Strigolactone Inhibition of Branching Independent of
Polar Auxin Transport1[OPEN]
Philip B. Brewer, Elizabeth A. Dun, Renyi Gui 2, Michael G. Mason, and Christine A. Beveridge *
The University of Queensland, School of Biological Sciences, St. Lucia, Queensland 4072, Australia
ORCID IDs: 0000-0003-4871-9260 (P.B.B.); 0000-0003-4068-0349 (E.A.D.); 0000-0003-3098-2861 (R.G.).
The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for
sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals,
cytokinins and strigolactones, which can move into axillary buds. The other involves suppression of auxin transport/canalization from
axillary buds into the main stem and is enhanced by a low sink for auxin in the stem. In this theory, the relative ability of the buds and
stem to transport auxin controls bud outgrowth. Here, we evaluate whether auxin transport is required or regulated during bud
outgrowth in pea (Pisum sativum). The profound, systemic, and long-term effects of the auxin transport inhibitor N-1naphthylphthalamic acid had very little inhibitory effect on bud outgrowth in strigolactone-deficient mutants. Strigolactones can
also inhibit bud outgrowth in N-1-naphthylphthalamic acid-treated shoots that have greatly diminished auxin transport. Moreover,
strigolactones can inhibit bud outgrowth despite a much diminished auxin supply in in vitro or decapitated plants. These findings
demonstrate that auxin sink strength in the stem is not important for bud outgrowth in pea. Consistent with alternative mechanisms of
auxin regulation of systemic signals, enhanced auxin biosynthesis in Arabidopsis (Arabidopsis thaliana) can suppress branching in
yucca1D plants compared with wild-type plants, but has no effect on bud outgrowth in a strigolactone-deficient mutant background.
Shoot branching is a highly plastic developmental trait
that is greatly affected by genetic and environmental
factors. Branches develop from buds that often enter a
state of highly repressed growth, which can be rapidly
reactivated to stimulate branch development in response
to systemic cues from the plant, such as Suc (Mason
et al., 2014). The mechanism of auxin action as the hormonal component of the shoot tip that suppresses axillary buds has led to a model composed of two major
hypotheses, auxin transport/canalization and direct
hormone action (Fig. 1). Here, we address the relative
importance of these hypotheses in shoot branching.
In the auxin transport/canalization hypothesis,
auxin in the main stem is thought to act by inhibiting
the flow of auxin from buds (Fig. 1; Shinohara et al.,
2013; Waldie et al., 2014). According to this hypothesis,
the plant hormone strigolactone inhibits bud growth
1
This work was supported by the Australian Research Council
Discovery and Future Fellowship programs.
2
Present address: Nurturing Station of State Key Laboratory of
Sub-tropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China.
* Address correspondence to [email protected].
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 ([email protected]).
P.B.B., E.A.D., and C.A.B. conceived the project, designed and carried out the experiments, analyzed the data, and wrote the article;
R.G. and M.G.M. designed and carried out the experiments, analyzed
the data, and helped to write the article.
[OPEN]
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by dampening auxin transport and creating a reduced
sink for auxin in the auxin-transporting cells in the
vasculature of the main stem (Fig. 1). The case in
support of this hypothesis is compelling due to experiments showing that auxin in the main stem repels
bud vascular connections (Sachs, 1969), and that auxin
flow from a growing shoot tip correlates with the
repression of other buds or branches (Morris, 1977;
Bangerth, 1989; Bennett et al., 2006). Experiments
showing that strigolactone mutants can have enhanced
levels of auxin and auxin transport in the main stem
(Beveridge, 2000; Beveridge et al., 2000; Crawford et al.,
2010; Agusti et al., 2011) and that strigolactone can only
inhibit branching in vitro in the presence of an apical
auxin supply support the hypothesis that strigolactones
could act via repression of auxin canalization (Bennett
et al., 2006; Ongaro and Leyser, 2008; Crawford et al.,
2010; Liang et al., 2010). The polarization and localization
of PIN-FORMED (PIN) auxin export proteins on cell
membranes, which are important for polar auxin transport (Wisniewska et al., 2006), are thought to promote
the establishment of a channel of focused auxin flow that
coincides with new vasculature formation (Sauer et al.,
2006). As predicted, PINs are apolar in the vasculature of
repressed buds in intact plants, but 2 to 6 h after removal
of the main shoot tip (decapitation), auxin builds up in the
buds (Gocal et al., 1991), and buds commence polar auxin
transport as PINs polarize toward the stem (Balla et al.,
2011). In Arabidopsis (Arabidopsis thaliana), strigolactones
promote PIN internalization, reducing the abundance
of PIN proteins available for auxin transport (Fig. 1;
Shinohara et al., 2013). This is a major finding in support
of strigolactone action via auxin transport.
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Shoot Branching and Auxin Transport
Figure 1. Experimentally demonstrated relationships among key
branching signals and bud outgrowth. These relationships have been used
to support the direct action or auxin transport/canalization hypotheses of
bud outgrowth. Auxin, which moves basipetally in the stem, inhibits
cytokinin levels (Li et al., 1995; Tanaka et al., 2006) and promotes expression of strigolactone biosynthesis genes (Foo et al., 2005; Zou et al.,
2006; Arite et al., 2007; Hayward et al., 2009). These hormones, strigolactone and cytokinin, in addition to Suc (Mason et al., 2014), can
move acropetally into buds and regulate branching (Dun et al., 2012).
