Strigolactone acts downstream of auxin to regulate bud outgrowth in pea and Arabidopsis.

Abstract

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.

Full text

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Strigolactone Acts Downstream of Auxin to Regulate Bud
Outgrowth in Pea and Arabidopsis1[C][OA]
Philip B. Brewer2, Elizabeth A. Dun2, Brett J. Ferguson, Catherine Rameau, and Christine A. Beveridge*
University of Queensland, Australian Research Council Centre of Excellence for Integrative Legume Research
and School of Biological Sciences, St. Lucia, Queensland 4072, Australia (P.B.B., E.A.D., B.J.F., C.A.B.); and
Station de Ge ´ne ´tique et d’Ame ´lioration des Plantes, Institut J. P. Bourgin, UR254 INRA, F–78000 Versailles,
France (C.R.)
During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the
production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits
auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-
branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied
strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found
that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that
auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant
or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin
transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to
growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis
mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of
endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act
primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-
dependent production of strigolactones.
Classical decapitation and replacement experiments
by Thimann and Skoog (1933, 1934) suggested that the
plant hormone auxin originating from the shoot tip
acted as a repressive signal for axillary bud outgrowth
at nodes below the shoot tip. However, unraveling the
action of apically derived auxin has been challenging,
mainly because auxin was found to move strictly
downward in the vascular cambium of the stem (the
polar auxin transport stream) and apparently cannot
change direction to move upward to enter axillary
buds and branches (Snow, 1937; Hall and Hillman,
1975; Morris, 1977; Morris and Thomas, 1978; Bangerth,
1989; Prasad et al., 1993; Booker et al., 2003). In
addition, while apically derived auxin moved down-
ward through live cells (Morris and Thomas, 1978), the
inhibiting influence was able to be transmitted up-
ward through dead tissue (Snow, 1929). Thus, this
“secondary inhibiting influence” of auxin was pro-
posed to act via an inhibiting substance that moved up
into buds through the transpiration stream (Snow,
1929, 1937). However, despite attempts at identifying
the second messenger of auxin action, such a sub-
stance was never found (Bangerth, 1989).
Seminal work on auxin canalization by Sachs (1968,
1969) led to the idea that auxin saturation in the
transport stream of the main stem could block auxin
transport from lateral sources. It was found that the
direction of the vascular connections of new buds was
influenced by the presence or absence of the apex or
subtending leaf (Sachs, 1968). This meant either that
auxin depletion in an established vascular stream (e.g.
after decapitation by removal of the apex) attracted the
formation of new vasculature or that auxin levels in an
established vascular stream repelled the formation of
new vasculature (i.e. the new vasculature would join
other vasculature where there is less auxin). Sachs
(1970) proposed that the transport stream of an intact
stem, full of auxin, repelled the development of vas-
culature from buds and thus blocked their outgrowth.
Decapitation depleted the apically derived stream of
auxin and thus released the buds to grow.
The work of Sachs (1968, 1969) seemingly bypassed
the need for a second messenger, as auxin could
influence lateral auxin transport from a distance,
such as at the vascular junctions in the main stem.
This theory initially relied on the idea that a bud
needed to form a vascular connection before it could
1This work was supported by the Australian Research Council
Centre of Excellence for Integrative Legume Research.
2These 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 A. 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.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.134783
482Plant Physiology, May 2009, Vol. 150, pp. 482–493, www.plantphysiol.org ? 2009 American Society of Plant Biologists

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grow. However, large dormant buds that are not
actively growing were found to have functional vas-
cular connections (Ali and Fletcher, 1970; Peterson and
Fletcher, 1973). Indeed, dormant buds are highly de-
veloped and would presumably require vascular con-
nections for early development and growth before
they enter a stage of dormancy. Additionally, buds
could become active and later reenter a stage of
dormancy (Stafstrom and Sussex, 1992; Shimizu and
Mori, 1998). In contrast to these findings, Sorokin and
Thimann (1964) reported that xylem strands from an
axillary bud did not connect with those from the main
stem until after release from apical dominance. These
vascular strands strengthened with time after release
from apical dominance (Sorokin and Thimann, 1964).
However, Marr and Blaser (1967) showed that the
strengthening of vascular connections of induced ax-
illary buds occurred after, not before, visible bud
outgrowth. Sachs (1981) later refined his idea to sug-
gest that it was the increase in bud vasculature that
allowed buds to grow.
The studies of Sachs (1968, 1969) focused on vascu-
lar development; however, Bangerth (1989) showed
that auxin (in this case, indole-3-acetic acid [IAA])
export out of a branch was correlated with its ability to
repress the outgrowth of other buds or branches. Thus,
it was proposed that auxin export may be a require-
ment of bud outgrowth, and auxin transport from an
earlier developed branch might simply inhibit auxin
transport from later developed buds. This competition
was suggested to occur at the junctions where the
auxin transport streams meet (Bangerth, 1989).
Cytokinins are a class of phytohormone that pro-
motes cell division, and application of cytokinin to
buds can induce outgrowth (Sachs and Thimann,
1967). While likely a trigger for bud release, cytokinin
may also promote auxin production and basipetal
auxin transport out of growing buds, which conse-
quently represses the production of cytokinin lower in
the stem and limits its availability for other buds
(Bangerth et al., 2000; Tanaka et al., 2006; Shimizu-Sato
et al., 2009).
The studies reviewed above have almost exclusively
used decapitation and related techniques to induce
branching and investigate the role of auxin. Indeed,for
this reason, the term apical dominance encouraged a
focus on the shoot tip as the source of branching
regulation. Importantly, these experiments were valid
for investigating the natural phenomenon of the re-
sponse to decapitation, but the caveat is that auxin
may not be the only relevant factor affected by decap-
itation. Moreover, branching in intact plants may not
be regulated by the same processes as those induced
by decapitation (Dun et al., 2006). Until the isolation
and characterization of branching mutants in various
species, it was not possible to address these issues. In
recent decades, many branching mutants have been
isolated that have problems with auxin, cytokinin, or
brassinosteroids, for example, and show highly pleio-
tropic phenotypes typical for these hormones (Lincoln
et al., 1990; Azpiroz et al., 1998; Tantikanjana et al.,
2001). However, a class of mutants were isolated that
displayed a specific increase in bud outgrowth that
was not correlated with any known hormonal signal
(Beveridge et al., 1996, 1997). Paradoxically, the mu-
tants were found to have generally higher levels of
auxin and lower levels of xylem cytokinin, facts diffi-
cult to reconcile with ideas about the roles of auxin and
cytokinin in regulating bud outgrowth (Beveridge
et al., 1997). These mutants were ramosus (rms) in pea
(Pisum sativum), decreased apical dominance (dad) in
petunia (Petunia hybrida), more axillary growth (max) in
Arabidopsis (Arabidopsis thaliana), and particular dwarf
(d) mutants in rice (Oryza sativa; Beveridge et al., 1994;
Napoli, 1996; Stirnberg et al., 2002; Ishikawa et al.,
2005). Grafting studies demonstrated that increased
bud outgrowth in some of the mutants was caused by
the loss of a long-distance mobile signal (termed SMS;
Beveridge, 2006) that moved upward from lower
tissues (Beveridge et al., 1994; Napoli, 1996; Foo
et al., 2001; Turnbull et al., 2002). Mutant phenotypes
were rescued by grafting with wild-type tissue, even
in interstock grafts where small pieces of wild-type
stem tissue were grafted between mutant rootstock
and shoot tissue (Napoli, 1996; Foo et al., 2001). Other
mutants were not rescued by grafting but were instead
suggested to lack response to SMS (Beveridge et al.,
1996; Booker et al., 2005). Grafting studies also showed
that outgrowth induced by decapitation in SMS
mutant plants cannot be inhibited by IAA applied
to the stump unless a wild-type rootstock is present
(Beveridge et al., 2000). Thus, SMS production some-
where in the plant is required for IAA to prevent buds
growing after decapitation. This means that auxin can
move down into the roots and promote the production
of SMS and suggests that SMS might in fact be the
second messenger for auxin.
Breakthroughs in cloning revealed that rms1, max4,
dad1, and d10 SMS synthesis mutant phenotypes were
caused by mutations in an orthologous gene, CAROT-
ENOID CLEAV AGE DIOXYGENASE8 (CCD8; Sorefan
et al., 2003; Snowden et al., 2005; Arite et al., 2007).
Likewise, RMS5, MAX3, and D17/HTD1 were found
to encode CCD7 (Booker et al., 2004; Johnson et al.,
2006; Zou et al., 2006). In contrast, the SMS response
mutants, max2, rms4, and d3, were found to be mutated
in an orthologous gene encoding an F-box protein
(Stirnberg et al., 2002; Ishikawa et al., 2005; Johnson
et al., 2006). Recently, the testing of putative carotenoid-
derived compounds led to the discovery that SMS is a
strigolactone or downstream product (Gomez-Roldan
et al., 2008; Umehara et al., 2008). Strigolactones are a
group of related molecules, thought to be derived from
carotenoids (Matusova et al., 2005), that are also in-
volved in promoting arbuscular mycorrhizae symbio-
sis and parasitic weed seed germination (Cook et al.,
1972; Akiyama et al., 2005). It is likely that CCD7 and
CCD8 are enzymes that are involved in the production
of strigolactones, as the mutants were found to be
deficient in strigolactones (Gomez-Roldan et al., 2008;
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009483

