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Light Regulation of Gibberellin Biosynthesis in Pea Is Mediatedthrough the COP1/HY5 Pathway W
James L. Weller,1 Valerie Hecht, Jacqueline K. Vander Schoor, Sandra E. Davidson, and John J. Ross
School of Plant Science, University of Tasmania, Hobart, Tasmania 7001, Australia
Light regulation of gibberellin (GA) biosynthesis occurs in several species, but the signaling pathway through which this
occurs has not been clearly established. We have isolated a new pea (Pisum sativum) mutant, long1, with a light-dependent
elongated phenotype that is particularly pronounced in the epicotyl and first internode. The long1mutation impairs signaling
from phytochrome and cryptochrome photoreceptors and interacts genetically with a mutation in LIP1, the pea ortholog of
Arabidopsis thaliana COP1. Mutant long1 seedlings show a dramatic impairment in the light regulation of active GA levels
and the expression of several GA biosynthetic genes, most notably the GA catabolism gene GA2ox2. The long1 mutant
carries a nonsense mutation in a gene orthologous to the ASTRAY gene from Lotus japonicus, a divergent ortholog of the
Arabidopsis bZIP transcription factor gene HY5. Our results show that LONG1 has a central role in mediating the effects of
light on GA biosynthesis in pea and demonstrate the importance of this regulation for appropriate photomorphogenic
development. By contrast, LONG1 has no effect on GA responsiveness, implying that interactions between LONG1 and GA
signaling are not a significant component of the molecular framework for light–GA interactions in pea.
INTRODUCTION
Light has profound effects on plant growth and development,
and there is widespread interest in understanding the molecular
basis of these effects. One general mechanism by which light
acts is the regulation of plant hormone production (Symons and
Reid, 2003), and changes in hormone levels occur in several
different light responses. Examples include changes in gibber-
ellin (GA) and abscisic acid levels during light-regulated seed
germination (Oh et al., 2006; Seo et al., 2006) and changes in
auxin levels in response to simulated shade (Tao et al., 2008).
In deetiolating seedlings, changes in expression of hormone
biosynthetic genes in response to light or specific photoreceptor
activity have been identified in several transcriptional profiling
studies inArabidopsis thaliana (Ma et al., 2001; Tepperman et al.,
2001; Folta et al., 2003), and this is generally understood to imply
that changes in actual hormone levels also occur during, and
contribute to, the process of deetiolation. However, the clearest
example of light-regulated hormone biosynthesis during deetio-
lation comes from pea (Pisum sativum), where transfer of etio-
lated pea seedlings to light induces a rapid drop in levels of the
active GA (GA1), to <10% of dark levels within 4 h (Ait-Ali et al.,
1999; Gil andGarcıa-Martinez, 2000; O’Neill et al., 2000; Symons
and Reid, 2003). This drop is associated with changes in tran-
script levels of GA biosynthesis genes and depends on the
activity of the phyA and cry1 photoreceptors (Reid et al., 2002;
Foo et al., 2006). A similar but somewhat less dramatic drop in
active GA (GA4) content has recently been shown to occur in
Arabidopsis (Zhao et al., 2007; Symons et al., 2008) and is also
temporally correlated with changes in expression of GA biosyn-
thesis genes (Zhao et al., 2007; Alabadı et al., 2008) .
Despite the apparent importance of these changes for the
inhibition of elongation in deetiolating seedlings, relatively little is
known about how they are achieved. One of the earliest molec-
ular responses to light is the transcriptional induction or repres-
sion of a small group of genes encoding transcription factors,
including the PIF family of bHLH proteins, the bZIP protein HY5,
and the myb protein CCA1 (Tepperman et al., 2001, 2004).
Transcriptional profiling shows that these genes in turn regulate
the expression of a much broader range of genes, including a
significant number involved in hormone biosynthesis or signal-
ing. Among these, auxin signaling genes are particularly prom-
inent, but genes for ethylene, cytokinin, andGAbiosynthesis also
feature (e.g., Monte et al., 2004; Sibout et al., 2006; Lee et al.,
2007). These observations suggest that light effects on hormone
synthesis and signaling depend on these master regulators.
Several recent reports have identified specific interactions be-
tween light and hormone signaling pathways (Khanna et al.,
2007; Alabadı et al., 2008; Chen et al., 2008; de Lucas et al.,
2008; Feng et al., 2008), but relatively few studies have ad-
dressed the mechanisms by which hormone biosynthesis is
regulated.
Oneof themain insights into light regulation ofGAbiosynthesis
to date has come from studies of germinating Arabidopsis seeds
by Oh et al. (2006), who showed that induction of GA biosynthe-
sis by light is achieved through degradation of the bHLH tran-
scription factor PIF1/PIL5, a transcriptional repressor of GA
biosynthetic genes. As PIF proteins are bound to and targeted for
degradation by activated phytochromes (Castillon et al., 2007),
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: James L. Weller([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.063628
The Plant Cell, Vol. 21: 800–813, March 2009, www.plantcell.org ã 2009 American Society of Plant Biologists
this represents a relatively direct mechanism in which light
activates GA biosynthesis through repression of a repressor. In
deetiolating seedlings, however, light acts to repress rather than
activate GA biosynthesis (Symons et al., 2008), and the mech-
anism underlying this effect is still unclear.
The Arabidopsis HY5 protein has an important role in light-
regulated development. Mutants for HY5 were first isolated on
the basis of a long-hypocotyl phenotype under several different
monochromatic light conditions and were subsequently shown
to impair a range of light responses in shoot and root (Koornneef
et al., 1980; Oyama et al., 1997). More recently, HY5 has also
been implicated in the light regulation of hormone responses,
based on findings that HY5 and the related protein HYH regulate
numerous genes involved or implicated in signaling from plant
hormones, including auxin, cytokinin, and abscisic acid (Holm
et al., 2002; Cluis et al., 2004; Sibout et al., 2006; Chen et al.,
2008). Several studies have also reported the reciprocal regula-
tion of HY5 by plant hormone action, including the regulation of
HY5 protein by cytokinin (Vandenbussche et al., 2007) and by
members of the DELLA family of GA signaling proteins (Alabadı
et al., 2008).
In this article, we report on the isolation and functional
characterization of the pea LONG1 gene. This gene is a repre-
sentative of a group of divergent legume HY5 orthologs previ-
ously characterized from soybean (Glycine max), fava bean
(Vicia faba), and Lotus japonicus (Cheong et al., 1998; Nishimura
et al., 2002b). We used a presumed null long1mutant containing
a premature stop codon to show that LONG1 has roles in
deetiolation, flowering, and root system development and
that it interacts genetically with LIP1, the pea ortholog of
Arabidopsis COP1 (Sullivan and Gray, 2000). We also examined
the possibility that elongation of the long1 mutant results from
elevated production of GAs. Our results show that LONG1 has a
major role in mediating the light regulation of GA biosynthesis
that occurs in deetiolating seedlings and thus provide a new
insight into the mechanisms by which light and GAs interact
during deetiolation.
