Roles for Auxin, Cytokinin, and Strigolactone in

Regulating Shoot Branching1[C][W][OA]

Brett J. Ferguson and Christine A. Beveridge*
School of Integrative Biology and Australian Research Council Centre of Excellence for Integrative Legume
Research, University of Queensland, St. Lucia, Queensland 4072, Australia

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by
the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other
growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be
involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and
perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin
treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching
independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive
effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants
compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these
treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component.
Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not
always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin
biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for
apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the
progression of buds through different control points from dormancy to sustained growth.

Regulating shoot architecture is important for plant
adaptation, survival, and competition. It provides the
plant with the flexibility to respond to environmental
factors, such as light and herbivory, while optimizing
its resources. In part, this regulation occurs via a
process called apical dominance, in which the shoot
apex inhibits the outgrowth of lateral buds. However,
there are a number of questions remaining to be
resolved for different circumstances of bud outgrowth.
Are changes in apical dominance always the underly-
ing cause of bud outgrowth? Can changes indepen-
dent of the shoot tip cause bud outgrowth? Can
changes in the level or transport of signals produced
in roots and stems cause bud outgrowth without
affecting the supply of signals from the shoot tip?
Here, we address these questions, discuss different
forms of branching control, and suggest how they may
interact.
1
This work was supported by the Australian Research Council
and the University of Queensland.
* 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.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a sub-
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.135475
Plant Physiology, April 2009, Vol. 149, pp. 1929–1944, www.plantphysiol.org Ó 2009 American Society of Plant Biologists
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A complete apical dominance phenotype (Cline,
1997) is typical of many wild-type varieties of garden
pea (Pisum sativum), where a total inhibition of lateral
bud outgrowth is observed during vegetative devel-
opment. Removal of the shoot apex (i.e. decapitation)
alleviates this inhibition, allowing for the outgrowth of
lateral buds. Decapitation also reduces the endoge-
nous levels of indole-3-acetic acid (IAA), a bioactive
form of the phytohormone auxin (Thimann and
Skoog, 1933, 1934; van Overbeek, 1938; White et al.,
1975; Morris et al., 2005). This correlation between
shoot IAA level and bud outgrowth led to what is
commonly referred to as the classical hypothesis of
apical dominance (reviewed by Dun et al., 2006).
Although IAA can suppress branching after decapita-
tion, it is often unable to completely inhibit lateral bud
outgrowth (Li et al., 1995; Beveridge et al., 2000; Cline
et al., 2001; Cline and Sadeski, 2002; Morris et al.,
2005). In fact, it was recently discovered that IAA
depletion after decapitation is not the first trigger
for bud outgrowth. By decapitating tall pea plants,
Morris et al. (2005) demonstrated that the rate of IAA
depletion along the stem was too slow to cause the
initial outgrowth response exhibited by buds located
at a substantial distance from the treatment site. A
decapitation-induced signal that moves more rapidly
than IAA, therefore, induced the initial outgrowth of
these buds, and further data showed that IAA acts
afterward to inhibit continued growth. In addition,
Morris et al. (2005) showed that a change in IAA content
can occur in the stem without stimulating bud out-
1929
Ferguson and Beveridge
growth, as auxin transport inhibitor treatment to intact
plants caused an IAA depletion similar to that caused
by decapitation yet did not cause bud outgrowth.
Numerous signaling elements in addition to IAA are
now known to be required for bud outgrowth. For
a decade, we have known that RAMOSUS (RMS)
branching genes in pea regulate a graft-transmissible
branching inhibitor (Beveridge et al., 1997; for review,
see Beveridge, 2006). To date, homologous pathways
have been identified in pea, Arabidopsis (Arabidopsis
thaliana), and petunia (Petunia hybrida) and are regu-
lated by RMS, MORE AXILLARY GROWTH (MAX),
and DECREASED APICAL DOMINANCE (DAD)
genes, respectively (for review, see Beveridge, 2006;
Dun et al., 2006). Orthologs have also been identified
in rice (Oryza sativa; Ishikawa et al., 2005; Arite
et al., 2007), revealing the highly conserved nature of
the regulatory process across plant species. Recently,
Gomez-Roldan et al. (2008) and Umehara et al. (2008)
provided convincing evidence that the signal regu-
lated by these genes is a strigolactone. Strigolactones
were discovered as the branching signal deficient in
rms1, rms5, and max4 mutants, following the lead that
these genes encode carotenoid cleavage dioxygenases
(Matusova et al., 2005; Gomez-Roldan et al., 2008). A
similar approach was also taken using rice (Umehara
et al., 2008). Although it may be some time before the
bioactive strigolactone or product is unequivocally
identified, we shall refer to the novel branching inhib-
itor here as a strigolactone.
Branching inhibition has been restored in particular
mutant rms, max, and dad shoots by grafting to wild-
type rootstocks or interstocks (Beveridge et al., 1994,
1997; Napoli, 1996; Foo et al., 2001; Booker et al., 2005).
In addition to auxin and the rapid decapitation-
induced signal mentioned above, this suggests that
shoot branching is also regulated by signaling from the
roots and stem below the buds. Indeed, a pulse of
strigolactone in the vasculature stream can inhibit
branching at nodes a few centimeters above (Gomez-
Roldan et al., 2008).
The strigolactone may be part of a mechanism that
contributes to apical dominance. IAA regulates the
expression of at least two of the genes responsible for
the biosynthesis of strigolactones (Sorefan et al., 2003;
Bainbridge et al., 2005; Foo et al., 2005; Johnson et al.,
2006). In pea, these are RMS1 and RMS5 (Foo et al.,
2005; Johnson et al., 2006). Whereas the application of
IAA can slightly increase the expression of these genes
(Sorefan et al., 2003; Bainbridge et al., 2005; Foo et al.,
2005), reducing the stem IAA content (e.g. via decap-
itation or application of auxin transport inhibitors)
results in a strong decrease in their expression (Foo
et al., 2005; Johnson et al., 2006). rms1 shoots lack the
branching inhibition response to exogenous IAA, but
this can be restored by grafting to wild-type rootstocks
(Beveridge et al., 2000), indicating that the strigolac-
tone may act downstream of auxin. Thus, in intact
wild-type plants, changes in the endogenous auxin
content in the stem, mediated by the shoot tip, may act
1930
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at least partly via strigolactone to regulate bud out-
growth as part of apical dominance. Consequently,
auxin signaling could be central to shoot branching,
channeling its effects through strigolactone. For this to
be the case, changes in auxin level or signal transduc-
tion in intact plants would be correlated with bud
outgrowth. Alternatively, in the absence of such
changes in auxin level or signaling, strigolactone
function in intact plants may be essentially separate
from apical dominance. Classical apical dominance
control by auxin would then apply in decapitated or
auxin-depleted systems. If this were the case, branch-
ing in intact rms mutants would not necessarily be
correlated with changes in auxin signaling. In any
case, if decapitation and auxin depletion act to trigger
bud outgrowth entirely through strigolactone, addi-
tive effects of decapitation in the branching mutants
would not be expected. In contrast, if decapitation
induces an auxin-independent rapid trigger for out-
growth, as Morris et al. (2005) suggested, we might
expect an additive effect of decapitation on branching
in the rms mutants.
Additional hypotheses have described the mecha-
nism of apical dominance from an entirely different
perspective. The IAA transport hypothesis suggests
that apical dominance is determined directly by the
flow of IAA in the plant (Morris, 1977, Bangerth, 1989;
Bennett et al., 2006; Mouchel and Leyser, 2007; Ongaro
and Leyser, 2008). According to this theory, a shoot
meristem, whether it be apical or axillary, can only
grow when it actively exports IAA basipetally, in what
is known as the polar auxin transport stream (PATS).
The PATS involves a number of trafficking proteins
that channel IAA cell to cell. In the PATS of the shoot,
the flow of IAA is typically dictated by IAA efflux
carriers, particularly PIN-FORMED1 (PIN1), which
generate a unidirectional flow of the hormone basip-
etally from the apex through the stem (Friml et al.,
2003). The IAA transport hypothesis proposes that a
bud must export IAA into the PATS of the main stem
in order for that bud to initiate outgrowth. Bennett
et al. (2006) expanded on this notion, suggesting that
the MAX pathway of Arabidopsis acts to control the
expression of the PIN transporters to essentially reg-
ulate apical dominance by regulating IAA transport.
The authors argue that in a plant exhibiting a complete
apical dominance phenotype, the PATS of the main
stem is functioning at maximum capacity or somehow
limiting an auxin transport property, and thus the
PIN1 transporters are unable to cope with additional
IAA entering from the buds (Bennett et al., 2006;
Mouchel and Leyser, 2007; Ongaro and Leyser, 2008).
In this instance, IAA transported from the main shoot
apex acts as the critical regulator of bud outgrowth,
impeding the flow of IAA from the bud, thus prevent-
ing the outgrowth of that bud. Only when this imped-
iment is alleviated (for instance, when IAA levels are
reduced or the number of IAA transporters is in-
creased) would IAA export from the bud commence
and subsequent outgrowth be achieved. This stipula-
Plant Physiol. Vol. 149, 2009
 Plant Hormones and Shoot Branching

