Interactions between Auxin and Strigolactone in Shoot

Branching Control1[C][OA]
Alice Hayward, Petra Stirnberg, Christine Beveridge2*, and Ottoline Leyser2
University of Queensland, School of Biological Sciences, Australian Research Council Centre of Excellence for
Integrative Legume Research, Queensland 4072, Australia (A.H., C.B.); and Department of Biology, University
of York, York YO10 5YW, United Kingdom (A.H., P.S., O.L.)

In Arabidopsis (Arabidopsis thaliana), the carotenoid cleavage dioxygenases MORE AXILLARY GROWTH3 (MAX3) and MAX4
act together with MAX1 to produce a strigolactone signaling molecule required for the inhibition of axillary bud outgrowth.
We show that both MAX3 and MAX4 transcripts are positively auxin regulated in a manner similar to the orthologous genes
from pea (Pisum sativum) and rice (Oryza sativa), supporting evolutionary conservation of this regulation in plants. This
regulation is important for branching control because large auxin-related reductions in these transcripts are associated with
increased axillary branching. Both transcripts are up-regulated in max mutants, and consistent with max mutants having
increased auxin in the polar auxin transport stream, this feedback regulation involves auxin signaling. We suggest that both
auxin and strigolactone have the capacity to modulate each other’s levels and distribution in a dynamic feedback loop required
for the coordinated control of axillary branching.

Shoot branching is dependent on both the formation
of axillary meristems in the axils of leaves and the
precise control of their outgrowth by genetically, hor-
monally, and environmentally regulated signals (for
review, see Schmitz and Theres, 2005; Dun et al., 2006;
Ongaro and Leyser, 2007). Mutations in the Arabidop-
sis (Arabidopsis thaliana) MORE AXILLARY GROWTH
(MAX) genes and orthologous genes in garden pea
(Pisum sativum), rice (Oryza sativa), and petunia (Petu-
nia hybrida) all lead to increased branching (Beveridge,
2000; Stirnberg et al., 2002; Sorefan et al., 2003; Ishikawa
et al., 2005; Snowden et al., 2005; Zou et al., 2005, 2006;
Johnson et al., 2006; Arite et al., 2007; Simons et al.,
2007). Two of these genes, MAX3 (AtCCD7) and MAX4
(AtCCD8), encode plastid-targeted carotenoid cleav-
age dioxygenases (CCDs) that together with the cy-
tochrome P450 family member, MAX1, control the
production of an upwardly mobile signal that inhibits
1
This work was supported by the Australian Research Council
Centre of Excellence for Integrative Legume Research, by the Bio-
technology and Biological Sciences Research Council, and by an
Australian Postgraduate Award, a Travelling Fellowship from the
Company of Biologists (Development), and a University of Queens-
land Graduate School Research Travel Award to A.H.
2
These authors contributed equally to the article.
* Corresponding author; e-mail c.beveridge@uq.edu.au.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Christine Beveridge (c.beveridge@uq.edu.au).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[OA]
Open Access articles can be viewed online without a sub-
scription.
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axillary bud outgrowth (Turnbull et al., 2002; Sorefan
et al., 2003; Booker et al., 2004, 2005; Auldridge et al.,
2006).
This mobile signal appears to be a strigolactone or a
derivative (Gomez-Roldan et al., 2008; Umehara et al.,
2008). Strigolactones are carotenoid-derived terpenoid
lactones previously shown to stimulate mycorrhizal
hyphal branching and seed germination of the plant
parasitic weeds Striga and Orobanche (Cook et al., 1972;
Akiyama et al., 2005; Humphrey and Beale, 2006). ccd7
and ccd8 mutants of rice and pea are deficient in
strigolactones, and the strigolactone analog, GR24
(Johnson et al., 1981), can restore branching inhibition
in these mutants as well as in max1, max3, and max4
(Gomez-Roldan et al., 2008; Umehara et al., 2008). In
the shoot, strigolactone signal transduction probably
involves the F-box Leu-rich repeat protein, MAX2
(Stirnberg et al., 2002, 2007; Booker et al., 2005). Ac-
cordingly, branching in max2 plants is not repressed by
GR24. F-box proteins function in Skp1-Cul1/Cdc53-
F-box (SCF) E3 ubiquitin ligase complexes, which
target proteins for ubiquitination, often triggering
subsequent degradation by the 26S proteosome (for
review, see Petroski and Deshaies, 2005).
A second hormone central to branching control is
auxin, which was first shown to inhibit branching over
seven decades ago (Thimann and Skoog, 1934). Auxin
also signals via SCF-mediated targeted protein degra-
dation. The F-box protein, TRANSPORT INHIBITOR
RESPONSE1 (TIR1), and closely related family mem-
bers (AFBs) act as auxin receptors (Dharmasiri et al.,
2005a, 2005b; Kepinski and Leyser, 2005). Auxin bind-
ing stabilizes the interaction between TIR1/AFBs and
members of the Aux/IAA family of transcriptional
repressors (Tan et al., 2007). This induces Aux/IAA