RESULTS
Genetic Analysis of long1, a New Elongated Pea Mutant
In screens of ethyl methanesulfonate–mutagenized pea seed-
lings of wild-type cv Torsdag under white light, we isolated a
new, recessive mutant with an extremely elongated epicotyl and
early internodes (Figures 1A and 1B). This mutant was not allelic
with other elongated mutants and apparently defined a novel
locus, which we designated LONG1. In various crosses, we
observed tight linkage between LONG1 and the ST locus and
weaker linkage of LONG1with other group III classical markersB
and DNE. This indicated a position near the LA gene (Weeden
et al., 1998), recently shown to encode a GA-signaling DELLA
protein (Weston et al., 2008), and we therefore considered the
possibility that long1 and la might be allelic. However, the
presence of several recombinants in a cross segregating for
long1 and la excluded this possibility and suggested a LA-
LONG1 distance of;4 centimorgans.
Shoot Phenotype of the long1Mutant
The pronounced effect of long1 on the length of the first two
internodes ismost similar to that of the constitutive GA-response
double DELLA mutant la cry-s (Potts et al., 1985; Weston et al.,
2008) and the GA-overproducing sln mutant, which has a muta-
tion in the GA catabolism gene GA2ox1 (Reid et al., 1992; Lester
et al., 1999) (Figures 1A and 1B). By contrast, the phyB mutant,
although equivalently elongated over later internodes, has only a
weak effect on elongation of early internodes (Figures 1A and
Figure 1. Shoot Phenotypes of long1, a New Elongated Pea Mutant.
(A), (B), and (E) Phenotypes of elongated mutant seedlings under
glasshouse conditions. Appearance of representative seedlings (A),
internode lengths (B), and stem chlorophyll (chl) content (D).
(C) and (E) Photoperiod response of wild-type and long1 mutant plants
for node of flower initiation (E) and stem length between nodes 1 and 3
(C). All plants received an 8-h photoperiod of natural daylight either with
(LD) or without (SD) a 16-h extension with low-irradiance cool-white
fluorescent light.
Values represent mean 6 SE for n = 8 plants except for (D), where n = 4.
HY5/ASTRAY Ortholog Function in Pea 801
1B). All four mutants also had reduced chlorophyll content in
internodes 1 and 2 (Figure 1D) but differed in their relative effects
in an internode-specific manner. In internode 1, the effect of
long1wasmuch stronger (80% reduction comparedwith thewild
type) than any of the other three mutants (30 to 50% reduction).
However, in internode 2, both long1 and phyB had stronger
effects (54 and 68% reduction, respectively) than the GA-related
mutants la cry (23% reduction) and sln (27% reduction). Thus,
long1 seedlings have a characteristic phenotype distinguished
from these other elongated mutants by strong effects on both
elongation and chlorophyll content in the first internode, regard-
less of whether chlorophyll content is expressed relative to fresh
weight (Figure 1D) or per internode (3.9 6 0.3 mg in wild type
versus 2.56 0.3mg in long1). These observations suggested that
LONG1 might act on both light signaling and GA synthesis/
response in early seedling development.
In Arabidopsis, increased elongation also results from muta-
tions affecting the circadian clock, such as elf3 (Zagotta et al.,
1996) and elf4 (Doyle et al., 2002), but this is photoperiod
dependent and associated with defects in photoperiodic flower-
ing. However, the effect of long1 on elongation was similar in
both long and short photoperiods (Figure 1C), and long1 had no
effect on photoperiodic flowering (Figure 1E), suggesting that
long1 was unlikely to have a defect in the circadian clock or the
photoperiod response mechanism.
LONG1 Participates in Signaling from
Multiple Photoreceptors
long1 seedlings grown in complete darkness have an etiolated
appearance similar to the wild type, with elongated epicotyl and
internodes, a normal apical hook, and strong suppression of leaf
development (Figures 2A and 3C). The elongated phenotype of
long1 seedlings grown under white light (W) is therefore light
dependent and different from that of theGA-related la cry and sln
mutants despite the similar internode length profile (Figure 1B)
(Reid, 1988). Mutant long1 seedlings were also much longer than
the wild type under monochromatic blue (B) or far-red (FR) light
and slightly longer under red (R) (Figure 2A), suggesting that
LONG1 affects signaling from multiple photoreceptors.
In pea seedlings, phyA and phyB both contribute to deetiola-
tion under R and act together with cry1 under B (Platten et al.,
2005). To examine which specific photoreceptor signals were
impaired by long1, we generated long1 phyA and long1 phyB
double mutants. Figure 2B shows that the small effect of long1
under R is retained and even slightly enhanced in the phyA and
phyB backgrounds. Because the phyA phyB double mutant is
insensitive to R (Weller et al., 2001), this shows that long1 affects
both phyA and phyB signaling under R. It also shows that
phytochrome signaling under R is not completely dependent on
LONG1 because both the phyA long1 and phyB long1 double
mutants are more responsive to R than the phyA phyB double
mutant. Under B, the phyA long1 double mutant was less
responsive than any of the three photoreceptor double mutants
(Figure 2B), and as the phyA phyB cry1 triplemutant is essentially
insensitive to B (Platten et al., 2005), this implies that long1
affects signaling from all three photoreceptors in blue light.
The long1Mutation Is Epistatic to lip1
The light-dependent nature and lack of spectral specificity of the
long1 elongated phenotype are similar to the phenotypic char-
acteristics of mutants for Arabidopsis genes HY5, HFR1, and
STH2 genes, which all encode transcription factors whose
stability is regulated through interaction with COP1 ubiquitin
ligase (Osterlund et al., 2000; Kim et al., 2002; Datta et al., 2007).
A direct molecular interaction between HY5 and COP1 was
initially suggested by allele-specific epistatic relationships be-
tween hy5 and cop1 mutants (Ang and Deng, 1994). More
recently, both hfr1 and sth2mutants have been shown to partially
overcome the effects of cop1 (Kim et al., 2002; Datta et al., 2007).
We therefore tested the genetic interaction of long1 with lip1, a
Figure 2. LONG1 Functions in Phytochrome and Crytochrome Signal-
ing.
(A) Internode elongation of wild-type and long1 seedlings grown from
sowing in darkness (D) or under continuous irradiation with far-red (FR),
red (R), or blue (B) light (15 mmol m�2s�1) or white light (100 mmol m�2s�1)
(B) Internode elongation and leaflet area of long1 mutant seedlings in
different photoreceptor-deficient genetic backgrounds under continuous
red (R) or blue (B) light (15 mmol m�2s�1).
Values represent mean 6 SE for n = 8 to 10 plants.
802 The Plant Cell
loss-of-function mutant of the pea COP1 ortholog (Sullivan and
Gray, 2000), which when grown in W has a dark-green, dwarf
phenotype opposite to that of long1 (Frances et al., 1992). In
seedlings grown under continuous W, long1 was almost com-
pletely epistatic to lip1 (Figures 3A and 3B) and also strongly
suppressed the lip1 phenotype in dark-grown seedlings, al-
though long1 itself had little effect in darkness (Figures 3C and
3D). This suppression was almost complete for early stem
elongation, which was similar in long1 and long1 lip1 double
mutants, but only partial for leaf expansion, which in the long1
lip1 double mutant was intermediate between long1 and lip1
single mutants (Figure 3D).