tion that IAA export from the bud triggers outgrowth phloem transport and the PATS, while allowing for
is in direct contrast to the bud transition hypothesis acropetal xylem transport and continued shoot tip
discussed below, in which IAA export is considered a growth (Snow, 1929; Davies and Wareing, 1965; Patrick
characteristic of sustained growth, functioning as a and Wareing, 1978; Thomas, 1983).
consequence, rather than a cause, of bud release (Dun
et al., 2006).
Correlative inhibition is another concept that can RESULTS
apply the IAA transport hypothesis described above to
competition between growing shoots (Bangerth, 1989; Stem Girdling Blocks Auxin Transport But Does Not
Bangerth et al., 2000; Bennett et al., 2006; Mouchel and Always Cause Bud Outgrowth
Leyser, 2007; Ongaro and Leyser, 2008; Ongaro et al., Girdling the stem with hot wax (Fig. 1A) was found
2008). In this case, rather than referring primarily to to kill the tissue at the treatment site in pea (Fig. 1B).
bud release, two growing shoots inhibit one another Consequently, pathways relying on living cells to
based on the extent of auxin transport in each shoot. actively transport signals in the stem would be
Blocking auxin transport from one shoot will stop its blocked. This includes the PATS, which is localized
growth and will enhance the growth of the other shoot. to living cells of the vasculature. In contrast, acropetal
The mechanism for this may be as described above. transport via the xylem may not be greatly affected.
Alternatively, even in shoots with well-established
vascular connections, it may relate to the ability of
auxin flow from a shoot to attract nutrients or signals
and hence to remain competitive with other shoots.
Indeed, shoot architecture models can mimic apical
dominance and correlative inhibition from a purely
source-sink perspective (Allen et al., 2005).
Stafstrom and colleagues proposed the bud transi-
tion hypothesis of branching control; this hypothesis
defines various stages at which a bud can reside,
including dormancy, transition, and sustained out-
growth (Stafstrom and Sussex, 1988, 1992; Stafstrom,
1995; Stafstrom et al., 1998; for review, see Napoli et al.,
1999; Shimizu-Sato and Mori, 2001; Dun et al., 2006).
According to this hypothesis, a dormant bud must first
enter into a state of transition before it can undergo
sustained growth. Hence, the induction of a signaling
pathway that promotes bud outgrowth, or the sup-
pression of one that inhibits it, would initially be
required to activate a bud from dormancy into transi-
tion, followed by additional steps to sustained out-
growth. This hypothesis provides a simple framework
for the interaction of multiple signals in branching
control.
In this report, we present findings from experiments
in which we evaluated the effects of stem girdling,
decapitation, and bud removal on branching in rms
mutants to determine whether apical dominance, cor-
relative inhibition, and the RMS/strigolactone path-
way are independent or interconnected mechanisms.
Decapitation and the application of auxin transport
inhibitors are techniques commonly used to reduce
IAA levels and stimulate bud outgrowth. However,
decapitation is extreme, involving the removal of plant
tissues that affect numerous signals other than IAA,
and disturbs plant turgor and source-sink relation- Figure 1. Effects of stem girdling in wild-type pea. A, Seven days
following treatment, bud outgrowth was exhibited below, but not
ships. Moreover, results from studies using auxin
above, the girdle. B, Removal of the girdle exposes dead tissue
transport inhibitors are often variable (Panigrahi and compared with an untreated internode (far left). Swelling and callus-
Audus, 1966; Tamas et al., 1989; Morris et al., 2005), like distortions were exhibited directly above the girdle site. C, [3H]IAA
and these pharmacological approaches may also affect uptake/transport in stem segments 8 h following application to the
other signals. We investigated whether stem girdling shoot apex. Data are means 6 SE (n = 5). The top of the girdle was
could be used as a reliable method for studying IAA positioned at about 40 mm from the apex. DPM, Disintegrations per
transport and bud outgrowth relations, as it disrupts minute. [See online article for color version of this figure.]

