Plant, Cell and Environment (2008) 31, 227–234
doi: 10.1111/j.1365-3040.2007.01759.x
The ABA1 gene and carotenoid biosynthesis are required
for late skotomorphogenic growth in Arabidopsis thaliana
JOSÉ MARÍA BARRERO1*, PEDRO L. RODRÍGUEZ2*, VÍCTOR QUESADA1*, DAVID ALABADÍ2,3,
MIGUEL A. BLÁZQUEZ2, JEAN-PIERRE BOUTIN4, ANNIE MARION-POLL4, MARÍA ROSA PONCE1 &
JOSÉ LUIS MICOL1
1
División de Genética and Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Alicante, Spain, 2Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior
de Investigaciones Científicas, 46022 Valencia, Spain, 3Fundación de la Comunidad Valenciana para la Investigación
Agroalimentaria (Agroalimed), 46113 Moncada, Valencia, Spain and 4Seed Biology Laboratory, UMR 204
INRA-AgroParisTech, Jean-Pierre Bourgin Institute, 78026 Versailles Cedex, France
ABSTRACT
INTRODUCTION
Several plant hormones, including auxin, brassinosteroids
and gibberellins, are required for skotomorphogenesis,
which is the etiolated growth that seedlings undergo in the
absence of light. To examine the growth of abscisic acid
(ABA)-deficient mutants in the dark, we analysed several
aba1 loss-of-function alleles, which are deficient in zeaxanthin epoxidase. The aba1 mutants displayed a partially
de-etiolated phenotype, including reduced hypocotyl
growth, cotyledon expansion and the development of true
leaves, during late skotomorphogenic growth. In contrast,
only small differences in hypocotyl growth were found
between wild-type seedlings and ABA-deficient mutants
impaired in subsequent steps of the pathway, namely nced3,
aba2, aba3 and aao3. Interestingly, phenocopies of the partially de-etiolated phenotype of the aba1 mutants were
obtained when wild-type seedlings were dark-grown on
medium supplemented with fluridone, an inhibitor of phytoene desaturase, and hence, of carotenoid biosynthesis.
ABA supplementation did not restore the normal skotomorphogenic growth of aba1 mutants or fluridone-treated
wild-type plants, suggesting a direct inhibitory effect of
fluridone on carotenoid biosynthesis. In addition, aba1
mutants showed impaired production of the b-carotenederived xanthophylls, neoxanthin, violaxanthin and antheraxanthin. Because fluridone treatment of wild-type plants
phenocopied the phenotype of dark-grown aba1 mutants,
impaired carotenoid biosynthesis in aba1 mutants is probably responsible for the observed skotomorphogenic phenotype. Thus, ABA1 is required for skotomorphogenic
growth, and b-carotene-derived xanthophylls are putative
regulators of skotomorphogenesis.
The control that light exerts upon a plant ranges from its
use as an energy source to its effects on specific developmental programs (Chen, Chory & Fankhauser 2004). One
of the earliest events after germination involves the establishment of a morphogenic developmental plan. In the
absence of light, seedlings grow in an etiolated form in a
process known as skotomorphogenesis, which is characterized by enhanced cell expansion resulting in longer hypocotyls, and the maintenance of an apical hook with folded
cotyledons. In contrast, light triggers the switch to photomorphogenesis, which is accompanied by a cessation of
cell expansion, the formation of new organs and massive
changes in gene expression, including the induction of
genes involved in photosynthesis and chloroplast function
(Casal et al. 2004).
In Arabidopsis thaliana, photomorphogenesis is
repressed in the dark by a mechanism dependent on the
activity of COP1 (CONSTITUTIVE PHOTOMORPHOGENESIS 1), an E3 ubiquitin ligase that triggers the degradation of a battery of transcription factors necessary for
the expression of light-regulated genes (Hoecker 2005).The
repression of photomorphogenesis and the promotion of
skotomorphogenesis are also under hormonal control
(Kim, Kim & von Arnim 2002). For instance, brassinosteroid (BR)-deficient and BR-insensitive mutants display a
de-etiolated phenotype in the dark, that is, shorter hypocotyls, cotyledon unfolding, and derepression of lightregulated genes such as CAB2 (CHLOROPHYLL A/B–
BINDING PROTEIN2) and RbcS (RubisCO smallsubunit) (Clouse & Sasse 1998; Schumacher & Chory 2000).