LONG1 Belongs to a Group of Divergent Legume Orthologs
of Arabidopsis HY5
In view of the physiological similarities between LONG1 and
COP1-interacting transcription factors HY5, HYH, STH2, and
HFR1, we searched for legume sequence information that might
help narrow the range of candidate genes. Close legume homo-
logs of HFR1 were not identified, and a Medicago truncatula
STH2 ortholog (AC183777_9/TC101468) was excluded as a
candidate by its location on chromosome 1 (syntenic with pea
linkage group II). Several HY5-like legume genes are known,
including VFBZIPZF from V. faba, STF1 from soybean, and BZF/
ASTRAY from L. japonicus (Cheong et al., 1998; Nishimura et al.,
2002b), and aMedicago EST contig (TC123156) also belongs to
this group. As amap positionwas not available for this sequence,
we isolated a cDNA covering the entire predicted coding se-
quence of the corresponding gene from pea and mapped this
gene to the middle of linkage group III near the position previ-
ously determined for LONG1. Sequencing results from the long1
mutant identified a G-to-A substitution in exon 3, causing a
nonsense mutation of the codon specifying Trp-124 (TGG) to a
stop codon (TGA) (Figure 4B), which cosegregatedwith the long1
phenotype, strongly supporting the conclusion that LONG1 is the
pea ortholog of Lotus ASTRAY. An alignment of LONG1 and
related proteins is shown in Supplemental Figure 1 online.
As previously reported for STF1 and ASTRAY (Cheong et al.,
1998; Nishimura et al., 2002b), the C-terminal bZIP domain of
LONG1 is highly similar to Arabidopsis HY5 and clusters with the
other legume genes in a distinct HY5 clade (Figure 4A). However,
the 189–amino acid N-terminal region encoded by the first four
exons of LONG1 contains a Zn-finger domain and an acidic
domain not present in Arabidopsis HY5, HYH, or HY5 homologs
from other species (Nishimura et al., 2002b). This region is similar
to the N-terminal region of cellulose synthase A subunits and is
most similar to Arabidopsis CESA1 (At4g32410). Intron/exon
boundaries are well conserved and suggest that the legume
genesmay represent a fusion of the first three exons ofCESA1 to
the last three exons of HY5 (Figure 4B). As the long1 mutation
would eliminate the entire HY5-homologous region (Figure 4B),
Figure 3. The long1 Mutation Is Epistatic to lip1.
(A) and (B) Interaction of long1 and lip1 in control of development in plants grown under continuous white light.
(A) Appearance of representative seedlings.
(B) Stem length between nodes 1 and 3 (n = 8 to 10) and chlorophyll content of internode 1 (n = 4 plants). Bars represent SE.
(C) and (D) Interaction of long1 and lip1 in control of development in plants grown in continuous darkness.
(C) Appearance of representative seedlings.
(D) Stem length between nodes 1 and 3, and leaflet area (n = 8 to 10). Bars represent SE.
HY5/ASTRAY Ortholog Function in Pea 803
the long1 mutant thus appears likely to be null for HY5-like
function. Other HY5-related sequences present in soybean
(CX517746) and Medicago (AC146793_14) seem to represent
more conventional orthologs of Arabidopsis HYH (Figure 4A).
LONG1 Has Light-Dependent Effects on Root
System Development
In addition to its effects on photomorphogenesis, the hy5mutant
also affects the root system, developing longer and more nu-
merous lateral roots and an increased lateral root angle (Oyama
et al., 1997; Cluis et al., 2004; Sibout et al., 2006). The astray
mutant in L. japonicus has similar effects on lateral root elonga-
tion and angle, enhanced primary root elongation, and preco-
cious initiation of nodules (Nishimura et al., 2002a, 2002b). We
therefore examined root phenotypes of the long1 mutant. In
seedlings grown in pots, the wild type and long1 developed very
similar root systems with respect to primary root length and the
length, number, and angle of lateral roots (see Supplemental
Figure 2 online). This growth system approximates natural con-
ditions in which the root system is underground and not exposed
to light. By contrast, root phenotypes of hy5 and astray have
been assessed on agar plates with root systems developing in
the light. To test whether long1 seedlings might show a light-
dependent root phenotype, we grew pea seedlings on inclined
agar plates. As observed for plants grown in soil, there was little
difference in the root systems of wild-type and long1 seedlings
grown in complete darkness (data not shown). However, when
grown under continuous white light, wild-type seedlings showed
decreased elongation and increased thickness of both primary
and lateral roots and pronounced chlorophyll accumulation in the
primary root (see Supplemental Figure 2 online). By contrast, the
root systems of long1mutants grown in the light were very similar
to those of dark-grown plants, showing that long1 impairs the
response of pea roots to light. Other traits affected by hy5 and
astraymutations, such as the timing of emergence or number of
lateral roots, were not apparently influenced by long1.
LONG1Mediates the Rapid Effects of Light on GA Economy
Transfer of etiolated pea seedlings to light is rapidly followed by a
dramatic decrease in the content of the active gibberellin GA1
(O’Neill et al., 2000; Symons and Reid, 2003), and we next tested
whether LONG1 was also required for this response. Figure 5A
shows that when 7-d-old wild-type seedlings were transferred to
continuous W for 4 h, the GA1 content of the apical portion
(including the apical bud and 20 mm of expanding internode)
dropped by more than 95%, consistent with the recent report of
Symons et al., (2008). In equivalent long1 mutant seedlings,
however, this did not occur (Figure 5A). The 4-h light treatment
was alsomuch less effective for inhibition of internode elongation
in long1 seedlings than in the wild type (Figure 5B). Light transfer
also caused a 50% drop in GA19 and GA20 content and a 2-fold
increase inGA8 content in thewild typewithin 4 h (Figure 5A), and
these changes were also largely blocked by the long1 mutation.
By contrast, IAA levels were similar in wild-type and long1
seedlings in darkness and after light exposure (Figure 5C).
In parallel with GA measurements, we also monitored tran-
script levels of shoot-expressed GA biosynthesis genesGA3ox1
(LE), GA2ox1 (SLN), and GA2ox2. GA3ox genes catalyze the
conversion of inactive GA20 to the active form GA1, whereas the
GA2ox genes convert GA1 to the inactive product GA8 (Figure
5A). GA2ox1 but not GA2ox2 also has the ability to convert GA20
to another inactive product GA29 (Lester et al., 1999). Figure 5D
shows that the clearest effect of light was seen for GA2ox2. In
expanding shoot tissue from wild-type seedlings, GA2ox2 ex-
pression increased 9-fold after light treatment, but only 2.4-fold
in long1 seedlings, despite similar dark expression levels, sug-
gesting that the increased GA1 content of light-exposed long1
seedlings mainly resulted from a reduction inGA2ox2-dependent
catabolism. In comparison, expression levels of other genes
showed relatively minor changes in response to light or LONG1
action (Figure 5D). For example, the long1 mutant showed a
small (40%) decrease inGA3ox1 expression and a small increase
(2.4-fold) inGA2ox1 expression, which would oppose the effects
of the strong decrease in GA2ox2 expression and would tend to
increase rather than decrease the amount of GA1 after transfer.