Plant Physiol. Vol. 149, 2009 1931

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Ferguson and Beveridge
This is supported by the fact that the region of the
shoot located above the girdle continues to grow,
demonstrating that it is receiving water and nutrients
from below.
To confirm the blockage of the PATS, the transport of
radiolabeled [3H]IAA was examined in the stem of
girdled and untreated control plants 8 h after its
application to the apex. The [3H]IAA loaded into the
PATS and moved basipetally in a wave-like manner,
having traveled slightly over 8 cm in untreated control
plants (Fig. 1C). This rate of transport is similar to
the 1 cm h21 speed described previously for pea
(Goldsmith, 1977; Kotov, 1996; Beveridge et al., 2000;
Morris et al., 2005). However, in plants that had been
girdled, no [3H]IAA was detected in stem segments
located below the site of the girdle, despite the major-
ity of the wave having moved past this location in
the nongirdled control plants (Fig. 1C). Instead, the
[3H]IAA was found to accumulate directly above the
girdle site (Fig. 1C). This demonstrates conclusively
that stem girdling causes a complete block of the PATS.
Interestingly, the location of the [3H]IAA accumulation
correlated precisely with stem-swelling and callus-
formation events observed on girded plants (Fig. 1B),
indicating that these features may be the result of a
buildup of IAA.
To identify the effect of stem girdling on bud out-
growth, plants with nine leaves expanded were gird-
led at an upper internode. One week after treatment,
untreated control plants displayed a complete apical
dominance phenotype, whereas those girdled at an
upper internode exhibited a strong outgrowth re-
sponse below the treatment site (Fig. 1A). This is
consistent with the concept that inhibitory signals
emanating from the shoot apex are involved in apical
dominance.
The relationship between girdle position and bud
outgrowth was investigated by girdling or decapitat-
ing at different internodes along the stem (Fig. 2).
Although decapitation at different positions always
induced outgrowth, girdling at progressively lower
positions (Fig. 2, B–F) resulted in fewer and shorter
branches; plants girdled between nodes 3 and 4 did
Figure 2. Effects of decapitation and girdle position on bud outgrowth in wild-type pea. Bud lengths were recorded 9 d following
treatment at each node of control (intact; A) or girdled or decapitated pea plants having nine leaves expanded at the time of
treatment (B–G). Arrows represent treatment sites. Data are means 6 SE (n = 8).
1932
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not exhibit any bud outgrowth (Fig. 2B). Thus, the bud
outgrowth response depends on the treatment type
and, in the case of stem girdling, on the location of the
treatment along the stem. This brings into question the
role of IAA in the processes of bud outgrowth and
apical dominance, as stem girdling completely blocks
IAA transport (Fig. 1C) but only induces bud out-
growth when the girdle is situated at upper internodes
(Fig. 2).
To better understand the effects of stem girdling and
decapitation on bud outgrowth and to determine
whether the girdle may prevent a decapitation-
induced signal, bud lengths were assessed using
plants that were girdled and decapitated simulta-
neously. This experiment is important, as Morris
et al. (2005) suggest that decapitation induces a rapid
signal perceived by axillary buds before any changes
in auxin level or transport are apparent. In the first
instance, we focused on girdling at a lower internode
where this treatment does not promote bud outgrowth
(Figs. 2B and 3). Using these plants, we tested whether
decapitation at an upper internode would induce
outgrowth below the low girdle as it does in non-
girdled, decapitated plants. Results from these exper-
iments were intriguing, as decapitation-induced bud
outgrowth was observed both above (data not shown)
and below the low girdle site (Fig. 3). Moreover, up to
4 h following decapitation, this outgrowth response
occurred irrespective of when the girdle was placed on
the stem, including prior to decapitation (Fig. 3A; for
individual bud lengths, see Supplemental Fig. S1).
However, at these early time points, the degree of bud
outgrowth observed in this region was significantly
less compared with that in plants that were decapi-
tated only (Fig. 3A; Supplemental Fig. S1). These
results suggest that a stem girdle is unable to prevent
decapitation-induced bud outgrowth from occurring
but significantly reduces subsequent growth. That is,
dormant buds were triggered by decapitation but their
lengths were suppressed by the girdle treatment. In
contrast, when the girdle was applied 72 h after
decapitation, the degree of bud outgrowth below
was comparable with that of decapitated control
Plant Physiol. Vol. 149, 2009

Figure 3. Total bud lengths at nodes 1 to 3 of wild-type pea 9 d
following decapitation between nodes 8 and 9 and/or girdling between
nodes 3 and 4. Decapitation (Decap) occurred at the same time for all
treatments (time 0), with the timing of stem girdling (Girdle) staggered
across early (A) or late (B) time points following decapitation. Data are
means 6 SE (n = 6–8).
plants (Fig. 3B). This may indicate that these buds had
reached a stage of active growth at which they were no
longer responsive to the effects of stem girdling.
We then tested whether girdling at an upper inter-
node, which induces branching, is additive to the
decapitation response. A similar degree of bud out-
growth was observed in these plants compared with
those girdled or decapitated alone at that location,
indicating that the effects are not additive (data not
shown).
To identify whether a lack of IAA coming from the
apex contributes to bud outgrowth following stem
girdling, as it does following decapitation, we applied
IAA directly below the treatment site of tall wild-type
plants girdled between nodes 8 and 9 (Fig. 4). Previ-
ously, Morris et al. (2005) used tall pea plants to
demonstrate that IAA, at concentrations as high as 3
mg g21 in lanolin, diminished the amount of bud
outgrowth following decapitation but was unable to
completely prevent outgrowth from occurring. This
high concentration not only restores IAA depletion but
also increases the endogenous IAA content to above
that of intact control plants (Morris et al., 2005). Lower
concentrations are less effective at inhibiting bud
outgrowth (Beveridge et al., 2000). Using this same
rather high concentration, we found that IAA mark-
edly reduced the amount of outgrowth in girdled
plants yet again was unable to prevent outgrowth
from occurring (Fig. 4). The trend observed was sim-
ilar to that using decapitated plants (Fig. 4), suggesting
that IAA has a similar role in decapitation- and stem
girdling-induced bud outgrowth. These findings are
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Plant Hormones and Shoot Branching
consistent with those of Morris et al. (2005) and
indicate a role for stem IAA in inhibiting sustained
bud outgrowth but not in preventing initial out-
growth.
Does Defoliation Affect Bud Outgrowth?
Defoliation is known to deplete stem auxin levels in
pea (Jager et al., 2007). However, compared with
untreated control plants, defoliation did not induce a
discernible bud outgrowth response (19.31 6 2.37 mm
total lateral length in intact control plants compared
with 17.94 6 1.54 mm in defoliated plants, 7 d follow-
ing treatment of plants having nine leaves expanded).
Thus, in pea, the significant reduction in the stem IAA
content caused by defoliation is not sufficient to trigger
bud outgrowth. This is consistent with findings re-
ported by Morris et al. (2005) using similar plants
treated with the auxin transport inhibitor naphthyl-
phthalamic acid (NPA) to cause a depletion in IAA
content without causing bud outgrowth.
In the case of a plant girdled between nodes 3 and 4,
which fails to exhibit any bud outgrowth (e.g. Figs. 2B
and 3), the buds located below the treatment site are
separated from all but one true leaf (located at node 3).
These buds, therefore, would be expected to have less
available leaf-synthesized products, including photo-
assimilates. Therefore, we explored whether a reduced
supply of leaf-derived energy and/or signals could
prevent the growth of buds induced to grow out. This
was tested by defoliating plants whose buds were
triggered to grow by stem-girdling or decapitation
treatments at upper nodes. Although defoliation sig-
nificantly diminished the amount of bud growth, it
failed to prevent it from occurring in these plants, and
the pattern of outgrowth was similar to that of non-
defoliated decapitated or girdled control plants (Fig.
5). Moreover, plants decapitated between nodes 3 and
4, and therefore having only one true leaf, also
exhibited outgrowth (Fig. 2). These findings demon-
strate that the complete lack of bud outgrowth ob-
Figure 4. Total lateral length at nodes 1 to 8 of wild-type pea 7 d
following decapitation or stem girdling between nodes 8 and 9. Plants
were treated with or without 3 mg g21 IAA in lanolin at (decapitation
[Decap]) or below (girdling [Girdle]) the treatment site. Plants had nine
leaves expanded at the time of treatment. Data are means 6 SE (n = 8).
1933
Ferguson and Beveridge

The Impact of Stem Girdling on Plant Growth

In addition to bud outgrowth, a number of charac-

teristics were investigated to establish how stem gird-
ling affects the growth and overall vigor of the shoot.
One week following treatment, the shoot height was
found to be slightly reduced in girdled plants when
compared with untreated control plants (Fig. 7A). This

Figure 5. Average bud length at each node of wild-type pea 7 d

following decapitation and/or stem-girdling treatment with or without
defoliation of all fully expanded leaves. No significant difference was
detected between additional untreated control plants left intact (total
lateral length, 19.31 6 2.37 mm) or defoliated (17.94 6 1.54 mm).
Plants had nine leaves expanded at the time of treatment. The arrow
represents treatment sites. Data are means 6 SE (n = 6–7).

served in plants girdled between nodes 3 and 4 (Figs.

2B and 3) is not simply a consequence of a reduced
supply of leaf-derived compounds.

