Control of Endogenous Auxin Levels in Plant Root Development

In this review, we summarize the different biosynthesis-related pathways that contribute to the regulation of endogenous auxin in plants. We demonstrate that all known genes involved in auxin biosynthesis also have a role in root formation, from the initiation of a root meristem during embryogenesis to the generation of a functional root system with a primary root, secondary lateral root branches and adventitious roots. Furthermore, the versatile adaptation of root development in response to environmental challenges is mediated by both local and distant control of auxin biosynthesis. In conclusion, auxin homeostasis mediated by spatial and temporal regulation of auxin biosynthesis plays a central role in determining root architecture.


Introduction
Flowering plants have evolved a high level of developmental and morphological plasticity to accommodate adaptive growth in response to diverse environmental stimuli [1]. Unlike organ growth in animals, most plant organs are formed post-embryonically and emerge from inherent structures known as meristems [2]. Throughout the plant's life span, meristems remain present and provide the basis for the plant's developmental plasticity [3,4]. Initially at the embryonic state, two types of meristems occur: the shoot apical meristem (SAM), which gives rise to above ground tissues and organs and the root apical meristem (RAM), which establishes the root architecture. The root organ appears to have evolved secondary to the shoot allowing plants to invade land and facilitates the uptake of water for surviving the dry conditions [5]. The diversification of plants with the generation of varied plant forms, from tiny mosses to giant sequoia trees and from annual weeds to long-living perennials, was only possible after the emergence of a root system. Following the initiation of roots, features such as secondary growth, gravitropic responses and the development of lateral root branches are important additions that improved the function of the root system and required more complex cellular communication [5,6]. In particular, the roots of flowering plants have evolved highly specialized functions while maintaining developmental and morphological plasticity in order to cope with diverse environmental stimuli [1].
Plant roots explore the soil environment in search for nutrients and water required for growth, development and reproduction while containing a vascular system to provide organs at a distance with these necessary nutrients, water and hormones [7,8]. Generally, the root system consists of two principal root-types: the primary root (PR), which is formed embryonically [9] and secondary roots, which form post-embryonically. These secondary roots encompass both lateral roots (LR), which develop as branches of the primary root and adventitious roots (AR), which develop on non-root tissue such as the hypocotyl, stems and leaves [10,11]. The root architecture of monocots and dicots is highly distinct. Dicots have a tap-root system with a central primary root and lateral roots branching of, while monocots have a fibrous root system consisting of mainly crown or adventitious roots [12].
The plant growth regulator auxin is involved in basically all cellular processes including cell-division and -expansion with implications for all plant developmental processes [13]. Already at an early stage in embryogenesis, auxin determines cell division, tissue patterning and organ development. Likewise, at later developmental stages, auxin is involved in physiological growth responses such as gravitropism and shoot branching [6,[14][15][16]. Since cell-division and -expansion requires to be spatiotemporally regulated, an appropriate auxin distribution across the tissue is highly important to coordinate growth and tissue development. Auxin homeostasis is assured not only by appropriate transport but also involves de novo biosynthesis, conjugation, storage, oxidation and catabolism, all of which need to take place in a coordinate fashion [17][18][19][20][21][22].
As an omnipotent regulator of root development, auxin controls the development and architecture of the root system [6,14]. In response to environmental cues, root growth is adapted through the modulation of endogenous auxin levels [23,24]. The establishment of auxin gradients in the root requires the interplay of local auxin biosynthesis [25][26][27][28], transport [29][30][31][32][33], perception and signaling [34][35][36]. In this review, we focus on the plant's regulation of the endogenous auxin concentrations by biosynthesis, metaand catabolism and conjugation. We provide information on how these processes are important for root development, encompassing the embryonic root, the PR and the secondary LR and AR. In the next paragraph, we briefly describe the molecular aspects of auxin perception (more can be found in e.g. References [37,38]). For reviews regarding the transport of auxin controlling root development see [16,39].