This is mediated at least partly via regulation of BRANCHED1 (BRC1),
which occurs rapidly (Mason et al., 2014) and/or without the need for
protein synthesis (Dun et al., 2012). BRC1 encodes a transcription factor
required for branching inhibition (Doebley et al., 1997; Aguilar-Martı́nez
et al., 2007; Braun et al., 2012; Dun et al., 2012). These findings led to
the direct action hypothesis of bud outgrowth. An alternative, but not
necessarily mutually exclusive, hypothesis is that reduced auxin transport/
canalization from axillary buds suppresses their outgrowth. In support of
this, strigolactones can reduce auxin transport in the stem (Crawford
et al., 2010) and enhance PIN cellular internalization (Shinohara et al.,
2013). N-1-Naphthylphthalamic acid (NPA) used in this study inhibits the
function of PIN and ABCB auxin transport proteins (Kleine-Vehn et al.,
2006; Petrásek et al., 2006; Sauer et al., 2006; Blakeslee et al., 2007).
The direct action hypothesis of bud outgrowth
highlights the role of strigolactones independent of
auxin transport and the role of strigolactone acting
downstream of auxin (Fig. 1; Beveridge, 2000;
Beveridge et al., 2000; Brewer et al., 2009). Combined
grafting and hormone studies with strigolactone mutants led to the hypothesis that auxin acts indirectly via
other signals, cytokinins and strigolactones (Beveridge,
2000; Beveridge et al., 2000; Brewer et al., 2009), as
predicted by Snow (1937). In this hypothesis, cytokinins and strigolactones act as long-distance secondary
messengers for auxin, but do not require auxin to act.
Accordingly, strigolactone supplied directly to axillary
buds of garden pea (Pisum sativum) can inhibit
branching all along the stem of decapitated, auxindepleted plants (Brewer et al., 2009; Dun et al., 2013).
Moreover, unless given a strigolactone supply by
grafting, strigolactone-deficient mutants respond very
poorly to auxin added to the stump after decapitation
(Beveridge et al., 2000), indicating that auxin requires
strigolactones to elicit its inhibitory effect. Additionally, exogenous strigolactone can reduce branching in
auxin mutants and reduce growth of large axillary
buds without affecting radiolabeled auxin transport (Brewer et al., 2009). Higher auxin transport
in strigolactone mutants (Bennett et al., 2006) may
be a consequence of the feedback loop between
strigolactones and auxin (Hayward et al., 2009), rather
than the cause of branching, and is not always observed
in pea strigolactone mutants (Brewer et al., 2009).
The mechanism of the direct action of strigolactones
has not been fully elucidated, but includes the budspecific transcription factor BRC1 in pea and Arabidopsis,
TEOSINTE BRANCHED1 in maize (Zea mays), and FINE
CULM1 in rice (Oryza sativa; Doebley et al., 1997; Takeda
et al., 2003; Aguilar-Martínez et al., 2007; Finlayson, 2007;
Braun et al., 2012). In support of this, brc1 mutants are
highly branched (Doebley et al., 1997; Takeda et al., 2003;
Aguilar-Martínez et al., 2007; Finlayson, 2007; Braun
et al., 2012), and BRC1 is regulated by strigolactones
without the need for protein synthesis (Dun et al., 2012).
Indeed, BRC1 is broadly regarded as an integrator of
branching signals and the environment (Kebrom et al.,
2006; Aguilar-Martínez et al., 2007; Minakuchi et al.,
2010; Braun et al., 2012; Dun et al., 2012). In pea, BRC1 is
also regulated by cytokinins and Suc (Braun et al., 2012;
Dun et al., 2012; Mason et al., 2014), and it is possible
that cell cycle progression is repressed by BRC1 in the
bud (Martín-Trillo and Cubas, 2010).
To unravel the relative importance of these hypotheses, we first sought to explore the isolated-node in
vitro system that shows a strigolactone response only in
the presence of auxin (Bennett et al., 2006; Ongaro and
Leyser, 2008; Crawford et al., 2010; Liang et al., 2010),
because this might give information as to why apical
auxin has been required to inhibit bud outgrowth in
isolated nodes but is not required for the same response
in decapitated plants (Brewer et al., 2009; Dun et al.,
2013). We are also intrigued by the observation that
auxin can inhibit outgrowth in nodal segments from
strigolactone mutants in vitro (Young et al., 2014),
which lack shoot tip, other buds, and a root system, but
cannot do so in decapitated plants (Beveridge et al.,
2000), which lack only the shoot tip. We then conducted
a range of experiments, in vivo, to further test the auxin
canalization process in pea and, finally, in Arabidopsis.
In so doing, we tested NPA as an essentially qualitative,
systemic inhibitor of auxin transport in pea. NPA inhibits the transport function of PIN and ATP-binding
cassette subfamily B (ABCB) auxin transporters in a
synergistic way (Petrásek et al., 2006; Blakeslee et al.,
2007), but NPA does not remove PIN transporters from
the cell membranes (Kleine-Vehn et al., 2006; Sauer
et al., 2006). Thus, NPA would be expected to block auxin
transport downstream of the action of strigolactones on
PIN internalization. We then tested whether bud outgrowth could commence and persist under conditions of
severely impaired auxin transport, and whether this
prevented strigolactone action.