Page 3

Umehara et al., 2008). Exogenous strigolactone applied
to buds or supplied to the roots or the vascular stream
was able to rescue the branching phenotype of the ccd7
and ccd8 mutants but not that of the putative SMS
response mutants rms4, max2, and d3 (Gomez-Roldan
et al., 2008; Umehara et al., 2008).
Prior to the discovery of SMS as a strigolactone,
studies with Arabidopsis SMS mutants led to reinter-
pretation of the theories of Sachs and Bangerth and the
establishment of the current auxin transport hypoth-
esis, which proposed that axillary buds compete for
limited auxin transport capacity in the main stem
(Bennett et al., 2006; Ongaro and Leyser, 2008). This is
based on the premise that in order for an axillary bud
to grow it must be able to export auxin and that the
main stem of a wild-type plant is saturated with
apically derived auxin (Ongaro and Leyser, 2008).
A key result in support of the auxin transport
hypothesis is that the excessive branching phenotype
of Arabidopsis SMS mutant plants could be rescued
when grown on medium containing the auxin trans-
port inhibitor N-1-naphthylphthalamic acid (NPA;
Bennett et al., 2006). This indicated that auxin trans-
port may be crucial for branching. Bennett et al. (2006)
and Lazar and Goodman (2006) also demonstrated
that inflorescence stems and rosettes of bolting Arabi-
dopsis SMS mutants had increased expression of
genes encoding PIN-FORMED (PIN) polar auxin
transport efflux proteins, which correlated with in-
creased protein abundance. An excess of transporters
present in the stem at rosette nodes just prior to bud
release may encourage auxin to flow from lateral
sourcesandtriggerbudstocommencegrowth(Bennett
et al., 2006). SMS, therefore, was proposed to act as a
regulator of auxin transport by reducing the expres-
sion and/or plasma membrane localization of auxin
transporters; in the SMS mutants, this inhibition
failed to occur (Bennett et al., 2006). It is important
to note that because SMS is required for inhibition of
decapitation-induced branching by IAA (see above;
Beveridge et al., 2000), auxin must regulate SMS
production and, according to the auxin transport
hypothesis, SMS must then move upward to regulate
auxin transport at vascular connections.
Auxin is known to promote the expression of SMS
synthesis genes (Sorefan et al., 2003; Bainbridge et al.,
2005; Foo et al., 2005; Johnson et al., 2006; Arite et al.,
2007). Decapitation resulted in reduced IAA levels and
decreased expression of SMS synthesis genes (Sorefan
et al., 2003; Foo et al., 2005; Johnson et al., 2006).
Applying IAA to the decapitated plants rescued the
drop in gene expression (Sorefan et al., 2003; Foo et al.,
2005; Johnson et al., 2006). These data suggest that
auxin might regulate strigolactone biosynthesis to
mediate apical dominance (Beveridge, 2006; Dun
et al., 2006).
Because the control of bud outgrowth involves
inputs and regulatory loops from multiple signals,
efforts to unravel the hormone interactions have been
challenging (Dun et al., 2006). In particular, the exact
role of auxin content and transport in the regulation of
bud outgrowth has been questioned (Morris et al.,
2005; Dun et al., 2006; Ferguson and Beveridge, 2009).
Indeed, big and bud1 mutants in Arabidopsis have
reduced auxin transport and enhanced branching (Gil
et al., 2001; Dai et al., 2006), in contrast to Arabidopsis
SMS mutants, which have increased auxin transport
and enhanced branching (Bennett et al., 2006). Addi-
tionally, depletion of auxin content is not always
sufficient to induce bud outgrowth (Morris et al.,
2005; Ferguson and Beveridge, 2009). The recent iden-
tification of SMS provides the crucial missing link to
assess branching control in plants. We report here on
experiments designed to pin down the importance of
auxin in bud outgrowth regulation. We show that
branching induced by auxin depletion in the main
stem following decapitation of pea plants was com-
pletely blocked by strigolactone application and that
strigolactone reduced branching in Arabidopsis auxin
response mutant plants. In addition, the application of
an auxin transport inhibitor to pea buds slowed their
outgrowth, but only after several days, whereas the
response to strigolactone application was rapid. Taken
together with auxin transport experiments, our results
suggest that auxin transport frombuds is not the initial
trigger of bud release, although it may be crucial for
ongoing bud outgrowth, as we have suggested previ-
ously (Dun et al., 2006; Ferguson and Beveridge, 2009).
Rather, it seems likely that auxin promotes strigolac-
tone biosynthesis in the main stem, implying that
strigolactone acts as the classical second messenger in
apical dominance.
RESULTS
Strigolactone Completely Represses Bud Outgrowth
after Decapitation
In addition to causing bud outgrowth, decapitation
has been shown to deplete IAA levels in the main stem
(Foo et al., 2005; Morris et al., 2005), which in turn may
reduce strigolactone levels (Foo et al., 2005; Johnson
et al., 2006). If auxin in the stem inhibits bud out-
growth via strigolactones, then strigolactone applica-
tion to wild-type buds should prevent their outgrowth
regardless of decapitation. Indeed, repeated applica-
tion of GR24 (a synthetic strigolactone; Akiyama et al.,
2005) directly to the uppermost axillary bud of wild-
type pea plants following decapitation completely
inhibited bud outgrowth (Fig. 1, A and B). Untreated
buds at lower nodes grew out as normal (data not
shown; note branches in Fig. 1A). The fact that GR24
can block bud outgrowth after decapitation implies
that apically derived auxin is not required for strigo-
lactone action.
NPA Inhibits Sustained But Not Early Bud Outgrowth
NPA rescues the branching phenotype of SMS mu-
tants of Arabidopsis, which suggests that strigolac-
Brewer et al.
484Plant Physiol. Vol. 150, 2009

