The detailed characterization of SL branching mutants over many years has resulted in two, non-exclusive proposed mechanisms for SL action. The first is that SL signals through the transcriptional upregulation of particular genes in the TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTORS 1 and 2 (TCP) family in buds (Braun et al., 2012; Dun et al., 2012). The second is that SL systemically triggers the removal of PIN1 auxin efflux proteins from the plasma membrane (Bennett et al., 2006; Crawford et al., 2010; Shinohara et al., 2013). These two targets of SL signalling form the basis of the two theories for how SL regulates bud outgrowth. Both can be used to explain an important feature of the SL branching phenotype, namely the resistance of SL mutant buds to inhibition by apical auxin (Beveridge et al., 2000; Sorefan et al., 2003; Bennett et al., 2006; Arite et al., 2007). Auxin is exported from the primary shoot apex, transported down the main stem in the polar auxin transport stream (PATS) and inhibits bud activity (Thimann and Skoog, 1933; Goldsmith, 1977). This auxin acts indirectly, however, as it does not enter the bud in appreciable quantities (Hall and Hillman, 1975; Morris, 1977). SLs are required in some way for this indirect auxin-mediated inhibition.
The transcriptional model
Related members of the TCP domain transcription factor family have been shown to play a major role in shoot branching in diverse species. Loss-of-function mutations in these genes result in increased shoot branching, exemplified by teosinte branched 1 (tb1) in maize, fine culm 1 (fc1) in rice and branched 1 (brc1) in Arabidopsis and pea (Doebley et al., 1997; Takeda et al., 2003; Aguilar-Martínez et al., 2007; Finlayson, 2007; Braun et al., 2012). These genes are expressed almost exclusively in buds and their enhanced expression is associated with bud inhibition (Doebley et al., 1997; Takeda et al., 2003; Lewis et al., 2008). Where tested, bud activation in the loss-of-function mutants is SL resistant (Brewer et al., 2009; Minakuchi et al., 2010; Braun et al., 2012). This has led to the proposal that these genes are a downstream target for the SL pathway in bud inhibition. This hypothesis has been investigated in several species and has particularly strong support in pea.
In pea, the expression of BRC1 is closely and negatively correlated with bud activity. SL mutant buds show reduced BRC1 expression, and the application of SLs to buds causes upregulation within 6 h in a cycloheximde-independent manner (Dun et al., 2012). This suggests that SLs can alter BRC1 transcription without the need for new protein synthesis, a hallmark of a primary response gene.
This mode of action of SL can explain the auxin resistance of SL mutant buds, with the additional observation that the transcription of SL biosynthetic genes is upregulated by auxin (Foo et al., 2005; Johnson et al., 2006; Zou et al., 2006; Arite et al., 2007; Hayward et al., 2009; Liang et al., 2010; Zhang et al., 2010). The BRC1 model is therefore essentially a linear pathway in which auxin positively regulates SL synthesis in the main stem, and SL moves up into the bud where it upregulates BRC1 expression, thereby suppressing branching (Brewer et al., 2009; Braun et al., 2012; Dun et al., 2012, 2013). SL activity downstream of auxin is consistent with the observed auxin resistance of rms/max mutant buds. It is also consistent with the observed SL suppression of bud activity in auxin-signalling mutants, including axr1 and tir1 afb1 afb2 afb3 in Arabidopsis (Brewer et al., 2009).
Although this model neatly fits the transcriptional and bud outgrowth responses in pea, there is still the question of whether it is the primary mode of SL action. BRC1 is expressed specifically and quite widely in buds, and this only partially overlaps with the much wider expression domains of MAX2 and D14, which are also expressed and presumably function systemically in vascular-associated tissues (Aguilar-Martínez et al., 2007; Stirnberg et al., 2007; Arite et al., 2009; Gao et al., 2009). Furthermore, in several species the correlation between the expression of BRC1 family members and bud activity is weak. For example, FC1 expression in rice d mutants is not reduced, overexpression of FC1 in d3 can only partially rescue the d3 branching phenotype and the addition of SL does not induce FC1 expression (Arite et al., 2007; Minakuchi et al., 2010). Similarly, in maize, where TB1 expression is constitutively high as a result of changes to the promoter selected during domestication (Doebley et al., 1997), SLs do not affect TB1 transcript levels. Mutations in ZmCCD8 result in increased branching, and GR24 treatment is not associated with altered TB1 expression but can reduce branch length (Guan et al., 2012). The branching phenotype of maize ccd8 mutants has been described as being relatively weak, but it is similar to the increased branching observed in Arabidopsis max4/ccd8 mutants. The phenotype only appears weak in comparison with the tb1 loss-of-function mutant, which is much more dramatic because of the dual role of maize TB1 in regulating both branch activity and branch identity. Because of this, tb1 mutants have long branches tipped by tassels, instead of short ear shoots (Hubbard et al., 2002). Thus, in both maize and rice, SL and TB1/FC1 are at least partially independent in their effects. It is possible that these differing responses reflect differing modes of regulation amongst species; however, even in pea the increased bud outgrowth observed at most nodes in the rms1/ccd8 mutant differs to the pronounced outgrowth that is restricted to basal nodes in the Psbrc1 mutant, suggesting SL–mediated branching inhibition, independent of BRC1 (Braun et al., 2012).