RESULTS
Strigolactone Can Act Independently of Auxin in Vitro
One of the findings commonly used to support the
auxin transport/canalization model is the requirement
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Brewer et al.
for a competing auxin source for strigolactone-mediated
bud inhibition (Crawford et al., 2010; Shinohara et al.,
2013; Waldie et al., 2014). In a split-plate in vitro assay
similar to that used for Arabidopsis (Chatfield et al.,
2000; Bennett et al., 2006), we also found that pea buds
do not respond to a basal supply of GR24, a synthetic
strigolactone, unless they are also supplied with auxin
apically (Fig. 2, B and C). However, when we applied
GR24 directly to buds in this in vitro system, we observed repressed growth without an auxin supply in
both the wild type (Fig. 2D) and a strigolactonedeficient branching mutant (Fig. 2E). Consequently,
apical auxin is not essential for bud growth inhibition
by strigolactone in vitro or in vivo (decapitated plants;
Brewer et al., 2009; Dun et al., 2013).
However, experiments with a new in vitro method
gave strikingly contrasting results to the split-plate assay. Segments comparable with those used in the splitplate assay (Fig. 2) were inserted into individual, small,
open, agar-filled tubes (Fig. 3A) and exposed to the
same concentration of GR24 as the previous in vitro
experiment. These segments were then responsive to
GR24 in the medium, even in the absence of apical
auxin (Fig. 3B). This was repeated in independent experiments, including ones with a strigolactone-deficient
branching mutant (Fig. 3C). It is unclear exactly why
basally supplied strigolactone is ineffective in the split
plate (Fig. 2) but very effective in individual, open,
agar-filled tubes (Fig. 3). However, the new findings for
pea show that direct application of strigolactones to
buds in the split-plate assay and strigolactone supplied
basally in the individual tubes can indeed inhibit bud
outgrowth in isolated segments without an exogenous
auxin supply. This supports the hypothesis of direct
action, that strigolactones act downstream of auxin.
NPA Is a Rapid, Long-Lasting, and Systemic Inhibitor of
Auxin Transport
Figure 2. A, Strigolactone applied directly can inhibit bud growth in
pea in the classical isolated-node, split-plate assay. B, Strigolactone
(GR24, basal) requires auxin (naphthylacetic acid [NAA], apical) to
inhibit pea bud growth. C, Strigolactone-deficient ramosus1 (rms1)
mutant with 0 or 0.5 mM NAA (apical) and 0 or 10 mM GR24 (basal) in
the medium. B and C, Results are from the same experiment. D and E,
Daily application of 10 mL of 1 mM GR24 alone directly to buds (Dun
et al., 2013) suppressed growth in the wild type (WT) and the rms1
Although exogenous apical auxin is not required for
strigolactone to inhibit branching in pea in vitro (Figs.
2 and 3) or in decapitated plants (Brewer et al., 2009;
Dun et al., 2013), it is possible that fully intact systems
may somehow differ in strigolactone action and auxin
interplay. Furthermore, with respect to the canalization model, one might hypothesize that bud growth
inhibition via auxin canalization might still occur in
these systems without apical auxin due to exogenous
GR24 dramatically enhancing PIN removal in buds
(Waldie et al., 2014). We sought to test the prediction
that auxin transport is required for bud growth in vivo
(Shinohara et al., 2013; Waldie et al., 2014) by carefully
observing auxin transport and branching after pea
plants were exposed to a high dose of NPA. As bud
strigolactone-deficient mutant. Data are presented as the mean 6 SEM,
provided the SEM exceeds the size of the symbol; n = 12.
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Shoot Branching and Auxin Transport
outgrowth occurs over several days, and auxin canalization from buds may continue to be important during
this time, we sought to determine whether NPA could
act rapidly and over the long term. Radiolabeled auxin
(3H-indole-3-acetic acid [3H-IAA]) transport from the
shoot tip was measured over time after a single treatment of 0.1% (w/v) NPA in lanolin as a ring around the
stem above the highest expanded leaf. Very little auxin
transport was detected during the period of 2 h to
5 d after treatment with NPA (Fig. 4; Supplemental Fig.
S1). For example, on day 2, when strigolactone-deficient
mutant plants showed significant outgrowth, wild-type
and mutant plants treated with NPA transported 0.4%
or 0.3%, respectively, of the radiolabel from 3H-IAA
compared with control-treated plants (measured at 6
cm; Supplemental Fig. S1). The amount of polar auxin
transport in NPA-treated plants is likely to be considerably less than this, because the very small movements
of radiolabel from the 3H-IAA supplied to the NPAtreated plants do not show the classic polar transport
shape (Supplemental Fig. S1). The movement is too fast
to be caused by diffusion (Kramer et al., 2011) but is
certainly fast enough to be caused by phloem transport
(Morris et al., 1973), which is not a proposed target of
strigolactones. In contrast, strigolactones have been
shown to affect PIN localization (Shinohara et al., 2013),
which directly affects the polar auxin transport pathway (Wisniewska et al., 2006).
As NPA appeared to act at least within 2 h (Fig. 4A;
see also Thomson et al., 1973; Sussman and Goldsmith,
1981) and probably over distance (Supplemental Fig. S1),
we sought to gauge more carefully how quickly NPA
may systemically affect auxin transport in shoots in vivo
in pea, particularly over a long distance (e.g. 30 cm).