Effects of Nutrients on Bud Outgrowth

To test the effect of nutrient availability on bud
outgrowth, plants whose buds were induced to grow
via decapitation, stem girdling, or treatment with the
auxin transport inhibitor NPA were supplied with or
without our standard weekly nutrient solution. NPA
was used at an amount known to affect IAA transport
and to deplete endogenous IAA levels to at, or very
near, those of comparable decapitated plants (Morris
et al., 2005). Following decapitation or stem girdling
above node 5, lateral shoot lengths at nodes 2 and 5
were considerably greater in plants supplied with
nutrients compared with those without (Fig. 6, A
and B). In the case of stem girdling, the absence of
supplied nutrients reduced the node 5 bud length to
only twice that of the control plants. As reported
previously (Morris et al., 2005), NPA had little effect on
bud outgrowth; there was little or no effect of nutrients
on buds of NPA-treated or untreated control plants
Figure 6. Effects of nutrient supply on the lateral length of the bud at
(Fig. 6, A and B). Therefore, withholding nutrient node 5 (A) and node 2 (B) as well as plant height 6 d following
supply to the plants did not prevent the outgrowth of decapitation (Decap.), 10 mg g21 NPA treatment, or stem girdling
buds induced to grow by these treatments but did (Girdle) of wild-type plants (C). Plants were supplied with (+N) or
affect branch lengths. As expected, for all treatment without (2N) nutrient solution and Osmocote, as outlined in “Materials
types, plants supplied with nutrients were taller than and Methods.” Treatments were made above node 5 using plants
those without (Fig. 6C). having five leaves expanded. Data are means 6 SE (n = 8–10).

1934 Plant Physiol. Vol. 149, 2009

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 Plant Hormones and Shoot Branching

Figure 7. Effects of stem girdling and decapitation on various traits 7 d after treatment. A and B, Shoot height (A) and number of
fully expanded leaves (B) of plants having seven leaves expanded at the time of stem girdling. C to E, Root (C) and internode (D
and E) dry weights (DW) of plants having nine leaves expanded at the time of stem girdling or decapitation. Arrows represent
treatment sites. Data are means 6 SE (n = 6–8).

may be a repercussion of the girdle impeding the
phloem, hence cutting off the supply of photoassimi-
lates to the root system, which would be exacerbated
the lower the girdle was placed on the stem. Indeed,
the root system dry weight was found to be signifi-
cantly reduced by stem girdling, as it was following
decapitation (Fig. 7C). The root dry weight of plants
girdled or decapitated above node 3 was 30% that of
intact controls, whereas the root dry weight for those
treated above node 5 was about 50% that of the
controls.
No significant difference was detected in the num-
ber of expanded leaves of girdled plants compared
with untreated control plants (Fig. 7B). However,
internodes located directly above the girdle were
found to be significantly greater in dry weight than
comparable internodes of control plants (Fig. 7D). This
may be due to increased cell division and lateral
expansion resulting from the girdle blocking the
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PATS (Fig. 1C), causing the IAA level to build up
directly above the girdle site. In contrast, younger
internodes located closer to the apex were found to be
significantly reduced in dry weight following stem
girdling (Fig. 7D). This is likely due to reduced nutri-
ent uptake resulting from the stunted root system
biomass (Fig. 7C). Internodes located below the girdle
were similar in dry weight to comparable internodes
from decapitated and untreated control plants (Fig.
7D). These internodes were mature at the time of
treatment and thus fully expanded. Hence, their dry
weight would not be expected to be greatly altered
following treatment.
Apical Dominance, Correlative Inhibition, and the
RMS/Strigolactone Pathway Are Distinct Processes
We used two approaches to test the role of the RMS/
strigolactone pathway in the processes of correlative
1935
Ferguson and Beveridge
inhibition and apical dominance. For correlative inhi-
bition, we examined whether axillary shoots affect the
outgrowth of other axillary shoots in rms mutants. For
apical dominance, we determined whether rms mu-
tants exhibit enhanced branching in response to de-
capitation or girdling. We included rms2 mutants in
these experiments because RMS2 is involved in shoot-
to-root signaling for feedback regulation of strigolac-
tone biosynthesis (for review, see Beveridge, 2006).
Basal branches of rms mutants have considerably
more fully expanded leaves and are longer than aerial
branches (Arumingtyas et al., 1992). Moreover, the
basal branches of rms2 plants grown under long days
appear to prevent the outgrowth of buds at aerial
nodes (Beveridge et al., 1996). This is similar to the
correlative inhibition discussed by Bangerth (1989)
and Ongaro et al. (2008). We tested whether this
correlative inhibition occurs in other rms lines (Fig.
8). We included rms6 because it is reported to branch at
basal nodes only (nodes 0–3; Rameau et al., 2002).
RMS6 is thought to act mostly in the shoot, possibly
Figure 8. Effects of basal lateral removal on total lateral length (insets)
and lateral lengths at individual nodes of rms mutant and wild-type
(WT) plants that exhibit a range of branching phenotypes. Axillary buds
were removed as they appeared from nodes 0 to 3, and lateral lengths
were scored on a single day when the plant had 20 to 24 leaves
expanded. Data are means 6 SE (n = 5–10). TLL, Total lateral length.
1936
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
downstream of strigolactone perception. We included
rms3, which is a physiologically similar mutant to rms4
(for review, see Beveridge, 2000), because we had
available the additive double mutant rms3 rms6. We
used a relatively short photoperiod to encourage branch-
ing at all zones along the main stem (Arumingtyas
et al., 1992). Under these conditions, intact rms2 and
rms6 plants showed a very strong emphasis toward
basal branching, whereas rms3 and rms4 plants pro-
duced branches at every node (Fig. 8). The rms3 rms6
double mutant showed an additive effect of long
basal branches and branches occurring at every other
node (Fig. 8). These long basal branches resulted in
the total lateral length of the double mutant being
nearly twice that of the rms3 single mutant and
nearly seven times that of the rms2 single mutant
(Fig. 8). When the basal buds/branches were re-
moved from any of the lines investigated, the aerial
buds were stimulated to grow out such that the total
lateral length at the time of scoring was similar for
plants of intact and bud-removal treatments (Fig. 8).
The fact that aerial buds are normally suppressed or
considerably shorter in intact rms mutant plants but
can be released by basal branch removal demon-
strates that correlative inhibition acts more or less
independently of the RMS system.
To examine the role of the RMS network in apical
dominance, we tested the extent to which apical dom-
inance functions in the rms mutants. Stems of rms
mutant plants were girdled or decapitated between
nodes 8 and 9. At this stage of development, rms
mutants have vigorous basal branches, yet the lengths
of buds at the uppermost nodes are similar to those of
the wild type. As for wild-type plants (Figs. 2–5), stem
girdling and decapitation induced significant out-
growth below the treatment site in rms2, rms4, and
rms5 mutants, whereas control plants retained short
axillary buds at these upper nodes (Fig. 9). This
demonstrates that the upper buds of these mutants
were inhibited by signals coming from the shoot apex
(i.e. apical dominance), despite the presence of the
basal shoots (i.e. correlative inhibition) and the ab-
sence of a functional RMS pathway. The bud out-
growth response caused by girdling at upper nodes
was similar to that caused by decapitation in both
mutant and wild-type plants (Fig. 9). Branch lengths at
basal nodes of mutant plants were not significantly
affected by stem girdling or decapitation (Fig. 9).
Cytokinin Biosynthetic Gene Expression Correlates with
Bud Outgrowth
The expression of molecular markers for cytokinin
(CK) biosynthesis (ISOPENTENYL TRANSFERASE1
[IPT1] and IPT2), IAA response (IAA4/5), and the RMS
pathway (RMS1 and RMS5) was determined in nodal
stem segments, consisting of internode and bud tissue.
Samples were harvested 24 h following decapitation or
stem girdling between nodes 8 and 9 or nodes 3 and 4.
The expression of each gene at a given node was made
Plant Physiol. Vol. 149, 2009

