus3 seeds on 100 μMAgNO
3
for 2 days and
transferred the resulting seedlings to soil. Again, the
fus3 phase transition phenotypes in the presence of
AgNO
3
were similar to those seen in the fus3 ein2 dou-
ble-mutant (Figures 5C to 5E). Because in this experi-
ment fus3 embryonic development occurred in the
absence of the ethylene signaling inhibitor AgNO
3
,any
alterations in fus3 vegetative phase transition were due
to the transient inhibition of ethylene signaling during
germination. In conclusion, the inhibition of ethylene
signaling during early germination is able to partially
suppress the premature vegetative phase transitions
observed in the fus3 mutant.
Discussion
The FUS3 hormonal framework
Four lines of evidence suggest that FUS3 negatively reg-
ulates ethylene action. First, gain-of-function activation
of FUS3 dampens the expression of a collection of ethy-
lene-responsive genes, whereas a loss-of-function fus3
allele shows the opposite effect. Second, the ectopic
expression of these ethylene-responsive genes in light-
grown seedlings is dampened by the addition of ethylene
synthesis or signaling inhibitors. Third, loss-of-function
fus3 seedlings show common ethylene-response pheno-
types in the dark which are dependent on ethylene sig-
naling and also have increased stability of the key
positive regulator of ethylene signaling, EIN3.Finally,
the precocious vegetative phase variation observed in
fus3 mutants is suppressed if ethylene signaling is inhib-
ited genetically or pharmacologically.
Inhibition of ethylene biosynthesis does not rescue fus3
phenotypes in the dark, suggesting that alteration of ethy-
lene signaling in fus3 is not merely a consequence of
increased ethylene levels and requires functional ethylene
signaling. However, genetic manipulation of FUS3 did
influence the expression of ACS6, encoding for an enzyme
involved in ethylene biosynthesis. The altered expression
of this gene might reflect a feedback response to changes
in ethylene signaling. On this note, only a subset of ethy-
lene signaling genes was regulated by FUS3,whichsug-
gests that the relationship between FUS3 and ethylene is
complex. Many of the ethylene-responsive genes regulated
by FUS3 contain RY elements in their promoter, which
suggests that this transcription factor may bind these pro-
moters directly. A direct regulation of gene transcription
may explain why the addition of ethylene inhibitors did
not fully alleviate the ectopic expression of the ethylene-
responsive genes. Nevertheless, the negative effect of FUS3
on the regulation of ethylene signaling adds this hormone
to ABA and GA as those that are dependent on FUS3 dur-
ing germination [11].
Coordination of the synthesis and signaling of various
hormones is important in regulating overall plant growth
and development, and many examples of ethylene, ABA
and GA interactions have been reported [28-33]. In one
of the best-studied cases, initial submergence of deep-
water rice plants resulted in the accumulation of ethy-
lene, which in turn enhanced GA sensitivity of the
internodes to promote rapid growth [29]. The addition of
ABA had the opposite effect by decreasing the sensitivity
of internode tissues to GA [32]. With respect to leaf iden-
tity, interactions between ethylene and ABA have also
been implicated in semiaquatic plants that exhibit hetero-
phylly [34]. ABA induces terrestrial leaf development and
ethylene stimulates the formation of submerged leaves in
part by reducing ABA levels [30]. In Arabidopsis, where
genetic analysis can be applied, loss of ethylene response
does increase ABA levels in leaves, and, conversely, Ara-
bidopsis mutants deficient in ABA synthesis show
increased ethylene production [35-38]. The antagonistic
and seemingly common relationships between ABA and
GA and between ABA and ethylene imply that FUS3 acts
as a control point of these multiple hormone pathways.
Suppressing ethylene signaling in fus3 mutants par-
tially rescued the defects in juvenile leaf identities and
the accelerated vegetative phase variation of this mutant,
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 9 of 15
but did not rescue the embryonic leaf identity pheno-
types. Perhaps this is not surprising, because there are
major cellular differences beyond size and shape
between embryonic cotyledons and juvenile and adult
leaves. For example, genetic programs involved in sto-
rage reserves and desiccation appear to be embryo-spe-
cific. Related to this is that controlled FUS3 activation
outside seed development was not sufficient to upregu-
late most of the standard seed-specific marker genes
such as storage proteins, suggesting that other regulators
are also required. The absence of seed-specific marker
induction by the controlled activation of FUS3 in seed-
lings is similar to results obtained by Kagaya et al.[39],
who also utilized a transgene-based activation system.
