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Auxin signals — turning genes on and turning cells around Thomas Berleth, Naden T Krogan and Enrico Scarpella The extremely wide spectrum of the plant processes that are influenced by auxin raises the question of how signals conveyed by a single molecule can trigger such a variety of responses. Although many aspects of auxin function remain elusive, others have become genetically tractable. The identification of crucial genes in auxin signal transduction and auxin transport in the past few years has led to molecularly testable concepts of how auxin signals regulate gene activities in individual cells, and how the polar transport of auxin could impact on patterning processes throughout the plant. Addresses University of Toronto, Department of Botany, 25 Willcocks Street, Toronto M5S 3B2, Canada e-mail: [email protected] Current Opinion in Plant Biology 2004, 7:553–563 This review comes from a themed issue on Cell signalling and gene regulation Edited by Jen Sheen and Steven Kay Available online 31st July 2004 1369-5266/$ – see front matter # 2004 Published by Elsevier Ltd. DOI 10.1016/j.pbi.2004.07.016 Abbreviations ARF auxin-response factor ASK ARABIDOPSIS Skp1-LIKE Aux auxin BFA brefeldin A CUL CULLIN DBD DNA-binding domain E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme E3 ubiquitin-ligase GEF guanosine exchange factor GN GNOM IAA indole acetic acid MR middle region PIN PIN-FORMED QC quiescent center RUB1 RELATED TO UBIQUITIN1 SAM shoot apical meristem SCF Skp1–Cullin–F-box TIR1 TRANSPORT INHIBITOR RESISTANT1 Introduction Auxin has been implicated in a confounding number of processes in plants. The continuously extending list of auxin functions includes the relay of environmental signals such as light and gravity, the regulation of branching processes in shoots and roots and, more recently, the www.sciencedirect.com patterned differentiation of cells in meristems and immature organs. The fact that the distribution of auxin itself has remained largely invisible has added to the difficulty in imagining how a single molecule could convey such a variety of disparate signals. As a consequence, questions directly connected to the distribution, routes of transport and cellular sites of perception of the crucial molecule indole acetic acid (IAA; the most important auxin in higher plants) are still subject to debate. At the same time, however, auxin action has become tractable on at least two levels. First, large protein families have been implicated in the positive and negative transcriptional regulation of auxin-dependent gene expression, and the molecular mechanisms through which auxin can influence the composition of relevant transcriptional complexes have been established. Second, families of membrane proteins have been convincingly implicated in auxin influx and efflux. The cell biology of the polar localization of these proteins at crucial stages of development now provides a formidable tool for understanding apical–basal polarity acquisition and its impact on patterning processes. In this review, we exclusively focus on recent advances made in auxin-dependent gene regulation, auxin transport and associated patterning. Other aspects of auxin function are excellently covered in recent reviews [1–3]. Auxin-controlled gene expression Key to our present understanding of auxin-controlled gene expression are transcripts that are rapidly induced by auxin, which are collectively referred to as primary auxin-response genes [4]. In Arabidopsis, these include several families of well-characterized genes [5], including many of the 29 members of the Aux/IAA gene family. Aux/ IAA genes encode small nuclear proteins that have a common four-domain (I–IV) structure (Figure 1a). They are not only subject to auxin-mediated transcriptional regulation but are also involved in auxin signal transduction ([6]; reviewed in [5,7–9]). Through their conserved domains III and IV, Aux/IAA proteins can interact with each other and with similar domains of auxin-response factors (ARFs). The ARF family comprises 22 transcription factors in Arabidopsis that are characterized by an amino-terminal DNA-binding domain (DBD), a long middle region (MR), and domains III and IV near the carboxyl terminus (Figure 1a; reviewed in [5,10]). The DBDs of ARFs bind to conserved promoter elements that confer auxin-responsive gene expression and, depending on the structure of the MR, individual ARFs function as transcriptional activators or repressors [11,12,13]. The regulation of gene activation by ARFs is presently far Current Opinion in Plant Biology 2004, 7:553–563 554 Cell signalling and gene regulation Figure 1 (a) Low auxin Aux/IAA (b) AXR1 U E1 ARF ECR1 R RCE1 R X Repression U E2 RBX1 High auxin U U E3 U U U Aux/IAA ARF Activation AAAAAA AAAAAA R CSN CUL1/ AXR6 R ASK1/2 TIR1 ARF ARF Potentiation AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA 26S Aux/IAA , etc. Current Opinion in Plant Biology Auxin regulation of gene expression. (a) ARF activity is controlled through interaction with Aux/IAA proteins or other ARFs. Domain I (red) of Aux/IAA proteins functions as a transferable repression domain that is capable of suppressing gene activation conferred by nearby transcription factors of various kinds [14]. Upon auxin-mediated Aux/IAA proteolysis, ARF monomers can activate transcription, but activation seems to be potentiated through ARF–ARF dimerization [13]. Auxin acts antagonistically by simultaneously promoting Aux/IAA protein degradation and Aux/IAA gene transcription. This mechanism could keep Aux/IAA protein levels in a balance, and also allows for the rapid restoration of their abundance when auxin levels subside. Domain II is shown in blue, domain III in yellow, domain IV in green, and the ARF DBD in purple. (b) Depletion of Aux/IAA protein levels by proteolysis through the 26S proteosome is regulated through the E3 ubiquitin (U)-ligase function of SCFTIR1, which is composed of four subunits (ASK1/2–CUL1–RBX1–TIR1). Auxin critically influences interaction between the F-box substrate recognition subunit TIR1 and the targeted Aux/IAA protein, whose domain II (blue) appears to be necessary for this association [20,30,31]. SCFTIR1 function is dependent