relative to the expression of that same gene in the
corresponding node of intact control plants (Fig. 10).
Increased IPT1 and IPT2 expression correlated
strongly with bud outgrowth (Fig. 10). Compared
with control samples, the expression of both genes
was elevated in tissue taken below all decapitation and
stem-girdling treatment sites that cause bud out-
growth. This was greatest in samples harvested di-
rectly below these treatment sites, where increases of
just under 100-fold were detected. Smaller but signif-
icant increases, particularly in IPT1 expression, were
seen at all other nodes that would later show bud
outgrowth. Strong increases in IPT1 and IPT2 expres-
sion were not detected at nodes where bud outgrowth
failed to ensue (Fig. 10). This includes all nodes
analyzed above girdle sites and also in the node
harvested below the girdle situated between nodes 3
and 4. This strong correlation between bud outgrowth
and IPT1 and IPT2 expression, and thus presumably
CK biosynthesis (Miyawaki et al., 2004; Nordstrom
et al., 2004; Tanaka et al., 2006), supports previous
suggestions of an important requirement of CK in bud
outgrowth (Pillay and Railton, 1983; Medford et al.,
1989; Bangerth, 1994; Cline et al., 1997, 2006; Li and
Bangerth, 2003; Mader et al., 2003a, 2003b; Nakagawa
et al., 2005).
We further investigated whether CK was indeed
limiting to bud outgrowth. This was done by girdling
wild-type plants having nine leaves expanded be-
tween nodes 3 and 4 and then applying 5 mL of 50%
ethanol (control) or 10 mg mL21 of the bioactive CK,
6-benzylaminopurine dissolved in 50% ethanol, to the
bud at node 2. The treatments were repeated 4 d later,
and the bud length at node 2 was measured 7 d
following the initial treatment. CK was indeed found to
stimulate outgrowth, as 6-benzylaminopurine-treated
buds were significantly greater in length (11.56 6 2.00
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Plant Hormones and Shoot Branching
Figure 9. Bud outgrowth at every node
at 0 d (A–D) and 7 d (E–H) following
stem girdling or decapitation between
nodes 8 and 9 of wild-type (WT; A and
E), rms2-1 (B and F), rms4-1 (C and G),
and rms5-3 (D and H) plants. Plants
had nine leaves expanded at the time
of treatment. Data are means 6 SE (n =
6–8 plants per treatment).
mm) than control-treated buds, which remained dor-
mant (0.81 6 0.04 mm). This demonstrates that these
buds are in fact capable of growing out and further
implies that an insufficient CK content may be respon-
sible for their usual lack of outgrowth.
The IAA response gene IAA4/5 showed a decrease in
expression below the decapitation and girdle sites (Fig.
10). This reduction was greatest in nodes harvested
directly below the treatment sites and was as much as
10-fold less than that of comparable intact tissue. In
contrast, IAA4/5 expression was consistently increased
in nodes harvested above the girdle site (Fig. 10). These
results are highly consistent with our above-mentioned
findings that stem girdling blocks IAA transport (Fig.
1), resulting in a buildup of IAA above the treatment
site and a depletion in the hormone below. They are
also consistent with the well-documented decrease
in IAA content occurring below a decapitation site
(Thimann and Skoog, 1933, 1934; van Overbeek, 1938;
White et al., 1975; Morris et al., 2005).
The expression level of RMS1 and RMS5 decreased
below decapitation and girdle treatment sites (Fig. 10).
Compared with control samples, RMS1 expression
was reduced as much as 10,000-fold in these tissues,
whereas 10-fold reductions were typical for RMS5.
Such decreases are consistent with those previously
reported following decapitation (Foo et al., 2005;
Johnson et al., 2006). These decreases in gene expres-
sion below the girdle or decapitation site were gener-
ally correlated with bud outgrowth. However, one
notable exception was below the treatment site of
plants girdled between nodes 3 and 4 (Fig. 10), where
RMS1 and RMS5 expression substantially decreased
yet bud outgrowth was not observed (Figs. 2 and 3).
Similarly, in this same tissue, reduced IAA4/5 expres-
sion was not correlated with bud outgrowth. These
findings differ from that detailed above for IPT1 and
1937
Ferguson and Beveridge

Figure 10. Expression levels of various genes from plants decapitated or girdled between nodes 3 and 4 or 8 and 9. To aid with
interpretation of the gene expression results, illustrations of the bud outgrowth phenotypes caused by the various treatments (as
detailed in Fig. 2) are provided. Each arrow represents the bud of a particular node. Arrow size is representative of the amount of
growth 7 d following treatment, with no arrow representing a nongrowing, dormant bud. For gene expression data, nodes 2, 4, 8,
and 9 (as indicated on the stem representing the intact treatment), consisting of 1 cm of stem tissue including the bud, were
harvested 24 h following decapitation (Decap.) or stem-girdling (Stem Girdle) treatment. Quantitative real-time reverse
transcription-PCR expression levels of PsRMS1, PsRMS5, PsIPT1, PsIPT2, and PsIAA4/5 are relative to the expression of that
particular gene in the same node of the intact control, subsequent to all genes being normalized against the expression level of
Ps18S. Plants had nine leaves expanded at the time of treatment. Data for quantitative real-time reverse transcription-PCR are
means 6 SE (n = 2 replicates of six plants per replicate). [See online article for color version of this figure.]

IPT2, which completely correlated with bud out-
growth phenotypes. The reduction in RMS1 expres-
sion detected below the treatment site of plants girdled
between nodes 3 and 4 was not as strong as that
detected below the other treatment sites. If this reduc-
tion in RMS1 expression was insufficient to reduce
striogolactone levels, it could account for why the
buds of these plants failed to grow out. However, such
a direct effect is unlikely, as the reduction in RMS1
expression was greater than that at comparable nodes
of plants decapitated or girdled high and where
branching was promoted. As mentioned above, IPT
gene expression was high for these induced nodes in
other treatments. Interestingly, like IAA4/5, the expres-
sion of RMS1 and RMS5 was found to increase above
girdle sites, possibly in response to elevated IAA levels
caused by girdling (Fig. 10).
depletion caused by girdling, defoliation, or NPA
application is not sufficient to trigger bud outgrowth
in wild-type plants. Second, apical dominance and
correlative inhibition function in rms mutants, which
are deficient in the newly identified branching hor-
mone strigolactone. This suggests that these processes
are at least partially functioning independently of
strigolactones and that IAA and RMS signaling are
not necessarily in a simple linear pathway where
strigolactones act upstream (as suggested previously
by Bennett et al., 2006) or downstream of IAA (Beveridge
et al., 2000). Third, even where RMS gene expression
is suppressed and IAA and strigolactone levels are
depleted in wild-type plants, other factors, such as
localized CK production, may still be required for bud
outgrowth.