These changes are more consistent with those expected as part
of feedback regulation in response to the increased GA1 level
(Yamaguchi, 2008). Consistent with this interpretation, the dou-
ble DELLA mutant la cry had similar effects to long1 on GA3ox1
and GA2ox1 expression, but in contrast with long1, had
Figure 4. LONG1 Is a Divergent Ortholog of Arabidopsis HY5.
(A) Phylogenetic analysis of HY5-related genes. Deduced amino acid
sequences were aligned and HY5-homologous regions (amino acids 77
to 151 in AtHY5) were used to construct a neighbor-joining tree, shown
with a root between HY5 and HYH clades. Bootstrap values were
determined from 1000 replications and given above each branch as a
percentage. The alignment is shown in Supplemental Figure 1 online.
(B) Structure of the LONG1 gene and comparison with related Arabi-
dopsis genes HY5 and CESA1. Boxes represent exons, and shaded
regions within boxes represent coding sequence. The site of the long1-1
mutation is indicated.
804 The Plant Cell
increased rather than decreased expression of GA2ox2 (see
Supplemental Figure 3 online).
LONG1 and LIP1 Interact in the Regulation of GA
Biosynthesis during Deetiolation
The clear epistasis of long1 over lip1 in the control of stem
elongation prompted us to examine whether lip1 might also
affect GA1 content. Apical stem portions from 7-d-old dark-
grown lip1 seedlings contained 3- to 4-fold less GA1 than thewild
type (Figure 5E), and this was associatedwith a 4-fold increase in
transcript level of GA2ox2 and a doubling in expression level of
GA2ox1 relative to the wild type (see Supplemental Figure 4
online). Nevertheless, GA1 content in lip1 was still strongly
responsive to light, dropping to a very low level after 4 h, similar
to thewild type. Expression ofGA2ox andGA3ox geneswas also
similarly regulated by light in the wild type and lip1, with a strong
induction of GA2ox2, a moderate induction of GA2ox1, and a
moderate repression of GA3ox1 (see Supplemental Figure 4
online). The effect of 4 h of light on elongation was also
proportionately similar in the wild type and lip1 (50 and 46%
inhibition, respectively) (Figure 5F). Consistent with the epistasis
of long1 over lip1 in the control of elongation (Figures 3 and 5F),
lip1 did not affect GA1 content or GA gene expression in the
long1 genetic background (Figure 5E; see Supplemental Figure 4
online), showing that its effects in both dark- and light-exposed
seedlings are largely dependent on LONG1.
LONG1 Also Regulates GA Production in
Deetiolated Seedlings
The long1 mutant clearly impairs short-term regulation of GA
biosynthesis and inhibition of elongation. However, from the
single time point examined, we could not distinguish whether
Figure 5. LONG1 Regulates Stem Elongation and GA Biosynthesis after Short-Term Transfer to Light.
Two separate experiments were performed in which wild-type and long1 seedlings ([A] to [D]) or wild-type, long1, lip1, and long1 lip1seedlings ([E] and
[F]) were grown in darkness (D) from sowing for 7 d before exposure to continuous white light (100 mmol m�2s�1) for four h (4 h W).
(A) GA levels in expanding stem tissue of wild-type and long1 seedlings.
(B) Final stem length between nodes 1 and 3 of wild-type and long1 seedlings. Measurements were taken at 14 d after sowing. Plants given the light
treatment were returned to darkness for 7d following the treatment.
(C) Levels of indole-3-acetic acid (IAA) in expanding stem tissue of wild-type and long1 seedlings.
(D) Relative transcript levels of GA biosynthesis genes GA3ox1 (LE), GA2ox1 (SLN), and GA2ox2 in expanding stem tissue of wild-type and long1
seedlings.
(E) GA levels in expanding stem tissue of wild-type, long1, lip1, and long1 lip1seedlings.
(F) Final stem length between nodes 1 and 3 of wild-type, long1, lip1, and long1 lip1 seedlings.
Values represent mean6 SE for n = 3 biological replicates each consisting of material pooled from 8 to 10 plants, except (B) and (F), where n = 10 to 16
plants.
HY5/ASTRAY Ortholog Function in Pea 805
long1 mutant blocked or merely delayed the downregulation of
GA1 production. We therefore examined the effect of long1 over
a longer period following light transfer. In wild-type apical stem
segments, GA1 content was again strongly repressed by 4 h after
transfer and maintained at this low level for at least 12 h (Figure
6A). From 24 h after transfer, the GA1 level slowly increased and
by 72 h reached ;20% of the dark control level. By contrast,
downregulation of GA1 production did occur in long1, but this
was slower and much weaker than in the wild type, reaching a
minimum 20% of the dark level 8 h after transfer. After this point,
GA1 levels in long1 rapidly increased, reaching ;2.5 times the
dark level by 48 h, before returning to dark levels by 72 h (Figure
6A). The GA1 content in long1was therefore >20-fold higher than
in the wild type by 4 h, and this difference wasmaintained until at
least 48 h after transfer.
In this time course, GA2ox1, GA2ox2, and GA3ox1 transcripts
showed distinct temporal patterns of accumulation and were all
affected by long1 (Figure 6B). GA2ox2 was the most strongly
light-regulated in wild-type seedlings, reaching a peak of ex-
pression 40-fold higher than the dark level from 4 to 12 h after
transfer, before gradually returning to the dark level by 72 h. In
long1, it reached a maximum only 3-fold higher than the dark
control. The level of GA3ox1 transcript showed a small (50%)
drop by 4 h after transfer before rising to 2-fold higher than the
dark level by 12 h and returning to dark level by 48 h. In long1, the
initial drop was also evident, but the subsequent recovery and
induction was much weaker, such that the expression level did
not exceed the dark control at any point. GA2ox1 expression in
the wild type was induced more slowly than GA2ox2, reaching a
maximum at 12 h and subsequently maintained at a level;8- to
10-fold above dark levels until 72 h. In long1, the initial induction
was retained, but from 12 h onwards, GA2ox1 transcript levels
dropped to near dark levels at 48 h.
Internode length measurements of mature plants showed that
long1 and lip1 continue to function in control of elongation
throughout development and that, as in seedlings, long1 re-
mained largely epistatic to lip1 (see Supplemental Figure 5A
online). We also measured GA1 content in apical shoot tissue of
30-d-old plants, which contained expanding tissue from inter-
nodes 9, 10, and 11. Supplemental Figure 5B online shows that
long1 and lip1 also affect GA1 content in this tissue, with a
significant (P = 0.008) 2-fold increase in long1 and a 75%
decrease in lip1. Once again, long1 overrode the effect of lip1
on GA content, with the double mutant containing 3-fold higher
levels than in the wild type (P = 0.032) (see Supplemental Figure
5A online). However, in contrastwith seedlings, these differences
were not correlated with expression of GA biosynthesis genes
(see Supplemental Figure 5C online).
