Auxin Stimulates Its Own Transport by Shaping Actin Filaments

The directional transport of the plant hormone auxin has been identified as central element of axis formation and patterning in plants. This directionality of transport depends on gradients, across the cell, of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. The organization of actin, in turn, has been shown to be under control of auxin. By overexpression of the actin-binding protein talin, we have generated transgenic rice (Oryza sativa) lines, where actin filaments are bundled to variable extent and, in consequence, display a reduced dynamics. We show that this bundling of actin filaments correlates with impaired gravitropism and reduced longitudinal transport of auxin. We can restore a normal actin configuration by addition of exogenous auxins and restore gravitropism as well as polar auxin transport. This rescue is mediated by indole-3-acetic acid and 1-naphthyl acetic acid but not by 2,4-dichlorophenoxyacetic acid. We interpret these findings in the context of a self-referring regulatory circuit between polar auxin transport and actin organization. This circuit might contribute to the self-amplification of auxin transport that is a central element in current models of auxin-dependent patterning.In addition to its role as central regulator of growth, auxin is involved in pattern formation (Berleth and Sachs, 2001). Auxin-dependent patterning is linked to a directional flow of auxin, a cell-to-cell transport described by a modified chemiosmotic model (Lomax et al., 1995). The self-amplification of cell polarity by a polar auxin flow has been linked with directional intracellular traffic. Positive feedback of auxin on this traffic in combination with mutual competition of neighboring cells for free auxin are central for pattern formation (Merks et al., 2007). This so-called auxin canalization model has been originally deduced from an analysis of vascular bundles in regenerating stems (Sachs, 1969) but was successfully applied to venation in developing leaves (Sachs, 2000) and the patterning of leaf primordia (Reinhard et al., 2000). Thus, patterning would ultimately depend on the directionality of auxin transport.In the meantime, several plant-specific pin-formed (PIN) proteins have been identified as candidates for auxin-efflux carriers (for review, see Chen and Masson, 2006), and despite a long debate on the actual function of these proteins, the most recent results show that they are in fact rate-limiting for auxin efflux (Petrášek et al., 2006). PIN proteins undergo constitutive recycling between plasma membranes and endosomal compartments (Geldner et al., 2001; Paciorek et al., 2005). This recycling seems to be under control of small GTPases, the ADP-ribosylation factors (ARFs), and their associated guanine nucleotide exchange factors (Geldner et al., 2003). Mutation of one of these guanine nucleotide exchange factors is responsible for the phenotype of the Arabidopsis (Arabidopsis thaliana) mutant gnom causing a mislocalization of PIN1 that becomes trapped in intracellular compartments. This cellular mutant phenotype can be phenocopied by treatment of the wild type with brefeldin A, a fungal toxin that selectively blocks ARF-guanine nucleotide exchange factors (Geldner et al., 2001). This suggests that ARF-dependent vesicle trafficking is involved in the polar distribution of PIN proteins and, thus, in cell polarity.The internalization of PIN1 caused by brefeldin A is arrested by the actin inhibitor cytochalasin D (Geldner et al., 2001). Conversely, PIN3 is rapidly internalized upon treatment with cytochalasin (Friml et al., 2002). Moreover, the potent actin inhibitor latrunculin B (LatB) impaired the polar localization of PIN1 in protophloem cells, and with even higher sensitivity, of the auxin-efflux carrier AUX1 (Kleine-Vehn et al., 2006), and inhibition of myosin function with butane-2,3 monoxime inhibited basipetal auxin transport in flower stalks of Arabidopsis (Holweg, 2007). These findings suggest that actin participates in the cycling of some of the PIN proteins.The relation between actin and auxin seems to be bidirectional but complex; as early as 1937, Sweeney and Thimann (1937) demonstrated that auxin stimulates cytoplasmic streaming in oat (Avena sativa) coleoptiles. However, when streaming was inhibited by cytochalasin B, this delayed the onset of auxin transport but left the rate of auxin transport unaltered (Cande et al., 1973). The stimulation of coleoptile growth by auxin is accompanied by a debundling of actin bundles into finer strands (Waller et al., 2002; Holweg et al., 2004).Inhibition of auxin transport impaired the organization of actin in zygotes of the brown alga Fucus and inhibited signal-induced developmental polarity (Sun et al., 2004). Since the cycling of PIN proteins is regulated by auxin itself (Paciorek et al., 2005), there might be a feedback loop between actin and auxin. Consistent with this view, binding sites for 1-N-naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, have been found to cosediment with actin (Butler et al., 1998).However, models that link the polar localization of the PIN proteins to actin-dependent transport (Muday and Murphy, 2002; Blakeslee et al., 2005) are challenged by experiments where PIN proteins maintained their polar localization, although actin filaments had been eliminated (for instance, by cytochalasin D [Geldner et al., 2001], by low concentrations of LatB [Kleine-Vehn et al., 2006], or by the phytotropin NPA or artificial auxin 2,4-dichlorophenoxyacetic acid [2,4-D; Rahman et al., 2007]).On the other hand, a recent report (Dhonukshe et al., 2008) demonstrated that 2,3,5-triiodobenzoic acid (TIBA) and the phytotropin 2-(1-pyrenoyl) benzoic acid induced actin bundling not only in plants, but also in mammalian and yeast cells, i.e. in cells that are not to be expected to use auxin as signaling compound. This was interpreted as supportive evidence for a role of actin filaments in polar auxin transport. However, it was mentioned in the same work that NPA failed to cause actin bundling in nonplant cells, suggesting that its mode of action must be different. This is consistent with classical work demonstrating that different phytotropins act on different targets (for review, see Rubery, 1990). Summarizing, although actin seems to play a role for the polarity of auxin fluxes, this issue is, first, not simple and, second, far from being understood.The relationship between actin and auxin was studied in the context of patterned cell division using the tobacco (Nicotiana tabacum) cell line BY-2 (Maisch and Nick, 2007). In this cell line, cell division is partially synchronized within a cell file, leading to higher frequencies of files with even cell numbers compared with files with uneven cell numbers. This synchrony can be interrupted by low concentrations of NPA, an inhibitor of polar auxin flux. To address the role of actin in this synchrony, the actin-binding protein mouse talin was overexpressed in those cells, resulting in a bundled configuration of actin and a loss of synchrony similar to the effect of NPA (indicative for a reduced auxin transport). By addition of auxins that are transported in a polar fashion (but not auxin per se), both the normal organization of actin (with fine strands) and the synchrony of cell division could be restored. This demonstrated that debundled actin strands are necessary and sufficient for the synchrony of cell division. However, although being indicative for a functional auxin transport, this synchrony is not a direct measure of auxin transport.To measure auxin transport directly, it would be necessary to administer radioactively labeled auxin to one pole of the file and to quantify the radioactivity recovered in the opposite pole of the file. This is not possible in a tobacco cell culture that has to be cultivated as suspension in a liquid medium. We therefore have returned to the classical Graminean coleoptile system (for a classical review, see Goldsmith, 1977), where auxin has been discovered originally by its polar transport and where auxin transport can be easily measured by following the distribution of radioactively labeled indole-3-acetic acid (IAA) fed to the coleoptile apex. We generated transgenic rice (Oryza sativa) lines expressing the actin-binding protein talin to variable levels. In those lines, as a consequence of talin overexpression, actin filaments were bundled to variable extent. The bundling of actin filaments was accompanied by a reduced polar transport of auxin. We could restore a debundled configuration of actin by addition of exogenous auxin, and by this treatment we were able to restore auxin transport. This rescue was mediated by transportable auxin species, but not by the artificial auxin 2,4-D that lacks polar transport. Using this approach, we can now probe the causal relationship between actin configuration and polar auxin transport directly.     