Reduced IAA Levels in the Main Stem Do Not Trigger

DISCUSSION Bud Outgrowth

Our results lead to three major conclusions relating Our findings clearly demonstrate that reduced IAA
to apical dominance and bud outgrowth. First, IAA content in the main stem is not always correlated with
1938 Plant Physiol. Vol. 149, 2009
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.

bud outgrowth. Based on our IAA4/5 expression and
[3H]IAA transport data, stem girdling, like decapita-
tion, causes a substantial depletion in IAA content
below the girdle, affecting IAA in both the PATS and
the phloem as it destroys all living tissues across the
stem (Figs. 1 and 10). However, the position of the
girdle along the stem dictates the resulting outgrowth
response. Although girdling and decapitation block
IAA transport from the same apical source, decapita-
tion always induced branching, whereas in several
treatment positions, girdling typically failed to do so
(Fig. 2). This discrepancy between girdled and decap-
itated plants is reduced in plants treated at progres-
sively higher internodes. At lower nodes, the reduced
branching response to girdling compared with decap-
itation was unlikely due to differences in IAA signal-
ing, as the treatments are expected to have similar
effects on IAA content and IAA4/5 expression was
reduced equally in both treatments (Fig. 10). Defolia-
tion also reduces the IAA content of pea stems (Jager
et al., 2007) but had no significant effect on bud
outgrowth of intact pea plants (Fig. 5).
To investigate whether a reduced energy supply
was responsible for the lack of outgrowth observed in
plants girdled at lower internodes, we observed how
defoliated plants responded to reduced resource sup-
ply below a girdle at an upper node. Even in the
extreme case of removing all leaves, we were unable to
prevent bud outgrowth at any node in defoliated
plants that were decapitated or girdled at upper
nodes. Rather, only branch lengths were affected. In
contrast, girdling at nodes below the midpoint of the
main stem failed to induce outgrowth at some or all
nodes (Fig. 2, A–D). However, girdling at this location
72 h after decapitation induced a similar outgrowth
response to that of decapitation alone (Fig. 3). Thus,
the girdling response is not simply an energy issue, as
the girdle did not reduce the extent of bud outgrowth
following bud release. Overall, the energy/carbon
supply appears to affect the extent of growth rather
than the initiation of outgrowth (Fig. 5).
As we have suggested previously (Morris et al.,
2005), our new results demonstrate that IAA depletion
in the main stem is not in itself causal of bud out-
growth. Instead, the accumulation of IAA in a trig-
gered bud (van Overbeek 1938; Hall and Hillman,
1975; Gocal et al., 1991) and its export into the PATS of
the main stem (Bangerth et al., 2000; Ongaro and
Leyser, 2008) may orchestrate other aspects of meri-
stem function and bud outgrowth, such as directing
nutrients to the bud (Nakamura, 1964; Davies and
Wareing, 1965; Phillips, 1968; Jiang et al., 2001; Yang
et al., 2007). Indeed, Davies and Wareing (1965) ele-
gantly demonstrated that radiolabeled phosphorous
accumulates at the site of exogenously applied IAA
but fails to do so when auxin transport inhibitors are
also applied, indicating that polar IAA transport, as
opposed to IAA itself, directs the accumulation. In
Arabidopsis, this process may be mediated by canali-
zation leading to enhanced vascular development, as
Plant Physiol. Vol. 149, 2009
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Plant Hormones and Shoot Branching
suggested by Ongaro and Leyser (2008). However, in
pea, vascular development to buds, particularly at
node 2 but also at all nodes, is well established even in
nongrowing buds (data not shown). When branching
is triggered in pea, the bud located at node 2 typically
exhibits growth, whereas those located at other nodes
are more variable in their outgrowth response. This
often depends on the proximity of these buds to the
treatment site (e.g. how far the bud is from the site of
decapitation; Fig. 2; Husain and Linck, 1966). This
pattern of outgrowth is likely due to the bud at node 2
having a better established vasculature connection or
being larger than other buds, enabling it to respond
quickly when triggered, which subsequently allows it
to induce correlative inhibition over other buds.
Although girdling completely blocks IAA transport,
outgrowth only occurs below a basally located girdle
when the plant is decapitated above but not when the
main shoot is left intact (Fig. 3). As IAA levels would be
depleted similarly, if not sooner, in the girdled plants
than in plants that were decapitated, this provides
further evidence that IAA depletion is not the initial
trigger for outgrowth. Our findings are entirely consis-
tent with those reported by Morris et al. (2005), who
suggested that IAA depletion is not the initial trigger
for outgrowth in decapitated plants. The evidence was
based on several observations: IAA transported in the
PATS was too slow to account for the rapid outgrowth;
outgrowth commences prior to changes in the IAA
content of the adjacent stem; IAA inhibition of branch-
ing occurs after buds have already commenced growth;
and NPA treatment depletes IAA level to that caused by
decapitation but is not always adequate to induce
outgrowth. Here, we again showed that NPA-treated
plants exhibit little bud outgrowth (Fig. 6, A and B). In
addition, exogenously supplying IAA to decapitated or
girdled plants did not prevent initial bud growth but
greatly reduced branch lengths (Fig. 4; Morris et al.,
2005). We also show that diminished nutrient supply
reduces bud lengths (Fig. 6, A and B). Moreover,
Stafstrom and Sussex (1992) showed that molecular
markers for bud outgrowth are expressed in decapi-
tated plants, with or without auxin treatment, suggest-
ing that they are induced by factors other than auxin.
Collectively, these findings point to the existence of a
fast, IAA-independent signal acting as the trigger for
outgrowth after decapitation. The signal must be rapid,
as outgrowth events in buds located at a distance from
the decapitation site occur faster than would be ex-
pected from an actively transported signal, such as IAA
(Everat-Bourbouloux and Bonnemain, 1980; Morris
et al., 2005). Indeed, the signal might not be a cumber-
some molecule per se but possibly a physical response
to a change in turgor pressure (Urao et al., 1999;
Reiser et al., 2003) or electrical potential (Stahlberg
and Cosgrove, 1992; Fromm and Lautner, 2007). That
the decapitation-induced signal is perceived by buds
located below a girdled internode (Fig. 3A) supports
this notion, as it demonstrates that the signal does not
require living tissue for transport.
1939
Ferguson and Beveridge
Apical Dominance, Correlative Inhibition, and the RMS/
Strigolactone Pathway Are Distinct Mechanisms for
Regulating Bud Outgrowth
We demonstrate here that apical dominance, correl-
ative inhibition, and the RMS/strigolactone pathway
are all independent but interacting mechanisms for
regulating bud outgrowth. Correlative inhibition func-
tions in the absence of strigolactones, as basal laterals
inhibit the outgrowth of aerial buds in rms mutant
plants (Fig. 8). This implies that the systems have
independent components and demonstrates that cor-
relative inhibition does not require the RMS/strigo-
lactone pathway to operate. Using Arabidopsis plants,
Ongaro et al. (2008) reported that correlative inhibition
is partially dependent on the MAX pathway but that
the processes have independent components, which is
consistent with our findings. Similarly, decapitation
and girdling each caused enhanced branching in rms
mutants (Fig. 9), clearly showing that apical domi-
nance also operates in the absence of the RMS/
strigolactone network. This indicates that strigolac-
tone deficiencies and IAA depletion caused by decap-
itation or girdling are not simply acting in a linear
pathway to regulate bud outgrowth; additional inter-
actions must be involved. Similarly, rms plants exhibit
bud outgrowth despite the presence of the growing
shoot apices. Thus, in the absence of a functional RMS
network, apical dominance alone is not sufficient to
inhibit basal bud outgrowth. In contrast, the RMS/
strigolactone pathway appears to require apical dom-
inance to function, as the decapitation of wild-type
plants reduces RMS1 and RMS5 gene expression and
IAA levels, leading to bud outgrowth (Fig. 10; Foo
et al., 2005; Johnson et al., 2006).
That RMS genes are IAA regulated indicates that
some IAA branching effects are RMS dependent, pro-
viding a mechanism for cross talk within the system.
Further evidence for cross talk is provided by the fact
that the signals involved in regulating shoot architec-
ture are derived in the main shoot tip (apical domi-
nance), axillary shoot tip (correlative inhibition), or
rootstock and internode (RMS/strigolactone network).
We suggest that by having multiple interacting mech-
anisms, the plant can determine when and where to
branch based on its needs, conditions, and number of
existing shoots. This may be of particular importance
for poorly branched monopodial annual species in
which the need to respond rapidly to decapitation is
essential for competition and reproduction.
Expression of CK Biosynthetic Genes Correlates with
Bud Outgrowth
Tanaka et al. (2006) reported a direct correlation
between IPT1 and IPT2 expression and CK content
following decapitation in pea. It is also reported
that IAA can regulate the expression of these genes
(Miyawaki et al., 2004; Nordstrom et al., 2004; Tanaka
et al., 2006). Our results show that increased IPT1 and
1940
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
IPT2 gene expression following decapitation or stem
girdling was always associated with bud outgrowth
(Fig. 10). In the absence of this increase, bud outgrowth
failed to occur regardless of reductions in RMS1 and
RMS5 gene expression and/or IAA signal transduc-
tion (as indicated by IAA4/5 expression; Fig. 10).
However, in this event, outgrowth could be induced
by exogenously applying CK to the bud. Thus, sup-
pression of RMS1 and RMS5 gene expression or IAA
content alone was insufficient to promote outgrowth,
whereas increased CK biosynthesis was correlated and
therefore may be critical. We and others (Tanaka et al.,
2006) suggest that an increase in CK production typ-
ically occurs following the perception of an initial
outgrowth trigger. However, buds of the right devel-
opmental stage can be induced to grow by increasing
their CK levels directly via transgenic approaches
(Medford et al., 1989) or exogenous application (data
not shown; Pillay and Railton, 1983; King and van
Staden, 1988; Cline et al., 1997). Thus, increased CK
levels appear to be able to circumvent the need for an
initial outgrowth trigger. A similar situation exists for
legume nodule development, where activation of a CK
receptor initiates downstream nodulation events, even
in the absence of preliminary triggering/signaling
events (Gonzalez-Rizzo et al., 2006; Murray et al.,
2007; Tirichine et al., 2007).
Unlike decapitation or girdling at an upper inter-
node, girdling at a basal internode did not induce CK
biosynthesis genes below the treatment site. Never-
theless, RMS1, RMS5, and IAA4/5 gene expression was
reduced at this location, indicating the IAA level had
diminished, as should be expected following this
treatment (Foo et al., 2005; Morris et al., 2005; Johnson
et al., 2006). This suggests that another signal/path-
way is required to effectively induce IPT expression in
this treatment. A possible signaling candidate could
be nitrate, which can up-regulate IPT expression
(Miyawaki et al., 2004; Takei et al., 2004; for review,
see Sakakibara et al., 2006) and is suggested to regulate
bud outgrowth via CK (Cline et al., 2006). Reduced
nitrate uptake caused by severely stunted root growth,
as occurs by girdling at a basal internode (Fig. 7), could
inhibit the induction of IPT gene expression. In con-
trast, increased IPT gene expression and bud out-
growth in basally decapitated plants may occur
because the relative shoot demand for nitrate is de-
creased by decapitation, which is not the case for
girdling. It is important to note that our data do not
preclude the possibility of cross talk involving strigo-
lactone inhibition of CK production via modulation of
IPT gene expression in addition to the well-described
direct effects of auxin and nitrate as discussed above.
Model of Bud Outgrowth
We developed a new working hypothesis of bud
outgrowth that accounts for the stages, mechanisms,
and signals required to regulate shoot branching (Fig.
11). The origins of this model were described by
Plant Physiol. Vol. 149, 2009
 Plant Hormones and Shoot Branching

