The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport

BACKGROUND: Plants achieve remarkable plasticity in shoot system architecture by regulating the activity of secondary shoot meristems, laid down in the axil of each leaf. Axillary meristem activity, and hence shoot branching, is regulated by a network of interacting hormonal signals that move through the plant. Among these, auxin, moving down the plant in the main stem, indirectly inhibits axillary bud outgrowth, and an as yet undefined hormone, the synthesis of which in Arabidopsis requires MAX1, MAX3, and MAX4, moves up the plant and also inhibits shoot branching. Since the axillary buds of max4 mutants are resistant to the inhibitory effects of apically supplied auxin, auxin and the MAX-dependent hormone must interact to inhibit branching. RESULTS: Here we show that the resistance of max mutant buds to apically supplied auxin is largely independent of the known, AXR1-mediated, auxin signal transduction pathway. Instead, it is caused by increased capacity for auxin transport in max primary stems, which show increased expression of PIN auxin efflux facilitators. The max phenotype is dependent on PIN1 activity, but it is independent of flavonoids, which are known regulators of PIN-dependent auxin transport. CONCLUSIONS: The MAX-dependent hormone is a novel regulator of auxin transport. Modulation of auxin transport in the stem is sufficient to regulate bud outgrowth, independent of AXR1-mediated auxin signaling. We therefore propose an additional mechanism for long-range signaling by auxin in which bud growth is regulated by competition between auxin sources for auxin transport capacity in the primary stem.     

Response to strigolactone treatment in chrysanthemum axillary buds is influenced by auxin transport inhibition and sucrose availability

Axillary bud outgrowth is regulated by both environmental cues and internal plant hormone signaling. Central to this regulation is the balance between auxins, cytokinins, and strigolactones. Auxins are transported basipetally and inhibit the axillary bud outgrowth indirectly by either restricting auxin export from the axillary buds to the stem (canalization model) or inducing strigolactone biosynthesis and limiting cytokinin levels (second messenger model). Both models have supporting evidence and are not mutually exclusive. In this study, we used a modified split-plate bioassay to apply different plant growth regulators to isolated stem segments of chrysanthemum and measure their effect on axillary bud growth. Results showed axillary bud outgrowth in the bioassay within 5 days after nodal stem excision. Treatments with apical auxin (IAA) inhibited bud outgrowth which was counteracted by treatments with basal cytokinins (TDZ, zeatin, 2-ip). Treatments with basal strigolactone (GR24) could inhibit axillary bud growth without an apical auxin treatment. GR24 inhibition of axillary buds could be counteracted with auxin transport inhibitors (TIBA and NPA). Treatments with sucrose in the medium resulted in stronger axillary bud growth, which could be inhibited with apical auxin treatment but not with basal strigolactone treatment. These observations provide support for both the canalization model and the second messenger model with, on the one hand, the influence of auxin transport on strigolactone inhibition of axillary buds and, on the other hand, the inhibition of axillary bud growth by strigolactone without an apical auxin source. The inability of GR24 to inhibit bud growth in a sucrose treatment raises an interesting question about the role of strigolactone and sucrose in axillary bud outgrowth and calls for further investigation.     

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

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.     

Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport

Shoot branching is essential in ornamental chrysanthemum production and determines final plant shape and quality. Auxin is associated with apical dominance to indirectly inhibit bud outgrowth. Two non-mutually exclusive models exist for indirect auxin inhibition. Basipetal auxin transport inhibits axillary bud outgrowth by limiting auxin export from buds to stem (canalization model) or by increasing strigolactone levels (second messenger model). Here we analyzed bud outgrowth in treatments with auxin (IAA), strigolactone (GR24) and auxin transport inhibitor (NPA) using a split-plate bioassay with isolated chrysanthemum stem segments. Besides measuring bud length, dividing cell percentage was measured with flow cytometry and RT-qPCR was used to monitor expression levels of genes involved in auxin transport (CmPIN1) and signaling (CmAXR2), bud dormancy (CmBRC1, CmDRM1) and strigolactone biosynthesis (CmMAX1, CmMAX3). Treatments over a 5-day period showed bud outgrowth in the control and inhibition with IAA and IAA?+?GR24. Bud outgrowth in the control coincided with high dividing cell percentage, decreased expression of CmBRC1 and CmDRM1 and increased CmPIN1 expression. Inhibition by IAA and IAA?+?GR24 coincided with low dividing cell percentage and unchanged or increased expressions of CmBRC1, CmDRM1 and CmPIN1. Treatment with GR24 showed restricted bud outgrowth that was counteracted by NPA. This restricted bud outgrowth was still concomitant with a high dividing cell percentage and coincided with decreased expression of dormancy genes. These results indicate incomplete inhibition of bud outgrowth by GR24 treatment and suggest involvement of auxin transport in the mechanism of bud inhibition by strigolactones, supporting the canalization model.     