The auxin transport canalization model
An alternative explanation for the SL inhibition of bud outgrowth is based on the concept of auxin transport canalization (Bennett et al., 2006; Prusinkiewicz et al., 2009; Crawford et al., 2010; Shinohara et al., 2013). The auxin transport canalization hypothesis proposes that an initial passive flux of auxin between a source and a sink positively regulates and polarizes its own transport in the direction of the initial flux, and in doing so organizes its transport into files of cells, or canals, that efficiently move auxin from the source to the sink (Sachs, 1981, 2000). Under a canalization-based model for bud regulation, it is proposed that dormant buds must establish an efficient flow of auxin into the main stem PATS in order to activate (Li and Bangerth, 1999). There is good correlative evidence to support this (Morris, 1977; Balla et al., 2011). For example, in pea, the polarization of PIN1 auxin efflux carriers is observed between the bud and the main stem PATS following bud activation by decapitation of the apex, and this polarization is not observed when buds are inhibited by applying auxin to the decapitation site (Balla et al., 2011). One explanation as to why the establishment of a PATS out of the bud is necessary for bud activation is that auxin export away from incipient leaves on the flanks of the shoot apical meristem may be required for continued phyllotactic patterning and leaf initiation (Bayer et al., 2009).
According to the auxin transport canalization model for bud inhibition, the ability of a bud to canalize auxin flow into the main stem PATS would be competitively inhibited by other stronger sources of auxin, i.e. the primary apex and already active buds, supplying auxin into the main stem PATS and reducing its sink strength. In this way, auxin in the main stem PATS can inhibit bud activation indirectly by preventing auxin export from the bud.
Evidence for the role of SL in regulating buds through this mechanism comes from the analysis of their effects on the auxin export protein, PIN1. SL regulates the accumulation of PIN1 proteins on the plasma membrane, specifically by triggering their rapid depletion in a clathrin-dependent process that is likely to be endocytosis (Shinohara et al., 2013). The effect of SL on PIN1 depletion is independent of new protein synthesis but dependent on MAX2, and can be detected within 10 min of SL treatment in some cases. Consistent with this, the max mutants have increased stem auxin transport and stem PIN1 accumulation, accompanied by increased auxin levels in the PATS (Bennett et al., 2006). The vascular expression pattern of PIN1 (Gälweiler et al., 1998) matches well with that of D14 and MAX2 (Aguilar-Martínez et al., 2007; Stirnberg et al., 2007; Arite et al., 2009), but although MAX2 and D14 are nuclear, PIN1 depletion occurs at the plasma membrane. One hypothetical mechanism for SL action is that a protein that promotes PIN1 endocytosis is sequestered in the nucleus by a protein that is targeted for degradation in an SL-, D14- and MAX2-dependent manner. In line with this, there is some evidence for the release or exclusion of COP1 from the nucleus in response to SL treatment of seedlings (Tsuchiya et al., 2010).
An increased rate of removal of PIN1 proteins, conditioned by high SL, would dampen the positive feedback between auxin flux and PIN1 protein accumulation and polarization, thereby inhibiting the canalization of auxin transport out of buds. In this way, SL acts to enhance competition between buds and/or the primary apex for access into the main stem PATS (Ongaro et al., 2008; Crawford et al., 2010; Liang et al., 2010; Ward et al., 2013). According to this idea, SLs do not directly inhibit buds. They act systemically to dampen auxin transport canalization, thereby reducing the total number of buds that can be active. Consistent with this idea, solitary Arabidopsis buds on excised nodal stem segments can be inhibited by apical auxin supply, but not by basal SL supply; however, in excised stem segments bearing two buds, SL typically inhibits one bud but not the other. This result cannot be easily explained by a mode of action where SL locally regulates gene expression in buds, inhibiting their activity.