Stems were treated with 1% (w/v) NPA in lanolin about
30 cm from the shoot tip, and to capture any changes
over a short period, shoot tips were then treated at 2 and
4 h with 3H-IAA (Fig. 5A). A significant difference in
radiolabel from 3H-IAA was observed in NPA-treated
compared with control plants, at positions including
near the peak front (4 cm; P , 0.005), indicating that
NPA has a systemic effect as early as 2 h over a distance
of about 30 cm (Fig. 5A). To test whether NPA itself
moves systemically in shoots, we added 3H-NPA to the
1% (w/v) NPA lanolin mixture. Based on the radiolabel
recovered from 3H-NPA, up to 0.26 mg of NPA was
delivered to the shoot tip within 6 h (Fig. 5B). This
equates to at least 40 mM in a 20-mg shoot tip, which is
well within the range of the concentrations typically used
in vitro to affect auxin transport (Kleine-Vehn et al., 2006;
Petrásek et al., 2006; Sauer et al., 2006; Blakeslee et al.,
2007). These findings of the rapid and systemic effects of
NPA validate its use in the investigation of the role of
auxin transport in axillary bud outgrowth.
Auxin Transport Is Not Correlated with Shoot Branching
We included strigolactone-deficient plants in the
auxin transport experiments above (Fig. 4) to test
whether they are affected in auxin transport or in response to NPA. In this experiment, as in Brewer et al.
(2009), there was no enhancement of auxin transport
in strigolactone-deficient mutant control plants
compared with wild-type control plants (Fig. 4A;
Supplemental Fig. S1). This serves to highlight that
differences in auxin transport are not always observed
in strigolactone mutants of pea (e.g. Beveridge et al.,
2000; Brewer et al., 2009). If strigolactones were to
act primarily via auxin transport to regulate shoot
branching, we would expect to observe differences in
auxin transport that correlate with the timing of bud
outgrowth. Whereas buds remained small in wild-type
plants, branching occurred in strigolactone-deficient
mutant plants over the course of this experiment
(Fig. 4B) and during a period when we consistently
observed no enhancement of auxin transport in mutant control plants compared with the wild type.
Previous studies in pea have also shown that, unlike
strigolactone, which is highly effective on small buds,
1 mM NPA in solution administered directly to small
buds of a size comparable with inhibited intact wildtype plants (approximately 1 mm) has little or no effect
on bud outgrowth after decapitation (Mason et al.,
2014) or in strigolactone mutant plants (Brewer et al.,
2009). In contrast, little branches 1 cm in length also
show a small inhibitory growth reduction in response
to NPA (Brewer et al., 2009). As pointed out by Waldie
et al. (2014), this reduction of growth by NPA was
similar to the GR24 response, but this similarity is restricted to this advanced stage of bud growth and to a
period when GR24 is less able to inhibit bud growth
(Brewer et al., 2009; Dun et al., 2013). In contrast to
Arabidopsis, GR24 had no effect on auxin transport in
pea (Brewer et al., 2009). In Arabidopsis, GR24 causes
small effects on 3H-IAA transport compared with NPA
(Crawford et al., 2010), and low levels of NPA can
reduce bud growth measured at 4 weeks (e.g. 1 mM
NPA; Bennett et al., 2006). According to Waldie et al.
(2014), this may be attributed to the different molecular effects of GR24 and NPA on auxin transport.
However, this does not explain how the lack of a
measurable effect of GR24 on 3H-IAA transport in pea
can coincide with significant effects on growth via
auxin canalization, or why NPA is poorly active in
inhibiting smaller axillary buds of pea (Brewer et al.,
2009). Here, despite demonstrating substantially reduced auxin transport over an extended period, there
was still no effect of 0.1% (w/v) NPA on axillary bud
outgrowth in the strigolactone-deficient mutant (Fig.
4B). In the wild type, branching was promoted below
the NPA treatment site (Fig. 4B), consistent with other
studies showing bud growth promotion by inhibition
of auxin transport (Snyder, 1949; Tamas, 1987; Prasad
et al., 1989; Tamas et al., 1989). The likely cause of this
bud outgrowth may be reduced auxin content, as
reported by Tamas (1987) and Morris et al. (2005),
which would then enhance cytokinin and reduce
strigolactone content, which would act to promote
bud outgrowth. As discussed below, it is likely that
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Figure 3. A, An open-tube in vitro method enables a basal strigolactone supply to inhibit bud
outgrowth in the absence of an apical auxin
supply. Segments similar to the experiment in
Figure 2 are placed in agar as shown. GR24
supplied basally reduced bud outgrowth in wildtype (WT; B) and rms5 (C) strigolactone-deficient
plants. Data are presented as the mean 6 SEM;
n = 11 or 12 (B); n = 14 or 15 (C).
auxin transport was also reduced in these wild-type
branches, again not consistent with the auxin canalization model.
To address the issue of whether auxin transport is
actually required for bud outgrowth, we analyzed the
effects of 1% (w/v) NPA, which, as we have shown
above, reduces auxin transport to ,0.4% of untreated
wild type and below the level thought to be required
for auxin canalization-driven branching inhibition
(i.e. 30% of the wild type; Shinohara et al., 2013;
Waldie et al., 2014). To test whether a ring of 1% (w/v)
NPA on the main stem (e.g. Fig. 4) only affects auxin
transport in the main stem and not in axillary
branches, we promoted bud outgrowth in the wild
type by decapitation and simultaneously added the
NPA in lanolin to the stem at a position three internodes below. In so doing, we were able to evaluate
auxin transport in large branches that quickly developed above and below the NPA treatment site (Fig.