Auxin signals are perceived intracellularly by a family of AUXIN SIGNALING F-BOX (AFB) receptors of which (TIR1) TRANSPORT INHIBITOR RESPONSE1 is the founder. These F-box proteins are the substrate-binding subunit of SCF-type ubiquitin protein ligase complexes, named after their subunits Skp1, Cullin and an F-box [38]. In the presence of auxin, the SCF TIR1/AFB -complex is brought in close proximity to Aux/IAA (AUXIN/INDOLE-3-ACETIC ACID) proteins, which allows their ubiquitinylation and consequent targeting for proteasome degradation [40]. Normally, the Aux/IAA proteins act as transcriptional repressors by binding to Auxin Response Factor (ARF) transcription factors [41]. The removal of Aux/IAAs upon auxin perception hence results in the activation of ARFs, which on their turn activate the auxin responses. The model species Arabidopsis thaliana contains 6 AFBs [42,43], 29 AUX/IAAs [44] and 23 ARFs [45]. Different TIR1/AFB-Aux/IAA combinations may contribute to different transcriptional responses, depending on their presence in certain tissues or the physiological status of the plants. However, this field and the resulting variety of transcriptional responses is still rather ambiguous and predicting the function of a certain TIR1/AFB-Aux/IAA pair will require much more research [46]. Besides multiple combinations between receptors and Aux/IAAs, also the plenitude of ARFs might contribute to the specificity of the auxin response [38]. Some of the cellular responses to auxin take place at the cell surface. A candidate receptor of auxin at the plasma membrane is AUXIN BINDING PROTEIN 1 (ABP1) [34,36,47]. ABP1 is found at the plasma membrane and in the endoplasmatic reticulum and is shown to act via non-transcriptional processes [38,48]. Recently however, the contribution of ABP1 to auxin signaling and plant development has been questioned and we are awaiting its re-examination as a regulator of auxin perception [49,50].
The differential affinity of ABP1 and TIR1 for synthetic analogues of auxin suggests that different endogenous auxin analogues may contribute to a higher complexity of auxin signaling, possibly by separate ABP1 and TIR1/AFB modules [51]. So far four endogenous molecules-indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 4-chloroindole-3-acetic acid (4-Cl-IAA) and phenylacetic acid (PAA)-exhibit auxinic activity in plants [48,[52][53][54]. IAA is most-studied and omnipresent in plants [17]. In planta, IAA is likely converted to IBA in a reversible reaction catalyzed by IBA synthetase but IBA could also serve as an IAA-precursor or -conjugate. Reversible β-oxidation of IBA to generate active IAA is a slow process and this slow release of active auxin could be the reason for the long-lasting function of IBA in rooting powders. Candidates for IBA to IAA conversion are four peroxisomal enzymes, three INDOLE-3-BUTYRIC ACID RESPONSE (IBR) isozymes and one ENOYL-COA HYDRATASE2 (ECH2) [55][56][57]. IBA has been identified in a number of plant species such as maize (Zea mays), pea (Pisum sativum) and Arabidopsis thaliana but its inherent auxin-activity is still uncertain. Several studies provide evidence for a role of endogenous IBA in root development but a direct role in auxin signaling remains elusive [19,55,58,59]. The endogenous auxin-analogue PAA can bind to ABP1 but inhibits carrier-mediated auxin transport [48,60]. 4-Cl-IAA on the other hand has strong auxinic effects but also modulates processes that IAA does not affect and can be found exclusively in leguminous species [48,61]. Active auxins together with conjugated auxins constitute the auxin pool in a plant. In particular exogenous application of auxin leads to an increase of auxin-conjugating enzymes whereas conjugate hydrolases are activated to release stored inactive auxin in conjunction with initiation of growth such as during germination [62,63]. Besides the naturally occurring auxins, there are multiple synthetic auxin-derivatives, many of which were developed because of herbicide activity such as for example 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), dicamba and picloram [64].

Auxin Biosynthesis
Dissecting the auxin biosynthetic pathway has been proven difficult, largely because several auxin biosynthesis routes exist and the enzymes involved belong to gene families that show redundant functions [65][66][67]. Moreover, de novo auxin biosynthesis is not strictly confined to a specific tissue, although auxin is primarily synthesized in young, not fully expanded leaves [68,69]. Even though biosynthesis is the highest in the younger leaves, the largest auxin pools are measured in the root and in the expanding tissues where auxin is essential for the organ to grow. This underscores the importance of efficient transport between biosynthesis site and action site [68,70,71]. Other parts of the plant maintain the capacity to synthesize auxin as evidenced by the local production upon wounding or other local stimuli [25,69]. Below, we summarize recent findings on de novo auxin biosynthesis, which help to regulate the outcome of auxin homeostasis on root development.