Several lines of evidence implicate auxins in the regulation
of photomorphogenesis; for example, mutations in the
auxin-regulated gene FIN219 (FAR-RED INSENSITIVE
219) suppress the de-etiolated phenotype of cop1 in the
dark (Hsieh et al. 2000), and phytochromes have been
reported to phosphorylate auxin-signalling elements
(Colón-Carmona et al. 2000). The involvement of auxin in
photomorphogenesis is in consonance with a large overlap
in the regulation of gene expression by auxin and BRs
Key-words: ABA; aba1 mutants; skotomorphogenesis.
Correspondence: J. L. Micol. Fax: +34 96 665 85 11; e-mail:
[email protected]
*These authors contributed equally to this work.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
227
228 J. M. Barrero et al.
(Nemhauser, Mockler & Chory 2004). Gibberellins (GAs)
are also implicated as repressors at photomorphogenic
development. As one recent example, in the dark, impairment of GA biosynthesis and the activation of negative
GA-signalling elements provoked de-etiolation that could
be partially reverted by the exogenous application of BRs
(Alabadí et al. 2004).
Carotenoids are a large class of isoprenoid-derived
compounds that have critical roles in all photosynthetic
organisms (Hirschberg 2001). Xanthophylls, which are
oxygenated carotenes, are accessory pigments in the lightharvesting antennae of the chloroplast, and function to
transfer photon energy to chlorophylls. Two branches of
xanthophylls are generated from a- and b-carotene (Fig. 1).
In particular, aba1 mutants are impaired in the epoxidation
of zeaxanthin, a xanthophyll derived from b-carotene, to
antheraxanthin, and to all-trans-violaxanthin. Despite the
high concentration of carotenoids in etioplasts, and evidence showing that carotenoid biosynthesis affects chloroplast biogenesis (Park et al. 2002), little is known about the
actual role of carotenoids in etiolated growth.
We examined the phenotype of carotenoid and abscisic
acid (ABA) biosynthetic mutants grown in the dark and
found that the loss of function of the ABA1 gene, which
Phytoene
PDS
Fluridone
z− Carotene
Neurosporene
LUT2
Lycopene
g -Carotene
d -Carotene
b-Carotene
a-Carotene
OH
H 3C
H
H3C
C
H33
CH
C
H33
CH
CH33
Zeinoxanthin
CH
C
H33
ABA1
CH
C
H33
H33C
CH
C
H33
H 3C
C
H33
CH
C
H33
CH
CH
C
H33
OH
OH
O
O
CH
C
H33
CH
C
H33
HO
CH
C
H33
Antheraxanthin
ABA1
H 3C
OH
O
CH
C
H33
HO
H
H3C
C
C
H33
CH
C
H33
CH
C
H33
CH
CH
C
H33
Zeaxanthin
Lutein
H
H3C
C
H33C
CH
C
H33
CH
C
H33
HO
H33C
CH33
CH
C
H33
All-trans-violaxanthin
CH 3
H3C
CH3
H 3C
H 3C
CH 3
O
O
HO
H3C
CH3
OH
CH 3
H 3C
H 3C
CH3
9′-cis-neoxanthin
9-cis-violaxanthin
H 3C
CH3
H 3C
CH3
O
H 3C
CH 3
H 3C
HO
OH
H 3C
OH
NCED3
CH
CH33
C H33
CH
H 3C
O
CHO
H
HO
C H33
CH
Xanthoxin
ABA2
C
H33
CH
C H33
CH
H 3C
OH
CHO
O
CH
C
H33
Abscisic aldehyde
AAO3, ABA3
H3 C
H
3
C H3
3
C H3
3
OH
OH
COOH
O
C H3
3
ABA
Figure 1. Carotenoid and abscisic acid (ABA)
biosynthetic pathways. The inhibitory effect of
fluridone on phytoene desaturase (PDS) is indicated.
LUT2 is a e-cyclase. The steps in the ABA biosynthetic
pathway catalysed by ABA1, NCED3, ABA2, AAO3
and ABA3 are indicated.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
De-etiolation in aba1 mutants 229
encodes zeaxanthin epoxidase (ABA1), partially removes
the growth restraint imposed by the absence of light. This
leads to the promotion of cotyledon expansion and to organogenesis. This phenotypic defect was not reverted by exogenous ABA, indicating that xanthophylls are part of the
machinery that blocks plant growth in the absence of external stimuli.