5C). 3H-IAA added to the tips of the decapitationinduced branches, which were at least 4 cm in
length, indicated that auxin transport was severely
suppressed in these branches by the stem NPA treatment compared with control treatment. Little radiolabel
from 3H-IAA was recovered beyond the apical-most
Figure 4. NPA (0.1% [w/v]) acts profoundly on polar auxin transport for
a long duration and does not inhibit bud outgrowth. A, Total 3H-IAA
transported in the main stem of wild-type (WT) and rms1 plants was
assayed at different intervals over several days after control or 0.1% (w/v)
NPA treatment. The individual data from each time point are shown in
Supplemental Figure S1. B, Bud outgrowth at the node below the NPA
treatment site is shown for comparable plants. A and B, Most rms1
symbols are obscured by wild-type, NPA symbols. All data are from the
same experiment and are presented as the mean 6 SEM plotted on a log
scale; n = 5 or 6 (A); n = 7 to 10 (B). The times shown are the harvest (A)
or scoring time (B) from the NPA treatment at time 0.
1 cm of branches above or below the NPA site compared with branches of control decapitated plants,
which transported large quantities of radiolabel (Fig.
5C). Again, this study confirms that branches can grow
out in plants with dramatically reduced polar auxin
transport both within the stem and within the growing
branch itself.
Strigolactone Treatment Inhibits Bud Outgrowth
Regardless of Auxin Transport Status
If strigolactones regulate bud outgrowth by affecting
auxin transport from axillary buds (Shinohara et al.,
2013), then strigolactones should not be able to inhibit branching under dramatically reduced polar auxin
transport. Alternatively, if strigolactones act more
downstream or independently of auxin on axillary bud
outgrowth, then they ought to be able to inhibit
branching even when polar auxin transport is suppressed. Moreover, according to the direct action model,
strigolactones should inhibit bud outgrowth regardless
of auxin content or transport. Thus, we designed two
experiments in which branching would occur during
auxin transport inhibition by 1% (w/v) NPA. This included intact plants grown under growth-promoting
conditions of supplemented light (Fig. 6A) and decapitated plants with reduced auxin content (Fig. 6B). We
treated the buds of these NPA-treated plants directly
with the synthetic strigolactone, GR24, and observed a
significant reduction in growth in both intact and decapitated plants (Fig. 6). An approximately 50% inhibition by GR24 occurred regardless of control or NPA
treatment (Fig. 6B). Given the magnitude of reduction in
auxin transport by the 1% (w/v) NPA (approximately
0.4% of the wild type), this full inhibitory effect of GR24
is unlikely due to altered polar auxin transport (Figs. 4
and 5). This is consistent with the aforementioned finding where strigolactone-deficient mutants continue to
branch when polar auxin transport is suppressed (Fig.
4B). It is also consistent with strigolactones acting in a
more direct mechanism, such as via regulation of BRC1,
independent of auxin transport.
Auxin-Repressed Branching Requires Strigolactones
Having discovered that polar auxin transport is not
required for strigolactone inhibition of bud outgrowth,
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Shoot Branching and Auxin Transport
Figure 5. NPA acts rapidly and systemically to block polar auxin transport in the main stem and axillary shoots. A, IAA transport
from the shoot tip is rapidly blocked by 1% (w/v) NPA treated about 30 cm below the highest expanded leaf. Plants were
supplied with 3H-IAA to the shoot tip at 2 and 4 h after NPA or lanolin control treatment. Segments were harvested at 6 h. n = 6.
B, Within 6 h, NPA moves and accumulates to about 40 mM in the shoot tip 30 cm away. One percent NPA was supplemented
with approximately 62 kBq 3H-NPA per plant and was supplied as per A. Adjacent 1-cm segments were taken on either side of
the treatment site. The apical bud and two lower 1-cm sections were also taken. n = 6. C, IAA transport inhibition persists
systemically even though branches are able to form. Plants were decapitated below node 9, and 1% (w/v) NPA was added
below node 6. 3H-IAA was added to the apex of axillary shoot tips at node 7 (upper) or node 4 (lower). Branches were at least 4
cm in length (day 10). The total percent transport to segments at 1 to 4 cm was 0.8% and 0.3%, respectively, for the upper and
lower branches; at 1.5 cm, the fold drop was 560 to 1,100; n = 4. Controls from lanolin-treated decapitated plants were the
combined nodes 7 and 4; n = 2. Segments were harvested after 2 h. A to C, Stems were lightly abraded at the treatment site
before NPA or lanolin treatment in a ring around the stem. Data presented on a log scale are the mean 6 SEM, provided the SEM
exceeded the size of the symbol.