Page 4

tones may act similarly to auxin transport inhibitors
(Bennett et al., 2006). To test whether blocking auxin
transport out of a bud inhibits bud outgrowth in a
similar way to strigolactone treatment, we applied
inhibitive quantities of either NPA or GR24 to small
buds of SMS mutant (ccd8) and decapitated wild-type
pea plants and compared outgrowth over time (Fig. 1,
B and C). Buds were treated at a time when the ccd8
buds had not grown in comparison with the wild-type
buds (e.g. at day 0, wild-type buds at node 2 were
0.97 6 0.07 mm and ccd8 buds were 0.81 6 0.03 mm in
length). GR24 treatment completely inhibited bud
growth in ccd8 and decapitated wild-type plants
from the outset, while unexpectedly, comparable
NPA-treated buds grew normally for the initial days
before growth was suppressed compared with control
ccd8 and decapitated wild-type plants. It is possible
that bud swelling (as opposed to actual bud out-
growth) caused by an auxin buildup in NPA-treated
buds could have contributed to the initial increase in
size of NPA-treated ccd8 and decapitated wild-type
buds. However, this is unlikely, as no increase in bud
size was observed in comparable buds of intact wild-
type plants after NPA treatment (Fig. 1D).
Cytokinin application can trigger bud outgrowth
(Sachs and Thimann, 1967) in a similar way to decap-
itation and SMS deficiency (Fig. 1, B–D). Therefore, we
used a cytokinin to induce bud outgrowth in order to
further corroborate the decapitation and SMS mutant
results. A bioactive cytokinin, 6-benzylaminopurine
(BA), was applied with or without NPA to buds of
intact wild-type plants. Again, prior to slowing, buds
treated with BA and NPA grew initially like BA-only-
treated plants (Fig. 1D). These data imply that auxin
transport out of a bud is not required for their initial
growth but instead may be important for sustained
bud growth.
NPA Inhibits Auxin Transport out of Buds
We have shown that applying NPA directly to
axillary buds at the earliest possible stage did not
prevent early outgrowth caused by decapitation, ccd8
mutation, or BA application (Fig. 1). These buds are
too small to assess their polar auxin transport. There-
fore, to confirm that NPA affects auxin transport in
buds and to compare its ability with that of GR24, we
used larger (13.5 6 0.5 mm) growing buds that en-
abled [3H]IAA transport measurements and that still
exhibited a growth inhibition response to GR24 and
NPA treatment (Fig. 2). NPA treatment of these grow-
ing buds caused a 57% reduction in growth 3 d after
treatment, which was very similar to that of GR24-
treated buds of this size (Fig. 2C). We showed that
while the growth of these older buds was inhibited by
GR24 and NPA, only NPA affected the transport of
[3H]IAA supplied in the same solution (Fig. 2, A and
C). Indeed, whereas NPA almost completely blocked
the transport of [3H]IAA in the bud, the profile of
[3H]IAA transport in the GR24-treated buds was very
similar to that of control buds (Fig. 2A). Importantly,
the inhibition of transport by NPA demonstrated that
Figure 1. Effects of GR24 and NPA on initial bud
outgrowth in pea. A, The axillary bud at node 5 of
decapitated wild-type plants grows out when treated
with 0 mM GR24 (left plant) but is inhibited when
treated with 2 mM GR24 at daily intervals for 4 d (right
plant), while untreated buds grow out as normal at
lower nodes of both plants. Photograph was taken 9 d
after decapitation. Abbreviations: B, Bud inhibited at
node 5; Br, branch growing at node 5; D, decapitated
stump. B, Bud length at node 5 of wild-type (Torsdag)
plants that were left untreated (intact) or decapitated
above node 5 and treated with 0 or 2 mM GR24 or
1 mM NPA at daily intervals for 3 d from 13 d old.
Data are means 6 SE (n = 13–14). C, Bud length at
node 2 of rms1-1 (ccd8) plants treated at 9 d old with
either 0 or 2 mM GR24 or 3.4 mM NPA. Data are
means 6 SE (n = 15–16). At day 0, corresponding
wild-type buds at node 2 were 0.97 6 0.07 mm in
length. D, Bud length at node 5 of wild-type (Torsdag)
plants that were treated at 12 d old with 0 or 1 mM
NPA, 500 mM BA, or 1 mM NPA and 500 mM BA
applied to the bud at node 5 or decapitated above
node 5 with 0 mM NPA applied to the bud. Data are
means 6 SE (n = 12). [See online article for color
version of this figure.]
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009485

Page 5

the [3H]IAA measured in the GR24-treated and control
buds was transported in the polar auxin transport
stream. These results support the premise that auxin
transport is important for the growth of these larger
buds but provide no evidence that GR24 affects auxin
transport.
Auxin Addition to Buds Does Not Trigger Bud Release
Classical canalization experiments by Sachs (1969)
demonstrated that increased concentrations of later-
ally applied IAA, relative to the concentration of IAA
applied in the main stem, overcame the “inhibitory
effect” of the main stem and allowed the connection of
lateral vasculature to the vasculature of the main stem.
This was demonstrated by applying IAA to the side of
pea epicotyls and observing the ensuing formation of
vascular connections. Sachs (1981) hypothesized that
this inhibitory effect might also control bud growth. If
the auxin transport capacity of the main stem is a
limiting factor preventing outgrowth of axillary buds
and the same principles are involved, then applying a
relatively high dose of auxin to an axillary bud should
induce its outgrowth. However, we could not induce
any outgrowth in pea buds when we applied a rela-
tively high concentration of IAA (239 mM) to the buds
(control bud length, 1.17 6 0.09 mm, IAA-treated bud
length, 1.18 6 0.06 mm, at 12 d after treatment [n = 15–
16]).
Strigolactone Reduces Branching in Auxin Response
Increased Branching Mutant Plants
Auxin has been shown to promote the expression of
SMS synthesis genes (Sorefan et al., 2003; Bainbridge
et al., 2005; Foo et al., 2005; Johnson et al., 2006; Arite
et al., 2007). As a result, branching mutants defective
in auxin response may have reduced strigolactone
biosynthesis, which may be the cause of their in-
creased branching phenotype. If this is the case, and
auxin acts to repress bud outgrowth primarily by
promoting strigolactone production, then exogenous
strigolactone application should rescue the increased
branching phenotype of this class of mutants. To test
this, we applied GR24 to Arabidopsis axr1 mutant
plants, which are defective in auxin response and
exhibit an increased branching phenotype (Lincoln
et al., 1990). GR24 significantly reduced branching in
axr1 (Fig. 3A; P , 0.0001 by Student’s t test). Moreover,
the 47% reduction in branch number was similar to
that of comparable GR24-treated SMS-deficient plants
(Fig. 3A).
To further investigate the relationship between
auxin response and shoot branching, axr1 and tir1
afb1 afb2 afb3 quadruple auxin response mutant plants
(Dharmasiri et al., 2005b) were grown in plant culture
trays and their shoot-branching responses to GR24
supplied to the roots in the medium were measured
(Fig. 3B). GR24 again significantly reduced branching
in axr1 and also in tir1 afb1 afb2 afb3 mutant plants (P ,
0.001 by Student’s t test). Branching in the Arabidopsis
branched1/teosinte branched1-like1 (brc1/tbl1) mutant,
which is mutated in a gene encoding a TCP transcrip-
tion factor that is thought to function downstream
of auxin and SMS perception (Aguilar-Martı ´nez et al.,
2008; Finlayson, 2008), was not reduced by GR24
(Fig. 3B).
That GR24 was able to reduce branching in plants
with defective response to auxin implies that one
function of auxin is upstream of strigolactone in
shoot-branching regulation. Since strigolactone can
act when auxin response is defective, these results
demonstrate that strigolactone action does not require
auxin response to function and therefore might act
downstream of auxin. These results also support the
idea that auxin acts to inhibit bud outgrowth at least in
part by promoting strigolactone production.
Intact SMS Mutant Shoots Do Not Show Enhanced
Auxin Transport Capacity
Experiments by Sachs (1968, 1969) led to the idea
that auxin saturation in the stem vasculature may act
to directly block bud outgrowth. This hypothesis was
recently expanded to explain the increased branching
Figure 2. Effects of NPA and GR24 on auxin transport and bud growth.
A, [3H]IAA transport in axillary buds of pea. Growing buds at node 4 of
20-d-old rms1-1 (ccd8) plants were treated with solution containing
[3H]IAA and 0 or 10 mM GR24 or 1 mM NPA. Bud internode tissue
below the shoot tip of the axillary bud and above the leaf axil was
harvested into 1.57-mm segments and radioactivity was quantified.
Data are means 6 SE (n = 8). B, Photograph of a treated growing axillary
bud prior to harvest. Bar = 1 cm. C, Bud growth of axillary buds at node
4 of rms1-1 (ccd8) plants measured 3 d after treatment with solution
containing 0 or 10 mM GR24 or 1 mM NPA. Data are means 6 SE (n =
13–15). [See online article for color version of this figure.]
Brewer et al.
486Plant Physiol. Vol. 150, 2009