Interactions between GA and Light Signaling
during Deetiolation
Several recent reports in Arabidopsis have identified interactions
between light- and GA-signaling components. For example,
DELLA proteins were recently shown to bind to and interfere
with the transcriptional activation activity of the PIF3 and PIF4
transcription factors (de Lucas et al., 2008; Feng et al., 2008),
while other studies have reported light effects on DELLA tran-
script levels in seeds (Oh et al., 2006) and deetiolating seedlings
(Lopez-Juez et al., 2008). To examine whether crosstalk at the
transcriptional level between light and GA signaling might occur
in pea, we used long1, lip1, and la cry mutants to examine the
regulatory interactions of the corresponding genes. As shown in
Figure 7A and Supplemental Figure 4 online, neither light expo-
sure, long1, or lip1 had a significant effect on transcript levels of
LA andCRY. LONG1 expression inwild-type seedlings showed a
4-fold induction by 4 h (Figures 7A and 7B), which was sustained
for at least 48 h (Figure 7B), but there was no significant effect of
the lip1 mutation on LONG1 expression in either the dark (P =
0.26) or 4 h after transfer (see Supplemental Figure 4 online).
LONG1 expression was significantly lower in the la cry mutant
after 4 h light (Figure 7B; 20%decrease, P = 0.016), but the small
size of this change suggests that the GA pathway does not
interact substantially with LONG1 function through transcrip-
tional control.
Another recent study has shown that DELLA proteins also
influence elongation by promoting the stability of the HY5 protein
(Alabadı et al., 2008). We reasoned that if the GA signaling
pathway in pea seedlings acts through LONG1, then long1
should affect responsiveness to GA. However, we found no
difference in responsiveness of light-grown wild-type and long1
plants to applied GA3 after depletion of endogenous GA1 by a
saturating dose of the GA biosynthesis inhibitor paclobutrazol
(Figure 7C). There was also no significant effect of the la and cry
mutations on elongation in a long1 background (Figure 7D; P =
0.097). This indicates that loss of LONG1 does not significantly
impair GA signaling in the light and shows that the large differ-
ence in elongation of untreated wild-type and long1 plants is
mainly due to the large difference in GA content rather than
increased activation of GA signaling. We also examined how the
loss of LONG1 function might influence the development of GA-
limited seedlings grown in darkness. Figure 7E shows that wild-
type and long1 seedlings have a near-identical dose–response
relationship for the effect of paclobutrazol on elongation, sug-
gesting that LONG1 also has no clear role inmediating the effects
of DELLA proteins in darkness.
DISCUSSION
There is a growing consensus that the regulation of active GA
levels is an important part of the mechanism by which light
controls stem elongation. Effects of light on levels of bioactive
GA were first reported in lettuce (Lactuca sativa) (Toyomasu
et al., 1992) and have been firmly established for GA1 in pea
(Ait-Ali et al., 1999; Gil and Garcıa-Martinez, 2000; O’Neill et al.,
2000). Similar changes have also been inferred to occur in
Arabidopsis from measurements of GA biosynthesis gene ex-
pression (Achard et al., 2007; Alabadı et al., 2008), and this has
recently been confirmed by direct measurements of GA4 (Zhao
et al., 2007; Symons et al., 2008). In this study, we show that the
pea LONG1 gene is a divergent ortholog of the Arabidopsis bZIP
transcription factor HY5 and is necessary for effects of light on
GA biosynthesis during deetiolation. This result provides a new
insight into the deetiolation mechanism and the interaction
between light and GA signaling.
806 The Plant Cell
LONG1 Functions Similarly to Arabidopsis HY5 in Control
of Photomorphogenesis
Like the divergent HY5 orthologs previously identified in other
legume species, LONG1 is distinguished from HY5 by the
presence of an additional N-terminal domain with close similarity
to the N-terminal RING-type Zn-finger domain of the cellulose
synthase A subunit (Nishimura et al., 2002b; Song et al., 2008).
Despite this structural difference, however, LONG1 and HY5
appear to have largely similar functions in photomorphogenesis.
Similar to HY5, LONG1 is necessary for deetiolation under R, B,
and FR light, it acts downstream of phyA, phyB, and cry1
photoreceptors, and it interacts genetically with LIP1, the pea
ortholog of Arabidosis COP1, throughout development. This
provides strong evidence that HY5 function and its mechanisms
of regulation may be widely conserved across flowering plants,
extending the conclusions of more limited functional analyses of
HY5 orthologs in other species. RNA interference knockdown of
a conventional HY5 ortholog in tomato (Solanum lycopersicum)
resulted in increased hypocotyl elongation and reduced chloro-
phyll content in leaves and pericarp (Liu et al., 2004). Amutant for
the L. japonicus LONG1 ortholog ASTRAY shows increased
hypocotyl elongation and reduced chlorophyll and anthocyanin
content (Nishimura et al., 2002a). Expression of the soybean
ortholog STF1 in transgenic Arabidopsis complemented the hy5
mutant phenotype with respect to these three traits (Song et al.,
2008). In addition, STF1 and HY5 have a similar DNA binding
repertoire (Song et al., 2008). Although more detailed compar-
isons of the legume genes with Arabidopsis HY5 are needed,
there is little evidence so far that the additional N-terminal
domain of the legume HY5-like genes has any additional role.
Although LONG1 is clearly essential for full expression of
photomorphogenic responses, residual light responses in long1
indicate that other factors can mediate partial light responsive-
nesswhen LONG1 is absent. InArabidopsis, bothHYH andSTH2
contribute to the residual light response in the hy5mutant (Holm
et al., 2002; Datta et al., 2007), and it is possible that pea
orthologs may function in a similar manner. The fact that the
residual light response of the long1 mutant is enhanced by the
lip1 mutation does suggest that these LONG1-independent
responses are nevertheless regulated by LIP1 (Figure 8A).
LONG1 and Light Regulation of GA Biosynthesis
Previous studies in pea have identified two phases of GA
regulation during deetiolation: an initial rapid drop in GA1 content
followed by a gradual recovery to dark levels (Ait-Ali et al., 1999;
Gil and Garcıa-Martinez, 2000; O’Neill et al., 2000; Reid et al.,
2002; Symons and Reid, 2003). The results presented here show
that the long1 mutant cannot downregulate GA1 production in
response to light during this initial phase. Also, the fact that long1
seedlings exhibit normal sensitivity to exogenous GA shows that
Figure 6. Derepression of GA Biosynthesis in long1 Persists for Several
Days after Transfer to Light.
Wild-type and long1 seedlings were grown in darkness (D) from sowing
for 7 d before transfer to continuous white light (100 mmol m�2s�1).
Tissue was harvested immediately before transfer to light and at 4, 8, 12,
24, 48, and 72 h after transfer. As an additional control, tissue was also
harvested at the 72 h time point from plants maintained in continuous
darkness. The two dark control points are connected by a dashed line.
Values represent mean 6 SE for n = 3.
(A) Gibberellin A1 levels in expanding stem tissue. Values represent
mean 6 SE for n = 3 biological replicates each consisting of material
pooled from 8 to 10 plants.
(B) Relative transcript levels of GA biosynthesis genes LE (GA3ox1),
SLN (GA2ox1), and GA2ox2 in expanding stem tissue. Values represent
mean 6 SE for n = 3 biological replicates each consisting of material
pooled from three plants.