upon cyclic modification of the CUL1 subunit by RUB (R) [32], whereas de-modification is dependent on the function of the COP9 signalosome (CSN) [33,34]. Mutations in RUB-specific E1-activating (AXR1-ECR1) and E2-conjugating (RCE1) enzymes are associated with auxin-insensitivity [28,29]. Experimental evidence suggests a role for RBX1 in both ubiquitin and RUB E3 functions [29,32]. better understood than negative gene regulation, and it will be a major challenge for the coming years to understand the in-planta functions of ARFs that have repression MR domains. Domains III and IV not only enable interactions between ARF and Aux/IAA proteins but also mediate ARF–ARF dimerization (Figure 1a). Aux/IAA proteins do not seem to bind DNA directly but instead act as transcriptional repressors through domain I when interacting with ARFs [14]. Aux/IAAs are extremely short-lived, and their half-lives and abundance can be dramatically increased through the application of proteosome inhibitors or reduced by auxin [15,16]. How auxin can influence ARF activity and Aux/IAA Current Opinion in Plant Biology 2004, 7:553–563 abundance became apparent with the identification of genes that are defective in Arabidopsis auxin-sensitivity mutants (Table 1). Most of these genes encode ARFs, Aux/IAAs or proteins that are associated with ubiquitinmediated protein degradation [8,9]. As far as they have been characterized, loss-of-function mutations in ARF genes lead to reduced responses of auxin-regulated gene expression [17,18,19]. By and large, diminished auxin responses are also associated with gain-of-function mutations in Aux/IAA genes [8,9], consistent with the idea that Aux/IAA proteins typically function as negative regulators of ARFs (Figure 1a). Strikingly, all gain-of-function mutations in Aux/IAA www.sciencedirect.com Auxin signaling Berleth, Krogan and Scarpella 555 Table 1 Functionally characterized genes. Gene name Gene identifier Auxin-response factors ARF3/ETTa AT2G33860 ARF5/MP AT1G19850 ARF7/NPH4/TIR5/MSG1 AT5G20730 Aux/IAA genes IAA3/SHY2 AT1G04240 IAA6/SHY1 AT1G52830 IAA7/AXR2 AT3G23050 IAA12/BDL AT1G04550 IAA14/SLR AT4G14550 IAA17/AXR3 AT1G04250 IAA18 AT1G51950 IAA19/MSG2 AT3G15540 IAA28 AT5G25890 Protein-degradation pathway AXR1 AT1G05180 ECR1 AT5G19180 RCE1 AT4G36800 ASK1 AT1G75950 AXR6/CUL1 AT4G02570 TIR1 AT1G12820 RBX1a AT5G20570 Auxin transport AUX1/WAV5 AT1G77690 PIN1 AT1G73590 EIR1/PIN2/AGR1/WAV6 AT5G57090 PIN3 AT1G70940 PIN4 AT2G01420 PIN7 AT1G23080 Reference(s) [73] [74,75] [17,61,62,76,77] [78,79] [7,78] [80,81] [47,82] [83] [84,85] [7] [63] [86] [28,87] [88,89] [29,90] [91,92] [26,27] [25,77] [32] [93–95] [96,97] [94,98–102] [37] [38] [39] a An atypical ARF lacking domains III and IV, therefore not discussed in this review. AGR, AGRAVITROPIC; ARF, AUXIN RESPONSE FACTOR; ASK, ARABIDOPSIS Skp1-LIKE; AUX, AUXIN RESISTANT; AXR, AUXIN RESISTANT; BDL, BODENLOS; CUL, CULLIN; ECR, E1 C-TERMINUS-RELATED; EIR, ETHYLENE INSENSITIVE ROOT; ETT, ETTIN; IAA, INDOLE ACETIC ACID; MP, MONOPTEROS; MSG, MASSUGU; NPH, NON-PHOTOTROPIC HYPOCOTYL; PIN, PIN-FORMED; RBX, RING-BOX PROTEIN; RCE, RUB1CONJUGATING ENZYME; SHY, SUPPRESSOR OF HY2; SLR, SOLITARY ROOT; TIR, TRANSPORT INHIBITOR RESISTANT; WAV, WAVY GROWTH. genes affect specific sites in domain II, and lead to vastly extended life spans and presumably much greater abundance of the respective Aux/IAA proteins [15,20,21]. Therefore, both genetic and molecular data support the notion that auxin relieves ARF repression by promoting Aux/IAA proteolysis. Poly-ubiquitin tagging of proteins that are destined for proteolytic degradation occurs through the activity of three enzymes, referred to as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitinligase (E3) (Figure 1b). Poly-ubiquitinated proteins are then subject to degradation by the 26S proteosome. The Arabidopsis SCF-type of E3 ubiquitin-ligase is composed of four primary units: ASK (in yeast Skp1), CULLIN (CUL), RBX, and an F-box protein. Generally, the substrate specificity of E3 is thought to be mediated by its Fwww.sciencedirect.com box component (reviewed in [22–24]). Arabidopsis auxininsensitive mutants that define SCF subunits have been isolated (Table 1). TRANSPORT INHIBITOR RESISTANT1 (TIR1) was found to be an F-box protein that is crucial for substrate recognition in auxin responses [20,25]. Another component, CUL1, itself identified by an auxin-insensitive mutant axr6 [26] (which is defective in CUL1–ASK interaction [27]), needs to be reversibly modified by the ubiquitin-related protein RUB1 (Figure 1b). This modification involves RUB-specific activating (E1) and conjugating (E2) activities, through which mutations in one of the E1 subunits (AXR1) and in E2 (RCE1) affect auxin sensitivity [28,29]. Several Aux/IAA proteins have been shown to directly interact with SCFTIR1, suggesting that they constitute natural substrates for SCFTIR1 action [20]. This interaction is very rapidly promoted by IAA in a concentrationdependent manner, and Aux/IAA proteins have been shown to be more stable in axr1, rce1, tir1 and axr6 mutant backgrounds [20,27,29,30,31]. Further, the increased Aux/IAA stability conferred by gain-of-function mutations in domain II appears to result from a reduced association between TIR1 and the mutant protein [20,30,31]. How auxin promotes the interaction between SCFTIR1 and Aux/IAA proteins is less clear. Domain II of Aux/IAA proteins contains a 13-amino-acid sequence that functions as a transferable degradation signal, and is necessary and sufficient to define Aux/ IAA protein stability [16]. This sequence could be subject to auxin-mediated protein modification that affects SCFTIR1–Aux/IAA association. Although the degradation signal does not contain a phosphorylation site, there are conserved proline residues that could be targets of auxinmodulated hydroxylation or isomerization [30,31]. Alternatively, modification of F-box proteins (such as TIR1) or CUL could influence the interaction between Aux/IAA proteins and SCFTIR1. Specifically, CUL modification seems to be subject to complex regulation, as both RUB conjugation and deconjugation are necessary for normal auxin response [32]. RUB deconjugation is dependent on the activity of the CONSTITUTIVE PHOTOMORPHOGENIC9 (COP9) signalosome ([33]; reviewed in [34]). Finally, it is possible that SCFTIR1 function and Aux/IAA stability are controlled by further unknown proteins, which may be identified in genetic screens. Such a role has recently been established for SGT1b, a protein previously implicated in plant diseaseresistance signaling [35]. When attempting to pin down the role of auxin, it is interesting to note that its effect on interactions between Aux/IAA proteins and SCFTIR1 can be seen in crude plant extracts [30]. Therefore, auxin signal transduction through this important pathway seems to occur entirely through soluble factors. Future research will precisely assess the relevance of this pathway in natural auxin Current Opinion in Plant Biology 2004, 7:553–563 556 Cell signalling and gene regulation Figure 2 (a) Nucleus Golgi apparatus Actin filament (b) Current Opinion in Plant Biology Dynamic auxin transport and patterning. (a) Hypothetical cellular mechanism for PIN localization (redrawn from [43]). Top: basal localization of the PIN protein (red) depends on BFA-sensitive vesicle transport. In BFA-treated cells, PIN accumulates in undefined endosomal membrane structures (light blue) [40,42]. Polar auxin transport depends on an intact actin cytoskeleton, and a high-affinity 1-N-naphthylphthalamic acid (NPA)-binding protein (green), which is known to interact with actin, may connect the respective transport vesicles (yellow) with the actin tracks. Permanent cycling of PIN proteins between intracellular compartments and the plasma membrane could account for rapid relocation of PIN protein, which occurs in response to tropic stimuli [37,103]. Bottom: hypothetical intercellular stabilization of PIN protein localization. If the localization of PIN proteins (red) is responsive to external signals, it could also respond to auxin flow. This would generate a postulated feed-back mechanism [45] that would result in the integration of apical–basal cell polarity and, further, in the reinforcement of unevenness of auxin conductivity along certain lines of cells (as proposed in [44–46,53,57,60]). (b) In the two-cell stage embryo (left), auxin seems to be transferred from the basal to the apical cell, the progenitor cell of the embryo proper [39]. This leads to a transient auxin-response maximum (blue) in the apical cell. In the early globular embryo (middle), the direction of auxin flow seems to be reversed, leading to a persistent auxin maximum in the uppermost suspensor cell, which will give rise to the central part of the primary root meristem. In the triangular-stage embryo (right), the central procambial strand Current Opinion in Plant Biology 2004, 7:553–563 www.sciencedirect.com Auxin signaling Berleth, Krogan and Scarpella 557 responses and will functionally dissect the pool of soluble components. Auxin transport As is the case for auxin perception, the molecular understanding of auxin transport is far from comprehensive. Nevertheless, the past two years have seen the detailed characterization of several new members of the PIN family of putative auxin-efflux membrane proteins and the revelation of further details on how these proteins are localized (Figure 2a, Table 1). Consistent with their presumed function, all characterized proteins of the PIN family show polar localization in cells, reflecting the efflux pole of the presumed auxin flow direction in various instances [36–38,39]. The ability of cells to position and dynamically reposition PIN proteins has previously been attributed to their rapid cycling between the plasma membrane and an intracellular compartment [40]. The vesicle transport that is crucial for proper membrane localization of PINs has been shown to depend on the guanosine exchange factor (GEF) EMB30/GN [36]. In emb30/gn mutants, the localization of PIN1 is no longer polar; hence, these mutants display extremely strong polarity defects, similar to those caused by strong inhibition of auxin transport [36,41]. The pin1 pin3 pin4 pin7 quadruple mutant displays similar extreme polarity distortions, supporting a role for PIN genes in auxin transport and suggesting that EMB30/GN-dependent vesicle transport is crucial in the localization of all PIN proteins [39]. The GEF function of EMB30/GN is thought to act on GTPases of the adenosyl ribosylation factor class and is sensitive to the fungal metabolite brefeldin A (BFA), which causes severe defects in cell polarity [36]. It seems that BFA affects cell polarity primarily through EMB30/GN because the localization of PIN1 is no longer sensitive to BFA in plants expressing a modified, BFA-resistant version of EMB30/GN [42]. The cell biology of PIN1 localization displays striking similarities to other instances of polar membrane protein localization in higher eukaryotes [43]. As these parallels can serve as templates for future exploration, it can be predicted that the organismal polarity conveyed by auxin transport will soon find a firm molecular link to the cytoskeletal organization of the individual cell. Auxin in pattern formation For years, a role for auxin in tissue patterning was suggested by auxin application and vascular severance experiments, which led to a concept of auxin as an integrator of apical–basal plant cell polarity ([44,45]; Figure 2a). This basic concept has subsequently been supported by genetic [26,36,46,47] and pharmacological [41,48,49] evidence. More recently, detailed studies have investigated the role of directional auxin signals and cell polarity in pattern formation in early embryos and apical meristems, as described below. It has been revealed that strongly diminished auxin signal transduction is associated with incomplete vascular systems and, in extreme cases, with failure to differentiate hypocotyl and root meristem and to properly position cotyledons in the early embryo. This triple-defect is observed in seedlings in which the ARF5/MP, IAA12/ BDL or CUL1/AXR6 gene is mutated [26,46,47]. How are these seemingly disparate traits connected to diminished auxin signaling and impaired vascular development? Results from investigations of post-embryonic organs provide a plausible explanation for the distortions observed in early embryos (Figure 2b; reviewed in [50]). First, the specification of root meristem initials