Under their study conditions, vegetatively activated
FUS3 could induce ectopic seed storage gene expression
only with the addition of ABA. These observations are
in contrast to constitutive FUS3 misexpression experi-
ments, which result in storage reserve accumulation in
vegetative tissues. Genetically reducing ABA levels in
fus3
A
B
C
fus3 ein2
fus3 (– Ag)
fus3 (+ Ag)
D
E
fus3 + Ag (n=10)
fus3 – Ag (n=10)
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9
Blade/petiole length
Leaf position
Blade/petiole length
Leaf position
0
1
3
5
7
9
1 2 3 4 5 6 7 8 9
fus3 (n=12)
fus3 ein2 (n=12)
1 2 3 4 5 6 7
0 0 39 85 100 100 100
0 0 0 0
46 92 100
Genotype
fus3 ein2 (n=13)
0 0 54 91 100 100 100
0 0 0 43
92 100 100
fus3 – Ag (n=10)
fus3 +Ag (n=10)
fus3 (n=13)
Leaf position
Figure 5 Influence of ethylene signaling on fus3 vegetative phase transition.(A) Mature rosette leaf morphologies of fus3 and fus3 ein2-1
plants. Leaves from 12 plants were dissected, and a representative profile is shown. Scale bar = 1.0 cm. (B) Ratios of blade-to-petiole lengths of
mature individual rosette leaves from fus3 and fus3 ein2-1 plants. (C) Percentage of rosettes displaying trichomes on the abaxial surface at each
leaf position. (D) Rosette leaf morphology of fus3 germinated for 2 days in minimal medium (MS) in the presence (+) or absence (-) of 100 μM
AgNO
3
(Ag) and then transferred to soil. Leaves from ten plants were dissected, and a representative profile is shown. Scale bar = 1.0 cm. (E)
Ratios of blade-to-petiole lengths of individual fus3 rosette leaves of plants germinated in MS with (+) or without (-) 100 μM AgNO
3
(Ag).
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 10 of 15
plants that constitutively express FUS3 also attenuates
the ability of FUS3 to activate embryonic programs ecto-
pically, consistent with an ABA requirement for full
FUS3 function [11]. It appears that there is a limited
time during germination during which tissues are more
sensitized to FUS3 action. The presence of the tran-
scription factor ABI5 has been posited to define a
60-hour developmental window during germination dur-
ing which ABA can still arrest the growth of the emer-
ging embryo and induce late-embryogenesis gene
expression [40]. Whether this 60-hour checkpoint also
defines a window in which FUS3 can reactivate late
embryogenesis programming needs to be determined,
but our expression studies place postembryonic FUS3
within this developmental window.
FUS3 and vegetative phase change
Transitions from one leaf identity to the next depend on
the coordination of at least two independent processes:
the timing of leaf initiation and a program that deter-
mines the duration of a developmental phase [3]. In the
latter case, the genes that define the juvenile boundary
may repress the expression of adult leaf-promoting genes
above a certain threshold. One prediction of this model
is that these juvenile boundary regulators will decrease in
activity over time to allow adult phase change to occur
[41]. FUS3 transcripts are detected after germination, and
although they decrease, they can still be detected 5 days
postgermination at a time when at least four to five leaf
primordia have formed [42]. Because leaf 5 is considered
to be the first to show adult characteristics, the decreased
temporal domain of FUS3 expression correlates well with
decreasing juvenile leaf identity. A simple model would
suggest that the presence of FUS3 in leaf primordia con-
tributes to juvenile leaf identity and also that as FUS3
activity drops, the transition to adult leaf phases can
occur.
Although we have shown a vegetative role for FUS3,
embryonic expression of FUS3 also affects the leaf iden-
tity of the first two juvenile leaves. This is similar to stu-
dies involving LEC1, which suggests that the identity of
the embryonic foliar organs influences the identity of
subsequent vegetative organs despite their different
developmental origins [15]. Unlike fus3 mutants, how-
ever, aberrant vegetative phase transitions in lec1 plants
are corrected in later leaves [15]. The lack of a detectable
LEC1 signal after germination, as shown in public micro-
arrays [43], may explain the difference between these
mutants.