we next tested the other important aspect that contributes to the auxin sink in the stem: auxin level
(Shinohara et al., 2013). Auxin is produced predominantly in young leaves of growing apical shoots and
then transported in the polar auxin transport stream
(Snow 1937). Following the canalization hypothesis,
enhanced auxin content in the stem should suppress
auxin flow from buds due to decreased auxin sink
strength of the main stem (Fig. 1; Shinohara et al.,
2013). Consequently, both wild-type and strigolactone
mutant plants should therefore show some branching
inhibition under high stem auxin content. Alternatively, if the enhanced auxin content acts only via enhanced strigolactones, the wild type should respond to
enhanced auxin content, but a strigolactone mutant
would not. We therefore took advantage of the dominant, gain-of-function yucca1D (yuc1D) mutant, which
has enhanced auxin biosynthesis due to overproduction of a flavin monooxygenase (Zhao et al., 2001),
to examine the effect of enhanced auxin levels in
Arabidopsis. We compared the branching phenotype
of yuc1D and the strigolactone-deficient more axillary
growth3 (max3) yuc1D double mutant with the wild
type and strigolactone-deficient max3. Branching was
significantly suppressed in yuc1D compared with the
wild type (Fig. 7). In contrast, the double mutant
combination appeared identical to max3 single mutants in increased branching (Fig. 7). This indicates that
virtually all of the branching repression induced by
increased auxin production in yuc1D acts through
strigolactones, and therefore, there is little or no effect
of decreasing the auxin sink strength of the main stem.
DISCUSSION
Here, we discover that auxin transport does not
correlate well with shoot branching. Strigolactone
mutants of pea branch out during a period when there
is no difference in polar auxin transport between
strigolactone-deficient plants and wild-type plants. In
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Brewer et al.
Figure 6. Strigolactone does not require auxin transport to inhibit
branching. Plants were left intact under high light conditions (A) or
were intact or decapitated under standard conditions (B). A and B,
Plants were treated with 0% or 1% (w/v) NPA midway between nodes
6 and 7 when the plants had nine leaves expanded. Five microliters of
5 mM GR24 (Dun et al., 2009) applied to the bud at node 6 inhibited
bud outgrowth compared with control-treated buds. Branches were
measured on day 4. Data are the mean 6 SEM. A, Intact plants were
encouraged to branch by supplementing the standard greenhouse light
conditions with halogen lights, which provided an additional 140 to
200 mmol m22 s21, approximately, at the pot top and 40 cm above the
pot top, respectively. Control plants, n = 7; NPA-treated plants, n = 14
or 15. B, Plants were left intact or decapitated above node 9, and buds
were treated with control or GR24 solution daily. Intact control plants,
n = 3; all other treatments, n = 9. n.d., not determined.
addition, strigolactones can inhibit branching in vitro
or in vivo without an apical auxin supply. Moreover,
buds can be grown from a state of suppressed growth
to a well-developed branch with severely diminished
polar auxin transport. All of these findings can be
explained in terms of strigolactones acting directly on
bud outgrowth (Fig. 1). However, these findings are
not consistent with auxin canalization being required for bud outgrowth, as the auxin transport/
canalization hypothesis has been reported to require
an auxin source and a directional flow of auxin
(Shinohara et al., 2013; Waldie et al., 2014). Furthermore, consistent with the direct action model, enhanced auxin biosynthesis in the yuc1D mutant, and
any resultant change in stem auxin content, requires
strigolactones to inhibit branching.
The new branches on NPA-treated plants showed
some twisting and poor leaf expansion, but otherwise
grew remarkably well (Figs. 4–6; Supplemental Fig.
S2). This is consistent with established roles of auxin
transport in tropic shoot growth and is reminiscent of
the phenotypes of mutants that affect ABCB transporters (e.g. Blakeslee et al., 2007). The weak effect on
branch morphology may be due to the relatively short
length of treatment and prior organogenesis of axillary
buds. Inhibited axillary buds already have several leaf
primordia, small leaves and internodes, and contain
vasculature, all laid down during initial bud formation
(Pate, 1975; Stafstrom and Sarup, 2000; Kang et al.,
2003). In contrast to strigolactones, NPA sometimes
promotes bud outgrowth (e.g. Fig. 4B), sometimes reduces branch elongation (e.g. Fig. 6B; Brewer et al.
2009), and sometimes has little effect (e.g. Morris et al.,
2005; Mason et al., 2014). This might have been
explained by the canalization model in which different
states of auxin transport predict different branching
outcomes (Shinohara et al., 2013). However, this model
also predicts that greater than 30% of the wild type is
required for auxin canalization and bud outgrowth.
The considerable growth of buds into branches during
repression of auxin transport by 1% (w/v) NPA (Figs.
5C and 6) demonstrates that auxin transport is not
required for bud outgrowth. In contrast, the outgrowth
of axillary buds and their inhibition by strigolactone
under these conditions is consistent with the direct
action model.
The growth of branches during severely diminished
auxin transport occurs with various tissue and vasculature development (Supplemental Fig. S2). This is
consistent with previous findings. In the early studies
of auxin transport and phyllotaxis, which described
the development of a pin-like structure in NPA-treated
plants, plants were grown for much longer periods on
very high NPA concentrations (15–50 mM in medium)
and were able to develop some leaves and inflorescence stems with vascular tissues (Okada et al., 1991;
Gälweiler et al., 1998; Mattsson et al., 1999). Some
vasculature forms even in the presence of up to 160 mM
NPA (Mattsson et al., 1999). These data and modeling
experiments have led others to conclude that auxin
canalization during vascular development can occur
without polar auxin transport (Rolland-Lagan and
Prusinkiewicz, 2005).