degradation, allowing auxin-mediated up-regulation
of transcription (Gray et al., 2001; Tiwari et al., 2001;
Zenser et al., 2001; Dharmasiri and Estelle, 2002). As a
homeostatic mechanism, many Aux/IAA genes are
rapidly transcriptionally induced by auxin in an
SCFTIR1/AFB-dependent manner (Park et al., 2002). An-
other protein, AUXIN RESISTANT1 (AXR1), is neces-
sary for proper SCF function by facilitating the
conjugation of the ubiquitin-like protein, RUB1, to
the Cullin subunit (Leyser et al., 1993; Wu et al., 2000;
Schwechheimer et al., 2001; del Pozo et al., 2002).
Mutations in AXR1 cause changes in SCFTIR1/AFB-
dependent auxin-responsive gene expression and sub-
sequent defects in downstream auxin responses. AXR1
also appears to regulate SCF signaling in photomor-
phogenesis and jasmonate responses (Schwechheimer
et al., 2002; Tiryaki and Staswick, 2002). Phenotypes
conferred by axr1 include increased branching, agravi-
tropic root growth, fewer lateral roots and root hairs,
and reduced fertility due to poor stamen elongation
(Lincoln et al., 1990).
Buds of strigolactone pathway mutants are resistant
to inhibition by apically applied auxin, suggesting that
strigolactones are required for this inhibition (Bever-
idge, 2000; Sorefan et al., 2003; Bennett et al., 2006).
Additionally, GR24 can inhibit branching in auxin-
depleted decapitated pea plants and in auxin signaling
mutants, including axr1 and the tir1afb1afb2afb3 qua-
druple mutant (Brewer et al., 2009). In pea, the genes
orthologous to MAX3 and MAX4, RAMOSUS5
(RMS5) and RMS1, are highly auxin regulated, with
transcripts greatly diminished or slightly increased by
treatments shown to deplete or enhance auxin, respec-
tively (Foo et al., 2005; Johnson et al., 2006). Auxin
regulation was also shown for the rice orthologs,
HIGH TILLERING DWARF1 and DWARF10 (D10;
Zou et al., 2006; Arite et al., 2007). These data suggest
that the auxin regulation of strigolactone synthesis
genes could represent a conserved and important
mode of auxin action in branching control. In Arabi-
dopsis, the response of MAX3 and MAX4 transcripts
to auxin depletion has not been assessed in detail. A
MAX4 promoter:GUS fusion was responsive to auxin
treatments, particularly in the root elongation zone
and in hypocotyls, and this was dependent on AXR1
(Bainbridge et al., 2005). In contrast, up-regulation of
GUS by auxin was not detected in shoot tissues.
In strigolactone pathway mutants of pea, RMS1 and
RMS5 transcription is feedback up-regulated (Foo
et al., 2005; Johnson et al., 2006). This was also shown
for the MAX4 ortholog in rice and petunia (Snowden
et al., 2005; Arite et al., 2007; Simons et al., 2007). A
comparatively minor increase in MAX4 transcription
has been reported in Arabidopsis max2 hypocotyls
(Bainbridge et al., 2005). In pea, grafting experiments
indicate that feedback up-regulation of RMS1 and
RMS5 occurs both locally and systemically (Foo et al.,
2005; Johnson et al., 2006; for review, see Dun et al.,
2006). Local feedback regulation could be mediated by
a direct effect of strigolactone signaling on RMS gene
Plant Physiol. Vol. 151, 2009
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Auxin and Strigolactone Interactions in Branching
expression in a manner typical of many hormone
biosynthetic pathways (Wang et al., 2002; Yamaguchi,
2008). However, systemic feedback regulation requires
a signal in addition to strigolactone, since strigolac-
tones move upward while systemic feedback signaling
has been shown to move down the plant (Foo et al.,
2005; Johnson et al., 2006). In pea and Arabidopsis,
feedback signaling from the shoot also down-regulates
the levels of the branching promoter, cytokinin, in the
root xylem sap (Foo et al., 2007).
In Arabidopsis, mutations in the MAX genes result
in increased stem conductivity for auxin and increased
expression of several auxin transporters, including
some of the PIN family of auxin exporters (Bennett
et al., 2006; Lazar and Goodman, 2006; Brewer et al.,
2009). The stems of max mutants have increased ex-
pression from the synthetic auxin-responsive pro-
moter DR5, suggestive of increased auxin content.
Similarly, increased auxin levels have been observed in
strigolactone mutants of rice (Arite et al., 2007) and
sometimes, but not always, in pea (for review, see Dun
et al., 2006). Therefore, high auxin content in the stems
of max mutants is a good candidate for mediating
feedback regulation of the MAX pathway. Low strigo-
lactone would lead to increased auxin content, which
in turn would up-regulate strigolactone synthesis. In
pea, both auxin and another long-distance feedback
signal are suggested to be involved in feedback (for
review, see Dun et al., 2006). Therefore, auxin and
strigolactone signaling could interact in interlocking
feedback loops, with each hormone having the poten-
tial to regulate the levels of the other. Here, we
describe the results of experiments aimed at further
investigating the auxin-strigolactone interaction in
Arabidopsis and exploring its functional significance.
RESULTS
MAX3 and MAX4 Expression in the Shoot Is
Up-Regulated by Auxin in an AXR1-Dependent Manner
Auxin regulation of RMS1/MAX4 and RMS5/MAX3
in stems has been detected in pea but not in Arabi-
dopsis (Bainbridge et al., 2005; Foo et al., 2005; Johnson
et al., 2006). To determine if auxin up-regulation of
MAX3 and MAX4 can be detected in the shoot using
the more sensitive and quantitative quantitative real-
time PCR (qRT-PCR), and whether this effect is AXR1
dependent, a dose response of MAX3 and MAX4
expression to the natural auxin indole-3-acetic acid
(IAA) was undertaken in the basal cauline internode of
intact axr1-3 and wild-type plants. The INDOLE-
3-ACETIC ACID INDUCIBLE1 (IAA1) gene known to
be up-regulated by auxin via the AXR1/TIR1 pathway
(Park et al., 2002; Yang et al., 2004) was analyzed as a
control.
In pea, 3 and 19 mM IAA applied in lanolin to
decapitated plants can substantially enhance total
stem auxin content and slightly but significantly up-
401
Hayward et al.
regulate RMS1 and RMS5 expression (Foo et al., 2005;
Johnson et al., 2006). When tested here, 1 and 19 mM
IAA, but not 100 mM or below, significantly increased
MAX3 and MAX4 transcript levels in the basal cauline
internode of intact plants by at least 10-fold at 3 h (t test
P , 0.01; Fig. 1). These genes behaved similarly to
IAA1, although IAA1 was slightly more responsive to
IAA addition. For all genes, the response to IAA was
clearly diminished in the axr1-3 mutant (P , 0.01–
0.05). In untreated plants, expression levels were
slightly reduced in axr1-3 relative to the wild type
for MAX4 (P , 0.05) and IAA1 (P , 0.01), while this
was not significant for MAX3. Together, these data
suggest that, like IAA1, MAX3 and MAX4 transcripts
are induced by auxin in internode tissue in an AXR1-
dependent manner. As is typical with auxin responses
in axr1 mutants, the auxin dose-response curve is
shifted such that high auxin doses successfully induce
a response similar to that observed with lower doses in
the wild type.
MAX3 and MAX4 Expression Is Reduced by Auxin
Depletion Treatments
In pea, the largest changes in RMS5 and RMS1
expression occur following treatments that cause
auxin depletion (Foo et al., 2005; Johnson et al.,
2006). Thus, to investigate this for MAX3 and MAX4,
transcript levels were quantified following treatments
predicted to deplete auxin in a number of tissues.
Figure 1. Auxin dose response of MAX3, MAX4, and IAA1 expression
in wild-type (WT) and axr1-3 basal cauline internodes. IAA was applied
in a lanolin ring around the primary bolt of 5-week-old plants 15 mm
above the base, and the cauline tissue below the application site was
collected 3 h post application. Expression is shown relative to wild-type
controls for each gene; data are means of two biological pools of six
plants 6 SE. Asterisks indicate significant differences between axr1 and
the wild type.
402
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Young expanding leaves at the shoot apex are im-
portant sources of auxin (Thimann and Skoog, 1934;
Ljung et al., 2001). In 5-week-old wild-type plants, re-
moving these sources by decapitation, or applying the
auxin polar transport inhibitor 1-N-naphthylphthalamic
acid (NPA) in lanolin, significantly depleted MAX3
transcripts in tissues below (Fig. 2A; all P , 0.05).
Specifically, these treatments reduced MAX3 transcripts
in the primary bolting stem by 200- to 400-fold after
24 h. In hypocotyls and roots, the greatest reductions
in MAX3 transcript occurred when plants were de-
capitated, including removal of young rosette leaves,
or when NPA was applied below these leaves around
the top of the hypocotyls (all P , 0.05). Qualitatively
similar, but generally quantitatively smaller, changes
were seen for MAX4 transcripts (data not shown).
The reductions in MAX3 and MAX4 transcript levels
in hypocotyls following NPA treatment and decapita-
tion were generally not as great in max mutants as in
wild-type plants (Fig. 2, B and C). This was particu-
larly obvious for max2, the most severe max mutant,
where there was no significant difference in transcript
levels between intact and decapitated plants (P $ 0.1).
A similar effect was seen for IAA1, which while
relatively less responsive to decapitation in general
shows a significant reduction in transcript levels (P ,
0.05) except in max1 and max2 (Fig. 2D), possibly
reflecting the increased auxin content/signaling prop-
erties of these mutants (Bennett et al., 2006). In decap-
itated plants of all genotypes, transcript loss was
prevented by the application of 19 mM IAA to the
decapitation site.
Auxin-Related Down-Regulation of MAX3 and MAX4
Expression Can Activate Branching
Grafting studies revealed that wild-type roots can
restore the branching in max3 and max4 shoots to wild-
type levels while max3 roots cannot rescue max4 shoots
and visa versa (Turnbull et al., 2002; Sorefan et al.,
2003; Booker et al., 2005). This indicates that MAX3
and MAX4 are required in the same tissue for strigo-
lactone production and that their product can move
from the root to the shoot to inhibit branching. Previ-
ously, roots of the axr1-3 mutant were also shown to
restore the branching in max4 shoots to wild-type
levels, suggesting that the axr1-3 roots of these grafts
are not strigolactone deficient (Sorefan et al., 2003;
Bainbridge et al., 2005). These and other data (Bennett
et al., 2006) suggest a MAX-independent role for auxin
signaling in branching suppression, but they do not
rule out a role for AXR1/TIR1-mediated regulation of
MAX3 and MAX4 transcription in modulating branch-
ing levels. To investigate this possibility, we turned to
the bodenlos-2 (bdl-2) mutant. This is a semidominant
auxin response mutant that confers defective primary
root formation in the embryo and greatly increased
shoot branching in the adult plant, particularly obvi-
ous in heterozygous plants (Fig. 3). The bdl-2 allele was
isolated from an ethyl methanesulfonate-mutagenized
Plant Physiol. Vol. 151, 2009