Figure 11. Model of the developmental stages of bud outgrowth illustrating the regulatory roles of IAA, CK, strigolactone, and
nutrients. A, Dormant buds must be triggered to a responsive state where they are receptive to outgrowth signals. B, Loss of the
shoot tip (apical dominance) leads to a rapid trigger that is not IAA mediated. C, Growing axillary buds/branches (correlative
inhibition) affect whether buds respond to depleted strigolactone and also reduce nutrient availability for bud growth and
elongation. D, Strigolactone inhibits bud outgrowth, whereas CK promotes it. In intact plants, IAA negatively regulates bud
outgrowth by maintaining high strigolactone and low CK contents. E, The IAA content of responsive buds increases and is
exported into the stem, allowing the bud to develop a more effective and substantive vascular system and attract nutrients for
growth. The extent of subsequent bud/branch growth is then strongly dependent on nutrient availability. This model illustrates the
developmental stages of a growing bud/branch. The arrows shown between stages represent the progression toward outgrowth
but do not preclude the fact that bidirectional development can occur, where inhibition can cause buds to revert to a previous
stage. References are indicated as follows: a, Morris et al. (2005); b, Beveridge (2000); c, Foo et al. (2005); d, Tanaka et al. (2006);
e, Miyawaki et al. (2004); Takei et al. (2004); f, Stafstrom (1995); g, Napoli et al. (1999); h, Bangerth (1989); i, Davies and
Wareing (1965); j, Phillips (1968); k, Ongaro and Leyser (2008); l, this study. [See online article for color version of this figure.]

Napoli et al. (1999). First, a wild-type dormant bud
requires a trigger to become receptive to outgrowth
signals and initiate growth (Fig. 11). One such signal is
the non-IAA-mediated fast-decapitation signal in-
duced following the loss of the shoot tip (apical
dominance). Growing axillary buds/branches (correl-
ative inhibition) influence outgrowth by affecting the
IAA status and relative sink strength within the plant,
in addition to, directly or indirectly, affecting the bud
responsiveness to strigolactone (Fig. 8; Bangerth, 1989;
Bangerth et al., 2000; Ongaro et al., 2008). Strigolactone
inhibits bud outgrowth, whereas CK promotes it. In an
intact plant, IAA acts to inhibit bud outgrowth by
maintaining high strigolactone and low CK content
(Fig. 11; Miyawaki et al., 2004; Nordstrom et al., 2004;
Foo et al., 2005; Johnson et al., 2006; Tanaka et al.,
2006). Low nitrate levels can also maintain a low CK
level (Miyawaki et al., 2004; Takei et al., 2004; for
review, see Sakakibara et al., 2006), which would
prevent branching in nitrate-limited conditions (Fig.
11). To induce bud outgrowth, reduced strigolactone
either acts independently of CK or at or before in-
creased CK production (Fig. 11), as reductions in RMS
expression in the absence of an increase in IPT expres-
sion fail to promote outgrowth (Fig. 10). Once trig-
gered to grow, IAA export from the bud into the main
stem increases (Bangerth et al., 2000; Ongaro and
Leyser, 2008), which is likely to aid in developing
strong vasculature connections to attract nutrients
(Fig. 11). The extent of sustained growth is then limited
by factors such as the ability of buds to continually
attract nutrients and acquire and/or attract energy/
carbon (Fig. 6, A and B).
MATERIALS AND METHODS
Plant Material and Growth Conditions
Unless stated otherwise, pea (Pisum sativum) seeds were sown two per pot
in 15-cm pots containing a fertilized (approximately 2 g of Osmocote per pot;
Scotts) mix of pasteurized 1:1 (v/v) peat:sand, as described by Dodd et al.

