Auxin adjusts diverse features of plant growth and development. The most common of Auxin is indole-3-acetic acid (IAA), is a weak acid and its intercellular movement is facilitated by auxin influx and efflux carriers. Polarity of auxin movement is provided by asymmetric localization of auxin carriers (mainly PIN efflux carriers). PIN-FORMED (PIN) and P-GLYCOPROTEIN (PGP) family of proteins are major auxin efflux carriers whereas AUXIN1/LIKE-AUX1 (AUX/LAX) are major auxin influx carriers. Genetic and biochemical evidence show that each member of the AUX/LAX family is a functional auxin influx carrier and mediate auxin related developmental programmes in different organs and tissues. Of the four AUX/LAX genes, AUX1 regulates root gravitropism, root hair development and leaf phyllotaxy whereas LAX2 regulates vascular development in cotyledons. Both AUX1 and LAX3 have been implicated in lateral root (LR) development.
Plant hormones are natural compounds that, at very low concentrations, act as signaling molecules to activate physiological processes (Romanov, 2002; Gaspar et al., 2003). Not all signaling molecules act as hormones, and not all hormones act as signaling molecules, however. Plant hormones are set apart from their animal counterparts for two reasons. Plant hormones can be synthesized in any living cell in the plant, though there are generally specific organs that synthesize the majority of a hormone (Gaspar et al., 2003). Hormone response can occur in the plant cell they are synthesized in, or after transport to a target cell. Animal hormones, on the other hand, are synthesized in a specific organ, usually endocrine glands, and travel in the blood to the target cell, which responds to the hormone (Campbell, 1997). Plant hormones are responsible for a wide range of phenomena that occur during the growth, development, and reproduction of a vascular plant. Historically, there were five classes of phytohormones: indole-3-acetic acid (IAA or auxin), cytokinins, gibberellins, abscisic acid, and ethylene (Taiz and Zeiger, 1998). In the past 10 years several other signaling molecules present at low concentrations throughout the plant have been identified as hormones, such as brassinosteroids, jasmonic acid, and salicylic acid (Reviewed in Santner et al., 2009).
The most abundant naturally occurring auxin in plants is indole-3-acetic acid (IAA) (Bartel et al., 2001). IAA is synthesized by two main methods, Trp-independent biosynthesis, a pathway with no identified biosynthetic enzymes, and tryptophan (Trp)-dependent biosynthesis, which uses the amino acid tryptophan as a precursor to generate IAA through indole-3-glycerol phosphate followed by indole-pyruvic acid or indole-3-acetoldoxime (Bartel et al., 2001; Coruzzi and Last, 2000; Crozier et al., 2000; Normanly, 2010).
After IAA is synthesized, it is transported throughout the plant by two methods: polar auxin transport (PAT), and phloem transport (Friml and Palme, 2002).
Indole-3-acetic acid (IAA), is a critical regulator of all stages of plant development including embryogenesis, germination, cell differentiation, root and shoot development, vascular tissue patterning, meristem maintenance, and seed dispersal (Weijers et al., 2006; Benjamins and Scheres, 2008; Pï¿½ï¿½ret et al., 2009; Sorefan et al., 2009; Vanneste and Friml, 2009). Though these developmental processes require auxin, they are influenced by other regulators during specific stages of development such as seed germination and lateral root development (Grieneisen et al., 2007; Lucas et al., 2008).
Carrier proteins located in the plasma membrane facilitate the asymmetric distribution of auxin. When auxin distribution is disrupted, phenotypic abnormalities result (Morris, 2000; Tanaka et al., 2006; Vanneste and Friml, 2009). Isolation of carrier proteins involved in auxin distribution began when Arabidopsis seeds were mutagenized with ethyl methanesulfonate and screened for survival on high concentrations of the herbicide and auxin analog, 2,4- dichlorophenoxyacetic acid (2,4-D; Maher and Martindale, 1980). Individuals surviving this treatment were insensitive to 2,4-D and showed wild type primary root lengths even in the presence of inhibitory concentrations of the hormone, produced agravitropic roots, and were named auxin1 (aux1). Since the structure of IAA is similar to that of tryptophan it was theorized that AUX1 was an auxin transporter. Although many studies examined the physiological effects of the aux1 mutant, biochemical evidence of AUX1-mediated auxin transport was lacking until 2006 when Yang et al. demonstrated that AUX1 was a specific H+/IAA- symporter Fig1. By expressing AUX1 in Xenopus laevis oocytes, the authors showed that AUX1-mediated IAA uptake was blocked by the same amino acid substitutions that resulted in mutant phenotypes. The same group demonstrated that uptake occurred at physiological concentrations. In addition to AUX1, a root specific influx carrier (Stone et al., 2009), the Arabidopsis genome encodes three other auxin influx carriers (Swarup et al., 2008). Unlike AUX1, LAX1 (LIKE AUX1), and LAX2 (Parry et al., 2001; Bainbridge et al., 2008), the LAX3 auxin influx carrier is specifically present in the cortical and endodermal cells of the primary root. Auxin increases expression of LAX3 in these cells, causing increases in local auxin uptake, which in turn stimulates the production of cell wall remodeling enzymes, allowing the growing lateral root primordium to emerge from the primary root. Since AUX1 and LAX3 are confined to the roots (Marchant et al., 1999; Swarup et al., 2001; Swarup et al., 2008), the other auxin influx carriers must exist in aerial plant organs. Though this has not yet been described, mutation of auxin influx carrier genes causes aerial phenotypic aberrations (Pickett et al., 1990; Stone et al., 2008), demonstrating the importance of proper auxin distribution by PAT during developmental processes.