HY5/ASTRAY Ortholog Function in Pea 807
the increased elongation of the long1 mutant during seedling
growth is largely due to this elevatedGA content. Themain target
of LONG1-dependent light regulation appears to be GA2ox2,
consistent with the conclusions of Reid et al. (2002), although
smaller effects on other genes may also contribute. Induction of
Arabidopsis GA2ox genes by light has also recently been
reported (Achard et al., 2007; Zhao et al., 2007; Alabadı et al.,
2008), and Arabidopsis GA2ox1 was also found to be HY5-
regulated in transcript profiling studies (Sibout et al., 2006; Lee
et al., 2007). These findings suggest that Arabidopsis HY5 may
play a similar role in light regulation of the GA pathway, although
this has yet to be directly tested.
Previous studies in pea have shown that high GA levels are
necessary to maintain the etiolated phenotype of dark-grown
seedlings and that GA acts to promote internode elongation and
to repress leaflet expansion and expression of RbcS genes
(Alabadi et al., 2004). Epistasis of long1 over lip1 in control of
deetiolation (Figure 3) and effects of lip1 on GA1 content (Figure
5) and GA gene expression (see Supplemental Figure 4 online)
show that LIP1 is necessary formaintenance of highGA1 levels in
dark-grown seedlings in a LONG1-dependent manner. This may
partially reflect LIP1 regulation of LONG1 transcription, but these
effects are weak compared with the strong effects on GA2ox2
expression (see Supplemental Figure 4 online). This suggests
that LIP1 repression of LONG1 action most likely occurs through
a different mechanism that, by analogy with Arabidopsis, may
involve LIP1 control of LONG1 protein stability (Osterlund et al.,
2000). With respect to GA1 levels, the effect of lip1 in dark-grown
seedlings was much smaller than the effect of 4 h light on wild-
type seedlings even though the effect on elongation was greater,
suggesting that the lower level of GA1 in dark-grown lip1 seed-
lings may not be sufficient to explain the short internode phe-
notype. A GA-independent effect of LIP1 is also revealed in
light-exposed seedlings, where lip1 is shorter than the wild type
despite both genotypes showing a similar depletion of GA1 to
trace levels (Figure 5). However, the interaction with long1 shows
that this GA-independent effect of lip1 must nevertheless act
through LONG1 (Figure 8A). It is also interesting to note that light
exposure had proportionately similar effects on elongation in
both wild-type and lip1 seedlings but a much smaller effect in
Figure 7. Regulatory Interactions between LONG1 and GA Signaling.
(A) Transcript levels of DELLA genes LA, CRY, and LONG1 in expanding stem tissue relative to those of ACTIN (ACT). Samples analyzed were the same
as used in Figure 6B. Values represent mean 6 SE for n = 3 biological replicates each consisting of material pooled from three plants.
(B) Transcript levels of LONG1 in wild-type and la cry seedlings relative to those of ACT. Seedlings were grown in darkness (D) from sowing for 7 d
before exposure to continuous white light (100 mmol m�2s�1) for 4 h (4 h W). Wild-type samples analyzed were the same as used in Figure 5D. Values
represent mean 6 SE for n = 3 biological replicates each consisting of material pooled from three plants.
(C)Dose–response relationship for the effect of GA3 on internode elongation in light-grown wild-type and long1 seedlings. Varying amounts of GA3 were
supplied together with a saturating dose of the GA biosynthesis inhibitor paclobutrazol (20 mg) in a 5-mL drop of ethanol applied to the exposed
cotyledon surface of a dry seed. Control plants received ethanol only. Values represent mean 6 SE for n = 10 to 16 plants.
(D) Stem length between nodes 1 and 3 for wild-type, long1, la cry double mutant, and long1 la cry triple mutant seedlings grown under standard
glasshouse conditions. Values represent mean 6 SE for n = 6 to 8 plants.
(E) Dose–response relationship for the effect of paclobutrazol on internode elongation in dark-grown wild-type and long1 seedlings. Varying amounts of
paclobutrazol were applied in a 5-mL drop of ethanol to the exposed cotyledon surface of a dry seed. Control plants received ethanol only. Values
represent mean 6 SE for n = 10 to 16 plants.
808 The Plant Cell
the lip1 long1 double mutant, highlighting a potentially LIP1-
independent effect of LONG1 (Figure 8A).
One explanation for a GA-independent regulation of elonga-
tion through LONG1 and LIP1 could involve changes in levels of,
or response to, other hormones, such as auxin. Changes in the
level of auxins themselves are unlikely, as IAA content in
expanding stem tissue from long1 seedlings was not significantly
different from the wild type after 4 h of light exposure (Figure 6C),
and in Arabidopsis, the hy5 mutation has no obvious effect on
auxin content (Cluis et al., 2004). However, hy5mutants do show
several auxin-related phenotypes and the misregulation of nu-
merous auxin-related genes (Oyama et al., 1997; Cluis et al.,
2004; Sibout et al., 2006), suggesting that HY5 may influence
auxin signaling rather than levels. If auxin signalingwas regulated
in a similar manner by the LONG1/LIP1 pathway in pea, wemight
expect auxin-dependent elongation to be increased in long1 in
the light and decreased in lip1 in the dark. This might also help
explain the apparent differences in tissue sensitivity to endog-
enous GAs between light- and dark-grown plants (Reid, 1988).
LONG1 and Homeostatic Control of GA Biosynthesis
The longer deetiolation time course in Figure 6 shows that GA1
levels in long1 mutant are not completely unresponsive to light
but undergo aweak transient downregulation, accompanied by a
residual inhibition of elongation, indicating that other genes act
together with LONG1 to regulate GA levels and elongation. The
time course also showed that LONG1 has persistent effects on
the GA pathway for at least 3 d after transfer and thus acts well
into the recovery phase described above. The molecular basis
for this recovery is not clear but is likely to involve homeostatic
autoregulation of GA production. This has been noted in several
systems (Yamaguchi, 2008) and would tend to increase GA3ox1
expression and reduce GA2ox expression in response to the
initial GA1 depletion. All three of the genes examined here show
feedback control during deetiolation (see Supplemental Figure 3
online; Reid et al., 2002), but in wild-type seedlings only GA3ox1
showed a clear reversal in sign from an initial repression to a
subsequent induction (Reid et al., 2002), suggesting GA auto-
regulation during deetiolation may be mainly achieved through
GA3ox1.
Interestingly, long1 had little effect on the initial light-dependent
repression of GA3ox1, but the subsequent induction was
strongly impaired in the long1 mutant, an effect most simply
interpreted as a consequence of the elevated GA1 content in
long1 feeding back to maintain low GA3ox1 expression. By
contrast, from 24 h onwards, expression of bothGA2ox genes is
lower in long1 than in the wild type despite dramatically elevated
GA1 content, suggesting that LONG1 has a role in maintenance
of GA catabolism throughout the time period examined but also
that without LONG1, feed-forward upregulation of these genes in
response to high GA1 level cannot proceed. This raises the
possibility that LONG1 may be necessary for both light and GA1
effects on GA2ox2 expression. Studies in Arabidopsis may
provide some precedent for this scenario. Arabidopsis HY5
does not contain a transcriptional activation domain (Ang et al.,
1998), and DNA binding by HY5 alone is neither light regulated
nor sufficient to confer light-dependent transcriptional regula-
tion, suggesting that the transcriptional regulation activity of HY5
is likely to depend on other coregulators, such as the putative
transcriptional coregulator STH2 (Datta et al., 2007). It is there-
fore conceivable that the effect on GA production of factors such
as light and GA itself might be integrated at the promoters of GA
metabolism genes through different transcription factors acting
together with HY5/LONG1.