MATERIALS AND METHODS
Plant material and growth conditions
Wild-type (Ler, Col-0 and Ws-2) and mutant Arabidopsis
thaliana (L.) Heynh. plants were grown under sterile conditions on 150 mm Petri dishes containing 100 mL of agar
medium at 20 ⫾ 1 °C, 60–70% relative humidity, and
continuous illumination at 7000 lx (98 mmol m-2 s-1), as
described in Ponce, Quesada & Micol (1998). The nced3-1
and lut2-1 mutants were kindly provided by H. Koiwa
(Texas A&M University, College Station, TX, USA) and by
P. Jahns (Heinrich Heine University, Düsseldorf, Germany),
respectively. The ABA-deficient mutants aba1, aba2, aba3
and aao3, as well as the aba1-101::pBIN19-ABA1 transgenic
line, were described previously (Gonzalez-Guzman et al.
2002, 2004; Barrero et al. 2005, 2006).
Pigment analysis and quantification
Extractions were carried out on 6 mm leaf discs taken from
the rosette leaves of 3-week-old plants grown in soil in a
glasshouse (~22 °C, minimum 13 h photoperiod, maximum
light intensity of 500 mmol m-2 s-1), and in separate experiments, on 10- or on 21-day-old etiolated seedlings grown in
the dark. The pigments were extracted in acetone and separated by HPLC, as described in North et al. (2005). The
carotenoids and chlorophylls were separated by reversephase high-performance liquid chromatography (HPLC)
on a System Gold HPLC (Beckman-Coulter France,
Villepinte, France) using two Adsorbosphere HS C18
3 mm columns (Alltech, Carquefou, France) in series
(100 ¥ 4.6 mm; 150 ¥ 4.6 mm). The pigments were detected
using a photodiode-array detector (Beckman-Coulter
France) and identified by the comparison of retention times
and absorption spectra with published values (Britton 1995)
or commercially available standards [zeaxanthin, lutein,
b-carotene (ExtraSynthèse, Genay, France), and chlorophyll a and b (Fluka, Sigma-Aldrich Chimie, St. Quentin
Fallavier, France)]. The pigments were quantified by the
integration of their peak areas.
RESULTS
Hypocotyl growth is impaired in dark-grown
ABA-deficient mutants
Approximately 50–100 seeds of each genotype were sown
on Petri dishes containing non-supplemented medium or
medium supplemented with 50, 100 or 500 nm ABA (SigmaAldrich A1049, Sigma-Aldrich, St. Louis, MO, USA), or 5,
10 or 20 mm fluridone (Sigma-Aldrich 45511), or 50 nm ABA
and 10 or 20 mm fluridone. To measure hypocotyl growth, 20
etiolated seedlings of each genotype grown on Petri dishes
for 10 d in the dark were photographed and analysed using
the Image-J program (http://rsb.info.nih.gov/ij/docs/menus/
file.html). For the de-etiolation assays, the phenotype of
50–100 seedlings grown on Petri dishes for 21 d in the dark
was scored and photographed.
To further substantiate the proposed role of ABA in cell
expansion (Sharp et al. 2000; Sharp & LeNoble 2002;
Barrero et al. 2005), we analysed hypocotyl elongation in
dark-grown ABA-deficient and wild-type plants. ABAdeficient mutants showed reduced hypocotyl growth in the
dark compared to wild-type seedlings (Fig. 2). The exogenous application of ABA promoted hypocotyl growth in
ABA-deficient mutants, whereas it had no significant effect
on wild-type Columbia (Col-0) plants (Fig. 2). Conversely,
the application of fluridone, an inhibitor of carotenoid and
ABA biosynthesis, dramatically reduced hypocotyl growth
both in wild-type and ABA-deficient plants (Fig. 2).
Northern blots
Total RNA was extracted from frozen, whole 7-day-old
dark-grown seedlings using the RNeasy Plant Mini Kit
(Qiagen, Hilden, Germany). Two micrograms of total RNA
was used for Northern analysis with 32P-labelled CAB2 and
RbcS probes, synthesized as described in Alabadí et al.