Fig. 1: IAA- influx carrier (Plant and Soil Sciences eLibrary 2015.)
The auxin efflux carriers are much better understood than the influx carriers. Auxin efflux carriers are believed to be multi-component systems, consisting of transport, catalytic, and regulatory domains (Morris, 2000). The first identified auxin efflux carrier gene was the Arabidopsis PIN1 (PIN-FORMED; Galweiler et al., 1998). In pin1 mutants, PAT is perturbed in the inflorescences whose pinnate apices develop into only a few, if any flowers. PIN1 is localized to the basipetal region of apical cells, where it is involved in transporting auxin from leaves into the vascular bundle. Two other PIN genes (PIN2 and PIN3) were found that function in different organs in Arabidopsis. PIN2 was localized to meristematic and elongating regions of the primary root (Muller et al., 1998) while PIN3 was localized to the columella initial cells of the root tip and on lateral membranes of endodermal cells in young stems (Friml et al., 2002a). PIN4 localizes to developing and mature root apices where it is theorized to have a role in generating an auxin sink used for patterning in development (Friml et al., 2002b). PIN7 localization was found to drive apical to basal auxin gradients in Arabidopsis suspensor cells during embryogenesis. Localization and transport studies demonstrated that PIN proteins export auxin and are responsible for the formation of a graded distribution (gradient) of auxin to drive development (Friml et al., 2003). Further analysis of PIN proteins showed that subcellular localization to the basal or apical pole of cells determines auxin transport routes (Friml et al., 2004), that PINs are necessary for maintenance of the meristem zone in the root (Blilou et al., 2005), and perform a rate-limiting function in auxin efflux (Petr’ï¿½ek et al., 2006). In addition to controlling auxin gradients, PIN proteins are also partially functionally redundant. Double, triple, and quadruple pin mutants show additive embryonic defects, ranging from aberrant cotyledon formation in double mutants (pin4 pin7), to cotyledon fusion and limited root development in triple mutants (pin1 pin3 pin4), to production of globular, malformed inviable embryos in quadruple mutants (pin1 pin3 pin4 pin7; Friml et al., 2003). Since PIN proteins generate critical auxin gradients to drive development, several levels of control exist within plants to manipulate localization of this family of efflux proteins. PLT (PLETHORA) proteins regulate the expression of PIN while PIN proteins inhibit the expression of PLT, demonstrating the complexity of the interactions necessary for normal vascular patterning to occur. The AUXIN BINDING PROTEIN1 (ABP1) has been examined over the course of several years and was once thought to be a candidate auxin receptor (Bennett et al., 1996; Napier et al., 2002; Napier et al., 2004). It is now known that ABP1 affects PLT gradients to drive auxin efflux (Tromas et al., 2009) and is required for post-embryonic shoot development (Braun et al., 2008). Depending on the concentration of PID (PINOID), a protein kinase that 7 interacts with 3-phosphoinositide-dependent protein kinase 1 (PDK1; Zegzouti et al., 2006) in Arabidopsis, the localization of PIN within cells switches from basal to apical (Friml et al., 2004) controlling a process termed ‘polarity’. Sub-threshold amounts of PID result in basal localization of PIN while above-threshold amounts result in apical localization of PIN. Counteracting the kinase activity of PID is PROTEIN PHOSPHATASE 2A (PP2A; Michniewicz et al., 2007). Like PID, apical and basal localization of PINs are affected by changing the concentration of PP2A; however PP2A acts in opposition to PID (Michniewicz et al., 2007). In addition to control of phosphorylation status, which in turn controls PIN polarization, polar targeting signals in the PIN amino acid sequence itself affect subcellular localization, and alteration of these signals causes aberrant polarity (Wiï¿½ï¿½niewska et al., 2006). Additional proteins such as TINY ROOT HAIR 1 (TRH1), a root-specific potassium carrier, are also required for auxin transport (Vicente-Agullo et al., 2004). Plants with reduced TRH1 are impaired in auxin efflux from the stele, resulting