Plant Physiol. Vol. 149, 2009 1941

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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Ferguson and Beveridge
(2007). Plants were grown in a heated glasshouse (24°C/18°C day/night
regime) under an 18-h photoperiod (naturally lit, supplemented with 60-W
incandescent lights) and supplemented weekly with Aquasol (Hortico) nu-
trient solution. The wild-type cultivar used was ‘Torsdag’, and a description of
the nearly isogenic rms genotypes rms2-1 (K524), rms3-1 (K487), rms4-1 (K164),
rms5-3 (HL298), and rms6-2 (K586) can be found in Arumingtyas et al. (1992).
For basal lateral removal experiments, plants were grown under a natural 12-h
photoperiod to encourage branching. The buds located at nodes 0 to 3 were
removed as they appeared. Individual bud/branch lengths were scored when
the plants had 20 to 24 leaves expanded.
Stem-Girdling Treatments
A cup formed out of Blu-tack (Bostik) was placed around the internode to
be girdled. Using a pipette, candle wax (1–2 mL) heated to approximately
110°C was transferred to the cup. The heat of the wax kills the plant tissue
almost immediately, and the wax subsequently rehardens within minutes of
being applied. For decapitation studies, tissue was excised in the middle of the
internode or directly above the girdle using a sterile razor blade. In the case of
plants both girdled and decapitated, girdling was always performed first,
unless noted otherwise. For defoliation treatments, all expanded leaves,
including stipules, were removed above node 3. Bud outgrowth measure-
ments were scored using electronic calipers. Tissue dry weights were recorded
3 to 4 d after placing samples in an oven at 60°C. Internode dry weights
reported in Figure 4 consist of the entire internode and the node located
directly above it. Nodes were counted acropetally, with the cotyledonary
node as 0.
Analysis of Polar Auxin Transport
[3H]IAA was obtained from Amersham Pharmacia Biotech (specific activ-
ity, 25 Ci mmol21). [3H]IAA transport was analyzed as outlined by Morris et al.
(2005) following application of 8.5 kBq [3H]IAA in 2 mL of 50% ethanol. [3H]
IAA was applied to the apical bud of intact plants and allowed to be taken up
and transported for a period of 8 h. The leaves and stipules were then
removed, and the stem was divided into consecutive 3-mm segments. Radio-
activity was extracted directly into 6-mL PE vials (Perkin-Elmer) containing 2
mL of scintillant fluid (Ultima Gold; Packard BioScience). Samples were
shaken overnight to allow the [3H]IAA to leach out of the plant tissue and
were analyzed using a 1600 TR Tri-CARB Liquid Scintillation Analyzer
(Packard).
Hormone Treatments
For IAA treatments applied to the stem, plants having nine fully expanded
leaves were left intact or decapitated or girdled between nodes 8 and 9.
Immediately following, lanolin containing IAA (dissolved in ethanol) at a final
concentration of 3 mg g21 (final ethanol concentration of 10%) was applied
directly to the decapitated stump (as outlined in Morris et al., 2005) or in a ring
directly below the girdle site. Outgrowth measurements were recorded 7 d
following treatment.
For NPA treatments, plants having five fully expanded leaves were treated
between nodes 5 and 6 with a ring of lanolin containing 10 mg g21 NPA
dissolved in 100% ethanol (final lanolin ethanol concentration of 10%), as
outlined by Morris et al. (2005).
Quantitative Gene Expression Analysis
For gene expression studies, plants having nine fully expanded leaves
were left intact (control), decapitated, or girdled between nodes 3 and 4 or
nodes 8 and 9. Twenty-four hours later, 1-cm nodal stem segments, consisting
of internode and bud tissue, were harvested from nodes 2, 4, 8, and 9,
immediately frozen in liquid nitrogen, and stored at 280°C. Additional plants
were left unharvested to confirm bud outgrowth phenotypes.
Gene expression analysis was performed similar to that outlined by
Johnson et al. (2006). Total RNA was isolated using NucleoSpin RNA plant
kits (Machery-Nagel) and quantified using NanoDrop 1000. cDNA was
generated in a 20-mL volume with 1.6 to 5 mg of total RNA, 50 to 250 ng of
random hexamers (Invitrogen), 0.5 mM deoxynucleoside triphosphates, 5 mM
dithiothreitol (Invitrogen), 1.253 first-strand buffer, 40 units of RnaseOut
(Invitrogen), and 200 units of SuperScript III reverse transcriptase (Invitrogen)
1942
Downloaded from www.plantphysiol.org on March 20, 2015 - Published by www.plant.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
incubated at 50°C for 1 h. cDNA quality was checked via PCR (25 cycles) and
gel electrophoresis with actin primers: forward (5#-CAACTATGTTTCCCGG-
TATTG-3#) and reverse (5#-AAGTCTGTGCCTCGACATCC-3#). Transcript
levels using 16 ng of template cDNA were measured using a 7900 HT Fast
real-time PCR system (Applied Biosystems) with 5 mL of SYBR Green PCR
Master Mix (Applied Biosystems) and 1 mL each of 400 nM forward and
reverse primers of RMS1, RMS5, IAA4/5, IPT1, IPT2, and 18S. Primers used
were as follows: RMS1 forward (5#-AAGGAGCTGTGCCCTCAGAA-3#) and
RMS1 reverse (5#-ATTATGGAGATCACCACACCATCA-3#; Foo et al., 2005);
RMS5 forward (5#-CGGCATCTTAAAGACTCCGTACA-3#) and RMS5 re-
verse (5#-TGGATACGATCGGGAAGTTCA-3#; Johnson et al., 2006); IAA4/5
forward (5#-GTTCTTCTGCAGCCCCTCCTG-3#) and IAA4/5 reverse (5#-ACA-
AAGATCCCACCAACATCAGCC-3#), designed using Primer Express 2.0
software (Applied Biosystems); IPT1 forward (5#-ACCGTCTTGATGCTACG-
GAGGTTGTGC-3#) and IPT1 reverse (5#-TCTAATGGGTTACCCCTGCCA-
CAGACG-3#; Tanaka et al., 2006); IPT2 forward (5#-TGGCAGCAACAT-
CATCCTCTGCCTGC-3#) and IPT2 reverse (5#-ACCTGTGGCCCCCATTAT-
CACTAC-3#; Tanaka et al., 2006); and 18S forward (5#-ACGTCCCTGCC-
CTTTGTACA-3#) and 18S reverse (5#-CACTTCACCGGACCATTCAAT-3#;
Ozga et al., 2003). Relative expression was calculated based on primer
efficiencies calculated using LinRegPCR 7.5 software (Ramakers et al., 2003).
RMS1, RMS5, IPT1, IPT2, and IAA4/5 expression was measured against that of
18S. For all experiments, two or three biological replicates each consisting of at
least six plants were used, with error bars in the figures representing different
biological replicates.
Statistical Analysis
Where comparisons were made, means were discriminated using Stu-
dent’s unpaired t test (P # 0.05).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Mean lengths of individual buds located at
internodes 1 to 3 of wild-type pea 9 d following decapitation between
nodes 8 and 9 and/or girdling between nodes 3 and 4.
ACKNOWLEDGMENTS
We thank Tanya Brcich, Ji Kim, Steve Kazokov, and Kerry Condon for
technical assistance and Dr. Elizabeth Dun for helpful suggestions regarding
the manuscript. We give special thanks to Zheng Zhang for performing the
bulk of the experiments in Figures 1, 2, and 7 and to Dr. Mike Hay for helpful
discussions regarding girdling techniques.
Received January 8, 2009; accepted February 3, 2009; published February 13,
2009.
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Plant Physiol. Vol. 149, 2009
 200
1 Node 1
180
2 Node 2
160
3 Node 3
an Bud Lengtth (mm) 140

120

100

80
Mea

60

40

20

0
Intact
Intact Girdle
Girdle Decap
Decap Girdle
Girdle Decap
15 min Decap 15 min Decap
Decap 4h
Decap
15 min Girdle
15 min Girdle
4h
Treatment
Decap Girdle Girdle
Treatment

Supplemental Figure S1. Mean lengths of individual buds located at internodes 1-3 of WT pea 9 d following
decapitation
p between nodes 8 and 9 and/or ggirdling
g between nodes 3 and 4. Decapitation
p occurred at the
same time for all treatments (time 0), whereas the timing of stem girdling was staggered. Data are means ±
S.E.; n = 6 to 8.