LONG1 and GA Signaling
Recent discussions of light and GA in Arabidopsis have focused
mainly on interaction between signaling pathways. The HY5, PIF,
and GA signaling pathways initially emerged as independent
pathways for light control of elongation but are now believed to
Figure 8. Interactions between Light and GA Pathways.
(A) Physiologically and/or genetically distinct responses to light in pea identified in this study. Operators specify the direction but not the molecular
nature of the interaction.
(B) Model for interactions between light and GA pathways in dark- and light-grown seedlings. This diagram summarizes interactions identified from
studies in Arabidopsis and pea. Genes and operators shown in black are active under the indicated light conditions; those shown in gray are inactive.
The new interaction described in this study is shown as a dashed line.
(C) Model for interactions between light and GA pathways in Arabidopsis seeds. Genes and operators shown in black are active; those shown in gray
are inactive.
Arrows represent activation; lines with flat ends represent inhibition.
HY5/ASTRAY Ortholog Function in Pea 809
interact in several different ways (Figure 8B) (Alabadı et al., 2008;
de Lucas et al., 2008; Feng et al., 2008). One interaction occurs
between the DELLAs and members of the PIF family, PIF3 and
PIF4. PIF3 and PIF4 proteins are abundant in darkness and
necessary for skotomorphogenic development. After transfer to
light, they are destabilized through interaction with PHYB, and
their transcriptional activation activity is blocked through physical
interactions with the DELLA proteins (de Lucas et al., 2008; Feng
et al., 2008). A second interaction involves COP1-dependent
maintenance of low HY5 protein levels in the dark by GA signaling
(Alabadı et al., 2008). In both mechanisms, a GA signaling com-
ponent (DELLA) is proposed to regulate activity of a light-signaling
component (HY5 or PIF). However, it is important to note that both
mechanisms also invoke a primary effect of light on GA content
since the DELLA protein level is presumed to be determined as a
result of prior light regulation of GA levels (Alabadı et al., 2008; de
Lucas et al., 2008; Feng et al., 2008).
The identification of LONG1 as a key gene through which light
regulates GA level thus places it at an earlier step than either of
these mechanisms and stands in contrast with the emphasis on
ArabidopsisHY5asa target rather thana regulator ofGAsignaling.
However, as mentioned above, it does seem likely that HY5 also
participates in regulation of GA levels during deetiolation in
Arabidopsis. On the other hand, the fact that LONG1 acts in light
regulation of GA biosynthesis does not exclude the possibility that
the response to these light-dependent GA changes may occur
through DELLA effects on PIF and LONG1 activity, as proposed in
Arabidopsis. A comparison of transcript profiles in Arabidopsis
hy5,pif3/4, andGApathwaymutantsmay also helpdistinguish the
extent to which light acts through HY5 and PIF-dependent mech-
anisms as opposed to direct DELLA-dependent transcriptional
regulation. Interestingly, if DELLA-dependent stabilization of HY5
and HY5 regulation of GA biosynthesis are found to both occur in
the same species, this would mean that after the initial HY5-
dependent drop in GA level, two opposing DELLA-dependent
mechanisms would potentially act during deetiolation to influence
GA levels; onebeing the feedbackupregulationofGAbiosynthesis
and the other an indirect repression through increased HY5
stability (Figure 8B).
An Expanding Molecular Framework for
Light–GA Interactions
The role for LONG1 inGAbiosynthesis not only addsanother link in
the network of interactions controlling stem elongation, it also
provides a interesting contrast to the mechanism through which
light regulates theGApathway in germinating seeds (Figure 8C). In
Arabidopsis seeds, light acting through phyA or phyB induces de
novoGA biosynthesis through activation ofGA3ox and repression
ofGA2ox genes (Oh et al., 2006). ThePIF family genePIL5 (PIF1) is
a phyB-dependent negative regulator of germination that plays a
central role in this mediating these effects. PIL5 was also found to
bind the promoters and activate transcription of the DELLA genes
GAI andRGA, providing an alternative means bywhich light could
derepress DELLA-dependent inhibition of germination. In contrast
with this direct regulation of DELLA genes, PIL5 regulation of GA
metabolismgenes isapparently not direct becausebindingofPIL5
to promoters of these genes was not detected in specific chro-
matin immunoprecipitation analyses (Oh et al., 2007), and the
existence of an unknown intermediate has been proposed. A
possible role for HY5 in this system has not been tested, but may
warrant examination, given the positive regulation of germination
by light through HY5 and the abscisic acid signaling component
ABI5 (Chen et al., 2008). Moreover, light regulation of GA levels in
seeds may depend in part on prior light regulation of abscisic acid
synthesis (Seo et al., 2006), and under some conditions light
clearly regulates GA levels in a PIL5-independent manner (Oh
et al., 2007). These observations raise the possibility that HY5
could also contribute to photocontrol of GA biosynthesis in seeds.
METHODS
Plant Material, Mutagenesis, Measurements, and
Growth Conditions
The pea (Pisum sativum) mutant lines long1-1, phyA-1, and phyB-5 were
derived from ethyl methanesulfonate mutagenesis of cv Torsdag [WT
(TOR)] as previously described (Weller et al., 1997). Unless otherwise
specified, plants were grown in growth cabinets at 208C or in the
glasshouse using previously described growth media light sources and
conditions (Hecht et al., 2007). The la cry-s double mutant used was
derived from the second backcross of Hobart line 197 (LE la-1 cry-s) into
cv Torsdag. The original lip1 mutant (Frances et al., 1992) was back-
crossed three times into the cultivar Torsdag background before use. The
SLN (L309+) and sln near-isolines were derived by single plant selection
from a cross between line NGB6074 (Reid et al., 1992) and cv Torsdag.
Unless otherwise indicated, internode length was measured as the
distance between nodes 1 and 3 in 2-week-old seedlings, and leaflet
area was estimated as the product of the length and width of a single
leaflet from the first true foliage leaf (leaf 3). Chlorophyll content was
determined as previously described (Hiscox and Israelstam, 1979).
Genetic Analysis of long1 and Phylogenetic Analysis of LONG1
Allelism tests were performed by crossing the long1 mutant to other
elongated mutants phyB and sln and observing wild-type seedling
elongation phenotypes in the F1 and F2 generations. The la monogenic
mutation has no phenotype in an otherwise wild-type background, and
potential allelismof long1 and lawas therefore assessed in the F2, F3, and
F4 progeny of a cross between long1 and the la crydoublemutant. Briefly,
F3 families segregating for all three mutations were genotyped for la and
crymutations using molecular markers (Weston et al., 2008), and recom-
binants were identified as elongated plants that that were heterozygous
for the la mutation (implying a long1 long1 LA la genotype and the
contribution of a recombinant long1 la gamete) or as plants with a wild-
type phenotype that were homozygous LA LA (implying the presence of at
least one wild-type LONG1 allele and recombinant LONG1 LA gamete).