in an agravitropic phenotype (Vicente-Agullo et al., 2004). Control of auxin efflux, and therefore auxin gradient formation, is dependent on subcellular localization of PIN proteins. As previously outlined, polar localization of PINs is determined by phosphorylation status. PIN proteins are mobilized through clathrin-dependent endocytosis, a dynamic and constitutive process used to internalize the efflux proteins into endosomes (Dhonukshe et al., 2007). Following endocytosis, PINs are exocytosed to a specific pole of cell and inserted into the plasma membrane pole, dependent on phosphorylation status. This exocytotic recycling step requires GNOM, an ADP-ribosylation factor GTPase guanine nucleotide exchange factor (ARF-GEF; Geldner et al., 2001). Without functional GNOM, PIN proteins accumulate exclusively within endosomes and no polar auxin gradients form. The constitutive mobilization and re-mobilization of PIN proteins allows plant cells to alter their polar sorting of PIN proteins in response to developmental signals and auxin concentrations (Sauer et al., 2006). Endocytosis of PINs is a rapid process and enables cells to quickly redirect auxin gradients to control development (Tanaka et al., 2006) in situations such as embryonic development (Friml et al., 2002b), tropic responses (Friml et al., 2002a), and in root meristem maintenance (Friml et al., 2002a). Though the complete picture of auxin efflux has not yet been fully described, multiple levels of regulation integrate to rapidly modulate auxin gradients in response to both environmental and endogenous signals to ultimately control auxin distribution within developing organs.
Fig. 2: IAA- efflux carrier (Plant and Soil Sciences eLibrary 2015.)
Molecular mechanisms of auxin response:
Knowledge of the effects of auxin on both plant development and physiology is abundant, yet it is only within the past ten years that the molecular mechanisms of auxin perception and response have been uncovered. The discovery of a true auxin receptor, TRANSPORT INHIBITOR RESPONSE (TIR1), not only allowed advances in our understanding of how the hormone acts, but also represented the first plant hormone receptor with an elucidated signaling pathway (Ruegger et al., 1997; Ruegger et al., 1998; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). TIR1 is a member of the F-box protein family; a class of protein motif containing approximately 50 amino acids that functions as a site of protein-protein interaction (Kipreos and Pagano, 1995; Kipreos and Pagano, 2000). Proteins with this motif, including TIR1, are components of the SCF ubiquitin-ligase complex, named after the SKP1, Cullin, and F-box constituents of the complex (Bai et al., 1996). Assembly of this complex is dependent on SKP1, which acts as an intermediate, binding the F-box protein at its C-terminus, while binding Cullin at the N-terminus. The F-box protein associates with SKP1 only after it has bound its specific partner. Following F-box-partner binding, the F-box protein binds SKP1 resulting in recruitment of Cullin, which in turn dimerizes with RBX1, a RING-H2 finger protein that binds zinc (Deshaies, 1999; Freemont, 2000; Moon et al., 2004). The RBX1-Cullin dimer catalyses the transfer of activated ubiquitin from a ubiquitin-conjugating enzyme to the F-boxassociated protein, targeting the protein for degradation via the 26S proteasome (Ulmasov et al., 1997a). The pathway to auxin perception and recognition broadly follows this outline; however it deviates at several key points forming a truly elegant signaling mechanism.
Fig. 3: Schematic depiction of auxin-regulated gene expression. Intracellular auxin binds to its nuclear receptor from the TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) family of F-box proteins, which are subunits of the SCF E3-ligase protein complex (a). This leads to the ubiquitylation and the proteasome-mediated specific degradation of auxin Aux/IAA transcriptional repressors (b). Subsequently, the auxin response factors (ARFs) are derepressed and activate auxin-inducible gene expression (c) (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Among other auxin-responsive genes, all known auxin transporters are regulated by this feedback mechanism (d). Ub, ubiquity.