生长素极性运输研究进展

Recent advances in dissecting polar auxin transport, i.e., the physiological characteristics and regulation of polar auxin transport, the chemiosmotic hypothesis for polar auxin transport, and the role of polar auxin transport in plant growth and development were reviewed. The authors here focus on the progress of new supports-isolation and function analysis of the genes encoding putative auxin carriers, for the old model of polar auxin transport.     

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.The directed flow of auxin from cell to cell, through tissues and organs, from sites of synthesis to sites of action underlies the coordination of many processes during plant growth and development. Arabidopsis (Arabidopsis thaliana) PIN-FORMED (PIN) genes were the first found to be necessary for the phenomenon known as polar auxin transport (Okada et al., 1991; Chen et al., 1998; Gälweiler et al., 1998). Asymmetric localization of PIN proteins to the downstream ends of each cell in auxin-transporting tissues was correctly suggested to be a molecular component of the efflux mechanisms (Gälweiler et al., 1998) originally hypothesized as necessary for a directionally biased, or polar movement of auxin through tissues (Rubery and Sheldrake, 1974; Raven, 1975; Goldsmith, 1977; Goldsmith et al., 1981). Other members of the eight-gene PIN family in Arabidopsis were subsequently shown to affect auxin distribution in various tissues and stages of development (Křeček et al., 2009).Shortly after the breakthrough work on PIN1, members of the B subfamily of ATP-binding cassette (ABCB) transporters were discovered to be equally necessary for the phenomenon of polar auxin transport. They were originally called P-GLYCOPROTEIN1 (Dudler and Hertig, 1992; Sidler et al., 1998) and MULTIDRUG RESISTANCE1 (Noh et al., 2001) and ultimately renamed AtABCB1 and AtABCB19, respectively (Verrier et al., 2008). The connection between ABCB transporters and auxin transport was first made through the analysis of Arabidopsis knockout mutants. Polar auxin flow through abcb19 mutant stems is impaired by approximately 80% compared with the wild type and further reduced in abcb1 abcb19 double mutants (Noh et al., 2001). Resultant effects on development include abnormal hypocotyl tropisms (Noh et al., 2003) and the photomorphogenic control of hypocotyl elongation (Wu et al., 2010). Import of indole-3-acetic acid (IAA) to cotyledons through the petiole is reduced by 50% in abcb19 mutants, and this is correlated with an equivalent reduction in cotyledon blade expansion (Lewis et al., 2009). In roots, loss of ABCB19 greatly impairs auxin flow toward the tip without any detectable effect on shootward flow (Lewis et al., 2007). Surprisingly, the only defect detected in abcb19 primary roots associated with this major disruption of auxin transport is greater meandering of the tip during elongation down a vertical agar surface; gravitropism is unaffected (Lewis et al., 2007). Outgrowth of lateral roots, although not their initiation, depends significantly on ABCB19-mediated tipward auxin transport (Wu et al., 2007). The emergence of adventitious roots at the base of hypocotyls from which roots have been excised from Arabidopsis seedlings depends strongly on ABCB19-mediated auxin accumulation at the sites of primordium initiation (Sukumar et al., 2013).The ABCB19 protein is present predominantly in the central cylinder and cortex of the root, consistent with its role in rootward auxin transport (Lewis et al., 2007; Mravec et al., 2008), whereas the closely related ABCB4 is restricted to the lateral root cap and epidermis (Cho et al., 2007), where it functions in shootward auxin transport (Lewis et al., 2007). Loss of ABCB4 function alters the timing and spatial pattern of gravitropic curvature development, apparently because the gravity-induced auxin gradient across the root is less rapidly dissipated by normal shootward (basipetal) transport of the hormone through the elongation zone (Lewis et al., 2007). Root hairs are significantly longer in abcb4 mutants, a phenotype attributed to auxin accumulation due to impaired efflux (Cho et al., 2007). ABCB4 is reported to conduct auxin influx or efflux, depending on the prevailing external auxin concentration (Kubeš et al., 2012).Noh et al. (2001) originally isolated ABCB19 in a molecular screen for genes encoding an ion channel activity in Arabidopsis cells shown by patch-clamp electrophysiology to be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). The rationale for the screen was that a plant challenged with a channel blocker would overexpress the gene encoding the blocked activity. A hypothesis emerging from the Noh et al. (2001) study is that ABCB19 encodes such an ion channel, which is required for polar auxin transport. If true, NPPB would be established as a blocker of polar auxin transport.Pharmacological inhibitors, used for decades in auxin transport research, have some advantages over mutations. Mutations can create complicating pleiotropic effects by inhibiting the process throughout development, while inhibitors can be used to impose an effect at a specific time. 1-Naphthylphthalamic acid (NPA) is the most commonly used inhibitor of polar auxin transport (Katekar and Geissler, 1980), but others are being discovered (Rojas-Pierce et al., 2007; Kim et al., 2010; Tsuda et al., 2011). Inhibitors are especially useful when their targets are well defined, which would be the case if NPPB blocked ABCB19 and induced its expression as hypothesized. The experiments reported here were designed to test this hypothesis with electrophysiological measurements of ABCB19 transport activity, radiotracer measurements of polar auxin transport in roots, levels of fluorescently tagged ABCB19 proteins, auxin reporter expression patterns, and machine-vision measurements of a root growth response that depends on auxin redistribution.     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis

Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We characterized the auxin transport and gravitropic phenotypes of the pinoid-9 (pid-9) mutant of Arabidopsis (Arabidopsis thaliana) and tested the hypothesis that phosphorylation mediated by PID kinase and dephosphorylation regulated by the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1) protein might antagonistically regulate root auxin transport and gravity response. Basipetal indole-3-acetic acid transport and gravitropism are reduced in pid-9 seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor staurosporine phenocopies the reduced auxin transport and gravity response of pid-9, while pid-9 is resistant to inhibition by staurosporine. Staurosporine and the phosphatase inhibitor, cantharidin, delay the asymmetric expression of DR5∷revGFP (green fluorescent protein) at the root tip after gravistimulation. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine and cantharidin, respectively. The pid-9 rcn1 double mutant has a more rapid gravitropic response than rcn1. These data are consistent with a reciprocal regulation of gravitropism by RCN1 and PID. Furthermore, the effect of staurosporine is lost in pinformed2 (pin2). Our data suggest that reduced PID kinase function inhibits gravitropism and basipetal indole-3-acetic acid transport. However, in contrast to PID overexpression studies, we observed wild-type asymmetric membrane distribution of the PIN2 protein in both pid-9 and wild-type root tips, although PIN2 accumulates in endomembrane structures in pid-9 roots. Similarly, staurosporine-treated plants expressing a PIN2∷GFP fusion exhibit endomembrane accumulation of PIN2∷GFP, but no changes in membrane asymmetries were detected. Our data suggest that PID plays a limited role in root development; loss of PID activity alters auxin transport and gravitropism without causing an obvious change in cellular polarity.A variety of important growth and developmental processes, including gravity response, embryo and vascular development, and the branching of roots and shoots, are controlled by the directional and regulated transport of auxin in higher plants. Reversible protein phosphorylation is an important regulatory strategy that may modulate auxin transport and dependent processes such as root gravitropism, perhaps through action of the PINOID (PID) kinase (for review, see DeLong et al., 2002; Galvan-Ampudia and Offringa, 2007). PID is an AGC family Ser/Thr kinase (Christensen et al., 2000) and belongs to an AGC kinase clade containing WAG1, WAG2, AGC3-4, and D6PK/AGC1-1 (Santner and Watson, 2006; Galvan-Ampudia and Offringa, 2007; Zourelidou et al., 2009). PID activity has been demonstrated in vitro and in vivo (Christensen et al., 2000; Michniewicz et al., 2007), and several pid mutant alleles exhibit altered auxin transport in the inflorescence and a floral development defect resembling that of auxin transport mutants (Bennett et al., 1995). Overexpression of the PID gene results in profound alterations in root development and responses to auxin transport inhibitors, reduced gravitropism and auxin accumulation at the root tip (Christensen et al., 2000; Benjamins et al., 2001; Michniewicz et al., 2007), as well as enhanced indole-3-acetic acid (IAA) efflux in tobacco (Nicotiana tabacum) cell cultures (Lee and Cho, 2006) and altered PINFORMED1 (PIN1), PIN2, and PIN4 localization patterns (Friml et al., 2004; Michniewicz et al., 2007), consistent with PID being a positive regulator of IAA efflux. However, the effects of pid loss-of-function mutations on auxin transport activities and gravitropic responses in roots have not yet been reported (Robert and Offringa, 2008).In contrast, auxin transport and gravitropism defects of a mutant with reduced protein phosphatase activity have been characterized in detail. The roots curl in naphthylphthalamic acid1 (rcn1) mutation, which ablates the function of a protein phosphatase 2A regulatory subunit, causes reduced PP2A activity in vivo and in vitro (Deruère et al., 1999). Roots and hypocotyls of rcn1 seedlings have elevated basipetal auxin transport (Deruère et al., 1999; Rashotte et al., 2001; Muday et al., 2006), and rcn1 roots exhibit a significant delay in gravitropism, consistent with altered auxin transport (Rashotte et al., 2001; Shin et al., 2005). These data indicate that PP2A is a negative regulator of basipetal transport and suggest that if PID-dependent phosphorylation regulates root auxin transport and gravitropism, then it may act in opposition to PP2A-dependent dephosphorylation.In roots, auxin transport is complex, with distinct sets of influx and efflux carriers that define tissue-specific and opposing directional polarities (for review, see Leyser, 2006). IAA moves acropetally, from the shoot toward the root apex, through the central cylinder (Tsurumi and Ohwaki, 1978), and basipetally, from the root apex toward the base, through the outer layer of cells (for review, see Muday and DeLong, 2001). When plants are reoriented relative to the gravity vector, auxin becomes asymmetrically distributed across the root tip, as a result of a process termed lateral auxin transport (for review, see Muday and Rahman, 2008). Several carriers that mediate root basipetal IAA transport have been clearly defined and include the influx carrier AUXIN-INSENSITIVE1 (AUX1; Marchant et al., 1999; Swarup et al., 2004; Yang et al., 2006) and efflux carriers of two classes, PIN2 (Chen et al., 1998; Müller et al., 1998; Rashotte et al., 2000) and ATP-BINDING CASSETTE TYPE B TRANSPORTER4/MULTIDRUG-RESISTANT4/P-GLYCOPROTEIN4 (ABCB4/MDR4/PGP4; Geisler et al., 2005; Terasaka et al., 2005; Lewis et al., 2007). Lateral