Roles for Auxin, Cytokinin, and Strigolactone in Regulating Shoot Branching^,,,

Abstract

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 underlying cause of bud outgrowth Can changes independent 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.

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 development. Removal of the shoot apex (i.e. decapitation) alleviates this inhibition, allowing for the outgrowth of lateral buds. Decapitation also reduces the endogenous 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 decapitation, 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 outgrowth, 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 regulated 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 regulated 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 inhibitor 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 decapitation 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 strigolactone 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 at least partly via strigolactone to regulate bud outgrowth 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 transduction 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, branching 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, additive effects of decapitation in the branching mutants would not be expected. In contrast, if decapitation induces an auxin-independent rapid trigger for outgrowth, 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 mechanism 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 basipetally 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 regulate 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 preventing the outgrowth of that bud. Only when this impediment is alleviated (for instance, when IAA levels are reduced or the number of IAA transporters is increased) would IAA export from the bud commence and subsequent outgrowth be achieved. This stipulation that IAA export from the bud triggers outgrowth is in direct contrast to the bud transition hypothesis discussed below, in which IAA export is considered a characteristic of sustained growth, functioning as a consequence, rather than a cause, of bud release (Dun et al., 2006).

Correlative inhibition is another concept that can apply the IAA transport hypothesis described above to competition between growing shoots (Bangerth, 1989; Bangerth et al., 2000; Bennett et al., 2006; Mouchel and Leyser, 2007; Ongaro and Leyser, 2008; Ongaro et al., 2008). In this case, rather than referring primarily to bud release, two growing shoots inhibit one another based on the extent of auxin transport in each shoot. Blocking auxin transport from one shoot will stop its growth and will enhance the growth of the other shoot. The mechanism for this may be as described above. 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 transition hypothesis of branching control; this hypothesis defines various stages at which a bud can reside, including dormancy, transition, and sustained outgrowth (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 suppression of one that inhibits it, would initially be required to activate a bud from dormancy into transition, followed by additional steps to sustained outgrowth. 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, correlative inhibition, and the RMS/strigolactone pathway 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 relationships. Moreover, results from studies using auxin transport inhibitors are often variable (Panigrahi and Audus, 1966; Tamas et al., 1989; Morris et al., 2005), and these pharmacological approaches may also affect other signals. We investigated whether stem girdling could be used as a reliable method for studying IAA transport and bud outgrowth relations, as it disrupts phloem transport and the PATS, while allowing for acropetal xylem transport and continued shoot tip growth (Snow, 1929; Davies and Wareing, 1965; Patrick and Wareing, 1978; Thomas, 1983).

RESULTS

Stem Girdling Blocks Auxin Transport But Does Not Always Cause Bud Outgrowth

Girdling the stem with hot wax (Fig. 1A) was found to kill the tissue at the treatment site in pea (Fig. 1B). Consequently, pathways relying on living cells to actively transport signals in the stem would be blocked. This includes the PATS, which is localized to living cells of the vasculature. In contrast, acropetal transport via the xylem may not be greatly affected. 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.

Figure 1.

Effects of stem girdling in wild-type pea. A, Seven days following treatment, bud outgrowth was exhibited below, but not above, the girdle. B, Removal of the girdle exposes dead tissue compared with an untreated internode (far left). Swelling and callus-like distortions were exhibited directly above the girdle site. C, [³H]IAA uptake/transport in stem segments 8 h following application to the shoot apex. Data are means ± se (n = 5). The top of the girdle was positioned at about 40 mm from the apex. DPM, Disintegrations per minute. [See online article for color version of this figure.]

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 h⁻¹ 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 majority 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 outgrowth, plants with nine leaves expanded were girdled 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 response 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 decapitating 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 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 outgrowth when the girdle is situated at upper internodes (Fig. 2).

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 ± se (n = 8).

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 simultaneously. 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 nongirdled, decapitated plants. Results from these experiments 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 decapitated 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 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.

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 ± se (n = 6–8).

We then tested whether girdling at an upper internode, which induces branching, is additive to the decapitation response. A similar degree of bud outgrowth 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). Previously, Morris et al. (2005) used tall pea plants to demonstrate that IAA, at concentrations as high as 3 mg g⁻¹ 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 markedly reduced the amount of outgrowth in girdled plants yet again was unable to prevent outgrowth from occurring (Fig. 4). The trend observed was similar 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 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 outgrowth.