(2004). All procedures were carried out as previously
described (Alabadí et al. 2004).
Hypocotyl length (mm)
ABA and fluridone treatments
20
0
500 nM ABA
5
10
20 mM fluridone
16
12
8
4
0
Col-0
aba3-101 aba2-14
aao3-2
aba1-101
Cotyledon angle measurements
Figure 2. Effects of exogenous abscisic acid (ABA) and of
Seedlings were placed on an acetate sheet and scanned at a
resolution of 800 dots per inch. The angle between the
cotyledons was measured from the images obtained
(Alabadí et al. 2004).
fluridone on hypocotyl length in 10-day-old dark-grown wild-type
and ABA-deficient seedlings. The histogram shows the mean
(n = 20) for seedlings grown on medium supplemented with the
indicated concentration of ABA or fluridone. All seedlings were
homozygous for the indicated mutations.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
230 J. M. Barrero et al.
Because the aba1-101 mutant showed the greatest reduction in hypocotyl growth, we decided to analyse this phenotypic defect in greater detail in several aba1 alleles, which
exist in different genetic backgrounds. In all alleles examined, the aba1 mutants displayed reduced hypocotyls length
compared to their corresponding wild-type plants (Fig. 3a).
This phenotypic effect was particularly noticeable in aba1101/aba1-101 and aba1-102/aba1-102 seedlings, which are in
the Col-0 and Ws-2 genetic backgrounds, respectively.
(a)
otyl length (mm)
Hypoco
(b)
25
0
20
500 nM ABA
15
10
5
0
(c)
Hy
ypocotyl length (mm))
0
5
10
20 mM fluridone
15
10
5
Figure 3. Effects of abscisic acid (ABA) and fluridone on
hypocotyl length in 10-day-old dark-grown wild-type and aba1
mutant seedlings. (a) Representative seedlings are shown. (b,c)
Histograms of the mean (n = 20) for seedlings grown on medium
supplemented with the indicated concentration of ABA or
fluridone. All seedlings were homozygous for the mutations
indicated. The seedlings were scored 10 d after sowing. Scale
bar = 1 mm.
Supplementing the growth medium with exogenous ABA
increased the hypocotyls length of all of the aba1 mutants
examined (Fig. 3b). Conversely, fluridone inhibited hypocotyl elongation in all of the aba1 mutants and their corresponding wild-type plants (Fig. 3c).
The aba1 mutants exhibit a
de-etiolated phenotype
The short hypocotyl of dark-grown ABA-deficient mutants
could be the result of a defect in either ABA-induced cell
elongation, as mentioned earlier, or ABA regulation of the
transition between skotomorphogenesis and photomorphogenesis, as shown for BRs and GAs (Li et al. 1996; Szekeres
et al. 1996;Alabadí et al. 2004).To distinguish between these
two alternatives, we analysed a number of classical photomorphogenesis markers, including the angle between cotyledons, and the expression of CAB2 and RbcS genes. For
this, 7-day-old dark-grown ABA-deficient seedlings were
assayed. The angle between the cotyledons in all of the
ABA-deficient mutants was similar to that of the wild type,
and no change was observed in terms of sensitivity to
paclobutrazol (PAC), an inhibitor of GA biosynthesis
(Fig. 4a). Similarly, CAB2 and RbcS expression was unaffected in the ABA-deficient mutant seedlings (Fig. 4b), in
contrast to the reported effect of PAC on GA biosynthesis
(Alabadí et al. 2004). CAB2 and RbcS expression was
also unaffected in fluridone-treated wild-type seedlings
(Fig. 4c). These data collectively lead us to suggest that
ABA itself is not the major player in the transition from
skotomorphogenesis to photomorphogenesis.
We next found a requirement for ABA1 to repress photomorphogenesis in the dark. The aba1 mutants were found
to be partially de-etiolated when grown in the dark for 21 d.
They exhibited open cotyledons and developed true leaves
(Fig. 5a and Table 1). Unexpanded cotyledons were shown
in >89% of the dark-grown wild-type seedlings, but only in
<20% of the mutant seedlings homozygous for the severely
hypomorphic or null aba1-1, aba1-101 and aba1-102 mutations. In addition to the high percentage of expanded cotyledons, a significant proportion of aba1/aba1 plants bore
true leaves (10–20% compared to <3% in the wild type;
Table 1). These collective observations are consistent with a
partial photomorphogenic phenotype in the aba1 mutants.