Page 6

phenotype of Arabidopsis SMS mutants. These mu-
tants are reported to have increased expression and
localization of auxin transport carriers and increased
auxin transported in segments of their inflorescences
(Bennett et al., 2006). As a result, it was suggested that
the increased branching phenotypes of these mutants
were due to their auxin transport streams not being
saturated (Bennett et al., 2006; Ongaro and Leyser,
2008).
We tested this auxin transport hypothesis by apply-
ing increasing concentrations of unlabeled IAA,
spiked with fixed amounts of [3H]IAA, to the shoot
apex of young intact pea plants or the inflorescence
apex of young intact Arabidopsis plants. As shown
previously for pea, this treatment led to a wave of IAA
that moved down the stem at the speed of polar auxin
transport (1 cm h21; Fig. 4, A–E; Morris et al., 2005;
Ferguson and Beveridge, 2009). NPA applied in a ring
around the stem of pea plants prevented the move-
ment of this wave, indicating that the IAA applied
moved in the polar auxin transport stream (Fig. 5).
Following the same application method, IAA trans-
ported in Arabidopsis inflorescences also moved in a
distinct wave traveling at approximately 1 cm h21(Fig.
4, F and G).
Having established this auxin transport method for
inflorescences of intact Arabidopsis plants, compari-
sons of auxin transport were made between the wild
type and SMS synthesis branching mutants of both
species (ccd8 of pea and ccd7 of Arabidopsis) for
different auxin concentrations. For each species, the
lowest concentration was chosen to only marginally
increase the auxin supply to the stem. However, the
fact that we were able to get any exogenous auxin into
the polar auxin transport stream of wild-type plants
indicated that it was not saturated and therefore not
functioning at full capacity. When 0.14 to 0.6 mM (50–
210 ng) IAA was applied, there was no difference in
IAA transport between wild-type and ccd8 mutant pea
plants, and the profiles were similar to those at the
weakest concentration of applied IAA (0.023 mM; Fig.
4, A–C). Application of these concentrations provided
an additional 25% to 200% of the total endogenous
IAA content of about 0.41 ng across the whole segment
(Beveridge et al., 2000; Morris et al., 2005). At 2.9 mM
(1,007 ng) applied IAA, ccd8 mutants showed a clear
peak of auxin transport, similar to that observed using
lower IAA concentrations. While the wild type
showed a peak front at this same position, it had
greater amounts of IAA than the ccd8 mutant closer to
the shoot tip. This enhancement behind the peak front,
which presumably also occurred to some extent in the
mutant, may have been due somewhat to IAA diffu-
sion. However, the total amount of IAA taken up and
transported at this concentration was also greater in
the wild type than in the ccd8 mutant (Fig. 4J), which is
not easily explained by diffusion. Similarly, when 14
mM (5,000 ng) IAA was applied, the ccd8 mutant was
not able to transport more IAA than the wild type and
the peak front still occurred at the same position as for
other concentrations (Fig. 4, A–E and J). In this case,
the amount of IAA taken up and transported in the
peak front was about seven to eight times that of
the endogenous IAA across the whole segment. That
the position of the peak front was not different across a
wide range of IAA concentrations suggests that the
majority of this IAA was likely in the polar auxin
transport stream. These data suggest that pea stems
are not normally saturated and instead have the ca-
pacity to rapidly respond to, and transport, additional
quantities of IAA. Moreover, the pea ccd8 mutant does
not appear to have enhanced auxin transport proper-
ties.
Using a range of lower IAA concentrations suited to
the small Arabidopsis inflorescences, similar findings
were detected for Arabidopsis as for pea in that wild-
type and ccd7 inflorescence shoots could load and
transport exogenously applied IAA. Moreover, when
23 to 250 mM (4–44 ng) IAA was applied, little differ-
ence in IAA transport was observed between ccd7
mutant and wild-type shoots (Fig. 4, G–I). At the
lowest IAA concentration (2.3 mM), we repeated pre-
viously reported findings with isolated segments that
Figure 3. GR24 reduces bud outgrowth in increased branching auxin
responsemutantsof Arabidopsis.A, Treatmentsof 0 or 5 mM GR24 were
applied to the rosette axillary buds and leaf axils of wild-type (WT),
max3-11 (ccd7), max4-1 (ccd8), and axr1-3 plants. Data are means 6
SE (n = 16–20). B, Wild-type, axr1-3, tir1-1 afb1-1 afb2-1 afb3-1 (tir1-q),
and brc1-2/tbl1-1 (brc1 in figure; SALK 091920) plants were grown in
Phytatrays with 0 or 5.8 mM GR24. Data are means 6 SE (n = 8–19).
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009487

Page 7

SMS mutant inflorescences can load and transport
moreIAA than the wild type (Fig. 4, Fand I).However,
the findings at higher concentrations of applied IAA
clearly demonstrate that the lack of branching exhib-
ited by wild-type pea and Arabidopsis plants is not
directly related to saturation of their auxin transport
streams, as both species can take up and transport
additional IAA (Fig. 4).
Interestingly, for both species, the total amount of
IAA transported relative to the amount applied
steadily decreased with increasing concentration
(Fig. 4, I and J). For example, when 0.023 mM IAA
was applied to wild-type pea apices, 12% was trans-
ported, compared with only 1.4% transported when 14
mM was applied. Comparable percentage reductions
were observed here using intact Arabidopsis inflores-
cences, and similar findings have been reported pre-
viously using pea cuttings (Baadsmand and Andersen,
1984). This implies that the main mechanism for
loading and/or transporting IAA is not diffusion,
because the higher concentrations should not have
reduced the percentage uptake. Indeed, the lack of a
clear change in the rate of transport of the peak front at
different concentrations of applied IAA implies that
diffusion is not an important variable for at least this
part of the auxin wave (for review, see Kramer, 2008).
Independent experiments showed that IAA appli-
cation to the stem of intact plants had no effect on the
Figure 4. SMS mutants and wild-type plants have the
capacity to transport high levels of IAA. A to E, IAA in
segments of wild-type (WT) and rms1-1 (ccd8) pea
plants 4 h after treatment with mixtures of IAA and
[3H]IAA to a total of 0.023 mM (A), 0.14 mM (B), 0.6
mM (C), 2.9 mM (D), and 14 mM (E) IAA. F to H, IAA in
segments of wild-type and max3-11 (ccd7) Arabi-
dopsis plants 2.5 h after treatment with mixtures of
IAA and [3H]IAA to a total of 2.3 mM (F), 23 mM (G),
and 250 mM (H) IAA. I, Total auxin transported in
wild-type and max3-11 (ccd7) Arabidopsis plants. J,
Total auxin transported in wild-type and rms1 (ccd8)
pea plants.Dataare means6 SE (n = 4 [A–Eand J] and
n = 5 [F–I]). The endogenous IAA in the stem of pea at
about 3 cm from the shoot tip is about 0.4 ng per
segment (Morris et al., 2005).
Brewer et al.
488Plant Physiol. Vol. 150, 2009