is dependent upon a signaling source in the quiescent center (QC), which is represented in the embryo by the progenitor hypophyseal cell [51]. The position of this signaling source, in turn, seems to be strictly correlated with the location of an auxin-response reporter gene expression maximum formed at the basal end of the central vascular system [52], apparently in response to auxin supplied from apical sources. These findings link formation of the root apical meristem to apical–basal auxin signaling through a central vascular strand, a correlation that also seems to hold for the post-embryonic formation of lateral roots [53]. Second, independent evidence has demonstrated how the central vascular tissue serves as a signaling source for cell division and patterning of the overlying ground tissue through the action of the non-cell-autonomous SHORT-ROOT protein [54]. This could explain why basal domains fail to differentiate in highly auxininsensitive mutant embryos. Finally, the application of auxin to post-embryonic shoot apical meristems (SAMs) has implicated auxin not only in the initiation of lateral organ primordia but also in their proper spacing [55]. This emerging picture was largely confirmed and extended through the use of molecular probes, including the presumed auxin (response) distribution indicator DR5-GFP, and antibodies against IAA or against presumptive auxin-transport proteins, such as PIN1 or AUX1 (Figure 2 Legend Continued) (orange), which is possibly established through auxin canalization in the early globular embryo, seems to be a pre-requisite for three subsequent patterning processes. First, it may supply auxin to sustain the basal auxin maximum and the associated acquisition of QC identity by cells in this region [52]. Signals from the QC, in turn, confer stem-cell identity to the neighboring cells (black arrows). Second, the central procambial strand also serves as a signaling source for ground tissue (yellow) proliferation and patterning (blue arrows) [54]. Third, self-canalizing auxin transport (red arrows) positions lateral shoot organs (here cotyledons) and could establish phyllotactic patterns by generating lateral-inhibitory fields of auxin depletion around newly initiated primordia [57]. Directions of auxin flow are predicted from the intracellular positions of auxin-efflux proteins of the PIN family, whereas auxin concentrations are deduced from the expression levels of the DR5 auxin-response marker [39,52,57]. www.sciencedirect.com Current Opinion in Plant Biology 2004, 7:553–563 558 Cell signalling and gene regulation (Table 1). Auxin-response maxima in preglobular embryos have been visualized in the apical cell of the two-cell embryo and may be established by the apical flow of auxin from the basal cell, as suggested by the intracellular localization of the newly characterized PIN protein PIN7 ([39]; Figure 2b). Later, once a globular embryo proper has been established, an anatomically recognizable axis is associated with basally positioned PIN1 protein in central cells, presumably reflecting an apical–basal auxin flow that is associated with the emergence of procambium in the center of the embryo. It is at the bottom of these cell files, before the differentiation of functional vasculature, that the embryonic basal auxinresponse maximum emerges (Figure 2b). Visualization of auxin-transport membrane proteins, such as PIN1 or the presumed auxin-influx protein AUX1, also suggests routes for canalized auxin in the shoot meristem. On the basis of the intracellular position of the PIN1 protein (and supported by independent evidence of the importance of the epidermis in the initiation of shoot lateral organs [56]), a tip-directed flow in the epidermal layer seems to supply IAA to the SAM surface, from where it appears to become redirected to inner cells at distinct spots [57]. Lateral organs (leaves or flowers) are initiated at these sites. During organ outgrowth, PIN1 is first found in the organ center and then along the organ midline. The net result is the local concentration of initially dispersed auxin in emerging organs, which function as auxin sinks. This could result in depletion of auxin in surrounding regions and thereby generate a phyllotactic pattern through lateral inhibition, as proposed in various models [58,59]. Most excitingly, auxin, and the probable feed-back associated with its transport, seems to have an instructive role in assigning differential identities to distinct areas within the meristem [57]. In pin1 (auxin-transport defective) mutants, lateral organs can still be initiated by the local application of IAA, but PIN1 mRNA expression does not become restricted to individual spots, indicating that auxin transport feedsback on its own routes. Self-regulation of auxin transport may not end with the specification of organ initiation sites. Instead, reproducible patterns of auxin distribution (suggested by the expression of the DR5 auxin-response marker), with maxima at organ tips, are observed in various kinds of lateral shoot organs as well as in lateral root primordia [60]. The formation of these maxima is usually correlated with a corresponding polar intracellular localization of PIN proteins, and is obstructed in plants carrying multiple mutations in PIN genes. Separation of auxin pathways Genetic dissection of signaling pathways should eventually determine whether auxin directly regulates the extreme diversity of functions that it is alleged to medCurrent Opinion in Plant Biology 2004, 7:553–563 iate. For example, are there specific auxin signal transduction pathways for patterning events as opposed to other kinds of auxin responses? Although the fairly small number of mutants that have auxin-related patterning defects might suggest the involvement of only a few specific genes in patterning, more recent genetic studies point in the opposite direction. Embryonic and postembryonic patterning defects associated with loss of ARF5/MP function are enhanced by mutations in ARF7/NPH4 [19], although the phenotype of the nph4 single mutant implicates the NPH4 gene specifically in auxin-controlled cell expansion [61,62,63]. Conversely, ARF5/MP functions in the post-embryonic control of cell expansion. Further, an increasing number of