These observations lead us to suggest that, although ABA
could participate in the control of the developmental transition to de-etiolated growth, its timing differs from that of
GAs, BRs and the COP1 pathway. However, we found that,
in similarly treated ABA-deficient mutants affected in steps
downstream of ABA1 (e.g. nced3-1, aba2-14, aba3-101 and
aao3-2), and in ABA-insensitive mutants (e.g. abi1-1, abi2-1
and abi4-1), a de-etiolated phenotype was not observed
(Table 1). Therefore, the de-etiolated phenotype of aba1
mutants appears to be a particular consequence of the loss
of function of ABA1, rather than a defect in ABA biosynthesis. Consistent with this, supplementing growth medium
with exogenous ABA did not suppress the de-etiolated phenotype of aba1-101/aba1-101 seedlings (data not shown),
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
De-etiolation in aba1 mutants 231
An
ngle between cotyledon
ns (deg)
(a)
100
0
80
1 mM PAC
60
40
20
0
(b)
Col-0
1 mM PAC –
Col-0
+
–
–
–
+
–
–
RbcS
CAB2
1991; Marin et al. 1996). As a consequence, they accumulate
zeaxanthin, and are severely impaired in the production
of zeaxanthin-derived xanthophylls, in addition to downstream metabolites of the ABA biosynthetic pathway. To
further document the effect of aba1 mutations, we analysed
the carotenoid profile in green and etiolated tissues of aba1101/aba1-101 and aba1-102/aba1-102 mutant seedlings. We
found that both mutant alleles had, compared to the wild
type, undetectable amounts of neoxanthin, violaxanthin and
antheraxanthin (Table 2). In contrast, more zeaxanthin was
accumulated in the green tissues and etiolated seedlings of
the aba1-101 and aba1-102 mutants than in those of the wild
type (Table 2). Beta-carotene, which is the immediate precursor of zeaxanthin, was more abundant in the aba1-101
and aba1-102 seedlings than in the wild type. The wild-type
ABA1 allele restored the levels of these metabolites to
those found in wild-type plants (Table 2). In contrast, other
ABA-deficient mutants (aba2-14, aba3-101 and aao3-2)
showed minor differences in their levels of neoxanthin, violaxanthin, antheraxanthin and zeaxanthin, compared to the
wild type (Table 2).
rRNA
rRNA
The inhibition of carotenoid biosynthesis by
fluridone treatment leads to a partially
de-etiolated phenotype
(c)
Col-0
10 mM fluridone –
CAB2
rRNA
+
Col-0
–
+
RbcS
rRNA
Figure 4. Early photomorphogenic markers are not altered in
aba mutants. (a) Angle between the cotyledons of wild-type
(Col-0), aba1, aba2, aba3 and aao3 seedlings grown in darkness
for 7 d, with or without 1 mm paclobutrazol (PAC). The histogram
shows the means (n = 20). (b) The aba1 and aba2 mutations do
not affect CAB2 or RbcS expression in 7-day-old dark-grown
seedlings. For comparison, note elevated CAB2 expression in
PAC-treated wild-type seedlings, which are de-etiolated. (c)
Fluridone treatment does not affect CAB2 or RbcS expression in
7-day-old dark-grown seedlings. All seedlings were homozygous
for the mutations indicated.
whereas the restoration of ABA1 function in the aba1-101
mutant by a transgene containing the wild-type ABA1 allele
resulted in normal skotomorphogenic growth (Table 1).