The genetic distance between LONG1 and LA was estimated from this
segregation data using JOINMAP software (vanOoijen, 2006). Amino acid
sequences of proteins related to LONG1 were aligned using ClustalX
(Thompson et al., 1997). Distance and parsimony-based methods were
used for phylogenetic analyses in PAUP*4.0b10 (http://paup.csit.fsu.
edu/) using the alignment shown in Supplemental Figure 1 online.
Sequence Isolation, Mapping, and Molecular Markers
Partial sequence of a pea gene homologous to Lotus japonicus ASTRAY
was isolated by PCR using genomic DNA and cDNA from 2-week-old
tissue with degenerate primers designed from legume sequences (L.
japonicusAB092677, Vicia faba X97904,Medicago truncatula TC103975,
810 The Plant Cell
andGlycinemax L28003) by the CODEHOP strategy (Rose et al., 1998) as
described previously (Hecht et al., 2005). A full-length cDNA sequence for
LONG1was obtained by 59 and 39 rapid amplification of cDNA ends PCR
performed using the BD SMART RACE cDNA amplification kit (BD
Bioscience Clontech). PCR fragments were cloned in pGEM-T (Promega)
and sequenced at the Australian Genome Research Facility (Brisbane,
Australia). A single nucleotide polymorphism in intron 4 of LONG1
between lines JI281 and JI399 was converted to a cleaved amplified
polymorphic sequence (CAPS) marker (HaeII site in JI281) and used to
map the gene in a JI2813 JI399 recombinant inbred line population (Hall
et al., 1997). The long1-1 point mutation was converted into a derived
CAPS marker, introducing a diagnostic NcoI site. Molecular markers for
detection of the phyA-1, phyB-5, la-1, and cry-s mutations have been
described previously (Platten et al., 2005; Weston et al., 2008). Details of
all primers are provided in Supplemental Table 1 online.
Hormone Quantification and Application
IAA and GAs were extracted and quantified as described by Jones et al.
(2005) and Jager et al. (2005), respectively, with the following changes.
Samples were methylated in a 1:7.5 mixture of methanol and 0.2 M
trimethylsilyldiazomethane (Sigma-Aldrich) in diethyl ether at room tem-
perature for 30 min and then (after drying) either partitioned against
diethyl ether as before (Jones et al., 2005) or transferred in diethyl ether
(200mL) to a clean vial prior to trimethylsilylation. For gas chromatography–
selected ion monitoring analysis of GA1, a BPX608 column (SGE) was
used (25 m 3 0.32 mm i.d. 3 0.4 um), which resolved GA1 from two GA1
isomers present in pea extracts (GA29 and GA81), eliminating the need for
HPLC purification of GAs. The GC oven temperature was ramped from 60
to 2508Cat 308C·min21 and then at 108C·min21; the pressure was 15 p.s.i.
The other GAswere chromatographed on a Hewlett Packard HP1 column
as before (Jager et al., 2005). GA3 and paclobutrazol (Duchefa Biochemie)
were applied in a 5-mL drop of ethanol to the exposed cotyledon of a dry
seed.
Gene Expression Studies
Harvested tissue consisted of 20 mm of young stem immediately below
the apical bud. Samples were immediately frozen in liquid nitrogen and
total RNA extracted using the Promega SV total RNA isolation system
(Promega) with an on-column DNase treatment. RNA concentrations
were determined using Ribogreen RNA quantification reagent (Molecular
Probes) in a Picofluor fluorometer (Turner Biosystems). Reverse tran-
scription was performed in 20 mL with 1 mg of total RNA using the
ImPromII reverse transcriptase (Promega) according to the manufac-
turer’s instructions. RT-negative (no enzyme) control was performed for
each sample to monitor for genomic DNA contamination. Real-time PCR
were performed as described previously (Hecht et al., 2007). Details of
primers are presented in Supplemental Table 1 online. Transcript levels
were normalized to an ACTIN reference gene using nonequal efficiencies
(Pfaffl, 2001). All data shown represent the mean 6 SE of three biological
replicates, with each consisting of pooled material from three plants.
Accession Numbers
Genomic and cDNA sequences are deposited in GenBank under the
provisional accession numbers bankit 1141280 (LONG1 genomic) and
bankit 1141295 (LONG1 cDNA). GenBank accession numbers for other
sequences used are V. faba BZIPZF (CAA66478), L. japonicus BZF/
ASTRAY (BAC20318), G. max STF1 (AAC05017) and bZIP69 (ABI34671),
Populus trichocarpa HY5a (fgenesh4_pm.C_LG_XVIII000127 protein
809109), HY5b (estExt_Genewise1_v1.C_LG_VI0997 protein 717128)
and HYH (grail3.0102003601 protein 657788), Arabidopsis thaliana HY5
(NP_568246, At5g11260) and HYH (NP_850604, At3g17609), M. trunca-
tula HYH (ABE88841),Arabidopsis CESA1 (NM_119393, At4g32410), and
P. sativum ACTIN (X68649).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Sequence Alignment of LONG1 and Related
Proteins.
Supplemental Figure 2. Light-Dependent Effects of long1 in Roots.
Supplemental Figure 3. Effect of the Constitutive Activation of GA
Signaling on Expression of GA Metabolism Genes.
Supplemental Figure 4. Interaction of LONG1 and LIP1 in Regulation
of GA Metabolism and Signaling Genes in Deetiolating Seedlings.
Supplemental Figure 5. Interactions of LONG1 and LIP1 in the
Control of Internode Length, GA Levels, and the Expression of GA
Metabolism Genes in Mature Plants.
Supplemental Table 1. Primers.
ACKNOWLEDGMENTS
We thank Ian Cummings for all-around glasshouse support and assis-
tance with controlled environments, Tracey Winterbottom, Chris Black-
man, and Scott Taylor for plant maintenance, Rob Wiltshire for help with
photography, and Greg Symons for advice on tissue harvests. We also
thank undergraduate students Emmeline Tan and Albert Wong for
assistance with gene isolation, genotyping, and plant measurements.
This work was supported by the Australian Research Council through
Discovery Project Grant DP0770478
ReceivedOctober 9, 2008; revised February 19, 2009; acceptedMarch 9,
2009; published March 27, 2009.
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HY5/ASTRAY Ortholog Function in Pea 813
DOI 10.1105/tpc.108.063628; originally published online March 27, 2009; 2009;21;800-813Plant Cell
James L. Weller, Valérie Hecht, Jacqueline K. Vander Schoor, Sandra E. Davidson and John J. RossLight Regulation of Gibberellin Biosynthesis in Pea Is Mediated through the COP1/HY5 Pathway
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Supplemental Data /content/suppl/2009/03/12/tpc.108.063628.DC1.html
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