doublemutant combinations of auxin-response mutants, each with no apparent patterning defect, result in rootless, mp-like seedlings [20,29]. It therefore seems possible that common auxin signaling pathways successively operate in the same organ: first, to execute genetic patterning programs in the primordium, and then to relay auxin responses to environmental signals in the mature organ. Tracing in vivo interactions among ARF and Aux/IAA proteins will be crucial for dissecting the genetic complexity of auxin-mediated gene regulation in auxin responses. ARF–ARF interactions are already known to display some degree of specificity in yeast two-hybrid assays, whereas no discriminating interaction properties of Aux/IAA proteins are apparent in single-cell assays [19]. Future research will determine whether specific expression profiles, or other unknown influences, impart greater selectivity and correlate specific interactions to individual auxin responses. Conclusions Recent research has generated strong confidence that the details of auxin-mediated gene regulation, if not the entire signal transduction chain, and of the cell biological mechanisms underlying auxin transport will be explored in molecular detail within the next few years. Moreover, there is increasing evidence that similar principles apply across the angiosperms and beyond. The ARF gene family, for example, is highly conserved from Arabidopsis to rice [64], and an instrumental role of auxin transport in embryo polarity has been demonstrated even in the brown alga Fucus distichus [65]. This could also be true for other elements of auxin signal transduction [66,67,68,69,70] and auxin-regulated gene activities [71], some of which seem to affect morphogenesis through the control of cell proliferation [68,72]. Most importantly, it can be expected that analogies in the cell biology of the polarization processes in animals and fungi will strongly promote our understanding of auxin functions in plants. Ironically, therefore, research in the near future may rapidly elucidate the roles of the identified auxin signaling and transport proteins in their cell biological context, whereas IAA itself, as the genetically least www.sciencedirect.com Auxin signaling Berleth, Krogan and Scarpella 559 tractable element, may be the last piece to be integrated into the puzzle. Acknowledgements We thank Dinesh Christendat (Toronto) and Hanjo Hellmann (Berlin) for valuable comments on the manuscript. We apologize to colleagues whose results could not be included in the available space. The authors’ signal transduction research has been supported by grants from the Natural Science and Engineering Research Council of Canada (NSERC) to T Berleth, as well as by an NSERC long-term postgraduate fellowship and a Government of Ontario/Dr FM Hill Scholarship in Science and Technology (OGSST) to NT Krogan. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Napier RM, David KM, Perrot-Rechenmann C: A short history of auxin-binding proteins. Plant Mol Biol 2002, 49:339-348. 2. Swarup R, Parry G, Graham N, Allen T, Bennett M: Auxin cross-talk: integration of signalling pathways to control plant development. Plant Mol Biol 2002, 49:411-426. 3. Leyser O: Regulation of shoot branching by auxin. Trends Plant Sci 2003, 8:541-545. 4. Abel S, Theologis A: Early genes and auxin action. Plant Physiol 1996, 111:9-17. 5. Hagen G, Guilfoyle T: Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol 2002, 49:373-385. 6. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ: Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 1997, 9:1963-1971. 7. Reed JW: Roles and activities of Aux/IAA proteins in Arabidopsis. Trends Plant Sci 2001, 6:420-425. 8. Leyser O: Molecular genetics of auxin signaling. Annu Rev Plant Biol 2002, 53:377-398. 9. Liscum E, Reed J: Genetics of AUX/IAA and ARF action in plant growth and development. Plant Mol Biol 2002, 49:387-400. 10. Guilfoyle TJ, Hagen G: Auxin response factors. J Plant Growth Regul 2001, 20:281-291. 11. Ulmasov T, Hagen G, Guilfoyle TJ: Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci USA 1999, 96:5844-5849. 12. Ulmasov T, Hagen G, Guilfoyle TJ: Dimerization and DNA binding of auxin response factors. Plant J 1999, 19:309-319. 13. Tiwari SB, Hagen G, Guilfoyle T: The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 2003, 15:533-543. Chimeric effector and reporter constructs are used in plant protoplast transfection assays to characterize the domains of the auxin-response factor (ARF) family. Experimental results show that the amino-terminal DNA-binding domain is sufficient to direct binding to cis-regulatory auxinresponse elements. The middle region confers either transcriptional activation or repression characteristics. The carboxy-terminal domain that is shared with Aux/IAA repressor proteins, and that mediates interactions between the two classes of factors, confers auxin responsiveness alone. Furthermore, the authors present evidence that suggests that Aux/ IAA-mediated repression of ARF activity does not occur through the prevention of ARF DNA-binding, but through recruitment of Aux/IAA proteins to DNA-bound ARFs. 14. Tiwari SB, Hagen G, Guilfoyle TJ: Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell 2004, 16:533-543. A clear and transferable function is assigned to domain I of Aux/IAA repressor proteins. In protoplast transfection assays, domain I acts as a www.sciencedirect.com strong transcriptional repressor, and the presence of an LxLxL motif is necessary for this effect. Domain I repression is also shown to be epistatic over transcriptional activation domains when situated in close proximity, either on neighboring proteins or as part of the same DNA-bound protein. These observations add to the current understanding of Aux/IAA repression of ARF-regulated transcription. 15. Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J: Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant J 2000, 21:553-562. 16. Ramos JA, Zenser N, Leyser O, Callis J: Rapid degradation of auxin/indoleacetic acid proteins requires conserved amino acids of domain II and is proteasome dependent. Plant Cell 2001, 13:2349-2360. 17. Stowe-Evans EL, Harper RM, Motchoulski AV, Liscum E: NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis. Plant Physiol 1998, 118:1265-1275. 