The block of carotenoid biosynthesis in aba1
mutants is correlated with its partially
de-etiolated phenotype
The epoxidation of zeaxanthin to antheraxanthin and violaxanthin is impaired in aba1 mutants (Rock & Zeevaart
The results suggest to us that the lack of any immediate
products of ABA1 activity, or the accumulation of the
immediate precursor of such activity, could be the cause of
the de-etiolated phenotype observed in aba1 mutants. To
differentiate between these two alternatives, we examined
the effect of fluridone treatment on dark-grown wild-type
seedlings. We reasoned that if the phenotype was caused by
the accumulation of a precursor of ABA1 activity, fluridone
treatment of wild-type plants should not phenocopy this
aba1 phenotype. Interestingly, we found that the treatment of wild-type plants with fluridone mimicked the
de-etiolated phenotype observed in aba1 mutants (Fig. 5b,
Table 3). As the ABA-deficient mutants nced3-1, aba2-14,
aba3-101 and aao3-2 did not show this phenotype (Table 1),
this leads us to suggest that fluridone- and aba1-induced
de-etiolation is caused by the lack of production of a molecule(s) at a step in the pathway between intermediates
catalysed by ABA1 and NCED3. In agreement with this
idea, simultaneous treatment with fluridone and exogenous
ABA did not rescue the seedling de-etiolated phenotype of
the wild type nor the aba1 mutant (Fig. 5c,d).To rule out the
possibility that de-etiolation is caused not by the immediate
products of ABA1, but by their putative effect on the synthesis of lutein, which, together with violaxanthin, is the
major carotenoid in etiolated tissue (Park et al. 2002), we
analysed the dark-growth phenotype of the lutein-deficient
mutant lut2-1 (Pogson et al. 1996) (Table 1). The lut2-1
mutant seedlings showed an etiolated phenotype when
grown in the dark, indicating that a-carotene-derived xanthophylls are not involved in the control of this developmental transition. Furthermore, zeaxanthin was not
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
232 J. M. Barrero et al.
(a)
aba1-102 Ler
Ws-2
(b)
aba1-1
Col-0 aba1-101
10 mM fluridone
–
+
–
Ws-2
+
–
Ler
(c)
+
Col-0
100 nM ABA
20 mM
fluridone
20 mM
fluridone
Figure 5. Fluridone treatment phenocopies the de-etiolated
phenotype of the aba1 mutants. (a) Representative aba1 mutant
seedlings and their corresponding wild types grown in the dark
for 21 d. Most wild-type plants displayed unexpanded cotyledons,
as shown for the Ws-2, Ler and Col-0 accessions. Expanded
cotyledons and leaves were observed in most aba1-102/aba1-102,
aba1-1/aba1-1 and aba1-101/aba1-101 seedlings.
(b–d) De-etiolation induced by fluridone in wild-type plants.
(b) Representative wild-type seedlings (Col, Ws-2 and Ler
ecotypes) grown in the dark for 21 d with or without 10 mm
fluridone. (c,d) Exogenous ABA supplementation does not
suppress the de-etiolated phenotype induced by fluridone in
wild-type or aba1-101 seedlings. (c) Representative
aba1-101/aba1-101 and wild-type Col-0 seedlings grown in the
dark for 21 d, in the presence or absence of fluridone and/or
abscisic acid (ABA). (d) The histogram shows the means (n = 50)
for Col-0 and aba1-101/aba1-101 seedlings grown in the dark for
21 d on medium supplemented with 20 mm fluridone in the
presence (100 nm) or absence (0 nm) of exogenous ABA. All
seedlings were homozygous for the mutations indicated. UC,
unexpanded cotyledons; EC, expanded cotyledons; ECL,
expanded cotyledons and true leaves. Scale bars = 1 mm.
that the proper synthesis of certain b-carotene-derived xanthophylls (e.g. neoxanthin, violaxanthin and antheraxanthin) is a necessary part of the normal skotomorphogenic
program. This conclusion is mainly supported by the
de-etiolated phenotype induced by fluridone and aba1
mutation, as well as by the lack of such a phenotype in all
other ABA-deficient mutants and lut2-1, which is unable to
synthesize a-carotene-derived xanthophylls such as zeinoxanthin and lutein.
Exogenous supplementation of aba1 with the b-carotenederived xanthophylls lacking in the mutant, namely neoxanthin, violaxanthin and antheraxanthin, was not possible
Percentage of UC, EC + ECL
in 20 mM fluridone
Table 1. De-etiolated phenotype of aba1 mutants
(d)
120
80
60
40
20
0
UC
EC + ECL
0
Genotype
Col-0
aba1-101
aba1-101::pBIN19-ABA1
Ler
aba1-1
Ws-2
aba1-102
nced3-1
aba2-14
aba3-101
aao3-2
abi1-1
abi2-1
abi4-1
lut2-1
89.8
18.0
95.1
99.0
11.4
94.7
18.2
89.8
87.4
91.4
78.9
97.9
91.3
91.4
93.7
aba1-101
Col-0
100
Expanded
cotyledons
Unexpanded Expanded and true
cotyledons
cotyledons leaves
UC
EC + ECL
100 nM ABA
detectable in dark-grown Ler, Col-0 or Ws-2 wild-type seedlings exposed to 20 mm fluridone.