In contrast to animals, plant hormones can function in
many different tissues and at various times during devel-
opment, resulting in various developmental outcomes.
One of the mechanisms by which FUS3 prolongs the
juvenile phase appears to involve the downregulation of
ethylene action, which suggests that this hormone is
important in promoting adult-phase transitions (Figure
6). Morphometric analysis of leaf growth of etr1 mutants
have shown that this ethylene-insensitive plant produce
shorter, broader leaves, which are more akin to a juve-
nile leaf identity [37]. Consistent with this, the vegeta-
tive-to-floral transition in a collection of ethylene-
insensitive mutants is delayed compared to the wild
type [44]. In agreement with this, ML1:FUS3-GFP plants
that constitutively express FUS3 postembryonically also
show delayed flowering [11,26]. Using ethylene to
quicken vegetative phase transitions is consistent with
the role of this hormone in promoting germination
[36,38]. Perhaps the short vegetative pulse of FUS3
serves to dampen the action of this hormone after it has
stimulated germination so that the leaf identities do not
advance too quickly.
Conclusions
Herein we provide evidence that the embryonic regulator
FUS3 also plays an important role postembryonically by
negatively regulating vegetative phase transitions through
repression of ethylene action. This study also implicates a
role for ethylene in temporal patterning. Together with
previous findings showing that FUS3 controls the
embryonic-to-vegetative phase transition by modulating
the ABA/GA ratio, this highlights a pivotal role of FUS3
in controlling the timing of expression of embryonic and
vegetative programs through hormonal regulation. The
pivotal role of FUS3 in regulating hormone levels and
responses introduces another dimension to understand-
ing the transitions of leaf identity that occur throughout
the plant’s life cycle. ABA and ethylene levels are also
very dependent on both abiotic and biotic external sig-
nals, such as water availability, temperature and pathogen
attack. Hence, the phase transitions observed will be
dependent not only on developmental regulators such as
FUS3 but also on environmental changes that influence
hormone concentrations. In the future, it will be interest-
ing to see if some of phenotypic plasticity observed in
leaf shape can be linked not only to the relative timing of
developmental regulators like FUS3 but also to environ-
mental conditions, both of which impinge on ethylene,
ABA and GA synthesis and/or signaling.
Methods
Plant material, growth conditions and leaf profiles
fus3-3 (fus3) [7] and all other strains used in this study
were derived from a Columbia (Col) genetic background.
For all experiments, plants were grown at 20°C under con-
stant light. Seeds from all genotypes were generated from
parent plants grown under identical conditions and stored
under the same conditions for the same length of time. All
transformations were performed as previously described
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 11 of 15
[45]. DEX (Sigma-Aldrich, St Louis, MO, USA) was dis-
solved in DMSO. We performed leaf profiles according to
Telfer et al. [24], for which 10 to 15 seedlings were germi-
nated and grown in individual 4-inch round pots for
approximately 3.5 weeks. To determine if DEX had any
off-target effects on leaf heteroblasty, we performed blade-
to-petiole measurements and trichome appearance assays
on 10 wild-type plants and found that the chemical did
not influence either of these markers of phase transition
(Additional file 3, Figure S1). For the ein2 fus3 double-
mutant construction, ein2-1 homozygotes were identified
by screening for an altered triple-response phenotype in a
segregating F2 population. Positive ein2-1 plants were
grown and assayed for fus3 allele using a cleaved amplified
polymorphic sequence marker that marks the mutant
polymorphism [11]. For the pharmacological studies, 100
μM of the inhibitor AgNO
3
or 10 μM of the inhibitor
AVG were added to MS plates.
Cloning and generation of transgenic plants
The EIN3 cDNA was amplified and carboxy-terminally
fused to GFP under the control of the 35S promoter in
ve
g
etative phase transition
Transcriptional Cascade
ERF1
Ethylene
Receptor
FUS3
ERF2
EDF1
EDF4
EDF2
EIN3
Other ethylene
responsive genes
EIN2
Figure 6 Working model of phase change regulation by FUS3. During late embryogenesis and early germination, FUS3 negatively influences
a number of factors, including ethylene signaling. As a consequence, the EIN3 protein, a key positive regulator of ethylene signaling, is reduced,
thereby causing a decrease in the expression of downstream transcription factors such as ERFs and EDFs. A reduction of these ethylene-
dependent transcription factors prevents the premature transition from the juvenile to the adult phase of development. In the loss-of-function
fus3 mutant, ethylene signaling increases, which in turn accelerates vegetative phase transitions. The lack of full restoration of altered gene
expression by ethylene inhibitors and the observation that only a subset of ethylene responsive genes are affected by FUS3 also suggest that
FUS3 may have ethylene-independent effects. Consistent with this hypothesis, many ethylene-responsive genes contain FUS3-binding RY
sequences in their promoter elements. It is therefore possible that FUS3 also influences these genes and some aspects of phase transitions
through ethylene-independent mechanisms.