Strigolactone Acts Downstream of Auxin to Regulate Bud Outgrowth in Pea and Arabidopsis

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

Existing branches correlatively inhibit further branching in Trifolium repens: possible mechanisms

In Trifolium repens removal of any number of existing branches distal to a nodal root stimulates development of axillary buds further along the stem such that the complement of branches distal to a nodal root remains constant. This study aimed to assess possible mechanisms by which existing branches correlatively inhibit the outgrowth of axillary buds distal to them. Treatments were applied to basal branches to evaluate the roles of three postulated inhibitory mechanisms: (I) the transport of a phloem-mobile inhibitory feedback signal from branches into the main stem; (II) the polar flow of auxin from branches into the main stem acting to limit further branch development; or (III) the basal branches functioning as sinks for a net root-derived stimulatory signal (NRS). Results showed that transport of auxin, or of a non-auxin phloem-mobile signal, from basal branches did not influence regulation of correlative inhibition and were consistent with the possibility that the intra-plant distribution of NRS could be involved in the correlative inhibition of distal buds by basal branches. This study supports existing evidence that regulation of branching in T. repens is dominated by a root-derived stimulatory signal, initially distributed via the xylem, the characterization of which will progress the generic understanding of branching regulation.     

Effect of plant growth substances on the growth of axillary buds in cultured stem segments of Phaseolus vulgaris L.

The hormonal control of axillary bud growth was investigated in cultured stem segments of Phaseolus vulgaris L. When the stem explants were excised and implanted with their apical end in a solid nutrient medium, outgrowth of the axillary buds-located at the midline of the segment-was induced. However, if indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) was included in the medium, bud growth was inhibited. The exposure of the apical end to IAA also caused bud abscission and prevented the appearance of new lateral buds.In contrast to apically inserted segments, those implanted in the control medium with their basal end showed much less bud growth. In these segments, the auxin added to the medium either had no effect or caused a slight stimulation of bud growth.The IAA transport inhibitor N-1-naphthylphthalamic acid (NPA) relieved bud growth inhibition by IAA. This suggests that the effect of IAA applied at the apical end requires the transport of IAA itself rather than a second factor. With the apical end of the segment inserted into the IAA-containing medium, simultaneous basal application of IAA relieved to some extent the inhibitory effect of the apical IAA treatment. These results, together with data presented in a related article [Lim R and Tamas I (1989) Plant Growth Regul 8: 151–164], show that the polarity of IAA transport is a critical factor in the control of axillary bud growth.Of the IAA conjugates tested for their effect on axillary bud growth, indoleacetyl alanine, indoleacetic acid ethyl ester, indoleacetyl-myo-inositol and indoleacetyl glucopyranose were strongly inhibitory when they were applied to the apical end of the stem explants. There was a modest reduction of growth by indoleacetyl glycine and indoleacetyl phenylalanine. Indoleacetyl aspartic acid and indoleglyoxylic acid had no effect.In addition to IAA and its conjugates, a number of other plant growth substances also affected axillary bud growth when applied to the apical end of stem segments. Myo-inositol caused some increase in the rate of growth, but it slightly enhanced the inhibitory effect of IAA when the two substances were added together. Gibberellic acid (GA₃) caused some stimulation of bud growth when the explants were from younger, rather than older plants. The presence of abscisic acid (ABA) in the medium had no effect on axillary bud growth. Both kinetin and zeatin caused some inhibition of axillary buds from younger plants but had the opposite effect on buds from older ones. Kinetin also enhanced the inhibitory effect of IAA when the two were applied together.In conclusion, axillary buds of cultured stem segments showed great sensitivity to auxins and certain other substances. Their growth responded to polarity effects and the interaction among different substances. Therefore, the use of cultured stem segments seems to offer a convenient, sensitive and versatile test system for the study of axillary bud growth regulation.     

Relative importance of nodal roots and apical buds in the control of branching in Trifolium repens L.

Clonal species are characterised by having a growth form in which roots and shoots originate from the same meristem so that adventitious nodal roots form close to the terminal apical bud of stems. The nature of the relationship between nodal roots and axillary bud growth was investigated in three manipulative experiments on cuttings of a single genotype of Trifolium repens. In the absence of locally positioned nodal roots axillary bud development within the apical bud proceeded normally until it slowed once the subtending leaf had matured to be the second expanded leaf on the stem. Excision of apical tissues indicated that while there was no apical dominance apparent within fully rooted stems and very little in stems with 15 or more unrooted nodes, the outgrowth of the two most distal axillary buds was stimulated by decapitation in stems with intermediate numbers of unrooted nodes. Excision of the basal branches from stems growing without local nodal roots markedly increased the length and/or number of leaves on 14 distally positioned branches. The presence of basal branches therefore prevented the translocation of root-supplied resources (nutrients, water, phytohormones) to the more distally located nodes and this caused the retardation in the outgrowth of their axillary buds. Based on all three experiments we conclude that the primary control of bud outgrowth is exerted by roots via the acropetal transport of root-supplied resources necessary for axillary bud outgrowth and that apical dominance plays a very minor role in the regulation of axillary bud outgrowth in T. repens.     