population in the ecotype Columbia (Col-0) back-
ground (Stirnberg et al., 2002). It has a Pro-74-to-Ser
amino acid substitution in domain II of the Aux/IAA
transcriptional repressor IAA12 identical to that de-
scribed for bdl-1, which is in the Landsberg erecta
genetic background (Hamann et al., 1999, 2002). This
substitution results in auxin-resistant stabilization
of the IAA12/BDL protein and thus likely consti-
tutive repression of target auxin-up-regulated genes
(Hamann et al., 2002; Dharmasiri et al., 2005b). The
bdl-2 mutation caused increased bud outgrowth from
the rosette in both homozygotes and heterozygotes.
Shoot height was far more reduced in homozygotes,
which were severely dwarfed, than in heterozy-
gotes, which with moderately shorter stature and in-
creased branching somewhat resembled max mutants
(Fig. 3B).
Transcript abundance of MAX3 and MAX4 was
reduced by approximately 3 orders of magnitude in
bdl-2 homozygotes and also to a lesser extent in
heterozygotes (Fig. 3D). IAA1 expression was equal
to, or higher than, wild-type expression in bdl-2,
suggesting distinct Aux/IAA regulation of these
genes. To examine this hypothesis further, we searched
for coexpression of IAA1, IAA12, MAX3, and MAX4
using the Genevestigator V3 microarray database
(Zimmermann et al., 2004; Hruz et al., 2008). IAA12
and IAA1 were both corepresented with MAX3 and
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Auxin and Strigolactone Interactions in Branching
Figure 2. A, MAX3 expression in re-
sponse to auxin-depleting treatments
in various tissues of 5-week-old wild-
type (WT) plants relative to intact hy-
pocotyls. Plants were intact, treated
with 3 mg mL21 NPA in a lanolin ring
around the primary bolt 15 mm above
the base (NPA-treated bolt) or around
the top of the hypocotyl (NPA-treated
hypocotyl), or decapitated (Decap) 15
mm above the base of the primary
inflorescence with or without young
rosette leaves removed. “Bolt” refers
to the basal 1 cm of primary cauline
stem below the treatment site. B to
D, MAX3 (B), MAX4 (C), and IAA1
(D) expression in response to auxin-
depleting treatments in the hypocotyls
of 3-week-old wild-type and max mu-
tant plants. Plants were intact, treated
with 3 mg mL21 NPA in a lanolin ring
around the top of the hypocotyl, or
decapitated by removing the entire
primary bolt and youngest leaves with
or without 19 mM IAA applied to the
decapitation site. Tissues were col-
lected 24 h after treatments. Data are
means of two to three biological pools
of seven to 25 plants 6 SE. Asterisks
indicate significant differences from
intact plants per genotype/tissue.
MAX4 in the hypocotyls and inflorescence stems (data
not shown); however, IAA12, MAX3, and MAX4 all
showed highest differential expression in the hypo-
cotyl xylem, while IAA1 was expressed most highly in
callus tissue and the lateral root cap.
To investigate whether low MAX3 and MAX4 ex-
pression in bdl-2 mutants correlates with insufficient
graft-transmissible branching suppression, as ex-
pected if strigolactone levels have been affected, we
carried out grafting between bdl-2 heterozygotes,
max4-1, and the wild type. If bdl-2 mutants are strigo-
lactone deficient, two results were expected. First, bdl-2
mutant roots should not rescue branching in max4-1
shoots; second, branching in bdl-2 shoots should
be rescued by wild-type but not max4-1 rootstocks.
Self-grafted bdl-2 heterozygous mutants produced sig-
nificantly more rosette branches than self-grafted
max4-1 mutants (P , 0.01; Fig. 3E), and both mutants
branched more than self-grafted wild-type plants
(P , 0.001). As expected, branching in max4-1 scions
was completely restored to wild-type levels by graft-
ing to wild-type rootstocks (Sorefan et al., 2003; Fig. 3,
E and F). However, bdl-2 heterozygote rootstocks did
not restore the branching in max4-1 shoots, suggesting
that BDL affects root strigolactone production. Fur-
thermore, wild-type rootstocks inhibited branching in
heterozygote bdl-2 scions by over 50% relative to bdl-2
heterozygote self grafts (P , 0.001) and compared with
403
Hayward et al.

Figure 3. Rosette branching and gene

expression in bdl mutants. A to C,
Representative rosette phenotypes of
5-week-old wild-type plants (WT; A),
bdl-2 heterozygotes (B), and bdl-2 ho-
mozygotes (C). Blue arrows indicate
the primary inflorescence stem; white
arrows indicate example axillary ro-
sette branches/buds. D, Relative ex-
pression levels of MAX3, MAX4, and
IAA1 in the basal cauline internodes of
5-week-old bdl-2 homozygotes and
heterozygotes. Data are means of two
biological pools of eight to 10 plants 6
SE; asterisks indicate significant differ-
ences from the wild type. E, Number of
rosette branches 5 mm or longer in
reciprocally grafted wild-type plants,
max4-1 mutants, and bdl-2 heterozy-
gotes 6 SE; n = 4 to 10. F, Photographs
of representative max4-1 grafted plants
(scion/rootstock). G, Number of rosette
branches 5 mm or longer per rosette
leaf in wild-type plants, max2-1 and
max4-1 mutants, and bdl-2 heterozy-
gotes with or without GR24; n = 13 to
21. Data are means 6 SE; asterisks in-
dicate significant inhibition of branch-
ing by GR24. [See online article for
color version of this figure.]

only 20% inhibition by max4-1 rootstocks (P , 0.05).
These data support the hypothesis that increased
branching in bdl-2 mutants is partly due to insufficient
strigolactones but also that there is a strigolactone-
independent component to the increased branching
observed in bdl-2 heterozygous plants.
We also determined the ability of exogenous strigo-
lactone to inhibit branching in bdl-2. The synthetic
strigolactone analog, GR24, was applied to rosette
axils every 2 to 3 d as described by Gomez-Roldan
et al. (2008). This reduced rosette branching in max4-1
and bdl-2 heterozygotes by a similar degree (39% and
34%, respectively; P , 0.001; Fig. 3G), but branching
remained increased relative to the wild type for both
genotypes, particularly in bdl-2. Branching was un-
changed in both the wild type and, as expected for a
putative strigolactone signal transduction mutant, in
max2-1 (Booker et al., 2005; Stirnberg et al., 2007;
Gomez-Roldan et al., 2008; Umehara et al., 2008).
404
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Auxin Signaling Contributes to Feedback Up-Regulation
of MAX3 and MAX4 Expression in max Mutants
Sixfold to 10-fold increases in MAX3 and MAX4
expression were observed in hypocotyls of all max
mutants relative to the wild type, indicative of feed-
back up-regulation (Fig. 2, B and C). Given that auxin
levels and/or signaling are increased in max mutant
stems (Bennett et al., 2006), MAX3 and MAX4 tran-
scription is auxin regulated in an AXR1-dependent
manner (Bainbridge et al., 2005; this study), and auxin
is downwardly mobile, it was hypothesized that this
feedback up-regulation may be in part mediated by
auxin. To investigate this possibility, we determined
the role of AXR1 in feedback signaling using the axr1-
3max2-1 double mutant (Bennett et al., 2006).
In the basal cauline internodes of 5-week-old plants,
MAX3, MAX4, and IAA1 transcripts accumulated to
significantly higher levels in max2-1 mutants than in
Plant Physiol. Vol. 151, 2009