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 12 of 15
the pEGAD vector using the primers EIN3-BamHI for-
ward 5’-ATA GGA TCC ATG ATG TTT AAT GAG
ATG GGA AAT G-3’;EIN3-BamHI reverse 5’-ATA
GGA TCC GAA CCA TAT GGA TAC ATC TTG C-3’;
and the BamHI enzyme [46] to generate 35S:GFP-EIN3.
The GFP-EIN3 translational fusion reporter fully rescues
ein3 loss-of-function ethylene-dependent phenotypes
(data not shown).
RT-PCR analysis
Thirty PCR cycles were performed with 250 ng of total
RNA to amplify ethylene-related genes identified
through microarrays using the following primers: ERF1
forward 5’-GTA TCC TCA ACG ACG CCT TTC AC-
3’;ERF1 reverse 5’-CTT CAC CGT CAA TCC CTT
ATC C-3’;EDF4/RAV1 forward 5’-GTA CAG GTT
CCA TCT GTG AAA CC-3’;EDF4/RAV1 reverse 5’-
CTC GTC TTC GTC CAT CTT CAC GTC-3’;EDF2/
RAV2 forward 5’-GCA TAG ACG AGA TAA GTT
CCT CCA C-3’;EDF2/RAV2 reverse 5’-GTT CTT GAA
GTT GAC GAC GGC GTC G-3’;ACS6 forward 5’-
ACA TCC AGA AGC TTC GAT TTG TAC-3’;and
ACS6 reverse 5’-CTC GTC TAC AAA CTC TTC ATC
GGA-3’. These primers were also used to check for
expression of genes involved in ethylene synthesis and
signaling in dry wild-type (Col) and fus3 immature
seeds. ACTIN7 was amplified using the following pri-
mers: ACT7 forward 5’-GGT GAG GAT ATT CAG
CCA CTT GTC TG-3’and ACT7 reverse 5’-TGT GAG
ATC CCG ACC CGC AAG ATC-3’and was used as
the control. Seeds were imbibed as described for 12, 24
and 48 hours.
Quantitative RT-PCR
RNA extraction and qRT-PCR were performed as pre-
viously described [26]. Sequences of gene-specific pri-
mers were designed to amplify 190-bp products. These
included the following: ACT7
190
forward 5’-TCA CAG
AGG CAC CTC TTA ACC-3’; ACT7
190
reverse, 5’-CCC
TCG TAG ATT GGC ACA G-3’;FUS3
190
forward, 5’-
TGT GAA TGC TCA TGG TCT GC-3’;andFUS3
190
reverse, 5’-GGA GGA GAA GAT CGT TAA CCA C-3’.
Histochemistry and microscopy
Detection of GUS activity and sectioning was performed
as described by Donnelly et al.[47].Leaftissueswere
cleared in 70% ethanol and 8:2:1 chloral hydrate:gly-
cerol:water, then mounted in the same solution on
microscope slides.
Microarray and bioinformatic analysis
Wild-type Columbia seeds containing the ML1:FUS3:GR
construct [11] were imbibed for 5 days at 4°C on filter
paper placed on MS plates. To minimize the effects of
biological variation, we followed a pooling strategy
according to that described by Zhu et al.[48]inwhich
approximately 100 individual plants per replicate experi-
ment were harvested and pooled before RNA extraction
and hybridization. The seedlings were then transferred
to 20°C for 5 days. Shoot tissues from seedlings were
collected for RNA isolation (day 5). The remaining seed-
lings on the filter paper were transferred to either MS
plates consisting of 1 μM DEX or the same concentra-
tion of DMSO. Two and four days after the transfer,
shoot tissue was collected from the seedlings. The above
protocol was repeated to obtain a second biological
replica.