The first enzymes identified to be engaged in the biosynthesis of auxin were from bacterial origin. The genes iaaM and iaaH were discovered in Pseudomonas and Agrobacterium and these enzymes respectively encode tryptophan-2-monooxygenase, which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM) and a hydrolase that releases IAA [66,72]. Klee et al. [73] engineered petunia plants to express iaaM and this resulted in approximately a 10-fold increase in endogenous IAA-levels. Plants overproducing IAA showed typical auxin phenotypes such as excessive xylem and phloem development and bigger epidermal and palisade cells, which together with an extended intercellular space give rise to thicker leaves. Other bacterial IAA-biosynthetic pathways were soon thereafter discovered and for example, IPyA decarboxylase (IPDC) was identified to synthesize indole-3-pyruvic acid (IPyA) as an intermediate of auxin biosynthesis [74][75][76]. Orthologous of iaaM and iaaH have not been found in plants whereas the IPyA-mediated IAA-biosynthetic pathway was shown to occur in plants [25,67].
The predominant auxin biosynthesis route uses tryptophan as the main precursor. This was concluded in feeding experiments with labelled tryptophan (Trp), resulting in the production of labelled IAA, which indicated that plants use Trp as a precursor and contain the enzymes to convert Trp into IAA [77,78]. Tryptophan is synthesized from chorismate-the final product of the shikimate pathway-via intermediate indole-3-glycerol phosphate (IGP) in the chloroplasts [79]. In Arabidopsis thaliana, ASA1 and ASA2 encode the subunits of anthranilate synthase (AS), the enzyme necessary for the first step of tryptophan synthesis. Indole-3-glycerol phosphate synthase (IGS) is then responsible for the generation of IGP, a key intermediate in auxin biosynthesis because it can either form tryptophan or be used as a precursor for the tryptophan-independent auxin biosynthetic pathway [80]. Further conversion to tryptophan occurs through a heterotetrameric a 2 b 2 complex consisting of Trp synthase a (TSA1) and Trp synthase b (TSB, in Arabidopsis there are 2 closely related versions of this gene: TSB1 and TSB2) [81]. A tryptophan-deficient Arabidopsis mutant, defective in anthranilate phosphoribosyl-transferase, accumulates anthranilic acid derivatives and these interfere with IAA-biosynthesis [82]. The observed morphological defects, including slow growth, small crinkled leaves and reduced apical dominance, also occur in mutants deficient in auxin signaling such as the auxin-resistant axr1, a gene encoding a subunit of the RUB1-activating enzymes regulating SCF-complex-mediated protein degradation [83]. The four most well-characterized auxin pathways use Trp as a precursor and are discussed below. Even though one of these pathways-the IPyA pathway-is considered the main route for auxin biosynthesis, the other routes act in their own way, in parallel or in a developmentally/environmentally-regulated manner.

The Indole-3-Pyruvic Acid (IPyA) Pathway
The existence of an IPyA auxin biosynthesis pathway was predicted based on the detection of certain intermediates and radiolabeling of tryptophan-derived metabolites [84]. The instability of IPyA at room temperature and its occurrence in two tautomer forms hampered the identification of enzymes involved [85]. The IPyA auxin biosynthesis enzymes were discovered only recently via genetic screens directed to identify regulators of organ development, ethylene response and shade avoidance. Defects in the corresponding genes lead to poor adaptation to low light conditions indicating the importance of this route of auxin biosynthesis in developmental plasticity. The IPyA pathway is a two-step reaction involving tryptophan aminotransferases (TAA1 and TARs) and Flavin-containing monooxygenases of the YUCCA family (YUC1-11) [86][87][88][89] (Blue Pathway in Figure 1). The Trp aminotransferase required for the first step were discovered by screening for ethylene insensitivity in roots, identifying wei8 (weak ethylene insensitive 8) [25] and by screening for lack of a shade avoidance response, identifying sav3 (shade avoidance 3) [67]. Wei8 and sav3 encode an aminotransferase that was renamed TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1). TAA1 catalyzes the transfer of the amino group from Trp to pyruvate or to α-ketoglutarate generating IPyA and the amino acids alanine or glutamine. Both the sav3 and wei8 mutants contain approximately 60% of the wild-type IAA-levels and show a strong impairment in dark induced hypocotyl elongation [67]. Another short hypocotyl mutant TRANSPORT INHIBITOR RESPONSE2 (TIR2) was shown to be allelic to TAA1 [26]. Overexpression of TAA1 causes modest auxin phenotypes indicating that this enzyme does not catalyze a rate-limiting biosynthesis step. The Arabidopsis TAA1 belongs to a small gene family that includes the homologues TAR1 to 4 (TRYPTOPHAN AMINOTRANSFERASE RELATED 1-4), which have overlapping functions [25]. Pea contains three TAA1 homologs: PsTAR1-3, which were shown to synthesize 4-Cl-IAA in addition to the canonical IAA [90].