10.2
60.2
4.9
1.0
79.3
2.6
59.7
10.2
10.5
2.2
21.1
2.1
8.7
7.5
6.3
0.0
21.6
0.0
0.0
9.1
2.6
21.9
0.0
2.1
6.4
0.0
0.0
0.0
1.1
0.0
DISCUSSION
We analysed several mutants impaired at different points in
the carotenoid and ABA biosynthetic pathways and found
Values indicate the percentage of seedlings that exhibited the indicated traits after 21 d of growth in the dark. All seedlings were
homozygous for the mutations indicated.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
t-N, trans-neoxanthin; c-N, cis-neoxanthin; t-V, trans-violaxanthin; c-V, cis-violaxanthin; A, antheraxanthin; L, lutein; Z, zeaxanthin; b-C, b-carotene; ND, not detected. The carotenoid content was
determined as described in the Methods section. Each value represents the mean ⫾ SE (in pmol cm-2 for 21-day-old light-grown plants, and pmol (mg fresh weight)-1 for 10-day-old etiolated
seedlings) of three independent samples from each genotype. All of the plants and seedlings analysed were homozygous for the mutations indicated.
0.09 ⫾ 0.00
0.21 ⫾ 0.01
0.11 ⫾ 0.13
0.16 ⫾ 0.01
0.12 ⫾ 0.00
0.31 ⫾ 0.13
ND
7.49 ⫾ 0.23
ND
ND
ND
16.24 ⫾ 0.61
2.85 ⫾ 0.14
4.12 ⫾ 0.15
3.37 ⫾ 0.15
4.21 ⫾ 0.32
2.96 ⫾ 0.28
8.75 ⫾ 0.39
0.17 ⫾ 0.01
ND
0.33 ⫾ 0.03
0.25 ⫾ 0.04
0.13 ⫾ 0.01
ND
1.10 ⫾ 0.04
ND
1.09 ⫾ 0.06
1.70 ⫾ 0.14
1.67 ⫾ 0.16
ND
0.10 ⫾ 0.01
ND
0.12 ⫾ 0.00
0.13 ⫾ 0.01
0.23 ⫾ 0.08
ND
0.15 ⫾ 0.00
ND
0.16 ⫾ 0.01
0.26 ⫾ 0.02
0.25 ⫾ 0.04
ND
1214 ⫾ 56
2012 ⫾ 81
1254 ⫾ 47
1402 ⫾ 70
1522 ⫾ 63
1522 ⫾ 63
1038 ⫾ 75
1408 ⫾ 76
56 ⫾ 10
3951 ⫾ 103
62 ⫾ 1
87 ⫾ 11
ND
53 ⫾ 8
56 ⫾ 5
3247 ⫾ 245
2490 ⫾ 109
3004 ⫾ 117
2477 ⫾ 106
3085 ⫾ 153
3219 ⫾ 214
2742 ⫾ 46
1909 ⫾ 153
1973 ⫾ 176
101 ⫾ 15
ND
91 ⫾ 8
167 ⫾ 22
101 ⫾ 6
134 ⫾ 10
88 ⫾ 8
ND
37 ⫾ 6
ND
24 ⫾ 3
69 ⫾ 5
33 ⫾ 8
37 ⫾ 4
28 ⫾ 11
ND
790 ⫾ 80
ND
803 ⫾ 31
1131 ⫾ 49
920 ⫾ 105
1039 ⫾ 22
628 ⫾ 82
ND
557 ⫾ 28
ND
592 ⫾ 21
594 ⫾ 27
799 ⫾ 43
584 ⫾ 13
453 ⫾ 30
ND
21-day-old plants (minimum 13 h photoperiod)
Col-0
181 ⫾ 12
aba1-101
ND
aba1-101:: pBIN19ABA1
139 ⫾ 7
aba2-14
232 ⫾ 17
aba3-101
193 ⫾ 26
aao3-2
149 ⫾ 12
Ws-2
142 ⫾ 25
aba1-102
ND
10-day-old etiolated seedlings grown in the dark
Col-0
0.20 ⫾ 0.01
aba1-101
ND
aba2-14
0.20 ⫾ 0.01
aba3-101
0.26 ⫾ 0.02
Ws-2
0.32 ⫾ 0.02
aba1-102
ND
c-N
t-N
Genotype
Table 2. Carotenoid content of wild type and aba mutants
t-V
c-V
A
L
Z
b-C
De-etiolation in aba1 mutants 233
because of the highly hydrophobic nature of these compounds and their subcellular localization in the chloroplast.