The standard Affymetrix labeling and hybridization
protocols were used on an ATH1 Affymetrix GeneChip
microarray encompassing 22, 814 probe sets (Affymetrix
Inc,SantaClara,CA,USA).Dataweregloballynormal-
ized using the MAS5.0 global normalization algorithm
with a target value of 500. The resulting data were fil-
tered to eliminate MAS5.0 “marginal”and “absent”calls
(Present requires both sets of replicates for treatment
and control samples). In addition, the rvalues for the
correlation of the expression values between the two
replicate set of experiments was greater than 0.95. The
complete data set has been submitted to the EMBL-EBI
ArrayExpress database under the accession number
E-MEXP-3465 in compliance with MIAME standards.
The average expression value for each gene was calcu-
lated for two replicate samples at each time point. The
Cluster and TreeView software programs were used for
cluster analysis [49]. The data set was filtered to identify
genes that exhibited more than a twofold increase or
decrease in expression level [50]. Cluster analysis was
performed using average linkage hierarchical clustering
with the centered Pearson correlation coefficient as the
similarity metric. RY motif (CATGCA) and p-values
were obtained using the “Motif Analysis”tool on TAIR
http://www.arabidopsis.org/tools/bulk/motiffinder/index.
jsp.
Temporal activation of FUS3
The FUS3:FUS3-GR construct was made by replacing
the ML1 promoter in the ML1:FUS3-GR construct with
the FUS3 promoter from the FUS3:FUS3-GFP construct
previously described [11]. For FUS3:FUS3-GR activation
during seed development, DEX was diluted to 30 μMin
dH
2
O containing 0.05% Triton X-100 and sprayed on
flowering plants twice weekly until the plants had
senesced.
Confocal microscopy
To study EIN3 stability in a fus3 background, we
crossed fus3 with an ein3 35S:GFP:EIN3 transgenic line.
The stability of the EIN3 protein in the fus3 background
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 13 of 15
was compared to non-fus3 mutant plants from the same
F1 plant. At least 20 seedlings were observed for each
genotype. A Nikon inverted fluorescence microscope
equipped with a Nikon water immersion objective
(Nikon Instruments, Inc, Melville, NY, USA) and a Bio-
Rad Radiance 2000 confocal head (Bio-Rad Laboratories,
Hercules, CA, USA) was used to detect GFP fluores-
cence. The same confocal settings were used in all
experiments.
Additional material
Additional file 1: Table S1 Genes upregulated by ectopic FUS3
activation. Values for fold changes in expression after 2 and 4 days (d)
of FUS3 activation with dexamethasone (+DEX) are the averages of two
replicates. The presence of RY promoter motifs (CATGCA) in the 500 bp
(0.5 K), 1, 000 bp (1 K) and 3, 000 bp (3 K) upstream regions of each
gene is included. p-values are 3.06 × 10
-4
, 1.77 × 10
-4
and 2.57 × 10
-2
,
respectively.
Additional file 2: Table S2 Genes downregulated by ectopic FUS3
activation. Values for fold changes in expression after 2 and 4 days (d)
of FUS3 activation with dexamethasone (+DEX) are the averages of two
replicates. Genes involved in hormone metabolism or response are in
bold. Ethylene response genes that were downregulated less than
twofold by FUS3 activation at both 2 and 4 days are in italics. The
presence of RY promoter motifs (CATGCA) in 500 bp (0.5 K), 1, 000 bp (1
K) and 3, 000 bp (3 K) upstream regions of each gene is included. p-
values are 4.27 × 10
-2
, 6.06 × 10
-3
and 3.46 × 10
-2
, respectively.
Additional file 3: Figure S1 Phenotypic analysis of wild-type plants
exposed to dexamethasone (DEX). Wild-type seeds were germinated
in 10 μM DEX for 2 days and then transferred to soil. The increase in the
blade-to-petiole ratio and the appearance of the abaxial trichomes on
leaf 5 is comparable to wild-type profiles shown in Figure 3. (A) Ratios of
blade-to-petiole lengths of individual wild-type rosette leaves. (B)
Percentage of wild-type rosettes that formed abaxial trichomes at each
leaf position.
Abbreviations
bp: base pair; GFP: green fluorescent protein; RT-PCR: reverse transcriptase
polymerase chain reaction.