Tryptophan feeding experiments identified a group of flavin monooxygenase-like enzymes-named YUCCA (YUC)-that catalyze the hydroxylation of the amino group of tryptamine, a rate-limiting step in IAOx tryptophan-dependent auxin biosynthesis pathway [91]. YUCs use NADPH and molecular oxygen to catalyze the oxidative decarboxylation of IPyA to IAA, using flavin as a cofactor [92,93]. Single yucca gene knockout mutants in Arabidopsis thaliana show wild-type growth phenotypes, likely because of redundancy among the 11 homologs [28]. Maize however possesses only 2 yucca copies and single mutants produce strong auxin-deficient phenotypes [94,95]. Overexpression of YUCs results in very limited effects on the IAA-levels, due to the strong homeostatic control of the active IAA-concentration but differences in IAA-metabolites can be clearly measured [87,93]. The YUC genes are mainly expressed in meristems, young primordia and reproductive organs, corresponding to sites of high auxin biosynthesis [69]. YUCs can also catalyze the decarboxylation of phenyl-pyruvate (PPA) to phenyl-acetic acid (PAA) [54]. The TAAand YUC-family genes are regulated spatiotemporally and contribute to the uneven distribution of IAA in plants [25,26,66,67,96]. Homologs of the Arabidopsis YUC gene family have been found from several other plant species, suggesting a widespread occurrence of this branch throughout the plant kingdom.
Besides the linear pathway from IPyA to IAA, using the YUCs, indole-3-acetaldehyde (IAD) might be formed as an intermediate, like it happens in plant growth promoting rhizobacteria [97]. Whereas, the enzyme that could cause the conversion of IPyA to IAD has not been discovered in plants yet, the downstream aldehyde oxidases (AO) necessary to generate IAA out of IAD are present [98] (Blue Pathway in Figure 1).

The Indole-3-Acetamide (IAM) Pathway
IAM is the key intermediate in the bacterial auxin biosynthesis pathway of Agrobacterium tumefaciens. IAM is also found in many plant species including Arabidopsis, Citrus and Prunus, rice and maize [99][100][101]. By analogy with the bacterial synthesis route, the putative plant IAM-pathway would also encompass a two-step reaction with in the first step the conversion of Trp to IAM, which may be executed by a trypthophan-2-monooxygenase. Next, IAM is converted to IAA by the action of IAM HYDROLASE. Searching for orthologs of the bacterial iaaH in Arabidopsis thaliana has resulted in the identification of AtAMI1 (AMIDASE 1) as a candidate plant IAM hydrolase [102] (Yellow Pathway in Figure 1). Also, Nicotiana tabacum harbors an iaaH orthologue, NtAMI1, which is required for growth of BY2 cultures on medium, in which NAA is depleted but indole-3-acetamide is supplemented as alternative auxin-source [103]. Homologs of AMI have been identified in other plant species including the monocot rice and algae, suggesting that this auxin biosynthesis route is evolutionary conserved [104][105][106]. AMI1 homologs can convert phenyl-2-acetamide (PAM) into PAA, giving an explanation for the occurrence of auxinic molecules other than IAA in plants [107].
IAM may also be produced from indole-3-acetaldoxime (IAOx) because the IAM-levels are significantly decreased in mutants defective in the cytochrome P450 enzymes CYP79B2 and CYP79B3 (cyp79b2cyp79b3), involved in the IAOx pathway [98,108,109] (Yellow Pathway in Figure 1). However, in rice, maize or tobacco, species that lack the CYP79B2/3 homologs, IAM is still detected, which indicates that in these species the role of this pathway's contribution to auxin biosynthesis is not entirely clear yet.