In contrast with the findings here, other studies have
shown that NPA partially inhibited bud outgrowth in
Arabidopsis and rice strigolactone-deficient mutants
(Bennett et al., 2006; Lin et al., 2009). However, those
results were obtained from plants exposed to continual, long-term NPA treatments in the growth medium
that affected overall plant growth, and it is likely that
NPA and/or associated suppressed auxin transport
Figure 7. Reduced branching due to increased auxin biosynthesis requires strigolactone production. The yuc1D mutant of Arabidopsis,
which has increased auxin production (Zhao et al., 2001), also has
decreased branching (P , 0.05, two-tailed t test; n = 7–12). This effect
of decreased branching is not expressed in the strigolactone-deficient
max3-11 background.
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Shoot Branching and Auxin Transport
may have had secondary effects that affect branching.
In this study, we have affected auxin transport specifically during bud release and outgrowth and
observed no reduced branching in pea strigolactonedeficient mutants compared with control-treated
plants (Fig. 4B). In this and other studies in which an
auxin transport inhibitor has been supplied in a ring
around the stem in wild-type plants, increased, rather
than decreased, branching has been observed below
the site of application (Snyder, 1949; Prasad et al., 1989;
Figs. 4B and 6A). This can be best interpreted in terms
of effects of NPA on hormone levels below the site of
application (Morris et al., 2005).
Another important piece of evidence in support of
the auxin transport/canalization model, and which
needs interpreting in terms of the direct action model,
is that a very low dose of strigolactone can promote
branching in the transport inhibitor resistant3 (tir3)
mutant (Shinohara et al., 2013). The highly pleiotropic
tir3 mutant is hypersensitive to strigolactone inhibition
of root growth (Shinohara et al., 2013), which could
increase available Suc for shoot and axillary bud
growth as well as decrease cytokinin contents, both of
which could explain this enhanced shoot branching via
direct action (Fig. 1). Moreover, the function of the
TIR3 protein, otherwise known as BIG, is not fully
established at the molecular level, despite being well
known as affected in auxin transport (Leyser, 2010). In
addition, the weakest concentration at which max4
mutants respond to strigolactone for branching inhibition is the same concentration that is toxic to tir3, causing extremely stunted overall plant growth (Shinohara
et al., 2013).
If strigolactone instead functions almost entirely via
direct action to regulate bud outgrowth, then why
does strigolactone promote the cellular internalization
of PIN auxin transporters (Shinohara et al., 2013) and
reduce expression of auxin signaling and transport
genes in the main stem (Bennett et al., 2006; Hayward
et al., 2009; Waters et al., 2012)? Strigolactones have
various functions in leaves, stems, and roots, and
some of these have been attributed to the effects of
strigolactones on auxin transport (for review, see
Brewer et al., 2013). In addition, strigolactones were
recently shown to alter tiller branch gravitropism in
rice by inhibiting auxin biosynthesis (Sang et al., 2014).
It is therefore possible that strigolactone regulation of
PINs relates to these functions and not to the role of
strigolactones in inhibiting bud outgrowth. Moreover,
we have long proposed that strigolactone depletion
enhances auxin levels and/or transport as part of a
long-distance feedback mechanism (Beveridge et al.,
1997, 2000; Beveridge, 2000; Foo et al., 2005; Brewer
et al., 2009; Hayward et al., 2009). New flow of auxin
from stimulated buds and branches presumably increases strigolactone levels and suppresses cytokinin
levels in the stem (Fig. 1; Dun et al., 2009) forming part
of the competition system between buds and branches.
This competition would be further amplified or accelerated under reduced strigolactone levels in buds and
branches by either increased auxin biosynthesis/
signaling or increased PINs at the cell membranes.
Future studies should therefore explore the role of
auxin export from buds in terms of within-plant
competition because, although this may not be a
cause of bud outgrowth, it may be a mechanism that
enhances competition among growing shoots (Snow,
1937; Crawford et al., 2010; Shinohara et al., 2013;
Mason et al., 2014; Waldie et al., 2014).
MATERIALS AND METHODS
Plant Material, Growth Conditions, and Branching Assays
For garden pea (Pisum sativum) experiments, the lines used were wild-type
cv Torsdag (line L107; Figs. 2, 3, 5, and 6; Supplemental Figs. S1 and S2) and
mutants rms1-2T (Fig. 2; Supplemental Fig. S1) and rms5-3 (line BL298; Fig. 3)
in Torsdag, and wild-type Parvus and mutant rms1-1 (line WL5237; Fig. 4;
Supplemental Fig. S1). Unless otherwise stated, pea plants were grown at two
per 2-L pot under greenhouse conditions (24°C day/18°C night, photoperiod
extended to 18 h by incandescent lighting), as described by Ferguson and
Beveridge (2009). Pots contained potting mix (7:2:1 pine bark fines:peat blend:
sand [Fig. 4; Supplemental Fig. S1] or 7:3 0–5-mm composted pine bark:coco
peat, with 1 kg m23 Yates FlowTrace, 1 kg m23 iron sulfate heptahydrate,
0.4 kg m23 superphosphate, 0.03 kg m23 copper sulfate, and 1 kg m23 gypsum
[Figs. 5 and 6]) with 2 g of Osmocote (Scotts); Flowfeed EX7 (Grow Force) was
supplied weekly. Nodes were numbered acropetally from the first scale leaf as
node 1, and lengths of lateral branches and buds were recorded using digital
calipers.