Acknowledgements
We are grateful to the Gazzarrini and McCourt research groups for
discussion and to K Breitkreuz for critical reading of the manuscript. We
thank J Kieber (University of North Carolina) for the eto1-1 seed. This work
was funded by a Natural Sciences and Engineering Research Council of
Canada (NSERC) grant (to SG) and an NSERC Industrial Research Chair grant
(to PM).
Author details
1
Department of Cell and Systems Biology (CSB), University of Toronto, 25
Willcocks Street, Toronto, ON, M5S 3B2, Canada.
2
Department of Biological
Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto,
ON, M1C 1A4, Canada.
3
Centre for the Analysis of Genome Evolution and
Function (CAGEF), University of Toronto, 25 Harbord Street, Toronto, ON,
M5S 3G5, Canada.
Authors’contributions
PM and SG designed the experiments and wrote the manuscript. SL and SG
performed the biological part of the microarray study and the data analysis
of the microarray experiment (Figure 1 and supplementary tables). SL and
YT performed RT-PCR. SL performed the experiments shown in Figures 3
and 5. QSL and SG performed the experiments shown in Figure 4. FD and
JH constructed and selected 35S:GFP-EIN3 transgenics. YT constructed and
selected FUS3-GR constructs. NJP performed cluster analysis. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 7 October 2011 Accepted: 20 February 2012
Published: 20 February 2012
References
1. Akam M: The molecular basis for metameric pattern in the Drosophila
embryo. Development 1987, 101:1-22.
2. Thummel CS: Molecular mechanisms of developmental timing in C.
elegans and Drosophila.Dev Cell 2001, 1:453-465.
3. Kerstetter RA, Poethig RS: The specification of leaf identity during shoot
development. Annu Rev Cell Dev Biol 1998, 14:373-398.
4. Henderson IR, Dean C: Control of Arabidopsis flowering: the chill before
the bloom. Development 2004, 131:3829-3838.
5. Poethig RS: Small RNAs and developmental timing in plants. Curr Opin
Genet Dev 2009, 19:374-378.
6. Bäumlein H, Miséra S, Luerßen H, Kölle K, Horstmann C, Wobus U, Müller AJ:
The FUS3 gene of Arabidopsis thaliana is a regulator of gene expression
during late embryogenesis. Plant J 1994, 6:379-387.
7. Keith K, Kraml M, Dengler NG, McCourt P: fusca3: a heterochronic
mutation affecting late embryo development in Arabidopsis. Plant Cell
1994, 6:589-600.
8. Meinke DW, Franzmann LH, Nickle TC, Yeung EC: Leafy cotyledon mutants
of Arabidopsis. Plant Cell 1994, 6:1049-1064.
9. West MAL, Matsudaira Yee K, Danao J, Zimmerman JL, Fischer RL,
Goldberg RB, Harada JJ: LEAFY COTYLEDON1 is an essential regulator of
late embryogenesis and cotyledon identity in Arabidopsis. Plant Cell
1994, 6:1731-1745.
10. Nambara E, Hayama R, Tsuchiya Y, Nishimura M, Kawaide H, Kamiya Y,
Naito S: The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase
transition from late embryo development to germination. Dev Biol 2000,
220:412-423.
11. Gazzarrini S, Tsuchiya Y, Lumba S, Okamoto M, McCourt P: The
transcription factor FUSCA3 controls developmental timing in
Arabidopsis through the hormones gibberellin and abscisic acid. Dev Cell
2004, 7:373-385.
12. Curaba J, Moritz T, Blervaque R, Parcy F, Raz V, Herzog M, Vachon G:
AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis,
is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3
in Arabidopsis. Plant Physiol 2004, 136:3660-3669.
13. Lotan T, Ohto M, Matsudaira Yee K, West MAL, Lo R, Kwong RW,
Yamagishi K, Fischer RL, Goldberg RB, Harada JJ: Arabidopsis LEAFY
COTYLEDON1 is sufficient to induce embryo development in vegetative
cells. Cell 1998, 93:1195-205.
14. Stone SL, Kwong LW, Matsudaira Yee K, Pelletier J, Lepiniec L, Fischer RL,
Goldberg RB, Harada JJ: LEAFY COTYLEDON2 encodes a B3 domain
transcription factor that induces embryo development. Proc Natl Acad Sci
USA 2001, 98:11806-11811.