wild-type plants (P , 0.01; Fig. 4A). In the axr1-3max2-1
background, the expression of all genes in this tissue
was reduced to levels not statistically significantly
different from the wild type or the single axr1-3 mu-
tant. Thus, axr1 is required for feedback up-regulation
in the shoot. In the hypocotyls of the same plants, the
up-regulation of MAX3 and MAX4 in max2-1 was less
than in cauline tissue (significant at P , 0.05; Fig. 4B),
and this was not abolished in the axr1-3 background.
This might indicate that the observed elevated expres-
Figure 4. Expression of MAX3, MAX4, and IAA1 in wild-type (WT),
axr1-3, max2-1, and axr1-3max2-1 plants. A and B, Expression in the
basal cauline internodes (A) and hypocotyls (B) of 5-week-old plants. C,
Expression in the hypocotyls of vegetative 2-week-old plants; samples
marked with “N” were treated with 3 mg mL21 NPA around the top of
the hypocotyls (note log scale). Data are means of two biological pools
of five to 10 plants 6 SE. Asterisks indicate a significant effect of axr1 on
feedback in max2.
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Auxin and Strigolactone Interactions in Branching
sion in max2-1 hypocotyls is not related to auxin;
however, like MAX3 and MAX4, IAA1 and additional
auxin-responsive Aux/IAA genes (IAA5, IAA19, and
IAA29; data not shown) showed increased expression
in max2-1 that was not significantly affected by the
axr1-3 mutation.
In the hypocotyls of vegetative 2-week-old max2-1
plants, MAX3, MAX4, and IAA1 transcripts were again
slightly but significantly up-regulated (P , 0.05; Fig.
4C). At this developmental stage, the axr1-3 back-
ground prevented the up-regulation of IAA1, but not
MAX3 or MAX4, in max2-1 hypocotyls. This could
suggest auxin-independent feedback specific to MAX3
and MAX4. However, we cannot rule out an auxin-
dependent relationship, because NPA treatment of
these plants abolished the up-regulation of MAX3
and MAX4 in axr1-3max2-1 relative to axr1-3 (Fig. 4D),
and our results with bdl-2 above suggest that differ-
ences in the auxin responsiveness of these genes could
be due to different Aux/IAAs involved in their regu-
lation.
Graft Transmissibility of Feedback Signals
To test if feedback regulation of MAX3 and MAX4
expression can occur over long distances, as shown in
pea (Foo et al., 2005; Johnson et al., 2006), we recipro-
cally grafted max2-1 and wild-type plants.
Feedback in the Shoot
As shown for pea, a local feedback effect was noted
in the shoot, as evidenced by the increased expression
of MAX3, MAX4, and IAA1 in the shoots of max2/
wild-type (scion/rootstock) grafts compared with
wild-type/wild-type grafts (Fig. 5A). For all grafts,
the expression of all genes in the shoot was dependent
only on the shoot genotype; therefore, similar to re-
sults in pea, no upwardly mobile feedback signaling
was detected (Foo et al., 2005; Johnson et al., 2006).
Feedback in the Root
The expression of MAX3, MAX4, and IAA1 was
higher in the rootstocks of wild-type/max2 grafts
relative to wild-type/wild-type grafts, supporting a
local feedback effect also in the root (Fig. 5B). How-
ever, MAX3, MAX4, and IAA1 expression was not
increased in the rootstocks of max2/wild-type grafts
relative to wild-type/wild-type grafts. Therefore, in
contrast with results in pea (Foo et al., 2005), a down-
wardly mobile feedback signal from max2-1 shoots,
able to up-regulate gene expression in wild-type roots,
was not detected. Nonetheless, the increased expres-
sion of MAX3 and MAX4, but not IAA1, in max2
rootstocks was reduced by approximately 40% when
grafted to a wild-type shoot compared with a max2
shoot (Fig. 5B; P , 0.05). This is suggestive of some
long-distance effect on MAX3 and MAX4 expression
in the rootstock depending on the shoot genotype.
405
Hayward et al.
Figure 5. Expression of MAX3, MAX4, and IAA1 in the shoot (A) and
the rootstock (B) of grafted wild-type (WT) and max2-1 plants. Expres-
sion was measured in vegetative plants 2 weeks after transfer to soil;
values are shown relative to wild-type/wild-type (scion/rootstock)
rootstocks. Data are means of two biological pools of five to eight
plants 6 SE. Bars with asterisks are significantly different from each
other due to a long-distance effect.
As growing shoot apices export auxin, it was con-
sidered that auxin, or additional, downwardly mobile
feedback signals may be enhanced by the presence of
actively growing branches in max mutants. Thus,
grafts were repeated and grown until plants had
primary bolting stems 15 to 30 cm in length and
rosette branches. MAX3, MAX4, and IAA1 were
up-regulated in the rootstocks of branching max2
self-grafts by a similar degree as in vegetative max2
self-grafts and again were not up-regulated in wild-
type rootstocks grafted to max2 shoots (data not
shown). Therefore, while there appears to be both a
local and a long-distance feedback effect of strigolac-
tone signaling on MAX3 and MAX4 transcript abun-
dance in Arabidopsis, it appears that, in contrast to
pea, this long-distance effect is relatively minor.
DISCUSSION
Auxin Can Affect Strigolactone-Mediated Branch
Inhibition via MAX3 and MAX4 Regulation
Previous analyses have shown that auxin may in-
teract with the strigolactone pathway in both mono-
406
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
cots and dicots by regulating the transcription of
MAX3 (CCD7) and MAX4 (CCD8) orthologs (Sorefan
et al., 2003; Foo et al., 2005; Johnson et al., 2006; Zou
et al., 2006; Arite et al., 2007). Here, we reveal that
auxin positively regulates both MAX3 and MAX4
expression in Arabidopsis more extensively than pre-
viously thought. This further supports conservation,
and hence possible functional significance, of this
regulation throughout evolution.
In the shoot, auxin enhanced MAX3 and MAX4
transcript levels within 3 h, and this was AXR1
dependent (Fig. 1). However, high concentrations
(.1 mM) of IAA in lanolin were required to elicit this
response, even for the IAA1 gene, which in etiolated
seedlings can be induced by as little as 1 mM IAA in
solution over a similar time frame (Abel et al., 1995).
This may suggest reduced permeability or sensitiv-
ity of intact light-grown cauline tissue to IAA ap-
plied in lanolin. Similarly to pea, rapid and large
reductions in MAX3 and MAX4 transcript levels
were observed following treatments predicted to
deplete endogenous auxin, and these reductions
were prevented by auxin addition (Fig. 2). Together,
these data imply that the basal levels of MAX3 and
MAX4 transcripts in intact wild-type plants are
substantially maintained by auxin. Consistent with
this idea, the levels of MAX3 and MAX4 transcript in
the auxin response mutant axr1-3 were in general
lower than in the wild type, although these differ-
ences were not always statistically significant (Figs.
1 and 4). This is likely due in part to reduced
feedback inhibition on auxin synthesis in the axr1-3
mutant, resulting in increased auxin levels (Romano
et al., 1995), which may partially compensate for
reduced auxin sensitivity. Furthermore, AXR1 acts
partly redundantly with an additional AXR1-like
protein, AXL1 (Dharmasiri et al., 2007), and the axr1-3
allele used in this work is not a null allele. AXR1-
dependent auxin regulation of MAX3 and MAX4 is
consistent with the existence of auxin-response cis-
elements in the promoter regions of these genes,
including four and two copies, respectively, of the
TGTCTC ARF-binding motif (Ulmasov et al., 1999)
within 3 kb upstream.
In contrast to the mild effect of the axr1-3 mutant on
MAX3 and MAX4 transcript levels, the highly
branched bdl-2 mutant (Fig. 3), expressing the
IAA12 transcriptional repressor that is resistant to
AXR1/TIR1-mediated destabilization (Hamann et al.,
2002; Dharmasiri et al., 2005b), had transcript levels
similar to those of plants treated to deplete endoge-
nous auxin (Figs. 2 and 3D). Thus, an attractive
hypothesis, also suggested by Brewer et al. (2009), is
that substantial auxin depletion results in increased
branching at least in part by reducing strigolactone
synthesis. Consistent with this, branching in bdl-2
shoots appears to involve strigolactone deficiency.
First, shoot branching in max4 mutants can be re-
stored to wild-type levels by grafting to wild-type
rootstocks but not bdl-2 rootstocks (Sorefan et al.,
Plant Physiol. Vol. 151, 2009

2003; Bainbridge et al., 2005; Fig. 3E), suggesting that
bdl-2 rootstocks do not produce sufficient strigolac-
tone. Second, wild-type rootstocks rescued over half
of the branching in bdl-2 shoots, while strigolactone-
deficient max4-1 rootstocks did not, suggesting this
strigolactone deficiency may be at least partly re-
sponsible for the increased shoot branching in bdl
(Fig. 3, E–G). Consistent with this idea, GR24 sup-
pressed branching by a similar degree in bdl-2 and
max4-1. An interaction between BDL/IAA12 and
MAX3 and MAX4 is consistent with their preferential
expression in the xylem and the localization of a BDL:
GUS fusion protein in vascular tissues (Zimmermann
et al., 2004; Dharmasiri et al., 2005b; Hruz et al., 2008).
Although strigolactone appears to be involved in the
shoot-branching phenotype of bdl-2 mutants, it is
unlikely to play a role in the embryonic phenotype
(Hamann et al., 1999, 2002; Hardtke et al., 2004), since
mutants for the strigolactone pathway do not show
similar pleiotropic abnormalities.
Taken together, our results suggest that auxin-
regulated MAX expression can be blocked by stabi-
lization of BDL/IAA12, and this can modulate the
degree of branching. Conversely, consistent with max4
shoot branching being rescued by grafting to axr1 roots
(Bainbridge et al., 2005), the mild down-regulation
of MAX3 and MAX4 transcripts observed in axr1-3
mutant roots may not be sufficient for strigolactone-
dependent modulation of branching. The increased
branching observed in axr1-3 mutants, the additive
bud auxin-insensitivity and branching phenotype of
axr1-3max double mutants relative to the single mu-
tants, and the normal levels of auxin transport in
axr1-3 mutants also point to mutually independent
roles for MAX and auxin signaling in suppressing
branching (Bainbridge et al., 2005; Bennett et al., 2006).
Although some effects of axr1 mutants may not be
related solely to auxin signaling (Schwechheimer et al.,
2002; Tiryaki and Staswick, 2002), an additional role
for auxin signaling in branching is supported here
by the fact that branching in bdl-2 shoots was par-
tially suppressed by max4-1 roots compared with bdl
self-grafts, suggesting that some of the branching in
bdl-2 cannot be explained by strigolactone deficiency
(Fig. 3).
An additional mode of auxin action is likely to
involve down-regulated synthesis of the branching
promoter cytokinin (Wickson and Thimann, 1958,
Sachs and Thimann, 1967; Bangerth, 1994; Li et al.,
1995; Nordström et al., 2004; Ferguson and Beveridge,
2009). Apically derived auxin has been shown to
down-regulate cytokinin levels in the xylem exudate
of pea and bean (Phaseolus vulgaris), and in pea it can
inhibit the expression of ISOPENTYL-TRANSFERASE
cytokinin biosynthesis genes in the stem (Bangerth,
1994; Li et al., 1995; Tanaka et al., 2006; Ferguson and
Beveridge, 2009). In Arabidopsis, the auxin-mediated
repression of cytokinin synthesis requires AXR1
(Nordström et al., 2004). Thus, differences in branch-
ing between bdl-2 self-grafts and bdl-2 shoots grafted
Plant Physiol. Vol. 151, 2009
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Auxin and Strigolactone Interactions in Branching
with max4 roots could involve differences in cytoki-
nin levels, caused by auxin insensitivity of bdl-2
root and shoot tissues. However, while local produc-
tion of cytokinin in shoot tissues can affect branch-
ing, the importance of root-derived cytokinin to
axillary branching remains controversial (Chaudhury
et al., 1993; Beveridge et al., 1997a; Faiss et al., 1997;
McKenzie et al., 1998; Catterou et al., 2002; Schmülling,
2002; Sakamoto et al., 2006).
The Mechanisms of Strigolactone Feedback Regulation
in Arabidopsis
We show that both MAX3 and MAX4 transcripts are
up-regulated in all max mutants, suggesting feedback
control (Fig. 2, B and C). The degree of up-regulation
varies between tissues and is of a similar magnitude to
that for orthologous genes in rice and petunia, being
substantially less than that for RMS1 in pea, where
transcripts can accumulate to more than 1,000-fold in
rms4 mutants (Foo et al., 2005; Snowden et al., 2005;
Arite et al., 2007; Simons et al., 2007; Umehara et al.,
2008). There are at least three not mutually exclusive
potential mechanisms for the up-regulation of MAX3
and MAX4 gene expression in max mutants: (1) it is a
direct effect of the strigolactone signaling pathway; (2)
it is associated with the demonstrated effect of auxin
on MAX3 and MAX4 expression, coupled with the
effect of the MAX pathway on auxin transport/levels
and thus auxin distribution; and (3) it is associated
with an additional downwardly mobile strigolactone-
regulated signal, as proposed for pea (Foo et al., 2005;
Dun et al., 2006).
Direct Effects of Strigolactone Signaling on Strigolactone
Biosynthesis Gene Expression
The best evidence for a direct feedback effect of
strigolactone signaling comes from studies of rice
roots, where up-regulated D10 expression in strigo-
lactone pathway mutants can be restored to wild-type
levels by strigolactone application in the biosynthesis
mutant but not the signaling mutant (Umehara et al.,
2008). However, this was determined 24 h after strigo-
lactone application and thus remains of limited value
in distinguishing a direct effect from one mediated
by another signal such as auxin, considering that
auxin alters gene expression within 3 h (Foo et al.,
2005; Fig. 1).
In this study, we included parallel observations of
MAX gene expression and the auxin-responsive gene
IAA1. We have also analyzed the axr1-3 mutant, which
has known defects in auxin-responsive gene expres-
sion. This allows some discrimination between a direct
effect of strigolactone on MAX3 and MAX4 expression
and an indirect effect mediated by changes in auxin
levels in the mutants. As discussed below, our findings
suggest that the major portion of feedback regulation
in Arabidopsis is indirectly mediated by auxin sig-
naling.
407
Hayward et al.