In principle, the accumulation of zeaxanthin observed
in aba1 might also be responsible for the observed
de-etiolated phenotype; however, fluridone treatment
inhibited carotenoid biosynthesis and hence zeaxanthin
production, and phenocopied the aba1 mutant phenotype
in wild-type seedlings. Therefore, a common effect produced both by fluridone treatment and a loss of function in
ABA1 is the biosynthetic blockade of certain b-carotenederived xanthophylls. Another trait shared by aba1 mutants
and fluridone-treated wild-type plants is impaired biosynthesis of downstream metabolites such as xanthoxin, abscisic aldehyde and endogenous ABA. However, this is
not responsible for the observed phenotype, because
ABA-deficient mutants that are negatively affected in the
biosynthesis of these metabolites display normal skotomorphogenic growth.
Why does the impairment of b-carotene-derived xanthophylls disturb skotomorphogenesis? As shown by Park et al.
(2002), carotenoids play a key role in dark-grown tissues
and have a well-established function in photosynthetic
tissues. Carotenoids, particularly lutein and violaxanthin,
are present at significant levels in etiolated seedlings,
although their functions have not been fully elucidated.
Park et al. (2002) demonstrated that carotenoid biosynthesis is required for the formation of prolamellar bodies
(PLB), the lattices of tubular membranes that define an
etioplast. Therefore, the presence of carotenoids in darkgrown tissue may facilitate chloroplast development during
seedling establishment and photomorphogenesis. Based on
our results, we propose that the absence of violaxanthin, a
major carotenoid together with lutein in etiolated tissue,
might disturb etioplast development, as well as plastid-tonucleus signalling. Studies that used mutant plants impaired
in carotenoid biosynthesis have demonstrated that the
expression of certain nuclear-encoded photosynthetic genes
is reduced in the absence of functional chloroplasts (Nott
et al. 2006). Likewise, norflurazon inhibits the expression of
several nuclear genes encoding chloroplast-localized proteins in light-grown seedlings (Nott et al. 2006). Thus, both
genetic and chemical impairment of carotenoid biosynthesis disturbs plastid-to-nucleus signalling and the photoautotrophic lifestyle. By analogy, we speculate that the
impairment of carotenoid biosynthesis at the step catalysed
by ABA1 might generate an unknown signal that disturbs
normal skotomorphogenic growth.
ACKNOWLEDGMENTS
We thank J.M. Serrano, V. García and T. Trujillo for technical assistance. This work was supported by a fellowship (to
J.M.B.) and research grants (BIO2000-1082 to J.L.M. and
BIO2005-01760 to P.L.R.) from the Ministerio de Educación y Ciencia of Spain. D.A. was supported by a Ramón
y Cajal research contract and the Fundación de la Comunidad Valenciana para la Investigación Agroalimentaria
(Agroalimed).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234
234 J. M. Barrero et al.
Table 3. De-etiolation induced by fluridone treatment
Fluridone (mm)
0
5
10
20
Accession
UC
EC
ECL
UC
EC
ECL
UC
EC
ECL
UC
EC
ECL
Ws-2
Ler
Col-0
94.7
96.8
100
2.6
1.0
0
2.6
1.5
0
58.3
42.4
11.4
41.6
45.4
85.7
0
12.1
0
27.1
8.8
9.3
54.0
44.4
78.1
16.2
46.6
12.5
8.3
9.1
12.1
77.1
56.3
66.6
14.5
34.5
21.2
The values indicate the percentage of seedlings that exhibited the indicated features after 21 d of growth in the dark.
UC, unexpanded cotyledons; EC, expanded cotyledons; ECL, expanded cotyledons and true leaves.
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Received 5 October 2007; accepted for publication 17 October 2007
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 227–234