transport at the root tip may be mediated by PIN3, an efflux carrier with a gravity-dependent localization pattern (Friml et al., 2002; Harrison and Masson, 2007).Gravitropic curvature of Arabidopsis (Arabidopsis thaliana) roots requires changes in IAA transport at the root tip (for review, see Muday and Rahman, 2008). Auxin transport inhibitors (Rashotte et al., 2000) and mutations in genes encoding basipetal transporters, including aux1 (Bennett et al., 1996), pin2/agr1 (Chen et al., 1998; Müller et al., 1998), and abcb4/mdr4/pgp4 (Lin and Wang, 2005; Lewis et al., 2007), alter gravitropism. Auxin-inducible reporters exhibit asymmetric expression across the root tip prior to differential growth, and this asymmetry is abolished by treatment with auxin transport inhibitors that prevent gravitropic curvature (Rashotte et al., 2001; Ottenschläger et al., 2003). Additionally, the pin3 mutant exhibits slightly reduced rates of gravitropic curvature (Harrison and Masson, 2007), and PIN3 is expressed in the columella cells, which are the site of gravity perception (Blancaflor et al., 1998; Friml et al., 2002). The PIN3 protein relocates to membranes on the lower side of columella cells after gravitropic reorientation, consistent with a role in facilitating asymmetric IAA transport at the root tip (Friml et al., 2002; Harrison and Masson, 2007).The available data suggest a model in which PID and RCN1 antagonistically regulate basipetal transport and gravitropic response in root tips (Fig. 1). In this model, the regions with the highest IAA concentrations in the epidermal and cortical cell layers are indicated by shading, and the arrows indicate the direction and relative amounts of basipetal auxin transport. Our previous work suggests that elevated basipetal IAA transport in rcn1 roots impairs gravitropic response, presumably due to the inability of roots either to form or to perceive a lateral auxin gradient in the context of a stronger polar IAA transport stream (Rashotte et al., 2001). Enhanced basipetal transport may increase the initial auxin concentration along the upper side of the root, impeding the establishment or perception of a gradient in rcn1 and cantharidin-treated wild-type roots (Fig. 1, right). Based on the published pid inflorescence transport data (Bennett et al., 1995), we hypothesize that pid seedling roots and staurosporine-treated wild-type roots have reduced basipetal auxin transport (Fig. 1, left). Upon reorientation of roots relative to the gravity vector, the reduced basipetal IAA transport in pid may lead to slower establishment of an auxin gradient across the root. This model then predicts that cantharidin treatment of pid-9 or staurosporine treatment of rcn1 seedlings would enhance or restore gravitropism in these mutants. Similarly, a double mutant might be expected to exhibit a corrected gravitropic response relative to the single mutants.Open in a separate windowFigure 1.Auxin transport defects in pid-9 and rcn1 mutants alter auxin redistribution after reorientation relative to the gravity vector. This model predicts that differences in basipetal auxin transport activities of wild-type, pid-9, and rcn1 roots will affect the formation of lateral auxin gradients. The shaded area in each root represents the region of highest IAA concentration in epidermal and cortical cells, with darker shading in the central columella cells, believed to be the auxin maxima. The direction and amount of basipetal IAA transport are indicated by arrows. The region of differential growth during gravitropic bending is indicated by the shaded rectangle. If auxin transport is reduced (as shown in the pid-9 mutant or in staurosporine-treated seedlings), this would lead to a slower formation of an auxin gradient in root tips. The rcn1 mutation (or treatment with cantharidin) has already been shown to lead to increased basipetal transport and a reduced rate of gravitropic bending, consistent with altered formation or perception of an auxin gradient. The antagonistic effects of kinase and phosphatase inhibition are predicted to lead to normal gravity responses in the pid-9 rcn1 double mutant as well as in pid-9 and rcn1 single mutants treated with the “reciprocal” inhibitor.The experiments described here were designed to test this model by examining gravitropism and root basipetal IAA transport in pid and staurosporine-treated seedlings. We investigated the regulation of gravity response by PID kinase and RCN1-dependent PP2A activities and observed antagonistic interactions between the rcn1 and pid-9 loss-of-function phenotypes that are consistent with reciprocal kinase/phosphatase regulation. We found that loss of kinase activity in the pid mutant and in staurosporine-treated wild-type plants inhibits basipetal auxin transport and the dependent physiological process of root gravitropism. Our results suggest that staurosporine acts to regulate these processes through inhibition of PID kinase and that PID effects are PIN2 dependent. In both wild-type and pid-9 roots, we observed polar membrane distribution of the PIN2 protein; unlike wild-type roots, though, pid-9 roots exhibited modest accumulation of PIN2 in endomembrane structures. Similarly, we detected asymmetric distribution and endomembrane accumulation of PIN2∷GFP in staurosporine-treated roots. Our data suggest that PID plays a limited role in root development; loss of PID activity alters PIN2 trafficking, auxin transport, and gravitropism without causing an obvious loss of cellular polarity. Together, these experiments provide insight into phosphorylation-mediated control of the gravity response and auxin transport in Arabidopsis roots.     