Involvement of Auxin in the Feedback Regulation of MAX3 rootstock) grafts was intermediate between wild-
and MAX4 type/wild-type and max2/max2 grafts (Fig. 5). This
suggests that MAX2 activity in either the shoot or the
Where there were large differences in MAX3 and root can affect MAX3 and MAX4 expression in the
MAX4 transcript accumulation between the wild type rootstock. Although wild-type shoots suppressed
and the max2 mutant, these were substantially reduced MAX3 and MAX4 gene expression in max2 roots
in the axr1-3 mutant background. In contrast, where by about one-third compared with max2 self-grafts,
the differences were small, these appeared to be un- they had no effect on IAA1 gene expression. Thus,
affected by the axr1-3 mutation (Fig. 4). Thus, it seems one possibility is a non-auxin long-distance signaling
likely that a major contributor to the elevated levels of effect. However, the extent of this long-distance regu-
MAX3 and MAX4 transcript in max mutant back- lation is considerably less than that in pea, where
grounds is increased auxin in some tissues, particu- 140-fold differences in RMS1 transcripts were ob-
larly cauline nodes. This is consistent with the very served for the comparable graft combinations (Foo
high DR5:GUS levels observed in the vasculature of et al., 2005). Also unlike comparable grafts of pea, we
max mutant stems and the expression of MAX genes in
vascular-associated cells where polar auxin transport
is occurring (Sorefan et al., 2003; Booker et al., 2005;
Bennett et al., 2006; Stirnberg et al., 2007).
A role for auxin in feedback is further suggested by
the strong correlation between IAA1 expression and
MAX3 and MAX4 expression across the feedback
assays used in this study (Figs. 2, 4, and 5), even in
cases where axr1 had no effect. For example, axr1 did
not suppress the elevated levels of MAX3 and MAX4
transcripts in mature max2 hypocotyls, yet the same
was observed for IAA1 transcripts and additional Aux/
IAAs (Fig. 4B; data not shown). A case for direct or
auxin-independent feedback on MAX3 and MAX4
expression (as in mechanisms 1 or 3 above) is better
suggested where the response of their transcripts to
feedback differs from IAA1. We found only two
such instances. First, in young hypocotyls, the up-
regulation of IAA1 expression in max2 was suppressed
in the axr1-3 background (axr1max2), as expected if
auxin was responsible for its up-regulation, while
MAX3 and MAX4 transcripts were expressed at
equally high levels in max2 and axr1max2 (Fig. 4C).
In this case, however, the enhanced MAX3 and MAX4
expression in axr1max2 relative to axr1 was abolished
by NPA treatment (Fig. 4D). This could again mean a
function for increased auxin in the polar auxin trans-
port stream (PATS) of max2 plants in this feedback
(Bennett et al., 2006) or that transcript levels are at their
lowest after NPA treatment, preventing an additional
effect of MAX2. The other case where IAA1 expression
levels did not reflect MAX3 and MAX4 expression was
in the rootstock of grafted plants, as discussed below Figure 6. Interactions between auxin and the MAX pathway. Regula-
(Fig. 5B). tory signals represented in blue were investigated in this study. Step 1,
Auxin traveling in the PATS promotes MAX3 and MAX4 expression in
an AXR1-dependent manner, prevented if IAA12 is stabilized. Step 2,
Auxin-promoted MAX3 and MAX4 transcript levels lead to increased
Systemic Feedback Regulation of MAX3 and MAX4
strigolactone production, which leads to reduced bud outgrowth and
The data described above demonstrate that auxin, reduced auxin export from buds into the PATS. Step 3, If strigolactones
likely that traveling in the PATS, is a major component are low, branching is increased and auxin level increases, feedback up-
regulating MAX3 and MAX4 to increase strigolactone levels as in step
of feedback signaling on MAX3 and MAX4 transcript
1. Step 4, Strigolactone signaling may feedback down-regulate tran-
abundance, especially in cauline nodes. Therefore, at script levels independent of auxin; the dashed line indicates weak/
least in the shoot, auxin could act as a long-range putative interaction. Step 5, Auxin also down-regulates cytokinin
feedback signal for MAX action. synthesis, and cytokinin promotes branching. The environment/geno-
In this study, similar to those for pea (Foo et al., 2005; type/developmental program may influence each of these regulatory
Johnson et al., 2006), the expression of MAX3 and stages tissue specifically. [See online article for color version of this
MAX4 in the rootstocks of wild-type/max2 (scion/ figure.]

408 Plant Physiol. Vol. 151, 2009

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Copyright © 2009 American Society of Plant Biologists. All rights reserved.