Page 8

growth of buds below. Whereas the IAA (3 mg g21)
applied in a lanolin ring to an expanding internode of
ccd8 pea plants was absorbed and stimulated elonga-
tion of the internode (data not shown), the length of
the bud below was unaffected by the auxin treatment.
The mean bud lengths were 5.2 6 2.1 cm and 6.2 6 1.4
cm in control and auxin-treated plants, respectively
(17 d after treatment [n = 9]; plants in the Parvus
background had six leaves expanded at the time of
treatment). This supports the idea that additional
auxin transported in the main stem does not prevent
bud outgrowth in the absence of strigolactone pro-
duction in mutant plants.
DISCUSSION
The current auxin transport hypothesis promotes
the idea that SMS acts upstream of auxin by regulating
PIN-dependent auxin transport in the stem (Bennett
et al., 2006). In contrast, strigolactone completely
inhibited decapitation-induced bud outgrowth in pea
(Fig. 1, A and B), supporting the idea that strigolactone
actually functions downstream of auxin in the main
stem in its regulation of bud outgrowth in decapitated
plants (Fig. 6). This nicely fits the classical second
messenger theory of apical dominance (Snow, 1929,
1937) and is consistent with our previous models
of shoot branching (Beveridge, 2000; Ferguson and
Beveridge, 2009). Indeed, this apical dominance ex-
periment showed that any changes in auxin level,
movement, or signaling caused by removalof the main
auxin supply in the shoot in no way prevented the
inhibition of bud outgrowth by strigolactone applica-
tion to buds. Any action of strigolactone itself in buds,
therefore, seems to be independent of auxin content in
the stem.
Previous studies have shown that auxin positively
regulates SMS synthesis gene expression (Sorefan
et al., 2003; Bainbridge et al., 2005; Foo et al., 2005;
Johnson et al., 2006; Arite et al., 2007), also suggesting
that strigolactones could function downstream of
auxin to inhibit bud outgrowth. Auxin regulation of
the expression of the SMS synthesis gene CCD8 was
demonstrated to be AXR1 dependent in Arabidopsis
(Bainbridge et al., 2005). Direct application of GR24 to
the buds of the Arabidopsis auxin response mutant
axr1 reduced its increased branching phenotype by a
similar magnitude as SMS-deficient plants (Fig. 3).
Growth of axr1 and tir1 afb1 afb2 afb3 auxin response
mutant plants in medium containing GR24 also led to
an inhibition of shoot bud outgrowth, whereas the
tbl1/brc1 mutant, which is thought to act downstream
of strigolactone response (Aguilar-Martı ´nez et al.,
2008; Finlayson, 2008), showed no response to strigo-
lactone treatment, as expected (Fig. 3B). This implies
that the auxin responsemutants may branch, at least in
part, due to endogenous strigolactone depletion and
that auxin response is not necessarily required for
strigolactone to inhibit shoot branching. The reduced
branching in GR24-treated tir1 afb1 afb2 afb3 quadruple
mutant plants suggests that AXR1 regulation of stri-
golactone biosynthesis might be mediated by the
SCFTIR1/AFBubiquitin ligase complex, which functions
as an auxin receptor and targets proteins for ubiquiti-
nation (Dharmasiri et al., 2005a, 2005b; Kepinski and
Leyser, 2005). Therefore, we propose that one role for
auxin in mediating apical dominance is through auxin
inducing the expression of strigolactone biosynthesis
genes (Fig. 6). This could occur in vascular cambial
Figure 5. [3H]IAA transport is NPA sensitive in pea plants. Values
shown are per segment of wild-type (WT) and rms1-1 (ccd8) pea stems
treatedwith a lanolin ring containing either 0 (control)or 1 mg g21NPA
and measured 4 h after treatment with [3H]IAA. The arrow indicates the
position of the lanolin ring 10 mm below the oldest unexpanded leaf
applied at 2 h before treatment with [3H]IAA.
Figure 6. Pathway for auxin and strigolactone action in regulating axillary bud growth. Arrows represent promotion, while flat-
ended lines represent inhibition. Auxin promotes strigolactone biosynthesis gene expression. The MAX2/RMS4 F-box protein is
requiredfor strigolactoneinhibitionof bud release.Auxin transport out of an axillarybud, whichcanbe inhibitedbyNPA, is then
required for a bud that has been released to proceed to sustained bud growth.
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009489

Page 9

cells, through which auxin is transported down the
stem (Morris and Thomas, 1978). AXR1 and strigolac-
tone biosynthesis genes are indeed known to be ex-
pressed in those cells (Booker et al., 2003, 2005; Sorefan
et al., 2003). Whereas AXR1 may have additional
targets, TIR1 is thought to more directly affect auxin
responses. Once technologies are available, strigolac-
tone content should be measured in axr1 and tir1 afb1
afb2 afb3 shoots.
It is unlikely that the increased branching pheno-
type of axr1 and possibly tir1 afb1 afb2 afb3 quadruple
mutant plants is due entirely to strigolactone deple-
tion. It is likely that auxin response mutants have
increased cytokinin content due to reduced auxin
regulationofcytokinin
et al., 2004; Bennett et al., 2006). This may contribute
to the additive branching phenotype of axr1 and SMS
double mutant plants and, possibly in combination
with impaired feedback signaling (Dun et al., 2006), to
the poor response of axr1 shoots to grafting with wild-
type rootstocks. Auxin signaling might also be in-
volved in other aspects of bud outgrowth regulation,
particularly the continued elongation of the growing
bud once it has been released to grow. Nevertheless,
whatever the action of auxin and strigolactone, strigo-
lactone inhibition of branching in auxin response
mutants suggests that strigolactone function is at least
partly downstream of auxin and auxin response, con-
sistent with the second messenger role for strigolac-
tone.
Sachs (1969) demonstrated that a high concentration
of IAA in the stem repelled the formation of new
vascular connections from lateral sources. It was pro-
posed that auxin is at a saturation point in the stem
and, therefore, that there would be no more room in
the transport stream for lateral auxin to enter (Sachs,
1981). However, when a greater concentration of IAA
was applied to a lateral point, the repulsion by the
main stem could be overcome (Sachs, 1969). Indeed,
consistent with canalization theory, auxin was shown
to promote its own efflux by enhancing PIN activity
(Paciorek et al., 2005). However, direct application of
IAA did not induce bud outgrowth in wild-type pea.
This supports an earlier experiment where IAA ap-
plied to pea buds after decapitation actually blocked
outgrowth (Thimann, 1937). Note that the amount we
chose for this experiment is equivalent to a mid-range
application, in order to provide an amount signifi-
cantly higher than endogenous levels (Fig. 4J).
Based on differences between PIN protein abun-
dance in the wild type and SMS-deficient mutants,
Bennett et al. (2006) suggested that reduced PIN
abundance in the main stem leads to saturation of
the auxin transport stream and prevents bud out-
growth in wild-type plants, whereas the main stem of
SMS mutants is not saturated by auxin. Although
increased amounts of IAA were transported in intact
ccd7 mutant shoots of Arabidopsis compared with the
wild type at very low concentrations of applied IAA,
this did not occur in ccd8 mutants of pea compared
biosynthesis(Nordstro ¨m
with the wild type and was not observed for the high-
concentration applications in either species (Fig. 4).
Moreover, we found that in pea and Arabidopsis,
wild-type and SMS mutant plants could instantly
transport additional exogenous IAA in the main
stem and SMS mutants could not take up and trans-
port excess IAA above the level of the wild type when
high IAA concentrations were applied (Fig. 4). To-
gether with observing the appropriate rate for polar
auxin transport (1 cm h21) at different concentrations,
we used localized NPA treatment below the shoot tip
of pea to show that the applied auxin was indeed
moving in the polar auxin transport stream (Fig. 5).
Altogether, this demonstrates that the auxin transport
stream in the main stem of wild-type pea and Arabi-
dopsis plants is not functioning at saturation and that
SMS synthesis mutants do not have increased capacity
to transport higher levels of IAA. Therefore, it is
unlikely that overcoming saturation of the main stem
auxin transport stream is involved in triggering bud
outgrowth.
Arabidopsis and pea have the ability to transport
excess IAA, which, for example, may act as a buffer
duringcircadianfluctuationsinauxinlevels(Covington
and Harmer, 2007) or when new primordia or flower
buds produce and transport new auxin into the polar
auxin transport stream (Benkova ´ et al., 2003). More-
over, although a small difference in IAA transport was
observed between wild-type and ccd7 Arabidopsis
inflorescences when low concentrations of IAA were
applied (Fig. 4, F and I), it is unlikely that this is a
primary cause for branching in plants, because such a
difference is not always observed in pea (Figs. 4, A–E
and J, and 5; Beveridge et al., 2000). Multiple repeat
experiments found no difference between wild-type
and SMS mutant pea plants in the transport of low
concentrations of IAA (data not shown). One would
expect the primary mechanism of strigolactone func-
tion to be conserved across plant species, especially
due to high conservation of the biosynthetic pathway
and its regulation by auxin (Fig. 6; Sorefan et al., 2003;
Bainbridge et al., 2005; Foo et al., 2005; Zou et al., 2006;
Arite et al., 2007; Gomez-Roldan et al., 2008; Umehara
et al., 2008). Interestingly, bud vascular traces were
found to be repelled from leaf vasculature in the
Arabidopsis ccd8 mutant background (Ongaro et al.,
2008). This may indicate that the production of auxin
in young leaves is increased in the SMS mutants, but it
does not distinguish whether this is a cause, or feed-
back consequence, of the branching phenotype. It is
likely that higher endogenous auxin levels, caused by
feedback regulation of auxin content in a failed at-
tempt to synthesize more SMS to inhibit branching,
has led to these vasculature differences and an in-
crease in PIN abundance in ccd mutants of Arabidop-
sis (Dun et al., 2006).
If auxin transport out of a bud is critical for trigger-
ing bud release, then an auxin transport inhibitor like
NPA should completely block bud outgrowth from the
earliest stage. When we applied NPA to buds of an
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490Plant Physiol. Vol. 150, 2009