18. Mattsson J, Ckurshumova W, Berleth T: Auxin signaling in Arabidopsis leaf vascular development. Plant Physiol 2003, 131:1327-1339. The authors use the b-glucuronidase (GUS) reporter gene driven by the synthetic auxin-responsive promoter DR5 to show that leaf vascular differentiation occurs along paths of maximum auxin response. Furthermore, they provide direct evidence of the auxin-insensitivity of mp seedlings. The authors report the vascular expression pattern of Athb20, which encodes a novel member of the homeodomain-leucine zipper family of transcription factors. They further show that the expression of Athb20 and Athb8, another early vascular transcription factor gene, as well as the rapid induction of these genes by auxin, is dependent on the activity of MP/ARF5. The expression level of MP/ARF5 is also shown to be a limiting factor in the transcription of several auxin-inducible genes. 19. Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G, Tiwari SB, Hagen G, Guilfoyle TJ, Berleth T: Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 2004, 131:1089-1100. This study shows that defects in cell-axis alignment and vascular differentiation in plants lacking MP/ARF5 activity are further enhanced by mutations in NPH4/ARF7, suggesting that the functions of these genes overlap at early stages of organ development. Yeast two-hybrid and genetic data together suggest that both MP/ARF5 and NPH4/ARF7 interact in heterodimers, and jointly function in the non-redundant regulation of downstream genes and in auxin-regulated cell expansion in mature organs. Both ARFs interact similarly with several Aux/IAA genes, but mutual suppression of MP/ARF5 (overexpression) and BDL/IAA12 gain-of-function mutant genotypes indicates a specific antagonistic relationship between an ARF and an Aux/IAA gene in vivo. These observations suggest a greater level of selectivity in ARF–Aux/IAA in vivo interactions than that predicted from yeast two-hybrid analyses. 20. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M: Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 2001, 414:271-276. 21. Ouellet F, Overvoorde PJ, Theologis A: IAA17/AXR3: biochemical insight into an auxin mutant phenotype. Plant Cell 2001, 13:829841. 22. Dharmasiri S, Estelle M: The role of regulated protein degradation in auxin response. Plant Mol Biol 2002, 49:401-409. 23. Hellmann H, Estelle M: Plant development: regulation by protein degradation. Science 2002, 297:793-797. 24. Kepinski S, Leyser O: Ubiquitination and auxin signaling: a degrading story. Plant Cell 2002, 14 (Suppl):S81-S95. 25. Ruegger M, Dewey E, Gray WM, Hobbie L, Turner J, Estelle M: The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p. Genes Dev 1998, 12:198-207. 26. Hobbie L, McGovern M, Hurwitz LR, Pierro A, Liu NY, Bandyopadhyay A, Estelle M: The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development. Development 2000, 127:23-32. 27. Hellmann H, Hobbie L, Chapman A, Dharmasiri S, Dharmasiri N, del Pozo C, Reinhardt D, Estelle M: Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis. EMBO J 2003, 22:3314-3325. Current Opinion in Plant Biology 2004, 7:553–563 560 Cell signalling and gene regulation The authors report that the genetic defect in the auxin-resistant mutant axr6 maps to the SCF subunit CUL1. Insertional inactivation of the locus results in embryonic lethality, whereas seedling-defective axr6-1 and axr6-2 alleles are found to have missense mutations at the same amino-acid position that result in the disruption of stable SCF complexes. Consequently, auxin-mediated degradation of Aux/IAA proteins is compromized in these mutants, probably leading to their embryonic patterning defects. Transgenic lines that have decreased CUL1 activity reveal severe post-embryonic abnormalities, indicating that SCF-dependent protein degradation is an important process throughout Arabidopsis development. 28. Lincoln C, Britton J, Estelle M: Growth and development of the axr1 mutants of Arabidopsis. Plant Cell 1990, 2:1071-1080. 29. Dharmasiri S, Dharmasiri N, Hellmann H, Estelle M: The RUB/ Nedd8 conjugation pathway is required for early development in Arabidopsis. EMBO J 2003, 22:1762-1770. The study provides biochemical and genetic evidence that RCE1 acts as an E2 conjugase for RUB modification of CUL1, the cullin subunit of SCFTIR1. rce1 mutants show a decrease in the amount of modified CUL1 and are phenotypically deficient in terms of auxin response and Aux/IAA protein destabilization. Further, double mutants that are defective in both RCE1 and AXR1, another factor involved in RUB conjugation, resemble several auxin-response mutants that are seedling lethal, lack hypocotyl and root meristems, and exhibit vascular defects. Therefore, an important role for the RUB-conjugation pathway in auxin-mediated embryo patterning is apparent. 30. Dharmasiri N, Dharmasiri S, Jones AM, Estelle M: Auxin action in a cell-free system. Curr Biol 2003, 13:1418-1422. The direct addition of auxin to a cell-free Arabidopsis extract stimulates the association of the F-box protein TIR1 with the Aux/IAA proteins IAA7 and IAA17. The authors use modifications of the cell-free system to demonstrate that auxin-dependent degradation of Aux/IAA proteins exclusively involves soluble components. Further, this auxin response appears to depend on a parvulin-type peptidyl prolyl cis/trans isomerase (PPIase) because in vitro and in vivo auxin responses are attenuated in the presence of the PPIase inhibitor juglone. 31. Tian Q, Nagpal P, Reed JW: Regulation of Arabidopsis SHY2/ IAA3 protein turnover. Plant J 2003, 36:643-651. The study uses an in vitro protein turnover assay to show that SHY2/IAA3 protein degradation requires normal AXR1 and TIR1 activities, implicating SCFTIR1 in this regulation. Pull-down assays confirm that SHY2/IAA3 interacts with both phytochrome B and TIR1. The latter association is increased by auxin and decreased by juglone, a compound known to inhibit parvulin-type peptidyl-prolyl isomerases, implicating this class of enzymes in the regulation of SHY2/IAA3 protein turnover. 32. Gray WM, Hellmann H, Dharmasiri S, Estelle M: Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function. Plant Cell 2002, 14:2137-2144. 