15. Tsukaya H, Shoda K, Kim GT, Uchimiya H: Heteroblasty in Arabidopsis
thaliana (L.) Heynh. Planta 2000, 210:536-542.
16. Kushiro T, Okamoto M, Nakabayashi K, Yamagishi K, Kitamura S, Asami T,
Hirai N, Koshiba T, Kamiya Y, Nambara E: The Arabidopsis cytochrome
P450 CYP707A encodes ABA 8’-hydroxylases: key enzymes in ABA
catabolism. EMBO J 2004, 23:1647-1656.
17. Reidt W, Wohlfarth T, Ellerström M, Czihal A, Tewes A, Ezcurra I, Rask L,
Bäumlein H: Gene regulation during late embryogenesis: the RY motif of
maturation-specific gene promoters is a direct target of the FUS3 gene
product. Plant J 2000, 21:401-408.
18. Mönke G, Altschmied L, Tewes A, Reidt W, Mock HP, Bäumlein H, Conrad U:
Seed-specific transcription factors ABI3 and FUS3: molecular interaction
with DNA. Planta 2004, 219:158-166.
19. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P,
Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C,
Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N,
Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R,
Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE,
Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR:
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 14 of 15
Genome-wide insertional mutagenesis of Arabidopsis thaliana.Science
2003, 301:653-657.
20. Guo H, Ecker JR: Plant responses to ethylene gas are mediated by
SCF
EBF1/EBF2
-dependent proteolysis of EIN3 transcription factor. Cell 2003,
115:667-677.
21. Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C,
Genschik P: EIN3-dependent regulation of plant ethylene hormone
signaling by two Arabidopsis F box proteins: EBF1 and EBF2. Cell 2003,
115:679-689.
22. Schaller GE, Kieber JJ: Ethylene. In The Arabidopsis Book. Volume 1. Edited
by: Somerville C, Meyerowitz E. Rockville, MD: American Society of Plant
Biologists; 2002:e0071[http://www.bioone.org/doi/pdf/10.1199/tab.0071].
23. Tsukaya H: Leaf development. In The Arabidopsis Book. Volume 1. Edited by:
Somerville C, Meyerowitz E. Rockville, MD: American Society of Plant
Biologists; 2002:e0072[http://www.bioone.org/doi/pdf/10.1199/tab.0072].
24. Telfer A, Bollman KM, Poethig RS: Phase change and the regulation of
trichome distribution in Arabidopsis thaliana.Development 1997,
124:645-654.
25. Bollman KM, Aukerman MJ, Park MY, Hunter C, Berardini TZ, Poethig RS:
HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase
change and morphogenesis. Development 2003, 130:1493-1504.
26. Lu QS, Dela Paz J, Pathmanathan A, Chiu RS, Tsai AYL, Gazzarrini S: The C-
terminal domain of FUSCA3 negatively regulates mRNA and protein
levels and mediates sensitivity to the hormones abscisic acid and
gibberellic acid in Arabidopsis. Plant J 2010, 64:100-113.
27. Tsuchiya Y, Nambara E, Naito S, McCourt P: The FUS3 transcription factor
functions through the epidermal regulator TTG1 during embryogenesis
in Arabidopsis.Plant J 2004, 37:73-81.
28. Cox MCH, Benschop JJ, Vreeburg RAM, Wagemaker CAM, Moritz T,
Peeters AJM, Voesenek LACJ: The roles of ethylene, auxin, abscisic acid,
and gibberellin in the hyponastic growth of submerged Rumex palustris
petioles. Plant Physiol 2004, 136:2948-2960.
29. Kende H, van der Knaap E, Cho HT: Deepwater rice: a model plant to
study stem elongation. Plant Physiol 1998, 118:1105-1110.
30. Kuwabara A, Ikegami K, Koshiba T, Nagata T: Effects of ethylene and
abscisic acid upon heterophylly in Ludwigia arcuata (Onagraceae). Planta
2003, 217:880-887.
31. Benschop JJ, Jackson MB, Gühl K, Vreeburg RAM, Croker SJ, Peeters AJM,
Voesenek LACJ: Contrasting interactions between ethylene and abscisic
acid in Rumex species differing in submergence tolerance. Plant J 2005,
44:756-768.