did not detect a systemic feedback signal from max2
shoots that could up-regulate MAX3 and MAX4 ex-
pression in wild-type rootstocks (Fig. 5; Foo et al.,
2005). Interestingly, in petunia, the up-regulation of the
MAX4 ortholog, DECREASED APICAL DOMINANCE1,
was not found to be graft transmissible to the rootstock
(Simons et al., 2007).
The relatively minor effect of the shoot genotype,
wild type or max2, on MAX3 and MAX4 gene expres-
sion in the roots contrasts with clear evidence of
the long-distance regulation of xylem sap cytokinin
by strigolactone signaling in Arabidopsis shoots
(Foo et al., 2007). Auxin regulation of cytokinin content
is well documented (Nordström et al., 2004), and
decapitation leads to depletion of xylem sap cytoki-
nins, which can be restored with exogenous auxin
(Bangerth, 1994; Li et al., 1995). However, a feedback
signal in addition to auxin has been proposed for pea,
because feedback down-regulated xylem cytokinin is
not significantly increased by decapitation in the rms1,
rms3, and rms4 mutants over 24 h, and the up to 2,000-
fold increases in RMS1 expression observed in the
rms4 (Psmax2) mutant have not been achieved by
auxin addition (Foo et al., 2005, 2007; for review, see
Dun et al., 2009). More direct studies of auxin involve-
ment in shoot-to-root regulation of xylem sap cytoki-
nin levels in Arabidopsis are required to determine
whether a signal in addition to auxin affects xylem
cytokinin content.
Sources of Differences
In this and previous studies, similar experiments
have given quantitatively and sometimes qualitatively
different results. Our data suggest that some of the
variability is likely technical, such as the method of
hormone application or expression analysis, the hor-
mone concentrations used, and the tissue types in-
cluded in the analyses. Different phenotypes of species
dictate the use of different tissues for grafting and gene
expression analysis, epicotyl in pea versus hypocotyl
in Arabidopsis and petunia, and perhaps this could
influence the movement of, or degree of response to,
long-distance signals. Importantly, some interesting
biological differences are also emerging. In pea but not
Arabidopsis, mutant rms4 (max2) rootstocks are more
able than wild-type rootstocks to suppress branching
in wild-type and various rms mutant shoots, and this
has been associated with the small local feedback up-
regulation of strigolactone biosynthesis genes in mu-
tant rootstocks (Beveridge et al., 1996, 1997b; Morris
et al., 2001; Booker et al., 2005; Foo et al., 2005; Johnson
et al., 2006). While this could involve differences in the
strength of feedback regulation, perhaps strigolactone
levels could be more rate limiting in pea than in
Arabidopsis in standard conditions. As discussed
above, the balance of auxin and auxin-independent
feedback regulation on strigolactone synthesis genes
may also differ, the latter component appearing to be
greater in pea than in Arabidopsis (Foo et al., 2005,
Plant Physiol. Vol. 151, 2009
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Auxin and Strigolactone Interactions in Branching
2007). Although many similarities are apparent be-
tween species, differences in strigolactone pathways
could potentially reflect differences in developmental
programs and shoot architectures.
CONCLUSION
We demonstrate that the auxin regulation of strigo-
lactone biosynthetic genes is conserved in Arabidop-
sis. This involves AXR1/TIR1-regulated Aux/IAA
stability and contributes to branching inhibition. Fur-
thermore, this auxin regulation also acts as a feedback
mechanism, with increased auxin content in condi-
tions of low strigolactone acting as a downstream
communicator to increase strigolactone biosynthesis.
We suggest that auxin-regulated strigolactone biosyn-
thesis may form a conserved component of auxin-
mediated branching inhibition and that auxin and
strigolactone signaling may participate in an inter-
locking feedback loop, involving interplay with addi-
tional stimuli, to precisely control branching in plants.
A diagrammatic representation of the interactions
between auxin and the MAX pathway built upon by
this study is shown in Figure 6.
MATERIALS AND METHODS
Plant Growth and Materials
For Figures 1, 2, 3E to 3G, 4C, 4D, and 5, Arabidopsis (Arabidopsis thaliana)
seeds were sown on a mixture of California potting mix type C and vermic-
ulite (3:2, v/v) at a density of one to two per 5 cm2 or one per 1 cm2 (Fig. 2) and
stratified for 2 to 3 d at 4°C. Trays were transferred either to a temperature-
controlled growth room at 22°C 6 2°C/18°C 6 2°C for 18 h of light/6 h of dark
with fluorescent lighting (Philips 36 W/840) supplying approximately 120
mmol m22 s21 light or to a temperature-controlled glasshouse at 24°C 6 2°C/
18°C 6 2°C for 18 h of light/6 h of dark with the natural daylength extended
by incandescent light. For Figures 3A to 3D, 4A, and 4B, seeds were cold
treated and grown at a density of one per 4 cm2 in a temperature-controlled
glasshouse according to Bennett et al. (2006). All plant lines used were in the
Col-0 background as follows: Col-0 wild type, max1-1, max2-1, max3-11, max4-1,
axr1-3, axr1-3max2-1, and bdl-2 (see below). Vegetative 2-week-old plants had
approximately six to eight mature leaves expanded, and bolting plants were
grown until the primary bolt was 2 to 5 cm (3 weeks) or 15 to 30 cm
(approximately 5 weeks) as indicated in the text.
The bdl-2 Allele
The bdl-2 mutant allele was found in a screen for branching mutants of
the ethyl methanesulfonate-mutagenized Col-0 population described by
Stirnberg et al. (2002). Genetic analysis of the mutant individual recovered
showed that its max-like phenotype was caused by a semidominant mutation
in a heterozygous state. Homozygotes were severely dwarfed. The mutation
mapped between PVV4 and nga63 on chromosome I, an interval containing
four IAA genes: IAA3/SHY2, IAA17/AXR3, IAA10, and IAA12/BDL. As muta-
tions with semidominant inheritance were known for this class of genes but
homozygous or heterozygous shy2 or axr3 were unlike our mutant (Leyser
et al., 1996; Tian and Reed, 1999), IAA10 and IAA12 were considered as
possible candidates and their coding regions were amplified from genomic
DNA of homozygous mutant plants. Sequencing revealed no change in IAA10
but a CCA-to-TCA change in codon 74 of IAA12, predicting a Pro-to-Ser
substitution identical to the bdl-1 allele, which is in the Landsberg erecta
genetic background (Hamann et al., 1999, 2002).
409
Hayward et al.
Decapitation and Hormone Treatments
Three-week-old plants were decapitated by removing the entire primary
inflorescence (2–5 cm) and the youngest three to four expanding rosette leaves
using a pair of fine forceps. For 5-week-old plants, primary bolts (15–30 cm)
were decapitated 15 mm above the stem base using a sterile scalpel, with or
without the youngest rosette leaves removed as indicated in the figure
legends. For hormone treatments, hormones were dissolved in ethanol and
mixed with lanolin to give the various concentrations indicated with a final
ethanol concentration of less than 10%. NPA was applied in a ring either
around the top of the hypocotyls or around the primary bolt 15 mm above the
base as indicated. IAA was applied to the decapitation site or, for the dose-
response assay, in a ring around the primary bolt of intact plants 15 mm above
the base. Control treatments were as above minus hormone. For expression
analyses, tissues as indicated (approximately 2–5 mm of hypocotyl, the basal
10 mm of the primary inflorescence stem, and the first 5 cm of root tissue) were
harvested after the times indicated. Statistical P values for all phenotypic and
expression data were calculated using the t test.