Hormonal control of the outgrowth of axillary buds in <Emphasis Type="Italic">Alstroemeria</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

We study apical dominance in Alstroemeria, a plant with an architecture very different from the model species used in research on apical dominance. The standard explant was a rhizome with a tip and two vertically growing shoots from which the larger part had been excised leaving ca. 1 cm stem. The axillary buds that resumed growth were located at this 1-cm stem just above the rhizome. They were released by removal of the rhizome tip and the shoot tips. Replacement of excised tips by lanolin with indole-3-butyric acid (IBA) restored apical dominance. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-napthylphthalamic acid (NPA) reduced apical dominance. 6-Benzylaminopurine (BAP) enhanced axillary bud outgrowth but the highest concentrations (> 9 μM) caused fasciation. Thidiazuron (TDZ) did not show improvement relative to BAP. Even though the architecture of Alstroemeria and the model species are very different, their hormonal mechanisms in apical dominance are for the greater part very similar.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato

The enormous variation in architecture of flowering plants is based to a large extent on their ability to form new axes of growth throughout their life span. Secondary growth is initiated from groups of pluripotent cells, called meristems, which are established in the axils of leaves. Such meristems form lateral organs and develop into a side shoot or a flower, depending on the developmental status of the plant and environmental conditions. The phytohormone auxin is well known to play an important role in inhibiting the outgrowth of axillary buds, a phenomenon known as apical dominance. However, the role of auxin in the process of axillary meristem formation is largely unknown. In this study, we show in the model species Arabidopsis thaliana and tomato (Solanum lycopersicum) that auxin is depleted from leaf axils during vegetative development. Disruption of polar auxin transport compromises auxin depletion from the leaf axil and axillary meristem initiation. Ectopic auxin biosynthesis in leaf axils interferes with axillary meristem formation, whereas repression of auxin signaling in polar auxin transport mutants can largely rescue their branching defects. These results strongly suggest that depletion of auxin from leaf axils is a prerequisite for axillary meristem formation during vegetative development.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Non‐dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum (Sorghum bicolor). Here, we identified and functionally characterized a mutant of the Non‐dormant Axillary Bud 1 (NAB1) gene from an ethyl methanesulfonate‐mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map‐based cloning revealed that NAB1 encodes a carotenoid‐cleavage dioxygenase 7 (CCD7) orthologous to rice (Oryza sativa) HIGH‐TILLERING DWARF1/DWARF17 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non‐dormant basal axillary buds restored the wild‐type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild‐type plants. Finally, RNA‐seq showed that the expression of genes involved in multiple processes, including auxin‐related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.     

Auxin–cytokinin interactions in the control of shoot branching

In many plant species, the intact main shoot apex grows predominantly and axillary bud outgrowth is inhibited. This phenomenon is called apical dominance, and has been analyzed for over 70 years. Decapitation of the shoot apex releases the axillary buds from their dormancy and they begin to grow out. Auxin derived from an intact shoot apex suppresses axillary bud outgrowth, whereas cytokinin induced by decapitation of the shoot apex stimulates axillary bud outgrowth. Here we describe the molecular mechanisms of the interactions between auxin and cytokinin in the control of shoot branching.     

CONTROL OF SHOOT-RHIZOME DIMORPHISM IN THE WOODY MONOCOTYLEDON,CORDYLINE (AGAVACEAE)

In Cordyline terminalis negatively geotropic leafy shoots and positively geotropic rhizomes develop from single axillary buds on either shoots or rhizomes. All axillary buds have similar morphogenetic potential when released from apical dominance. Experiments in which the orientation of the apex is changed, organs removed, or growth regulators applied indicate that after a rhizome is initiated, it is maintained as a rhizome by auxin originating in the leafy shoot. When auxin levels are lowered by changes in the orientation of the axis or shoot removal, the rhizome apex becomes a shoot apex, which appears to be the stable state of the actively growing apex. Benzyl adenine when applied exogenously to the apex or lateral buds has the same effect as lowering the auxin level. Gibberellic acid has no effect on the apex or lateral buds. High levels of exogenous naphthaleneacetic acid cause bud release and development of rhizomes from previously inhibited axillary buds of the shoot. However, it was not possible to convert a shoot apex into a rhizome apex by auxin treatment. It is suggested that the release of buds on the lower side of horizontal branches and of buds directly above a stem girdle is caused by high auxin levels on the lower side or distal to the girdle. The experimental results are discussed in relation to naturally occurring shoot-rhizome dimorphism.