Effect of simulated microgravity on auxin polar transport in inflorescence axis of Arabidopsis thaliana.

The morphology, growth and development of higher plants are strongly influenced by environmental stimuli on the earth, which affect the changes in the dynamics of plant hormones in plants. Qualitative and quantitative changes in plant hormones are the most important internal factor to regulate plant growth and development. Among them, auxin (IAA) is of most significant. There are numerous reports concerning the physiological roles of auxin in plant growth and development (Matthysse and Scott 1984). One of the characteristics of auxin is to have the ability of polar transport along the vector of gravity on the earth (Schneider and Wightman 1978), suggesting that the activity of auxin polar transport is also important for the growth and development of plants. It has recently been reported that the normal activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana was required for flower formation (Okada et al. 1991, Ueda et al. 1992). Considering the above evidence together with the fact that gravity affects the morphology, growth and development of higher plants, gravity might affect the qualitative and quantitative changes in plant hormones including the activity of auxin polar transport. In this paper, we report the effect of microgravity condition simulated by a three-dimensional (3-D) or a horizontal clinostat on the activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana.     

Phenomenon of “Siamese embryos” in cereals in vivo and in vitro: Cleavage polyembryony and fasciations

The genesis of wheat microsporial polyembryoids in vitro was analyzed in detail. The nature of different phenotypes of cereal polymeric embryos was identified. They represent the class “multiple shoot meristems,” which results from a cleavage polyembryony and is accompanied by organ fasciations of all known types (radial, flat, or ring). The morphological nature of cereal embryonic organs has been clarified: shoot meristem—axial organ; scutellum—lateral outgrowth of this axis; coleoptile—derivative of shoot meristem but fused with scutellum; terminality of scutellum—the result of linear fasciation that occurred historically. An explanation is given on how the structural model of an auxin polar transport works during the establishment of bilateral symmetry in a cereal embryo that is associated with the inverted polarization of the carrier protein PIN1 on cell membranes and, correspondingly, with the inverted auxin transport performed by this carrier (Fischer-Iglesias et al., 2001; Forestan et al., 2010).     