32. Hoffmann-Benning S, Kende H: On the role of abscisic acid and
gibberellin in the regulation of growth in rice. Plant Physiol 1992,
99:1156-1161.
33. Gazzarrini S, McCourt P: Cross-talk in plant hormone signalling: what
Arabidopsis mutants are telling us. Ann Bot 2003, 91:605-612.
34. Nishii K, Kuwabara A, Nagata T: Characterization of anisocotylous leaf
formation in Streptocarpus wendlandii (Gesneriaceae): significance of
plant growth regulators. Ann Bot 2004, 94:457-467.
35. Chiwocha SDS, Cutler AJ, Abrams SR, Ambrose SJ, Yang J, Ross ARS,
Kermode AR: The etr1-2 mutation in Arabidopsis thaliana affects the
abscisic acid, auxin, cytokinin and gibberellin metabolic pathways
during maintenance of seed dormancy, moist-chilling and germination.
Plant J 2005, 42:35-48.
36. Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P:
Regulation of abscisic acid signalling by the ethylene response pathway
in Arabidopsis. Plant Cell 2000, 12:1117-1126.
37. LeNoble ME, Spollen WG, Sharp RE: Maintenance of shoot growth by
endogenous ABA: genetic assessment of the involvement of ethylene
suppression. J Exp Bot 2004, 55:237-245.
38. Beaudoin N, Serizet C, Gosti F, Giraudat J: Interactions between abscisic
acid and ethylene signaling cascades. Plant Cell 2000, 12:1103-1116.
39. Kagaya Y, Toyoshima R, Okuda R, Usui H, Yamamoto A, Hattori T: LEAFY
COTYLEDON1 controls seed storage protein genes through its
regulation of FUSCA3 and ABSCISIC ACID INSENSITIVE3.Plant Cell Physiol
2005, 46:399-406.
40. Lopez-Molina L, Mongrand S, Chua NH: A postgermination developmental
arrest checkpoint is mediated by abscisic acid and requires the ABI5
transcription factor in Arabidopsis.Proc Natl Acad Sci USA 2001,
98:4782-4787.
41. Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS: SGS3 and SGS2/
SDE1/RDR6 are required for juvenile development and the production of
trans-acting siRNAs in Arabidopsis.Genes Dev 2004, 18:2368-2379.
42. Hunter CA, Aukerman MJ, Sun H, Fokina M, Poethig RS: PAUSED encodes
the Arabidopsis exportin-t ortholog. Plant Physiol 2003, 132:2135-2143.
43. Toufighi K, Brady SM, Austin R, Ly E, Provart NJ: The Botany Array
Resource: e-Northerns, Expression Angling, and promoter analyses. Plant
J2005, 43:153-163.
44. Ogawara T, Higashi K, Kamada H, Ezura H: Ethylene advances the
transition from vegetative growth to flowering in Arabidopsis thaliana.J
Plant Physiol 2003, 160:1335-1340.
45. Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-
mediated transformation in Arabidopsis thaliana.Plant J 1998, 16:735-743.
46. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR: Random GFP::cDNA
fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency. Proc Natl Acad Sci USA 2000,
97:3718-3723.
47. Donnelly PM, Bonetta D, Tsukaya H, Dengler RE, Dengler NG: Cell cycling
and cell enlargement in developing leaves of Arabidopsis.Dev Biol 1999,
215:407-419.
48. Zhu T, Budworth P, Chen W, Provart N, Chang HS, Guimil S, Su G, Estes B,
Zou G, Wang X: Transcriptional control of nutrient partitioning during
rice grain filling. Plant Biotechnol J 2003, 1:59-70.
49. Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and display
of genome-wide expression patterns. Proc Natl Acad Sci USA 1998,
95:14863-14868.
50. DeRisi JL, Iyer VR, Brown PO: Exploring the metabolic and genetic control
of gene expression on a genomic scale. Science 1997, 278:680-686.
doi:10.1186/1741-7007-10-8
Cite this article as: Lumba et al.: The embryonic leaf identity gene
FUSCA3 regulates vegetative phase transitions by negatively
modulating ethylene-regulated gene expression in Arabidopsis. BMC
Biology 2012 10:8.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Lumba et al.BMC Biology 2012, 10:8
http://www.biomedcentral.com/1741-7007/10/8
Page 15 of 15