The Tryptamine (TAM) Pathway
Early reports of auxin activity in Avena curvature tests with tryptamine (TAM) suggest it acts as a precursor of auxin biosynthesis [105]. TAM is the first secondary metabolite derived from tryptophan in the synthesis of terpenoid indole alkaloids. In animals TAM is synthesized by tryptophan decarboxylase (TDC) in the cytosol [97] but a corresponding plant TDC-gene has hitherto not been found. TAM is converted to N-hydroxytryptamine in a rate-limiting step catalyzed by members of the YUCCA family [91] (Green Pathway in Figure 1). However, with the now completely described IPyA pathway, this double role of YUCs is questionable because N-hydroxytryptamine has not been detected in plants so far [89,93,110].

The Indole-3-Acetaldoxime (IAOx) Pathway
The IAOx route is unique to the Brassicaceae family [101]. Two homologous cytochrome P450 enzymes, CYP79B2 and CYP79B3 catalyze the synthesis of IAOx from Trp. This step takes place in the chloroplasts [108,109] (Pink Pathway in Figure 1). In the cyp79b2cyp79b3 double mutant, the IAOx levels were far below the detection limit, confirming the role of these enzymes [101]. In addition to its role as auxin precursor, IAOx is also shunted to several secondary metabolites such as indole glucosinolates (IG) and camalexin [111] (Pink Pathway in Figure 1). Mutations in glucosinolates biosynthesis genes SUR1 (superroot1), SUR2 or UGT74B1 (UDP-glucosyltransferase UGT74B1) resulted in elevated auxin accumulation [112][113][114]. The blocked IG biosynthesis may cause an increased flux of IAOx to IAA [28]. Because the levels of indole-3-acetonitrile (IAN) were also decreased in the cyp79b2cyp79b3 mutant, IAN is assumed to be a downstream intermediate of the IAOx pathway (Pink Pathway in Figure 1). Indeed, IAN can be formed by dehydration of IAOx and this reaction is catalyzed by CYP71A13 [115]. This IAN can be an intermediate for IAA but it is not found in all plant species [94]. Additionally, IAN is produced by hydrolysis of IG by myrosinases [116]. Auxin biosynthesis from IAN might involve nitrilases (NIT 1-4) but that process is not well understood yet [117,118] (Pink Pathway in Figure 1). The reduction of free IAA in the cyp79b2cyp79b3 is more significant at high temperature. This could indicate that the IAOx-pathway in Arabidopsis is involved in temperature-induced or -regulated IAA-formation [109].

Tryptophan-Independent Auxin Biosynthesis Pathways
Because tryptophan synthase-deficient mutants of Arabidopsis thaliana showed higher levels of IAA-conjugates, it was suggested that Arabidopsis harbors a tryptophan-independent auxin biosynthesis route [78]. Indeed, the intermediates IGP and indole may condense to form IAA, without Trp as an intermediate [82,119] (Gray Pathway in Figure 1). Decorticating the Trp-independent auxin biosynthesis route awaits further research. For reviews on this pathway see [17,80,85].

Auxin Biosynthesis Regulates Root Embryogenesis
The initiation of the apical-basal body plan of a plant embryo occurs during the initial stages of embryogenesis and depends on auxin gradients, transport and local biosynthesis [120][121][122][123]. Manipulation of auxin homeostasis by conditional expression of iaaM (indoleacetic acid-tryptophan mono-oxygenase) or the expression of a Pseudomonas syringae indoleacetic acid-lysine synthetase (iaaL) did not interfere with embryo-patterning [124]. Additionally, the sur mutants produced normal embryos but displayed auxin-overproduction phenotypes after germination, which indicates that the IAOx-biosynthetic route is not active during embryogenesis [125]. These results suggest that during embryogenesis there is a compensatory mechanism for biosynthesis-mediated changes in auxin homeostasis. On the other hand, inhibition of transport by 1-N-Naphthylphthalamic acid (NPA), led to fused cotyledons [126], which indicates that proper auxin distribution is critical during embryogenesis. Mutant studies attributed a major role to the PIN1 and PIN4 auxin transporters to establish and maintain the auxin gradient in the embryo [124]. But because of the feedback regulatory role of auxin itself on PIN-trafficking and -localization also auxin biosynthesis must be tightly regulated during embryogenesis [127][128][129].