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SMS synthesis mutant of pea, ccd8, we could not
phenocopy the early bud repression shown by GR24
(Fig. 1C). While GR24 application prevented bud
outgrowth in pea ccd8 mutants, NPA application
allowed early bud growth to occur, only inhibiting
the sustained bud growth that occurred in the control
ccd8 plants (Fig. 1C). This was also the case for buds of
decapitated wild-type pea plants (Fig. 1B). In addition,
NPA was unable to inhibit early bud outgrowth in-
duced by the cytokinin, BA (Fig. 1D). In contrast, NPA
seemed to slow bud growth only after several days
(Fig. 1, B–D). The early outgrowth seen in NPA-treated
buds (Fig. 1, B–D) was not due to swelling induced by
auxin accumulation, because intact wild-type NPA-
treated buds did not grow at all (Fig. 1D). In addition,
the later inhibitory effect on continued bud growth
was not due to NPA affecting strigolactone via the loss
of auxin in the stem, as ccd8 mutants are in any case
unable to produce strigolactone and decapitated
plants have had their major auxin supply removed.
The different outgrowth responses of buds treated
with either NPA or GR24 are also not due to GR24
being more efficient at inhibiting auxin transport, as
GR24 treated together with [3H]IAA had no effect on
the transport of [3H]IAA in a growing bud, yet NPA
did (Fig. 2). At this later developmental stage, both
treatments had a similar effect on bud outgrowth, re-
ducing it by about half (Fig. 2). By assessing [3H]IAA
transport from the main shoot tip, we have observed
that NPA remains effective at blocking [3H]IAA trans-
port for at least 7 d (data not shown), indicating that
the initial NPA treatment applied to tiny buds could
potentially have remained active after several days.
Apart from other possible side effects of NPA, these
data suggest that normal auxin transport may only be
required for ongoing bud growth rather than being the
initial trigger of bud release (Fig. 6; Morris et al., 2005;
Dun et al., 2006) and that strigolactone and NPA act
quite differently. This also provides an explanation for
strigolactone acting downstream of auxin, even
though NPA reduces branch lengths (Fig. 1; Bennett
et al., 2006).
Early stages of bud outgrowth stimulated by re-
duced strigolactone signaling would lead to enhanced
primordium development and growth of the bud,
increasing auxin supply and export to stems. What-
ever the precise action of strigolactone in preventing
the initial bud release, an outcome is that auxin trans-
port from inhibited buds will be reduced compared
with growing buds. Once growing, buds synthesize
auxin (Gocal et al., 1991) and its export may enhance
vascular connections and nutrient flow to further
stimulate the growing bud. Consequently, NPA sup-
presses continued bud growth (Bennett et al., 2006) but
does not suppress the earliest bud growth. An out-
come of the failure to inhibit bud release under
strigolactone deficiency in SMS mutants or decapi-
tated plants that have greatly suppressed CCD7 and
CCD8 gene expression is that stem auxin level can
increase along with enhanced auxin transport. This
hypothesis is consistent with previous findings that
cytokinin triggered bud release while auxin promoted
the subsequent elongation of buds (Sachs and
Thimann, 1967). What remains to be seen is how
strigolactones might interact with cytokinin to regu-
late shoot branching, especially since auxin and stri-
golactone deficiency is not always sufficient to
promote bud outgrowth unless cytokinin biosynthesis
genes are activated (Ferguson and Beveridge, 2009).
CONCLUSION
Our findings support previous ideas that auxin
induces the production of a second messenger to
regulate bud outgrowth. We propose that strigolactone
could act as a second messenger for auxin action and
that this messenger directly represses bud outgrowth
(Fig. 6). Auxin export from buds, however, seems to be
critical for ongoing bud growth, rather than as the
initial trigger (Fig. 6). In this case, it seems that auxin
has two actions: one is the involvement of auxin levels
in regulating bud release, while the other is via auxin
transport being necessary for sustained bud growth.
MATERIALS AND METHODS
Plant Material, Growth Conditions, and Treatments
For all garden pea (Pisum sativum) experiments, plant growth conditions
were as described by Ferguson and Beveridge (2009), except that plants were
sometimes grown in 9:1 composted fine slash:medium river sand potting mix
(Bassett Barks). Nodes were numbered acropetally from the first scale leaf,
and lengths of lateral buds and branches were recorded using digital calipers.
For Arabidopsis (Arabidopsis thaliana) experiments, unless otherwise stated,
plants were grown as reported by Gomez-Roldan et al. (2008).
For bud application studies in pea (Fig. 1), solutions of 5 mL contained 2%
polyethylene glycol 1450, 50% ethanol, 0.2% to 1% acetone, and 0.2% to 0.5%
dimethyl sulfoxide (DMSO). For Figure 1C, axillary buds at nodes 1 and 3
were removed to encourage the growth of the bud at node 2.
For direct applications in Arabidopsis, solutions were given in 0.1% Tween
20. For Arabidopsis root treatments, plants were germinated and grown in
plant culture trays (Phytatray II; Sigma) containing 0 or 5.8 mM GR24 in
standard Arabidopsis Murashige and Skoog growth medium at 24?C in a
growth chamber (Conviron) with fluorescent lighting and 18-h daylength. The
number of rosette branches longer than 5 mm was counted when the plants
were 63 d old.
IAA Transport in the Main Stem and Overloading of the
Polar Auxin Transport Stream
IAA transport in the main stem and the overloading of the polar auxin
transport stream were analyzed using methods similar to those outlined by
Beveridge et al. (2000) and Morris et al. (2005). For pea experiments, various
concentrations of IAA, each containing 34 kBq [3H]IAA (American Radiola-
beled Chemicals; specific activity, 20 Ci mmol21), were dissolved in 50%
ethanol. Total IAA concentrations of the solutions were 0.023 mM (8.04 ng),
0.14 mM (49.74 ng), 0.6 mM (209.04 ng), 2.9 mM (1,007.04 ng), and 14 mM
(4,998.04 ng). Two microliters of these solutions was applied to the apical bud
of 19-d-old wild-type (cv Parvus [L77]) or rms1-1 (ccd8) mutant (WL5237)
plants having seven leaves fully expanded. The radiolabel was taken up and
transported over a 4-h period. Following this, the internode tissue beginning
directly below the apical region was harvested from individual plants and
divided into 3-mm equal-length sections.
The same method was used to analyze IAA transport in the main
inflorescence stem of Arabidopsis wild-type and max3-11 (ccd7) plants (Co-
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009491