Plants were treated with GR24 (http://www.chiralix.com/) as described
by Gomez-Roldan et al. (2008) with modifications. For 5 mM-treated and 0 mM
control plants, solutions containing 0.05% Silwet and 0.05% acetone with or
without GR24 were applied to plants every 3 d starting at 18 d after transfer to
the growth room. At 24 d, the GR24 dose was doubled to 10 mM applied every
2 d for two treatments followed by an additional four treatments of 5 mM every
3 d. Rosette leaf numbers (minus cotyledons) were scored 1 week after bolting,
and branch numbers of 5 mm or greater were scored at 47 to 50 d.
Grafting
Transverse grafts were performed using a protocol adapted from Turnbull
et al. (2002). Sterilized cold-treated seeds were germinated vertically on plates
containing half-strength Murashige and Skoog basal salts, 1% Suc, and 0.8%
agar in an incubator at 24°C with an 18-h photoperiod. Seedlings were grown
for 4 to 5 d before careful transfer to sterilized petri dishes containing a
premoistened layer of Millipore nitrocellulose filter (type HA; pore, 0.45 mm)
on a single layer of Whatman No. 1 filter paper for grafting. Grafts were
returned to the incubator, and successful grafts were transferred to soil by 7 d
and grown in a temperature-controlled growth room as above. Grafts were
visually assessed, and phenotypic analyses were carried out at the cessation of
primary meristem activity. For expression analyses, hypocotyl and root tissues
below the graft union (approximately 2 cm) were collected as rootstock tissue
and rosette stem tissue (rosette stem plus approximately 0.5 mm of petiole
tissue) was collected as shoot tissue. For vegetative-stage plants, tissues were
collected 2 weeks after transfer to soil. For mature plants, tissues were
collected following phenotypic analysis.
qRT-PCR
Total RNA was isolated from pools of five to 25 plants using NucleoSpin
RNA plant kits (Machery-Nagel) and quantified using a NanoDrop 1000.
cDNA was synthesized in 20 mL with 100 ng to 1 mg of total RNA, 250 ng of
random primers (Promega), 250 ng of oligo(dT)15 (Promega), 0.5 mM deoxy-
ribonucleotide triphosphates, 5 mM dithiothreitol (Invitrogen), 13 first-strand
buffer, and 100 units of SuperScript III reverse transcriptase (Invitrogen) as
described in the Invitrogen SSIII RT protocol. Reverse transcriptase-minus
control reactions were performed for each RNA sample to assess DNA
contamination during qRT-PCR. Each qRT-PCR was performed in duplicate
using SYBR Green PCR Master Mix, 200 nM of each primer, and 2 to 15 ng of
cDNA in an ABI Prism 7900HT Sequence Detection System (Applied Biosys-
tems) with melt-curve analysis. No-template control reactions were per-
formed for each primer. Primer efficiencies (PE) of each gene were calculated
per run by LinRegPCR (Ramakers et al., 2003). Relative quantitation was
determined using the comparative cycle threshold (CT) method; CT values for
technical replicates for each gene were averaged (AvCT), and MAX3
(At2g44990), MAX4 (At4g32810), and IAA1 (At4g14560) expression was
normalized against 18S (At2g01010; Figs. 1, 2, B–D, and 3–5) or three b-actin
reference genes (At3g18780, At5g09810, and At1g49240; Fig. 2A) according to
Brown et al. (2003) using the formula PEgene2AvCTgene/PEreference2AvCTreference.
Resulting values were averaged for biological replicates and plotted with SE
values relative to a chosen sample (the calibrator), as indicated on the y axis.
Primers were designed using PrimerExpress 1.5 (Applied Biosystems) with
one primer across intron junctions where possible. The qRT-PCR for Figure 3
410
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
was performed using Power SYBR Green PCR Master Mix in an ABI
Prism 7300 Sequence Detection System (Applied Biosystems) with melt-curve
analysis. Gene expression was normalized to 18S using the comparative
CT method with the formula 22CTgene/22CT18S. Primer sequences are as fol-
lows: 18SF (5#-TTCCTAGTAAGCGCGAGTCATCA-3#), 18SR (5#-GAACA-
CTTCACCGGATCATTCAAT-3#), QRTMAX4F (5#-GAAAGATACCCACT-
TGGCTGAATG-3#) QRTMAX4R (5#-TGTGGAGTAGCCGTCGAAGAG-3#),
QRTMAX3F (5#-GATTCGTTGGTGAGCCCATG-3#), QRTMAX3R (5#-CAC-
CGAAACCGCATACTCGA-3#), QRTIAA1F (5#-GCTCCTCCTCCTGCAAA-
AACAC-3#), QRTIAA1R (5#-ACGGTTAGATCTCACTGGAGGC-3#); b-actin
primers were as described by Brown et al. (2003). The Genevestigator V3
Classic Metaprofile analysis tool (available at https://www.genevestigator.
ethz.ch/) was used to generate an anatomy heat map of IAA1 (245397_at),
MAX3 (266129_at), MAX4 (253398_at), and IAA12 (264605_at; data not
shown).
ACKNOWLEDGMENTS
At the University of Queensland, we thank Julia Cremer and the
Beveridge laboratory for assistance with the experiment in Figure 2, Dr.
Philip Brewer for assistance with the strigolactone application experiment,
Dr. Elizabeth Dun and Tanya Brcich for helpful discussions, Mr. Bob Simpson
for qRT-PCR advice, and Kerry Condon for technical assistance. At the
University of York, we thank horticultural staff for plant care, Dr. Karin van
de Sande for initial isolation of the bdl-2 mutant, and University of York
Technology Facility staff for qRT-PCR advice. We are also indebted to Dr.
Catherine Rameau (INRA, France), who provided the GR24.
Received February 24, 2009; accepted July 21, 2009; published July 29, 2009.
LITERATURE CITED
Abel S, Nguyen MD, Theologis A (1995) The PS-IAA4/5-like family of
early auxin-inducible mRNAs in Arabidopsis thaliana. J Mol Biol 251:
533–549
Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes induce
hyphal branching in arbuscular mycorrhizal fungi. Nature 435: 824–827
Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M,
Sakakibara H, Kyozuka J (2007) DWARF10, an RMS1/MAX4/DAD1
ortholog, controls lateral bud outgrowth in rice. Plant J 51: 1019–1029
Auldridge ME, Block A, Vogel JT, Dabney-Smith C, Mila I, Bouzayen M,
Magallanes-Lundback M, DellaPenna D, McCarty DR, Klee HJ (2006)
Characterisation of three members of the Arabidopsis carotenoid cleav-
age dioxygenase family demonstrates the divergent roles of this mul-
tifunctional enzyme family. Plant J 45: 982–993
Bainbridge K, Sorefan K, Ward S, Leyser O (2005) Hormonally controlled
expression of the Arabidopsis MAX4 shoot branching regulatory gene.
Plant J 44: 569–580
Bangerth F (1994) Response of cytokinin concentration in the xylem
exudate of bean (Phaseolus vulgaris L.) plants to decapitation and auxin
treatment and relationship to apical dominance. Planta 194: 439–442
Bennett T, Sieberer T, Willett B, Booker J, Luschnig C, Leyser O (2006) The
Arabidopsis MAX pathway controls shoot branching by regulating
auxin transport. Curr Opin Plant Biol 16: 553–563
Beveridge CA (2000) Long-distance signalling and mutational analysis of
branching in pea. Plant Growth Regul 32: 193–203
Beveridge CA, Murfet IC, Kerhoas L, Sotta B, Miginiac E, Rameau C
(1997a) The shoot controls zeatin riboside export from pea roots:
evidence from the branching mutant rms4. Plant J 11: 339–345
Beveridge CA, Ross JJ, Murfet IC (1996) Branching in pea: action of genes
Rms3 and Rms4. Plant Physiol 110: 859–865
Beveridge CA, Symons GM, Murfet IC, Ross JJ, Rameau C (1997b) The
rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced
root-sap zeatin riboside content but increased branching controlled by
graft-transmissible signals. Plant Physiol 115: 1251–1258
Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O (2004)
MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the
synthesis of a novel plant signalling molecule. Curr Biol 14: 1232–1238
Booker J, Sieberer T, Wright W, Williamson L, Willett B, Stirnberg P,
Turnbull C, Srinivasan M, Goddard P, Leyser O (2005) MAX1 encodes a
cytochrome P450 family member that acts downstream of MAX3/4 to
Plant Physiol. Vol. 151, 2009