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 g⁻¹ 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 ± se (n = 8).

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 ± 2.37 mm total lateral length in intact control plants compared with 17.94 ± 1.54 mm in defoliated plants, 7 d following 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 reported by Morris et al. (2005) using similar plants treated with the auxin transport inhibitor naphthylphthalamic 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 photoassimilates. 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 significantly 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 nondefoliated 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 demonstrate that the complete lack of bud outgrowth observed 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.

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 ± 2.37 mm) or defoliated (17.94 ± 1.54 mm). Plants had nine leaves expanded at the time of treatment. The arrow represents treatment sites. Data are means ± se (n = 6–7).

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 (Fig. 6, A and B). Therefore, withholding nutrient supply to the plants did not prevent the outgrowth of buds induced to grow by these treatments but did affect branch lengths. As expected, for all treatment types, plants supplied with nutrients were taller than those without (Fig. 6C).

Figure 6.

Effects of nutrient supply on the lateral length of the bud at node 5 (A) and node 2 (B) as well as plant height 6 d following decapitation (Decap.), 10 mg g⁻¹ NPA treatment, or stem girdling (Girdle) of wild-type plants (C). Plants were supplied with (+N) or without (−N) nutrient solution and Osmocote, as outlined in “Materials and Methods.” Treatments were made above node 5 using plants having five leaves expanded. Data are means ± se (n = 8–10).

The Impact of Stem Girdling on Plant Growth

In addition to bud outgrowth, a number of characteristics were investigated to establish how stem girdling 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 may be a repercussion of the girdle impeding the phloem, hence cutting off the supply of photoassimilates 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 significantly 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.

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 ± se (n = 6–8).

No significant difference was detected in the number 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 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 nutrient 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 inhibition and apical dominance. For correlative inhibition, we examined whether axillary shoots affect the outgrowth of other axillary shoots in rms mutants. For apical dominance, we determined whether rms mutants exhibit enhanced branching in response to decapitation or girdling. We included rms2 mutants in these experiments because RMS2 is involved in shoot-to-root signaling for feedback regulation of strigolactone 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 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 branching 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 produced 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 removed 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 demonstrates that correlative inhibition acts more or less independently of the RMS system.

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 ± se (n = 5–10). TLL, Total lateral length.

To examine the role of the RMS network in apical dominance, we tested the extent to which apical dominance 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 outgrowth 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 absence of a functional RMS pathway. The bud outgrowth 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).

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 ± se (n = 6–8 plants per treatment).

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 relative to the expression of that same gene in the corresponding node of intact control plants (Fig. 10).

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 ± se (n = 2 replicates of six plants per replicate). [See online article for color version of this figure.]

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 outgrowth. This was greatest in samples harvested directly below these treatment sites, where increases of just under 100-fold were detected. Smaller but significant increases, particularly in IPT1 expression, were seen at all other nodes that would later show bud outgrowth. Strong increases in IPT1 and IPT2 expression 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 between nodes 3 and 4 and then applying 5 μL of 50% ethanol (control) or 10 μg μL⁻¹ 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 ± 2.00 mm) than control-treated buds, which remained dormant (0.81 ± 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 responsible 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 expression below the girdle or decapitation site were generally 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 expression was not correlated with bud outgrowth. These findings differ from that detailed above for IPT1 and IPT2, which completely correlated with bud outgrowth phenotypes. The reduction in RMS1 expression 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 reduction 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 expression of RMS1 and RMS5 was found to increase above girdle sites, possibly in response to elevated IAA levels caused by girdling (Fig. 10).