Page 11

lumbia ecotype), except that the total IAA concentrations of the solutions were
2.3 mM (0.4 ng; including 1.85 kBq [3H]IAA mL21), 23 mM (4 ng), and 250 mM (44
ng; including 18.5 kBq [3H]IAA mL21), only 1 mL of the solution was applied to
apices of 4-week-old, newly bolted plants, and the radiolabel was transported
over a 2.5-h period.
Radioactivity was extracted directly from the segments of individual
samples in 2 mL of Ultima Gold liquid scintillant (Perkin-Elmer Life and
Analytical Sciences) gently shaken overnight as outlined by Morris et al.
(2005). Radioactivity was analyzed using a Packard Tricarb 1600 TR Liquid
Scintillation Analyzer (Packard Instruments) and recorded as dpm. For pea,
dpm was converted to ng of IAA based on 1 dpm being equivalent to 3.95 fg of
[3H]IAA. For Arabidopsis, dpm was converted to ng of IAA based on the dpm
readings from samples of known ng of [3H]IAA applied to each plant. Total
IAA transported was calculated from segments 0.6 to 4.5 cm (Arabidopsis)
and 0.6 to 9.0 cm (pea) from the shoot apex based on the assumption that the
same percentages of [3H]IAA and IAA were transported.
IAA Transport in NPA-Treated Plants
IAA transport in the main stem of NPA-treated plants was measured as in
the overloading experiment, except that a ring of lanolin containing either 0 or
1 mg g21NPA with 4 mL g21DMSO and 100 mL g21ethanol was applied
around the uppermost expanding internode, 10 mm below the oldest
unexpanded leaf, of 17-d-old wild-type (Parvus [L77]) and rms1-1 (ccd8)
mutant (WL5237) pea plants. Two hours after treatments were applied in
lanolin, 17 kBq [3H]IAA was applied to the apical bud and the resulting
transport was measured as described, except using 5-mm stem segments.
IAA Transport out of Axillary Bud Apices
rms1-1 (WL5237) plants were grown for 9 d before axillary buds were
removed from nodes 1 and 2 to encourage the growth of the axillary buds at
upper nodes. When the plants were 20 d old, plants with a growing bud at
node 4 that was 10 to 20 mm in length were selected. These buds had basal
internodes of 8 to 17 mm in length. Two microliters of solution containing 14.8
kBq (3.5 ng) of [3H]IAA, 1% acetone, 0.15% DMSO, 50% ethanol, and either 0
or 10 mM GR24 or 1 mM NPAwas applied to the bud inside the stipules of the
first two nodes of the unexpanded leaves. Plants were also treated, but
without [3H]IAA, for bud outgrowth measurements. [3H]IAA transport was
measured as described above, except the treatments were left for 1 h, after
which the bud internode was harvested into 1.57-mm segments.
ACKNOWLEDGMENTS
We thank A/Prof. Dolf Weijers for the kind gift of tir1-1 afb1-1 afb2-1 afb3-1
seeds, Alice Hayward for the kind gift of axr1-3 seeds, and the Salk Institute
Genomic Analysis Laboratory and the Nottingham Arabidopsis Stock Centre
for supplying brc1-2/tbl1-1 seeds. Special thanks to Dr. Marjolein McDonald
for developing experiments to test auxin transport capacity, Kerry Condon,
Shannon Dollery, and Heather Vickstrom for assistance with experiments,
and Dr. John Ross and Alice Hayward for comments on the manuscript.
Received December 23, 2008; accepted March 23, 2009; published March 25,
2009.
LITERATURE CITED
Aguilar-Martı ´nez JA, Poza-Carrio ´n C, Cubas P (2008) Arabidopsis
BRANCHED1 acts as an integrator of branching signals within axillary
buds. Plant Cell 19: 458–472
Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes induce
hyphal branching in arbuscular mycorrhizal fungi. Nature 435: 824–827
Ali A, Fletcher RA (1970) Xylem differentiation in inhibited cotyledonary
buds of soybeans. Can J Bot 48: 1139–1140
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
Azpiroz R, Wu Y, LoCascio JC, Feldmann KA (1998) An Arabidopsis
brassinosteroid-dependent mutant is blocked in cell elongation. Plant
Cell 10: 219–230
Baadsmand S, Andersen AS (1984) Transport and accumulation of indole-
3-acetic acid in pea cuttings under two levels of irradiance. Physiol Plant
61: 107–113
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 (1989) Dominance among fruits/sinks and the search for a
correlative signal. Physiol Plant 76: 608–614
Bangerth F, Li CJ, Gruber J (2000) Mutual interaction of auxin and
cytokinins in regulating correlative dominance. Plant Growth Regul
32: 205–217
Benkova ´ E, Michniewicz M, Sauer M, Teichmann T, Seifertova ´ D,
Ju ¨rgens G, Friml J (2003) Local, efflux-dependent auxin gradients as a
common module for plant organ formation. Cell 115: 591–602
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 Biol 16: 553–563
Beveridge CA (2000) Long-distance signalling and a mutational analysis of
branching in pea. Plant Growth Regul 32: 193–203
Beveridge CA (2006) Axillary bud outgrowth: sending a message. Curr
Opin Plant Biol 9: 35–40
Beveridge CA, Ross JJ, Murfet IC (1994) Branching mutant rms-2 in Pisum
sativum: grafting studies and endogenous indole-3-acetic acid levels.
Plant Physiol 104: 953–959
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 (1997) 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 signal(s). Plant Physiol 115: 1251–1258
Beveridge CA, Symons GM, Turnbull CGN (2000) Auxin inhibition of
decapitation-induced branching is dependent on graft-transmissible
signals regulated by genes Rms1 and Rms2. Plant Physiol 123: 689–697
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, Chatfield S, Leyser O (2003) Auxin acts in xylem-associated or
medullary cells to mediate apical dominance. Plant Cell 15: 495–507
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
produce a carotenoid-derived branch-inhibiting hormone. Dev Cell 8:
443–449
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 Chem Soc 94: 6198–6199
Covington MF, Harmer SL (2007) The circadian clock regulates auxin
signaling and responses in Arabidopsis. PLoS Biol 5: 1773–1784
Dai Y, Wang H, Li B, Huang J, Liu X, Zhou Y, Mou Z, Li Y (2006) Increased
expression of MAP KINASE KINASE7 causes deficiency in polar auxin
transport and leads to plant architectural abnormalities in Arabidopsis.
Plant Cell 18: 308–320
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, Lechner E, Yamada M, Hobbie L,
Ehrismann JS, Ju ¨rgens G, Estelle M (2005b) Plant development
is regulated by a family of auxin receptor F box proteins. Dev Cell 9:
109–119
Dun EA, Ferguson BJ, Beveridge CA (2006) Apical dominance and shoot
branching: divergent opinions or divergent mechanisms? Plant Physiol
142: 812–819
Ferguson BJ, Beveridge CA (2009) Roles for auxin, cytokinin, and strigo-
lactone in regulating shoot branching. Plant Physiol 149: 1929–1944
Finlayson SA (2008) Arabidopsis TEOSINTE BRANCHED1-LIKE 1 regu-
lates axillary bud outgrowth and is homologous to monocot TEOSINTE
BRANCHED1. Plant Cell Physiol 48: 667–677
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, Turnbull CGN, Beveridge CA (2001) Long-distance signalling
and the control of branching in the rms1 mutant of pea. Plant Physiol 12:
203–209
Brewer et al.
492Plant Physiol. Vol. 150, 2009