Initially, auxin is supplied by the maternal tissue but the mathematical models do not exclude that local auxin production takes place in the suspensor [129,130]. This theory is substantiated by specific expression of YUC3, YUC4 and YUC9 in the suspensor and the localization of PIN7 to direct the synthesized IAA towards the developing pro-embryo [122,131]. Afterwards, auxin production in the pro-embryo starts and TAA1 expression is observed in the top apical cells of the 16-cell stage embryos. At a later stage, YUC1 and YUC4 are expressed in this region, while YUC8 is expressed closer to the root pole [96,131,132]. While at the apical part of the developing embryo cotyledons are initiated, the hypophysis, the uppermost suspensor cell, develops into the root meristem with the stem cells and quiescent center (QC) and onset of radial tissue patterning necessary for post-embryonic root growth [3,14,133]. Mutants missing YUC1, YUC4, YUC8, YUC10 or YUC11 failed to develop a hypocotyl and a root meristem, which confirmed the role of auxin generated by the YUCCA flavin monooxygenases during embryogenesis [96]. The expression of the auxin biosynthesis genes (TAA1/TAR and/or different YUC) in different regions of the developing embryo is furthermore essential for vasculature development and the initiation of cotyledons [25,67]. A paralog of Tryptophan synthase α (TSA), indole synthase (INS) is thought to act in a pathway parallel to TAA1. Cellular divisions in the ins-1 mutant embryo are disturbed and this indicates a role for INS during embryogenesis. Moreover, INS is expressed even earlier than TSA in the embryo and suspensor. Lastly, Trp-independent auxin biosynthesis is considered an important source of the plant growth regulator during embryo development [134].

Local Auxin Accumulation Regulates Root Development and Branching
A typical root system of dicotyledonous plants is composed of a primary root (PR) and lateral roots (LRs), which are secondary root branches, formed post-embryonically from PR to enable the plant to exploit the soil environment [135,136]. Common to all roots is the presence of a small group of cells at the center of the tip of the root organ. These cells are the stem cell niche, which coordinates the establishment of various tissues present in the root. Without these stem cells, root initials and the QC, the root can no longer grown and add cells to the developing tissue files. Critical to the activity of the QC and the root initials is the presence of an auxin maximum centered at the stem cell niche [62,137]. The formation of the auxin maximum at the root tip has been attributed to directional transport of shoot-derived auxin moving down into the root tip where complex patterns of auxin efflux carriers result in auxin accumulation at the stem cell niche [6,[138][139][140]. Maintenance of the auxin maximum is however not solely the result of transport but also involves local auxin biosynthesis [141], which is possible in all parts of a root system [62,69]. Genes involved in Trp production (ASA1) and auxin biosynthesis genes encoding proteins of the IPyA pathway (TAA1, TAR2 and YUCCA) are expressed in the root tip [25,67,142]. The expression of YUC genes at the root tip depends on symplastic communication between the cells of the stem cell niche, suggesting that stem cell maintenance is intrinsically linked with the maintenance of a local auxin maximum [141]. Auxin derived from the shoot does not fully rescue the root growth and gravitropic responses characteristic for auxin-deficiency at the root tip, emphasizing the importance of local synthesis for root development [142]. In addition, locally synthesized auxin has been shown to rescue a local phenotype, yet does not show effects at a distance [66,96,143]. Also, root growth and patterning is strongly affected by deficiencies in local IPyA-mediated auxin biosynthesis. In rice, disruption of the FISH BONE gene, an orthologue of TAA1, affects many auxin-mediated processes including root development [144]. Antisense expression of YUCCA1 (OsYUC1) in rice results in a defective root, which phenocopies the root of auxin-insensitive mutants [145]. Similarly, loss of function mutants of the rice OsTDD1 gene, encoding anthranilate synthase β-subunit, have severe root defects but their phenotype can be rescued by overexpression of OsYUC1 [146]. In woodland strawberry (Fragaria vesca L.), silencing of YUCCA6 affected various post-embryonic organ developmental steps including root formation [147]. Many steps of Trp-synthesis involve transamination reactions, which require vitamin B6 as a cofactor. The pdx1 mutant, a vitamin B6 biosynthesis mutant with a short root, is defective in the generation of IAA from tryptophan. The root defects in this mutant are another indication that PR growth depends on Trp-dependent local auxin biosynthesis [148]. Other auxin biosynthetic pathways, such as the IAOx pathway are involved in root growth as well. For example, AtTGG4 and AtTGG5 act during the conversion of indole glucosinolates (IG) to the auxin precursor, indole-3-acetonitrile (IAN) and show defects in the establishment of an auxin gradient in the root tips resulting in a disturbed root growth [116]. Downstream nitrilase enzymes, such as NIT1, are involved in auxin biosynthesis in the root [149]. Furthermore, simultaneous inactivation of cyp79b2 and cyp79b3 results in a significant reduction of the auxin levels at the root tips [69,109]. However, due to redundancy in the different auxin biosynthetic pathways, most of the single biosynthetic mutations did not show dramatic root defects.