Further evidence that the effects of SL on auxin transport are causal in bud regulation includes the observation that the branching and auxin-resistant bud outgrowth phenotypes of the max mutants can be rescued by treatment with low levels of the auxin transport inhibitor 1–naphthylphthalamic acid (NPA) (Bennett et al., 2006). Similar effects have been reported in rice with d27 (Lin et al., 2009). This result is particularly striking because treatment of wild-type plants with the same levels of NPA results in increased branching, presumably because lower auxin in the main stem allows more buds to activate. Under this model, there are therefore two distinct ways to achieve a highly branched phenotype: the first with low PIN1 removal from the plasma membrane resulting in high PIN1 accumulation, high auxin transport and high auxin export from bud to stem; or the second with low PIN1 accumulation or activity, low auxin transport and lower auxin export from bud to stem. This leads to the prediction that the addition of SL to plants already compromised in their auxin transport ability would actually promote branching, a prediction that has been validated (Shinohara et al., 2013). This ability of SL to promote branching in some circumstances is also not easy to explain by the local regulation of gene expression in buds.
The strongest evidence against the idea that the primary mode of action of SL is enhancement of competition between buds via PIN1 depletion from the plasma membrane is the observation that all pea buds on a plant can be inhibited following decapitation and the simultaneous application of SL to buds at every node (Dun et al., 2013). Even so, some competition is observed, as the bud closest to the decapitation site still exhibits a small amount of outgrowth compared with intact plants, and this top bud remains fully inhibited if more basal branches are allowed to grow. Furthermore, under the canalization-based hypothesis, the application of sufficient quantities of SL directly to buds might be able to prevent canalization, even in the absence of a competing auxin source and hence strong stem sink strength. The ability to canalize depends on the relative rates of PIN1 insertion and PIN1 removal, and therefore particularly high levels of PIN1 removal may be sufficient to prevent canalization.
Further evidence against the central importance of SL effects on auxin transport in bud regulation comes from detailed comparisons of the application of NPA and GR24 on bud growth and auxin transport in recently activated buds. Here, GR24 and NPA were shown to have similar inhibitory effects on bud elongation, but NPA was much more effective than GR24 at inhibiting auxin transport, assessed by measuring the basipetal transport of co-applied radiolabelled auxin away from the GR24/NPA application site (Brewer et al., 2009). In interpreting these experiments it is important to remember that although both NPA and GR24 can reduce auxin transport, they act by completely different mechanisms and with different dynamics. SL is not strictly speaking an auxin transport inhibitor at all, but acts to reduce the level of PIN1 at the plasma membrane. In contrast, NPA is a strong pharmacological inhibitor of transport of both the PIN and ABCB family of auxin exporters (Yang and Murphy, 2009). In Arabidopsis stems treated with modest levels of NPA, only 10% of the auxin transport remains, whereas stems treated with extremely high concentrations of GR24 retain 70% of their auxin transport, compared with untreated controls (Crawford et al., 2010). Thus, co-application of NPA and radiolabelled auxin would prevent a large proportion of the auxin from entering the transport stream at the site of application, whereas GR24 would be predicted to have no such effect and would instead alter PIN accumulation dynamics at the plasma membrane. The inhibitory effect of these treatments on bud stem elongation could be caused by similarly different mechanisms: over-accumulation of auxin in the bud caused by auxin transport blockage with NPA treatment, or gradual depolarization of PINs in the bud with GR24 treatment.
It should be noted that the two models for SL action in bud inhibition are not mutually exclusive (Figure 3). Both could happen in parallel, or changes in BRC1 expression could result from SL–mediated changes in auxin transport. In this scenario, BRC1 could act as a stabilizer of bud repression, in which buds with low levels of auxin export, for example because of high SL levels, are kept in an off state by upregulation of BRC1. Interestingly, the majority of genes reported to be repressed following the treatment of seedlings with GR24 for 90 min are known or putative auxin-regulated genes (Mashiguchi et al., 2009), consistent with the idea that altered auxin transport is central to the subsequent transcriptional effects of SL.
To add to the complexity of SL–mediated branching inhibition, it was recently reported that OsTB1/FC1 may be involved in regulating the expression of the D14 gene (Guo et al., 2013). The transcription factor OsMADS57 is a transcriptional repressor of D14, but OsTB1/FC1 can interact with OsMADS57 to permit D14 expression. Additionally, it was shown that miRNAs contribute to this regulation, whereby OsMIR444a targets OsMADS57. These findings place BRC1 upstream of D14, or perhaps into a positive feedback loop, further locking buds into an inactive state. This may be important in the context of the canalization-based model for bud activation, which has at its heart a positive feedback loop, making it inherently unstable under stochastic fluctuations.