produce a carotenoid-derived branch-inhibiting hormone. Dev Cell 8:
443–449
Brewer P, Dun E, Ferguson B, Rameau C, Beveridge C (2009) Strigolactone
acts downstream of auxin to regulate bud outgrowth in pea and
Arabidopsis. Plant Physiol 150: 482–493
Brown RL, Kazan K, McGrath KC, Maclean DJ, Manners JM (2003) A role
for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of
Arabidopsis. Plant Physiol 132: 1020–1032
Catterou M, Dubois F, Smets R, Vaniet S, Kichey T, Van Onckelen H,
Sangwan-Norreel BS, Sangwan RS (2002) hoc: an Arabidopsis mutant
overproducing cytokinins and expressing high in vitro organogenic
capacity. Plant J 30: 273–287
Chaudhury AM, Letham S, Dennis ES (1993) amp1: a mutant with high
cytokinin levels and altered embryonic pattern, faster vegetative
growth, constitutive photomorphogenesis and precocious flowering.
Plant J 4: 907–916
Cook CE, Whichard LP, Wall ME, Egley GH, Coggon P, Luhan PA,
McPhail AT (1972) Germination stimulants. II. The structure of strigol: a
potent seed germination stimulant for witchweed (Striga lutea Lour.).
J Am Chem Soc 94: 6198–6199
del Pozo JC, Dharmasiri S, Hellmann H, Walker L, Gray WM, Estelle M
(2002) AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis
cullin AtCUL1 is required for auxin response. Plant Cell 14: 421–433
Dharmasiri N, Dharmasiri S, Estelle M (2005a) The F-box protein TIR1 is
an auxin receptor. Nature 435: 441–445
Dharmasiri N, Dharmasiri S, Weijers D, Karunarathna N, Jurgens G,
Estelle M (2007) AXL and AXR1 have redundant functions in RUB
conjugation and growth and development in Arabidopsis. Plant J 52:
114–123
Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L,
Ehrismann JS, Jürgens G, Estelle M (2005b) Plant development is regu-
lated by a family of auxin receptor F box proteins. Dev Cell 9: 109–119
Dharmasiri S, Estelle M (2002) The role of regulated protein degradation in
auxin response. Plant Mol Biol 9: 401–409
Dun EA, Brewer PB, Beveridge CA (2009) Strigolactones: discovery of the
elusive shoot branching hormone. Trends Plant Sci 14: 364–372
Dun EA, Ferguson BJ, Beveridge CA (2006) Apical dominance and shoot
branching: divergent opinions or divergent mechanisms. Plant Physiol
142: 812–819
Faiss M, Zalubilova J, Strnad M, Schmülling T (1997) Conditional trans-
genic expression of the ipt gene indicates a function for cytokinins in
paracrine signalling in whole tobacco plants. Plant J 12: 401–415
Ferguson BJ, Beveridge CA (2009) Roles for auxin, cytokinin and strigo-
lactone in regulating shoot branching. Plant Physiol 149: 1929–1944
Foo E, Bullier E, Goussot M, Foucher F, Rameau C, Beveridge CA (2005)
The branching gene RAMOSUS1 mediates interactions among two
novel signals and auxin in pea. Plant Cell 17: 464–474
Foo E, Morris SE, Parmenter K, Young N, Wang H, Jones A, Rameau C,
Turnbull CGN, Beveridge CA (2007) Feedback regulation of xylem
cytokinin content is conserved in pea and Arabidopsis. Plant Physiol
143: 1418–1428
Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP,
Letisse F, Matusova R, Danoun S, Portais JC, et al (2008) Strigolactone
inhibition of shoot branching. Nature 455: 189–194
Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin
regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Na-
ture 414: 271–276
Hamann T, Benkova E, Baurle I, Kientz M, Juergens G (2002) The
Arabidopsis BODENLOS gene encodes an auxin response protein
inhibiting MONOPTEROS-mediated embryo patterning. Genes Dev
16: 1610–1615
Hamann T, Mayer U, Jurgens G (1999) The auxin-insensitive bodenlos
mutation affects primary root formation and apical-basal patterning in
the Arabidopsis embryo. Development 126: 1387–1395
Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G,
Tiwari SB, Hagen G, Guilfoyle TJ, Berleth T (2004) Overlapping and
non-redundant functions of the Arabidopsis auxin response factors
MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Develop-
ment 131: 1089–1100
Hruz T, Laule O, Szabo G, Wessendrop F, Bleuler S, Oertle L, Widmayer P,
Gruissem W, Zimmermann P (2008) Genevestigator V3: a reference
expression database for the meta-analysis of transcriptomes. Advances
in Bioinformatics 2008: 420747
Plant Physiol. Vol. 151, 2009
Downloaded from www.plantphysiol.org on June 24, 2014 - Published by www.plant.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Auxin and Strigolactone Interactions in Branching
Humphrey AJ, Beale MH (2006) Strigol: biogenesis and physiological
activity. Phytochemistry 67: 636–640
Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J (2005)
Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant
Cell Physiol 46: 79–86
Johnson AW, Gowda G, Hassanali A, Knox J, Monaco S, Razavi Z,
Rosebery G (1981) The preparation of synthetic analogs of strigol.
J Chem Soc Perkin Trans 1 1981: 1734–1743
Johnson X, Brcich T, Dun EA, Goussot M, Haurogné K, Beveridge CA,
Rameau C (2006) Branching genes are conserved across species: Genes
controlling a novel signal in pea are coregulated by other long-distance
signals. Plant Physiol 142: 1014–1026
Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an
auxin receptor. Nature 435: 446–451
Lazar G, Goodman HM (2006) MAX1, a regulator of the flavonoid pathway,
controls vegetative axillary bud outgrowth in Arabidopsis. Proc Natl
Acad Sci USA 103: 472–476
Leyser HM, Lincoln CA, Timpte C, Lammer D, Turner J, Estelle M (1993)
Arabidopsis auxin-resistance gene AXR1 encodes a protein related to
ubiquitin-activating enzyme E1. Nature 364: 161–164
Leyser HMO, Pickett FB, Dharmasiri S, Estelle M (1996) Mutations in the
AXR3 gene of Arabidopsis result in altered auxin response including
ectopic expression from the SAUR-AC1 promoter. Plant J 10: 403–413
Li CJ, Herrera GJ, Bangerth F (1995) Effect of apex excision and replace-
ment by 1-naphthylacetic acid on cytokinin concentration and apical
dominance in pea plants. Physiol Plant 94: 465–469
Lincoln C, Britton JH, Estelle M (1990) Growth and development of the
axrl mutants of Arabidopsis. Plant Cell 2: 1071–1080
Ljung K, Bhalerao RP, Sandberg G (2001) Sites and homeostatic control of
auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28:
465–474
McKenzie MJ, Mett VV, Stewart Reynolds PH, Jameson PE (1998) Con-
trolled cytokinin production in transgenic tobacco using a copper-
inducible promoter. Plant Physiol 116: 969–977
Morris SE, Turnbull CGN, Murfet IC, Beveridge CA (2001) Mutational
analysis of branching in pea: evidence that Rms1 and Rms5 regulate the
same novel signal. Plant Physiol 126: 1205–1213
Nordström A, Tarkowski P, Tarkowska D, Norbaek R, Åstot C, Dolezal K,
Sandberg G (2004) Auxin regulation of cytokinin biosynthesis in Arab-
idopsis thaliana: a factor of potential importance for auxin-cytokinin-
regulated development. Proc Natl Acad Sci USA 101: 8039–8044
Ongaro V, Leyser O (2007) Hormonal control of shoot branching. J Exp Bot
59: 67–74
Park JY, Kim HJ, Kim J (2002) Mutation in domain II of IAA1 confers
diverse auxin-related phenotypes and represses auxin-activated expres-
sion of Aux/IAA genes in steroid regulator-inducible system. Plant J 32:
669–683
Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-ring
ubiquitin ligases. Nat Rev Mol Cell Biol 6: 9–20
Ramakers C, Ruijter JM, Lekanne Deprez LH, Moorman AFM (2003)
Assumption-free analysis of quantitative real-time PCR data. Neurosci
Lett 339: 62–66
Romano CP, Robson PR, Smith H, Estelle M, Klee H (1995) Transgene-
mediated auxin overproduction in Arabidopsis: hypocotyl elongation
phenotype and interactions with the hy6-1 hypocotyl elongation and
axr1 auxin-resistant mutants. Plant Mol Biol 27: 1071–1083
Sachs T, Thimann K (1967) The role of auxins and cytokinins in the release
of buds from dominance. Am J Bot 54: 136–144
Sakamoto T, Sakakibara H, Kojima M, Yamamoto Y, Nagasaki H, Inukai
Y, Sato Y, Matsuoka M (2006) Ectopic expression of KNOTTED1-like
homeobox protein induces expression of cytokinin biosynthesis genes in
rice. Plant Physiol 142: 54–62
Schmitz G, Theres K (2005) Shoot and inflorescence branching. Curr Opin
Plant Biol 8: 506–511
Schmülling T (2002) New insights into the functions of cytokinins in plant
development. J Plant Growth Regul 21: 40–49
Schwechheimer C, Serino G, Callis J, Crosby WL, Lyapina S, Deshaies RJ,
Gray WM, Estelle M, Deng XW (2001) Interactions of the COP9
signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin
response. Science 292: 1379–1382
Schwechheimer C, Serino G, Deng X (2002) Multiple ubiquitin-mediated
processes require COP9 signalosome and AXR1 function. Plant Cell 14:
2553–2563
411
Hayward et al.
Simons JL, Napoli CA, Janssen BJ, Plummer KM, Snowden KC (2007)
Analysis of the DECREASED APICAL DOMINANCE genes of petunia
in the control of axillary branching. Plant Physiol 143: 697–706
Snowden KC, Simkin AJ, Janssen BJ, Templeton KR, Loucas HM,
Simons JL, Karunairetnam S, Gleave AP, Clark DG, Klee HJ (2005)
The Dad1/PhCCD8 gene affects branch production and has a role in
leaf senescence, root growth and flower development. Plant Cell 17:
746–759
Sorefan K, Booker J, Haurogne K, Goussot M, Bainbridge K, Foo E,
Chatfield SP, Ward S, Beveridge CA, Rameau C, et al (2003) MAX4 and
RMS1 are orthologous dioxygenase-like genes that regulate shoot
branching in Arabidopsis and pea. Genes Dev 17: 1469–1474
Stirnberg P, Furner IJ, Leyser HMO (2007) MAX2 participates in an SCF
complex which acts locally at the node to suppress shoot branching.
Plant J 50: 80–94
Stirnberg P, van de Sande K, Leyser HMO (2002) MAX1 and MAX2 control
shoot lateral branching in Arabidopsis. Development 129: 1131–1141
Tan X, Calderon-Villalobos LI, Sharon M, Zheng C, Robinson CV, Estelle
M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiq-
uitin ligase. Nature 446: 640–645
Tanaka M, Takei K, Kojima M, Sakakibara H, Mori H (2006) Auxin
controls local cytokinin biosynthesis in the nodal stem in apical dom-
inance. Plant J 45: 1028–1036
Thimann KV, Skoog F (1934) On the inhibition of bud development and
other functions of growth substance in Vicia faba. Proc R Soc Lond B Biol
Sci 114: 317–339
Tian Q, Reed JW (1999) Control of auxin-regulated root development by
the Arabidopsis thaliana SHY2/IAA3 gene. Development 126: 711–721
Tiryaki I, Staswick PE (2002) An Arabidopsis mutant defective in jasm-
onate response is allelic to the auxin-signaling mutant axr1. Plant
Physiol 130: 887–894
Tiwari SB, Wang XJ, Hagen G, Guilfoyle TJ (2001) AUX/IAA proteins are
active repressors, and their stability and activity are modulated by
auxin. Plant Cell 13: 2809–2822
412
Downloaded from www.plantphysiol.org on June 24, 2014 - Published by www.plant.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Turnbull CG, Booker JP, Leyser O (2002) Micrografting techniques for
testing long-distance signalling in Arabidopsis. Plant J 32: 255–262
Ulmasov T, Hagen G, Guilfoyle TJ (1999) Activation and repression of
transcription by auxin-response factors. Proc Natl Acad Sci USA 96:
5844–5849
Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya
N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, et al (2008)
Inhibition of shoot branching by new terpenoid plant hormones. Nature
455: 195–200
Wang KLC, Li H, Ecker JR (2002) Ethylene biosynthesis and signalling
networks. Plant Cell (Suppl) 14: S131–S151
Wickson ME, Thimann KV (1958) The antagonism of auxin and kinetin in
apical dominance. Physiol Plant 11: 62–74
Wu K, Chen A, Pan ZQ (2000) Conjugation of Nedd8 to CUL1 enhances the
ability of the ROC1-CUL1 complex to promote ubiquitin polymeriza-
tion. J Biol Chem 275: 32317–32324
Yamaguchi S (2008) Gibberellin metabolism and its regulation. Annu Rev
Plant Biol 59: 225–251
Yang X, Lee S, So JH, Dharmasiri S, Dharmasiri N, Ge L, Jensen C,
Hangarter R, Hobbie L, Estelle M (2004) The IAA1 protein is encoded
by AXR5 and is a substrate of SCFTIR1. Plant J 40: 772–782
Zenser N, Ellsmore A, Leasure C, Callis J (2001) Auxin modulates the
degradation rate of AUX/IAA proteins. Proc Natl Acad Sci USA 98:
11795–11800
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004)
GENEVESTIGATOR: Arabidopsis microarray database and analysis
toolbox. Plant Physiol 136: 2621–2632
Zou J, Chen Z, Zhang S, Zhang W, Jiang G, Zhao X, Zhai W (2005)
Characterizations and fine mapping of a mutant gene for high tillering
and dwarf in rice (Oryza sativa L.). Planta 222: 604–612
Zou J, Zhang S, Zhang W, Li G, Chen Z, Zhai W, Zhao X, Pan X, Xie Q, Zhu
L (2006) The rice HIGH-TILLERING DWARF1 encoding an orthologue
of Arabidopsis MAX3 is required for negative regulation of the out-
growth of axillary buds. Plant J 48: 687–696
Plant Physiol. Vol. 151, 2009