Page 12

Gil P, Dewey E, Friml J, Zhao Y, Snowden KC, Putterill J, Palme K, Estelle
M, Chory J (2001) BIG: a calossin-like protein required for polar auxin
transport in Arabidopsis. Genes Dev 15: 1985–1997
Gocal GFW, Pharis RP, Yeung EC, Pearce D (1991) Changes after decap-
itation in concentrations of indole-3-acetic acid and abscisic acid in the
larger axillary bud of Phaseolus vulgaris L. cv Tender Green. Plant
Physiol 95: 344–350
Gomez-Roldan V, Fermas S, Brewer PB, Puech-Page `s 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
Hall SM, Hillman JR (1975) Correlative inhibition of lateral bud growth in
Phaseolus vulgaris L.: timing of bud growth following decapitation.
Planta 123: 137–143
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 X, Brcich T, Dun EA, Goussot M, Haurogne ´ 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
Kramer EM (2008) Computer models of auxin transport: a review and
commentary. J Exp Bot 59: 45–53
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
Lincoln C, Britton JH, Estelle M (1990) Growth and development of the
axr1 mutants of Arabidopsis. Plant Cell 2: 1071–1080
Marr C, Blaser HW (1967) The origin and development of induced
branches in Helianthus. Am J Bot 54: 498–504
Matusova R, Rani K, Verstappen FWA, Franssen MCR, Beale MH,
Bouwmeester HJ (2005) The strigolactone germination stimulants of
the plant-parasitic Striga and Orobanche spp. are derived from the
carotenoid pathway. Plant Physiol 139: 920–934
Morris DA (1977) Transport of exogenous auxin in two-branched dwarf
pea seedlings (Pisum sativum L.). Planta 136: 91–96
Morris DA, Thomas AG (1978) A microautoradiographic study of auxin
transport in the stem of intact pea seedlings (Pisum sativum L.). J Exp Bot
29: 147–157
Morris SE, Cox MCH, Ross JJ, Kristantini S, Beveridge CA (2005) Auxin
dynamics after decapitation are not correlated with the initial growth of
axillary buds. Plant Physiol 138: 1665–1672
Napoli CA (1996) Highly branched phenotype of the petunia dad1-1 mutant
is reversed by grafting. Plant Physiol 111: 27–37
Nordstro ¨m A, Tarkowski P, Tarkowska D, Norbaek R, A˚stot C, Dolezal K,
Sandberg G (2004) Auxin regulation of cytokinin biosynthesis in Arabi-
dopsis thaliana: a factor of potential importance for auxin-cytokinin-
regulated development. Proc Natl Acad Sci USA 101: 8039–8044
Ongaro V, Bainbridge K, Williamson L, Leyser O (2008) Interactions
between axillary branches of Arabidopsis. Mol Plant 1: 388–400
Ongaro V, Leyser O (2008) Hormonal control of shoot branching. J Exp Bot
59: 67–74
Paciorek T, Zazı ´malova ´ E, Ruthardt N, Petra ´sek J, Stierhof YD, Kleine-Vehn
J, Morris DA, Emans N, Ju ¨rgens G, Geldner N, et al (2005) Auxin inhibits
endocytosis and promotes its own efflux from cells. Nature 435: 1251–1256
Peterson CA, Fletcher RA (1973) Apical dominance is not due to a lack of
functional xylem and phloem in inhibited buds. J Exp Bot 24: 97–103
Prasad TK, Li X, Abdel-Rahman AM, Hosokawa Z, Cloud NP, LaMotte
CE, Cline MG (1993) Does auxin play a role in the release of apical
dominance by shoot inversion in Ipomoea nil? Ann Bot (Lond) 71: 223–229
Sachs T (1968) On the determination of the pattern of vascular tissue in
peas. Ann Bot (Lond) 32: 781–790
Sachs T (1969) Polarity and the induction of organised vascular tissues.
Ann Bot (Lond) 3: 263–275
Sachs T (1970) A control of bud growth by vascular tissue differentiation.
Israel J Bot 19: 484–498
Sachs T (1981) The control of the patterned differentiation of vascular
tissues. Adv Bot Res 9: 151–262
Sachs T, Thimann KV (1967) The role of auxins and cytokinins in the
release of buds from dominance. Am J Bot 54: 136–144
Shimizu S, Mori H (1998) Analysis of cycles of dormancy and growth in
pea axillary buds based on mRNA accumulation patterns of cell cycle-
related genes. Plant Cell Physiol 39: 255–262
Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin-cytokinin interactions in
the control of shoot branching. Plant Mol Biol 69: 429–435
Snow R (1929) The transmission of inhibition through dead stretches of
stem. Ann Bot (Lond) 43: 261–267
Snow R (1937) On the nature of correlative inhibition. New Phytol 36:
283–300
Snowden KC, Simkin AJ, Janssen BJ, Templeton KR, Loucas HM, Simons
JL, Karunairetnam S, Gleave AP, Clark DG, Klee HJ (2005) The
Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE
DIOXYGENASE8 gene affects branch production and plays 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 S, Ward S, Beveridge C, 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
Sorokin HP, Thimann KV (1964) The histological basis for inhibition of
axillary buds in Pisum sativum and the effects of auxins and kinetin on
xylem development. Protoplamsa 59: 326–349
Stafstrom JP, Sussex IM (1992) Expression of a ribosomal protein gene in
axillary buds of pea seedlings. Plant Physiol 100: 1494–1502
Stirnberg P, van de Sande K, Leyser HMO (2002) MAX1 and MAX2 control
shoot lateral branching in Arabidopsis. Development 129: 1131–1141
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
Tantikanjana T, Yong JWH, Letham S, Griffith M, Hussain M, Ljung K,
Sandberg G, Sundaresan V (2001) Control of axillary bud initiation and
shoot architecture in Arabidopsis through the SUPERSHOOT gene.
Genes Dev 15: 1577–1588
Thimann KV (1937) On the nature of inhibitions caused by auxin. Am J Bot
24: 407–412
Thimann KV, Skoog F (1933) Studies on the growth hormone of plants. III.
The inhibiting action of the growth substance on bud development. Proc
Natl Acad Sci USA 19: 714–716
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
Turnbull CGN, Booker JP, Leyser HMO (2002) Micrografting techniques
for testing long-distance signalling in Arabidopsis. Plant J 32: 255–262
Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya
N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, et al (2008) Inhi-
bition of shoot branching by new terpenoid plant hormones. Nature 455:
195–200
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 ortholog of
Arabidopsis MAX3 is required for negative regulation of the outgrowth
of axillary buds. Plant J 48: 687–696
Strigolactone Acts Downstream of Auxin in Bud Outgrowth
Plant Physiol. Vol. 150, 2009493