Role for Apyrases in Polar Auxin Transport in Arabidopsis

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [³H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.In both animals and plants, cells release nucleotides into their extracellular matrix, where they function as signaling agents, inducing rapid increases in the concentration of cytosolic calcium that are transduced into downstream changes in cell physiology (Kim et al., 2006; Burnstock, 2007; Roux and Steinebrunner, 2007; Tanaka et al., 2010a, 2010b; Demidchik et al., 2011). Prominent among these downstream changes in plants are changes in the growth of cells, including the growth of pollen tubes (Steinebrunner et al., 2003), root hairs (Clark et al., 2010b), and cotton (Gossypium hirsutum) fibers (Clark et al., 2010a). These results suggest the possibility that the signaling changes induced by extracellular nucleotides intersect with signaling changes induced by one or more of the hormones that regulate plant cell growth. Consistent with this possibility, Tang et al. (2003) showed that a concentration of applied nucleotides that inhibited the gravitropic growth of roots could block the transport of the growth hormone auxin and that this effect could not be attributed to either pH changes or chelation of divalent cations. Correspondingly, Clark et al. (2010a) showed that when the application of nucleotides to cotton ovules growing in culture altered the rate of cotton fiber growth, it also induced the production of ethylene, a hormone known to regulate the growth of cotton fibers.Given the potency of extracellular nucleotides to regulate cellular activities, it would be important for cells to control the concentration of these nucleotides. In both animals and plants, the principal enzymes that limit the buildup of extracellular ATP (eATP) and extracellular ADP are ectoapyrases (apyrase; EC 3.6.1.5). These enzymes, which are nucleoside triphosphate diphosphohydrolases, are characterized by apyrase-conserved regions whose peptide sequences are highly similar throughout the plant and animal kingdoms (Clark and Roux, 2009). Based on this structural criterion, there are seven apyrases in Arabidopsis (Arabidopsis thaliana; APY1–APY7), and two of these, APY1 and APY2, share 87% protein sequence identity but are less than 30% similar to the other five apyrases. These two apyrases partially complement each other’s function and play central roles in growth control in Arabidopsis, as judged both by genetic and biochemical criteria (Wolf et al., 2007; Wu et al., 2007). Polyclonal antibodies raised to APY1 (Steinebrunner et al., 2000) inhibit the apyrase activity released into the medium of growing pollen tubes, and when these antibodies were added to the culture medium of germinated pollen, they both blocked the growth of the pollen and raised the concentration of ATP in the medium (Wu et al., 2007). Similarly, treatment of cultured cotton ovules with antibodies that recognize cotton fiber apyrase both inhibits the growth of the fibers and increases the concentration of ATP in the medium, further establishing the link between apyrase activity and regulation of the extracellular ATP concentration ([eATP]) in growing tissues (Clark et al., 2010a).Because wild-type pollen tubes expressing active APY1 or APY2 and cultured cotton fibers with wild-type apyrase activity grow at a normal rate, and because the antibodies inhibit apyrase activity (Wu et al., 2007), the growth inhibition induced by the antibodies further implicated apyrase activity as critical for the growth of these tissues. The antibodies were unlikely to enter the pollen tubes or cotton fibers, so these results also suggested that the pollen and cotton apyrases were ectoapyrases. However, these data do not rule out a possible Golgi function for APY1 and APY2 and for the cotton APY(s), as discussed by Wu et al. (2007) and Clark and Roux (2011). In fact, there is strong evidence that APY1 and APY2 are localized in the Golgi and may function there to regulate protein glycosylation and/or affect polysaccharide synthesis (Chiu et al., 2012; Schiller et al., 2012).Although the suppression of APY1/APY2 or of apyrase activity has a dramatic effect on growth, overexpression of APY1 or APY2 has much less of an effect. Constitutive expression of APY1 induces a small but statistically significant increase in the growth of etiolated hypocotyls, while overexpressing APY2 has no effect on this growth (Wu et al., 2007). This is probably because the wild-type levels of apyrase expression are near optimal for growth (Roux and Steinebrunner, 2007).The double knockout apy1apy2 is sterile, because the pollen of this mutant does not germinate (Steinebrunner et al., 2003). However, when APY1 is suppressed only approximately 60% by an inducible RNA interference (RNAi) construct in apy2 null mutants, pollen of these mutants will germinate, permitting fertilization and subsequent normal development, although the adult plants of these mutants are dwarf (Wu et al., 2007). Suppression of ectoapyrase activity would be expected to raise the equilibrium concentration of eATP (Wu et al., 2007), and since higher levels of eATP can inhibit auxin transport in roots (Tang et al., 2003), it was reasonable to hypothesize that the suppression of apyrase by RNAi could suppress auxin transport. The experiments described in this report test this hypothesis. The results indicate that suppression of APY1/APY2 expression in an inducible RNAi line, R2-4A (Wu et al., 2007), results in a significant inhibition of polar auxin transport in Arabidopsis hypocotyls and roots, with a concomitant altered distribution of endogenous auxin. Consistent with this result and with the results of Tang et al. (2003), suppression of APY1/APY2 also blocks the asymmetric distribution of a GFP reporter encoded by a DR5:GFP construct in gravistimulated primary roots of Arabidopsis seedlings and diminishes the extent of the elongation zone in these roots. These results are consistent with the novel conclusion that inhibition of auxin transport is a key step in the signaling pathway that links the inhibition of apyrase expression to growth inhibition.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