DISCUSSION

Our results lead to three major conclusions relating to apical dominance and bud outgrowth. First, IAA 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 hormone 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 Bud Outgrowth

Our findings clearly demonstrate that reduced IAA content in the main stem is not always correlated with bud outgrowth. Based on our IAA4/5 expression and [³H]IAA transport data, stem girdling, like decapitation, 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, decapitation always induced branching, whereas in several treatment positions, girdling typically failed to do so (Fig. 2). This discrepancy between girdled and decapitated plants is reduced in plants treated at progressively higher internodes. At lower nodes, the reduced branching response to girdling compared with decapitation was unlikely due to differences in IAA signaling, as the treatments are expected to have similar effects on IAA content and IAA4/5 expression was reduced equally in both treatments (Fig. 10). Defoliation 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 supply 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 outgrowth. Instead, the accumulation of IAA in a triggered 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 meristem 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) elegantly 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 canalization leading to enhanced vascular development, as 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 consistent 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 branching 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 decapitated plants, with or without auxin treatment, suggesting 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 expected from an actively transported signal, such as IAA (Everat-Bourbouloux and Bonnemain, 1980; Morris et al., 2005). Indeed, the signal might not be a cumbersome 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.

Apical Dominance, Correlative Inhibition, and the RMS/Strigolactone Pathway Are Distinct Mechanisms for Regulating Bud Outgrowth

We demonstrate here that apical dominance, correlative inhibition, and the RMS/strigolactone pathway are all independent but interacting mechanisms for regulating bud outgrowth. Correlative inhibition functions 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 correlative inhibition does not require the RMS/strigolactone 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 dominance also operates in the absence of the RMS/strigolactone network. This indicates that strigolactone deficiencies and IAA depletion caused by decapitation or girdling are not simply acting in a linear pathway to regulate bud outgrowth; additional interactions 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 dominance 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, providing 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 architecture are derived in the main shoot tip (apical dominance), axillary shoot tip (correlative inhibition), or rootstock and internode (RMS/strigolactone network). We suggest that by having multiple interacting mechanisms, 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 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 transduction (as indicated by IAA4/5 expression; Fig. 10). However, in this event, outgrowth could be induced by exogenously applying CK to the bud. Thus, suppression 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 typically occurs following the perception of an initial outgrowth trigger. However, buds of the right developmental 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 internode, girdling at a basal internode did not induce CK biosynthesis genes below the treatment site. Nevertheless, 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/pathway 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 contrast, increased IPT gene expression and bud outgrowth in basally decapitated plants may occur because the relative shoot demand for nitrate is decreased 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 strigolactone 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 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 induced following the loss of the shoot tip (apical dominance). Growing axillary buds/branches (correlative 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 increased CK production (Fig. 11), as reductions in RMS expression in the absence of an increase in IPT expression fail to promote outgrowth (Fig. 10). Once triggered 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).

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.]

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. (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) nutrient 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 measurements 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 activity, 25 Ci mmol⁻¹). [³H]IAA transport was analyzed as outlined by Morris et al. (2005) following application of 8.5 kBq [³H]IAA in 2 μL 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. Radioactivity 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 g⁻¹ (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 g⁻¹ 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 −80°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-μL volume with 1.6 to 5 μg of total RNA, 50 to 250 ng of random hexamers (Invitrogen), 0.5 mm deoxynucleoside triphosphates, 5 mm dithiothreitol (Invitrogen), 1.25× first-strand buffer, 40 units of RnaseOut (Invitrogen), and 200 units of SuperScript III reverse transcriptase (Invitrogen) incubated at 50°C for 1 h. cDNA quality was checked via PCR (25 cycles) and gel electrophoresis with actin primers: forward (5′-CAACTATGTTTCCCGGTATTG-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 μL of SYBR Green PCR Master Mix (Applied Biosystems) and 1 μL 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 reverse (5′-TGGATACGATCGGGAAGTTCA-3′; Johnson et al., 2006); IAA4/5 forward (5′-GTTCTTCTGCAGCCCCTCCTG-3′) and IAA4/5 reverse (5′-ACAAAGATCCCACCAACATCAGCC-3′), designed using Primer Express 2.0 software (Applied Biosystems); IPT1 forward (5′-ACCGTCTTGATGCTACGGAGGTTGTGC-3′) and IPT1 reverse (5′-TCTAATGGGTTACCCCTGCCACAGACG-3′; Tanaka et al., 2006); IPT2 forward (5′-TGGCAGCAACATCATCCTCTGCCTGC-3′) and IPT2 reverse (5′-ACCTGTGGCCCCCATTATCACTAC-3′; Tanaka et al., 2006); and 18S forward (5′-ACGTCCCTGCCCTTTGTACA-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 Student'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.