33. Schwechheimer C, Serino G, Callis J, Crosby W, Lyapina S, Deshaies R, Gray W, Estelle M, Deng X-W: Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science 2001, 292:1379-1382. 34. Schwechheimer C, Deng X-W: COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol 2001, 11:420-426. 35. Gray WM, Muskett PR, Chuang H, Parker JE: Arabidopsis SGT1b is required for SCFTIR1-mediated auxin response. Plant Cell 2003, 15:1310-1319. A mutation in SGT1b/ETA3, a gene implicated in plant disease resistance signaling, is identified as an enhancer of the auxin-response mutation tir1. A role for the SGT1b gene in auxin-mediated protein turnover is suggested by the stabilization of an AXR3/IAA17-reporter translational fusion in an eta3 background, and by the increased stability of AXR2/IAA17 in the eta3 tir1 double mutant relative to either single mutant. Interestingly, overexpression of SGT1b results in partial suppression of the tir1 auxinresponse defect, suggesting that excess SGT1b can partially restore the diminished function of SCFTIR1 in tir1 mutants or can promote the function of related SCF complexes. 38. Friml J, Benkova E, Blilou I, Wisniewska J, Hamann T, Ljung K, Woody S, Sandberg G, Scheres B, Jürgens G, Palme K: AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell 2002, 108:661-673. 39. Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G: Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis. Nature 2003, 426:147-153. This study uses the subcellular localization of members of the PIN family of auxin efflux regulators, and both green fluorescent protein (GFP) driven by DR5 (a synthetic auxin responsive promoter) and anti-IAA antibodies, to monitor auxin flow and associated auxin distribution in early embryos of various stages. An apparent early auxin concentration maximum in the apical cell of the two-celled embryo is immediately followed by a basal maximum in the globular embryo. The authors further characterize a novel member of the PIN family of auxin efflux regulators, PIN7. They also address the issue of functional redundancy among structurally related PIN genes that are expressed during embryogenesis through double, triple and quadruple mutant analyses. Remarkably, pin1 pin3 pin4 pin7 quadruple mutant seedlings display defects that are very similar to those of emb30/gnom mutants. 40. Geldner N, Friml J, Stierhof JD., Jürgens G: Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 2001, 413:425-428. 41. Hadfi K, Speth V, Neuhaus G: Auxin-induced developmental patterns in Brassica juncea embryos. Development 1998, 125:879-887. 42. Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G: The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 2003, 112:219-230. Polar PIN1 localization depends on the presence of a functional GNOM (GN) protein, an exchange factor for adenosyl ribosylation factor GTPases that is sensitive to the fungal metabolite brefeldin A (BFA). The authors show that GN localizes to endosomes and is required for their structural integrity. Furthermore, they show that the localization of PIN1 is no longer sensitive to BFA in transgenic plants harboring an engineered BFA-resistant functional version of GN. By contrast, the trafficking of other membrane proteins that are unrelated to PIN1 remains BFA-sensitive in these plants. Finally, the authors show that the BFA-resistant version of GN confers insensitivity to BFA-mediated inhibition of auxin transport and of auxin transport-dependent responses in the root. These results suggest that GN is an important in vivo mediator of auxin transport. 43. Muday GK, Murphy AS: An emerging model for polar auxin transport regulation. Plant Cell 2002, 14:293-299. 44. Sachs T: The control of the patterned differentiation of vascular tissues. Adv Bot Res 1981, 9:152-262. 45. Sachs T: Cell polarity and tissue patterning in plants. Development 1991, 1 (Suppl):83-93. 46. 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Rockwill, MD: American Society of Plant Biologists; 2002. (Published online. http://www.aspb.org/publications/ Arabidopsis) 37. Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K: Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 2002, 415:806-809. 51. van den Berg C, Willemsen V, Hendriks G, Weisbeek P, Scheres B: Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 1997, 390:287-289. Current Opinion in Plant Biology 2004, 7:553–563 www.sciencedirect.com Auxin signaling Berleth, Krogan and Scarpella 561 52. Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B: An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 1999, 99:463-472. 61. Watahiki MK, Yamamoto KT: The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf. Plant Physiol 1997, 115:419-426. 53. Geldner N, Richter S, Vieten A, Marquardt S, Torres-Ruiz RA, Mayer U, Jürgens G: Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis. Development 2004, 131:389-400. The authors describe the post-embryonic expression pattern of the GNOM::GUS chimeric protein. They report the isolation of two novel, weak gn alleles, and characterize the root phenotypes of one of these alleles and of a trans-heterozygote of complementing strong gn alleles. The authors show that localized treatments with an auxin transport inhibitor mimic the collapsed root meristem of these weak gn lines, and that this phenotype can be ameliorated by exogenous auxin. Finally, in agreement with the proposed function of GN in PIN1 polar localization, they correlate defects in auxin-induced lateral root formation in a gn weak allele with its failure to properly re-localize PIN1 protein in response to auxin. 62. Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E: The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. Plant Cell 2000, 12:757-770. 54. Nakajima K, Giovanni S, Nawy T, Benfey P: Intercellular movement of the putative transcription factor SHR in root patterning. Nature 2001, 413:307-311. 55. Reinhardt D, Mandel T, Kuhlemeier C: Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 2000, 12:507-518. 