Modeling Auxin Transport and Plant Development

The plant hormone auxin plays a critical role in plant development. Central to its function is its distribution in plant tissues, which is, in turn, largely shaped by intercellular polar transport processes. Auxin transport relies on diffusive uptake as well as carrier-mediated transport via influx and efflux carriers. Mathematical models have been used to both refine our theoretical understanding of these processes and to test new hypotheses regarding the localization of efflux carriers to understand auxin patterning at the tissue level. Here we review models for auxin transport and how they have been applied to patterning processes, including the elaboration of plant vasculature and primordium positioning. Second, we investigate the possible role of auxin influx carriers such as AUX1 in patterning auxin in the shoot meristem. We find that AUX1 and its relatives are likely to play a crucial role in maintaining high auxin levels in the meristem epidermis. We also show that auxin influx carriers may play an important role in stabilizing auxin distribution patterns generated by auxin-gradient type models for phyllotaxis.     

Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices

The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.     

Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.Auxin plays a critical role in plant development and growth by forming local concentration gradients. Local auxin gradients, created by the polar cell-to-cell movement of auxin, are implicated in primary axis formation, root meristem patterning, lateral organ formation, and tropic movements of shoots and roots (for recent review, see Vanneste and Friml, 2009). The cell-to-cell movement of auxin is achieved by auxin influx and efflux transporters such as AUXIN-RESISTANT1 (AUX1)/LIKE-AUX1 for influx and PIN-FORMED (PIN) and the P-glycoprotein (PGP) of ABCB (ATP-binding cassette-type transporter subfamily B) for efflux. Since diffusive efflux of the natural auxin indole-3-acetic acid (IAA; pKa = 4.75) is not favorable and PINs are localized in the plasma membrane in a polar manner, PINs act as rate-limiting factors for cellular auxin efflux and polar auxin transport through the plant body. These PINs'' properties explain why representative physiological effects of auxin transport are associated with PINs.Auxin flows from young aerial parts all the way down to the root tip columella in which an auxin maximum is formed for root stem cell maintenance and moves up toward the root differentiation zone through root epidermal cells, where a part of it travels back to the root tip via cortical cells (Blilou et al., 2005). This directional auxin flow is supported by the polar localization of PINs: PIN1, PIN3, and PIN7 at the basal side of stele cells (Friml et al., 2002a, 2002b; Blilou et al., 2005), PIN4 at the basal side in root stem cells (Friml et al., 2002a), and PIN2 at the upper side of root epidermis and at the basal side of the root cortex (Luschnig et al., 1998; Müller et al., 1998). Another interesting aspect of PIN-mediated auxin transport is the dynamics in directionality of auxin flow due to environmental stimuli-directed changes of subcellular PIN polarity, as exemplified for PIN3, whose subcellular localization changes in response to the gravity vector (Friml et al., 2002b).An intriguing question is how different PIN proteins have different subcellular polarities, which might be attributable to PIN-specific molecular properties, cell-type-specific factors, or both. The different PIN subcellular polarities in different cell types seemingly indicate that cell-type-specific factors are involved in polarity. In the case of PIN1, however, both classes of factors appear to affect its subcellular localization because when expressed under the PIN2 promoter, PIN1 localizes to the upper or basal side of root epidermal cells, depending on the GFP insertion site of the protein (Wiśniewska et al., 2006). A recent study demonstrated that the polar targeting of PIN proteins is modulated by phosphorylation/dephosphorylation of the central hydrophilic loop of PINs, which is mediated by PINOID (PID; a Ser/Thr protein kinase)/PP2A phosphatase (Michniewicz et al., 2007). The central hydrophilic domain of PINs might provide the molecule-specific cue for PIN polarity, together with as yet unknown cell-specific factors. Different recycling behaviors of PINs, which show variable sensitivities to brefeldin A (BFA), also imply different molecular characters among PIN species. Most PIN1 proteins are internalized by BFA treatment, whereas considerable amounts of PIN2 remain in the plasma membrane in addition to internal accumulation after BFA treatment. Recycling and basal polar targeting of PIN1 is dependent on the BFA-sensitive guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEFs), GNOM, which is the major target of BFA. In contrast, apical targeting and recycling of PIN2 is independent of GNOM and controlled by BFA-resistant ARF GEFs (Geldner et al., 2003; Kleine-Vehn and Friml, 2008).In contrast to their distinct subcellular localizations, the differential auxin-transporting activities of PINs remain to be studied. The divergent primary structures of PIN proteins are not only indicative of differential subcellular polarity, but also would represent their differential catalytic activities for auxin transport. The auxin efflux activities of Arabidopsis (Arabidopsis thaliana) PINs have been demonstrated using Arabidopsis and heterologous