Lateral root development:
Prior to the formation of lateral roots, xylem associated cells in the basal meristem known
as xylem pole pericycle cells are ‘primed’ toward lateral root development, a process regulated by auxin (De Smet et al., 2007). Lack of auxin, caused either by application of a transport inhibitor such as N-1-naphthylphthalamic acid (NPA) or by mutation of the PIN efflux transporters blocks or inhibits lateral root formation (Himanen et al., 2002; De Smet et al., 2007; Laskowski et al., 2008). High auxin levels in the basal meristem, detected using the DR5::GUS reporter construct correlate with future sites of lateral root initiation. Furthermore, the amount of auxin in the basal meristem oscillates under constant light, reaching a maximum every 15 hours (De Smet et al., 2007). This temporally and spatially specific auxin maximum correlates with and is capable of stimulating lateral root initiation. Exogenous local application of auxin is also sufficient to induce lateral root formation (Boerjan et al., 1995; Dubrovsky et al., 2008), and the oscillatory action of auxin maxima explains that though every xylem pole pericycle cell is capable of forming a lateral root, not all are fated to become primordia. This oscillatory action also causes regular spacing of lateral roots, a mechanism unknown until recently. Initiation of lateral roots involves SOLITARY-ROOT (SLR), an Arabidopsis gene encoding IAA14, a transcriptional repressor within the Aux/IAA protein family (Fukaki et al., 2002). Gain-offunction slr plants, generated by mutating specific amino acids within the coding region of the gene and therefore stabilizing the repressor protein, completely lack lateral roots and are insensitive to applied auxin. Like other Aux/IAA proteins, IAA14 represses transcription of auxin-regulated genes by dimerizing with specific ARFs, identified as ARF7 and ARF19, both of which are transcriptional activators (Fukaki et al., 2005). In the presence of root-derived auxin (Bhalero et al., 2002), IAA14 is targeted to the 26S proteasome by SCFTIR1-mediated ubiquitination freeing the ARFs to activate auxin-regulated gene transcription, allowing lateral root initiation to proceed.
Lateral root emergence presents an interesting challenge: how does the root pierce the epidermis without water loss or pathogen exposure? Prior to root emergence, auxin originating from developing lateral roots creates local auxin maxima near the primordia (Swarup et al., 2008). These local maxima, in turn, activate expression of LAX3, an auxin influx carrier and homologue of AUX1 in the cortical and epidermal cells overlying the growing primordia (Swarup et al., 2008). Accumulation of auxin in the cortical and epidermal cells resulting from LAX3-mediated auxin influx activates expression of cell wall remodeling enzymes. Furthermore, activation of these enzymes is spatially restricted to cell layers overlying the growing primordia (Swarup et al., 2008).
Until auxin flux in pre-emergent lateral roots was examined the mechanism for emergence was
unknown. Auxin’s involvement in lateral root development pervades every step and is integral for initiation and emergence. Following emergence, the lateral root continues to elongate and is composed of similar cell layers as the primary root (Dolan et al., 1993).
Sequential action of PIN3 and LAX3 determines the specific expression pattern of LAX3 during lateral root emergence. (A) Regulatory network controlling LAX3 induction by auxin during lateral root emergence. (B) Auxin moves from the central xylem-pole pericycle file (XPP, red) towards the outer tissue and activates PIN3 in the cortex, resulting in a net flux of auxin towards the epidermis (red arrowheads). (C) The two files expressing PIN3 that make the most (indirect) contact with the cortex then accumulate enough auxin to induce LAX3 at a later stage (blue arrowheads). Accumulation of LAX3 then leads to further increases in auxin levels, which subsequently trigger cell separation and promote the passage of the LRP. Mol Syst Biol. 2013; 9: 699.
Lateral root are formed within the pericycle deep inside the primary root and have to emerge through the outer tissue, passing through the endodermal, cortical (blue), and epidermal cells (D). Mechanism proposed by Swarup et al. (2008) describing how auxin (IAA) entering the cortical cell induces the expression of LAX3. This generates the establishment of a positive feedback loop that triggers high auxin levels and subsequent induction of cell wall remodeling (CWR) genes, such as the polygalacturonase (PG) (E).
In plants, roots represent the major organ used for both water and mineral uptake. Despite the tremendous advances over the past decade in understanding how roots develop, we still do not understand how interplay between plant hormones modulates the myriad signals present in cells to regulate development.
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Lateral root (LR) formation and emergence in Arabidopsis thaliana. (A) Cross-section of an Arabidopsis root (during stages 0’I of LRP emergence) showing the different cell types, with the position of the cross-section shown in (B). Xylem-pole pericycle cells are grouped in three cell files and are in contact with several endodermal cell files, which in turn about several cortical cell files (highlighted cells). (B) Stages of LR formation. Between stages 0 and I, the XPP cells (from which the LRP originate) undergo several rounds of anticlinal division. Note that in the transverse direction cells vary in length and appear in a staggered formation. (C, D) LAX3 protein accumulation pattern was visualised using a functional pLAX3:LAX3YFP fusion in a tangential root section (C) or a cross-section (D) (with the position of the cross-section shown indicated by the dashed line in (C)). Mol Syst Biol. 2013; 9: 699.
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