For the Arabidopsis (Arabidopsis thaliana) experiment, Columbia-0, max3-11,
yuc1D, and double mutant plants were germinated and grown on University
of California potting mix supplemented with Flowfeed EX7 nutrients in an
Arabidopsis Chamber (Percival; 22°C day/18°C night, 16 h daylength). The
number of rosette branches longer than 5 mm was counted when the plants
were 41 to 45 d old.
A Student’s t test was used to test for statistical significance using
GraphPad Prism version 6.01 (www.graphpad.com).
In Vitro Excised Stem Segment Assays
The classical excised stem segment assay (Chatfield et al., 2000) was
adapted for pea. The agar growth medium contained 2.3 g L21 Murashige and
Skoog Basal Salt Mixture (PhytoTechnology Laboratories, M404), 0.5 g L21 MES
(Sigma-Aldrich, M2933), and 10 g L21 agar (Sigma-Aldrich, A1296) and was
adjusted to pH 5.7, sterilized, and poured into 10- 3 10-cm plastic square petri
dishes. A 2-cm trough was carefully cut removing agar across the middle of the
dish. The remaining agar parts were then injected with hormone or control solution to give the required final concentrations and left to rest for 72 h. In experiments where auxin was supplied apically, the apical agar segments
contained 0.56 mL mL21 ethanol, with or without naphthaleneacetic acid (NAA).
In experiments where strigolactone was supplied basally, basal agar segments
contained 1.11 mL mL21 acetone, with or without GR24. For plant material, peas
were grown at four or five per pot (6 3 6 3 9 cm) in a peat-sand blend potting
mix (Green Fingers B2 Potting Mix; www.greenfingerspottingmix.com) for
10 d in a growth cabinet (22°C, 18-h photoperiod). Plants with a fully expanded
leaf at node 4 were used. These plants, Torsdag and rms1-2T mutant, have a
repressed bud at node 3, which together with 1.5 cm of stem on either side of the
node, were excised, had their stipules, leaflets, and majority of petiole removed
(leaving only approximately 10 mm of petiole), and were carefully embedded as
a bridge between the agars, two per petri dish. No sterilizing of pea tissue was
required due to the absence of Suc in the medium. Enclosed petri dishes were
returned to the growth cabinet conditions, lying flat.
The open-tube method was adapted from above to provide less agar, less
humidity, and more airflow. Similar agar growth medium (3 g L21 Murashige
and Skoog Basal Salt Mixture, 0.5 g L21 MES, 10 g L21 agar, pH 5.9), containing hormone or control solutions added immediately prior, was pipetted
into sterilized 2-mL screw cap tubes, 1.5 mL per tube. Pea plant tissue was
used, as above, and the cut stems were carefully embedded into the agar. The
tubes were arranged upright with sticky tack (Bostik Blu-Tack) in empty 10- 3
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Brewer et al.
10-cm plastic square petri dishes, three per dish, as shown (Fig. 3A), and kept
upright in the growth cabinet.
Radiolabel Assays
The long-term effect of NPA on auxin transport and branching (Fig. 4;
Supplemental Fig. S1) was assessed as follows: 17-d-old wild-type and
rms1 strigolactone-deficient pea plants were supplied with a lanolin ring,
with or without 0.1% (w/v) NPA, to the stem in the upper end of the
oldest expanding internode, as per Brewer et al. (2009). Then, at certain
times after NPA treatment, 3H-IAA (American Radiolabeled Chemicals,
Inc.) was added to the shoot apex and allowed time to flow down through
the NPA treatment site. Radiolabel was then quantified from 0.5-cm stem
segments, as per Brewer et al. (2009). The total radiolabel was the sum of
all segments, excluding the first segment, and was measured as disintegrations per minute. Data from individual segments are shown in
Supplemental Figure S1. Branch length was measured at the node below
the NPA treatment site.
Other methods are described in the text and legends except for the 3H-NPA
transport analyses, in which 370 kBq [2,3,4,5-3H]-NPA (3H-NPA; American
Radiolabeled Chemicals, Inc.) in 60 mL (0.125 pmol) was added to 500 mL of
1% (w/v) NPA for NPA transport experiments, and the small amount of
background (21.77 DPM in Fig. 4; Supplemental Fig. S1; 17 DPM in Fig. 5) was
subtracted from the values before calculating mean and SEM. Radiolabeled
transport samples were analyzed as per Brewer et al. (2009).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. NPA acts profoundly on polar auxin (3H-IAA)
transport and for a long duration.
Supplemental Figure S2. NPA-treated wild-type plants develop branches
after decapitation similarly to the lanolin control despite huge reductions
in polar auxin transport.
ACKNOWLEDGMENTS
We thank Kerry Condon, Rosanna Powell, and Bethany Reid (University of
Queensland) for technical support; Jozef Mravec (University of Copenhagen)
for providing the yuc1D line; Steven Smith (University of Tasmania), Graeme
Mitchison (University of Cambridge), and Ottoline Leyser (University of Cambridge) for helpful discussions; and Jessica Bertheloot (INRA, Angers), Nathalie
Leduc (INRA, Angers), and Stephanie Kerr (University of Queensland) for comments on the article.
Received January 8, 2015; accepted June 24, 2015; published June 25, 2015.
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