systems: PIN1 and PIN5 in Arabidopsis cells (Petrásek et al., 2006; Mravec et al., 2009); PIN2, PIN3, PIN4, PIN6, and PIN7 in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Lee and Cho, 2006; Petrásek et al., 2006; Mravec et al., 2008); PIN1, PIN2, PIN5, and PIN7 in yeast (Saccharomyces cerevisiae) cells (Petrásek et al., 2006; Blakeslee et al., 2007; Mravec et al., 2009; Yang and Murphy, 2009); and PIN1, PIN2, and PIN7 in HeLa cells (Petrásek et al., 2006; Blakeslee et al., 2007). Among the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7, which share a similar molecular structure in terms of the presence of a long central loop (hereafter called long-looped PINs; Fig. 1A; Supplemental Fig. S1), have been shown to catalyze auxin efflux at the cellular level. On the other hand, PIN5 and PIN8 possess a very short putative central loop (hereafter called short-looped PINs). Although PIN5 was recently shown to be localized in the endoplasmic reticulum (ER) and proposed to transport auxin metabolites into the ER lumen, its cellular function regarding its intracellular auxin-transporting activity has not been shown, and the auxin-transporting activity of PIN8 has yet to be demonstrated. In spite of the same transport directionality (auxin efflux) and similar molecular structures, the long-looped PINs exhibit sequence divergence not only in their central loop, but also in certain residues of the transmembrane domains. This structural divergence of long-looped PINs might be indicative of their differential auxin-transporting activities, which have not yet been quantitatively compared.Open in a separate windowFigure 1.Differential activities of PINs in the Arabidopsis root hair. A, Two distinctive PIN groups with different central hydrophilic loop sizes. Topology of PIN proteins was predicted by four different programs as described in Supplemental Figure S1. Numbers above indicate the number of transmembrane helices for each N- and C-terminal region, and numbers below indicate the number of amino acid residues of the central hydrophilic domain. B, Representative root images of control (Cont; Columbia-0) and root-hair-specific PIN-overexpressing (PINox; ProE7:PIN-GFP or ProE7:PIN [−]) plants. Bar = 100 μm for all. C, Root hair lengths of control and PINox plants. Six to 12 independent transgenic lines (average = 8.3), and 42 to 243 roots (average = 86.8) and 336 to 2,187 root hairs (average = 727.8) per construct, were observed for the estimation of root hair length. Data represent means ± se. The root hair lengths of PIN5ox lines were significantly longer than those of the control (P = 0.016 for PIN5ox; P < 0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).To comparatively assess the cytological behaviors and molecular activities of different PIN members, it would be favorable to use a single assay system that provides a consistent cellular environment and enables quantitative estimation of PIN activity. In previous studies, we adopted the root hair single cell system to quantitatively assay auxin-transporting or regulatory activities of PINs, PGPs, AUX1, and PID (Lee and Cho, 2006; Cho et al., 2007a). Root hair growth is proportional to internal auxin levels in the root hair cell. Therefore, auxin efflux inhibits and auxin influx enhances root hair growth (Cho et al., 2007b; Lee and Cho, 2008). In addition, the use of a root-hair-specific promoter (Cho and Cosgrove, 2002; Kim et al., 2006) for expression of auxin transporters enables the transporters'' biological effect to be pinpointed to only the root hair cell, thus excluding probable non-cell-autonomous effects that could be caused by the general expression of auxin transporters.In this study, we expressed five long-looped PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and two short-looped PINs (PIN5 and PIN8) in root hair cells and compared their auxin-transporting activities and cytological dynamics. To directly measure the radiolabeled auxin-transporting activities of PIN5 and PIN8, we used an additional assay system, tobacco suspension cells. Our data revealed that PINs have differential molecular activities and pharmacological responses and that the short-looped and long-looped PINs have different subcellular localizations.     

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.     

Models of long-distance transport: how is carrier-dependent auxin transport regulated in the stem?

? This paper presents two models of carrier-dependent long-distance auxin transport in stems that represent the process at different scales. ? A simple compartment model using a single constant auxin transfer rate produced similar data to those observed in biological experiments. The effects of different underlying biological assumptions were tested in a more detailed model representing cellular and intracellular processes that enabled discussion of different patterns of carrier-dependent auxin transport and signalling. ? The output that best fits the biological data is produced by a model where polar auxin transport is not limited by the number of transporters/carriers and hence supports biological data showing that stems have considerable excess capacity to transport auxin. ? All results support the conclusion that auxin depletion following apical decapitation in pea (Pisum sativum) occurs too slowly to be the initial cause of bud outgrowth. Consequently, changes in auxin content in the main stem and changes in polar auxin transport/carrier abundance in the main stem are not correlated with axillary bud outgrowth.