56. Reinhardt D, Frenz M, Mandel T, Kuhlemeier C: Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristem. Development 2003, 130:4073-4083. The authors use laser ablation and microsurgical tissue removal to assess the functions of different parts of the tomato shoot apical meristem in the auxin-mediated initiation of lateral shoot organs. Interestingly, removal of the external L1 layer results in terminal differentiation of the subtending layers and the lack of lateral organ formation, a phenotype similar to that obtained through chemical or genetic interference with auxin transport or response. By contrast, ablation of a central domain does not arrest lateral-organ formation and is followed by the restoration of the central domain. 57. Reinhardt D, Pesce ER, Stieger P, Mandel T, Baltensperger K, Bennett M, Traas J, Friml J, Kuhlemeier C: Regulation of phyllotaxis by polar auxin transport. Nature 2003, 426:255-260. This study explores the routes of auxin flow during shoot lateral organ initiation and the requirements for auxin transport and auxin perception in this process. The authors show that direct application of auxin can trigger the formation of leaves and flowers in the auxin transport defective pin1 mutant, but not in the highly auxin-insensitive mutant mp. The expression pattern and intracellular localization of AUX1 and PIN1, the two central proteins implicated in auxin influx and efflux, respectively, suggest that auxin transport in the epidermis is required for auxin supply in the shoot meristem, and that auxin flow is directed towards the inner cells at sites of organ initiation. Interestingly, functional PIN1 protein is necessary for the generation of this pattern, indicating that auxin does not merely promote the emergence of an otherwise specified pattern, but that the selfregulation of auxin flow has an instructive patterning function in lateral organ positioning. 58. Kuhlemeier C, Reinhardt D: Auxin and phyllotaxis. Trends Plant Sci 2001, 6:187-189. 59. Reinhardt D, Kuhlemeier C: Plant architecture. EMBO Rep 2002, 3:846-851. 60. Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jürgens G, Friml J: Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 2003, 115:591-602. The study visualizes apparent common auxin transport and distribution features in shoots and roots. Local auxin maxima at the tips of organ primordia are visualized through both anti-IAA immunostaining and correlated maxima of (auxin-responsive) DR5 reporter gene expression. Furthermore, the correct establishment of these maxima depends on the presence of functionally regulated PIN proteins in a variety of genetic backgrounds. In agreement with these findings, the authors report a striking correlation between the asymmetric localization of PIN proteins during organ development and the localization of auxin accumulation maxima. These data suggest that PIN-mediated, local auxin distribution represents a common module for the formation of all plant organs, regardless of their morphology or ontogeny. www.sciencedirect.com 63. Tatematsu K, Kumagai S, Muto H, Sato A, Watahiki MK, Harper RM, Liscum E, Yamamoto KT: MASSUGU2 encodes Aux/ IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 2004, 16:379-393. Alleles of the dominant auxin-resistant mutant msg2 are shown to result from protein-stabilizing missense mutations in the conserved domain II of the Aux/IAA gene IAA19. This is consistent with the identified genetic lesions of other dominant Aux/IAA mutants. Induction of IAA19 gene expression by auxin is reduced in loss-of-function nph4/arf7 mutants, which share many common auxin-response defects with msg2. Along with biochemical evidence of ARF7–IAA19 protein interaction, the results of this study suggest that an IAA19-dependent negative feedback loop regulates ARF7-mediated transcription. 64. Sato Y, Nishimura A, Ito M, Ashikarin M, Hirano H-Y, Matsuokai M: Auxin response factor family in rice. Genes Genet Syst 2001, 76:373-380. 65. Basu S, Sun H, Brian L, Quatrano RL, Muday GK: Early embryo development in Fucus distichus is auxin sensitive. Plant Physiol 2002, 130:292-302. 66. Tao LZ, Cheung AY, Wu HM: Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression. Plant Cell 2002, 14:2745-2760. 67. Monroe-Augustus M, Zolman BK, Bartel B: IBR5, a dual specificity phosphatase-like protein modulating auxin and abscisic acid responsiveness in Arabidopsis. Plant Cell 2003, 15:2979-2991. The authors report the characterization of ibr5, a weak auxin-response Arabidopsis mutant that also shows decreased sensitivity to the phytohormone abscisic acid (ABA). Aberrant mutant phenotypes, including reduced expression of the DR5-GUS auxin-responsive reporter, suggest that IBR5 acts as a positive regulator of auxin signaling. Finally, IBR5 was shown to encode a gene that exhibits similarities to genes that encode dual-specificity mitogen-activated protein kinase (MAPK) phosphatases. 68. Ullah H, Chen J-G, Temple B, Boyes DC, Alonso JM, Davis KR, Ecker JR, Jones AM: The b-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. Plant Cell 2003, 15:393-409. The authors investigate the role of an Arabidopsis putative heterotrimeric G protein complex (consisting of Ga-, Gb- and Gg-subunits) in the control of auxin-induced cell division. Their results indicate that overexpression of Gb reduces the sensitivity of this process toward auxin, whereas mutation of Gb has the opposite effect, suggesting that the Gb-subunit acts to negatively regulate auxin-induced cell division. Consistent with this notion is the observation that the basal repression of many identified auxin-regulated genes is lost in the Gb mutant. 69. Zhao Y, Dai X, Blackwell HE, Schreiber SL, Chory J: SIR1, an upstream component in auxin signaling identified by chemical genetics. Science 2003, 301:1107-1110. The authors report the isolation and molecular characterization of the sir1 auxin-hypersensitive mutant of Arabidopsis. The sir1 mutant was identified because of its resistance to sirtinol, a small molecule that activates many auxin-inducible genes and promotes auxin-related developmental phenotypes. SIR1 is predicted to encode a protein containing a ubiquitinactivating enzyme (E1)-like domain and a domain that shares homology with prolyl cis/trans isomerases. This gene is proposed to function as a negative regulator of auxin signaling. 70. Cheng Y, Dai X, Zhao Y: AtCAND1, a HEAT-repeat protein that participates in auxin signaling in Arabidopsis. Plant Physiol 2004, 135:1020-1026. 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