LRs develop from pericycle cells, adjacent to the xylem poles [150]. Within these so-called xylem pole pericycle founder cells, cell cycle reactivation and subsequent divisions result in the formation of an LR primordium. Priming of the founder cells occurs in the basal root meristem where it requires oscillating auxin fluxes [151][152][153][154]. The generation of IAA out of IBA seems to play a critical role in these oscillation patterns [58,59]. Programmed cell death at the lateral root cap is a second cause for the oscillatory auxin responses because it releases pulses of auxin into the surrounding root tissues [155]. Upon priming, the pericycle cells proceed through two cycles of asymmetric divisions, which gives rise to the formation of a single layer lateral root primordium (LRP) [156]. Further periclinal divisions yield a dome-shaped multilayer LRP, ready to emerge from the main PR [157][158][159]. Without the endogenous priming in the root meristem, a random stimulation of auxin biosynthesis in a pericycle cell results in an increase of auxin, sufficient to start the formation of an LR [160]. The Arabidopsis superroot1 (sur1) and sur2 have excessive amounts of LRs, indicating that local accumulation of auxin is sufficient to induce LR and AR formation [98,112,161,162]. These findings indicate that auxin accumulation is a trigger to modify the developmental program of the pericycle cell [163]. In young seedlings, early after germination, LR emergence depends on shoot-derived auxin but the capacity to synthesize auxin increases with the age of seedlings, which in turn reduces the dependence on shoot-derived auxin for LR development and local auxin biosynthesis takes over to control LR emergence [70]. The local synthesis of auxin appears to depend on auxin transport because disruption of the GNOM-dependent auxin transport resulted in reduced expression of YUCCA genes (YUC2, YUC3, YUC5 and YUC6) and TAA1 and TAR2. In gnom this reduced auxin accumulation at the sites of LRP formation resulted in fewer LR. A complex regulatory network combining auxin transport and local biosynthesis hence plays an important role in the initiation and further development of LR [164].

Importance of Auxin Biosynthesis for Adventitious Root Development
Adventitious root (AR) emerge from aerial parts of the plant in response to wounding, flooding and other stresses, or as part of a root developmental program [165][166][167]. There are two types of ARs: roots originating from latent and ready-made initials and de novo initiated AR [168]. Generally, ARs are initiated from tissues close to the vascular tissues including the pericycle in the hypocotyl, phloem or xylem parenchyma cells, interfascicular cambium cells, procambium and vascular parenchyma cells [10,11,168,169].
Like many other developmental processes, AR formation is regulated by external cues such as light, temperature and nutrient availability, which generate endogenous hormone signaling events [168,170]. However, wounding is probably the most powerful signal that induces de novo rooting process, especially when the shoot organ is severed from the root. A primary response to wounding is the accumulation of auxin by means of directional auxin transport and local auxin biosynthesis. In petunia cuttings, IAA peaked at the base of the stem early post-excision accompanied by a fast induction of YUCCA-family members and two IAA-amino acid hydrolases [171]. Their combined activity leads to a higher IAA-concentration typical for a wound-response [172]. A similar study in Arabidopsis thaliana showed that following root pruning the endogenous auxin levels rise due to polar auxin transport and increased expression of YUCCA9 [173,174]. Also for AR induction on detached leaves, YUCCA-mediated auxin synthesis was associated with root initiation [174]. For wound responses, YUC1 and YUC4 play the most important roles but it is not known yet how wound signals trigger these biosynthetic genes [174,175]. TAA1 family members are not induced by wounding, yet mutants lacking TAA1 and its closest homologue TAR2 are required for formation of roots on detached leaves [176]. YUC6-mediated auxin biosynthesis is needed at the tip of the AR to stimulate post-emergence growth, similar to local auxin production in the PR and LR tips [62,177,178]. This local auxin biosynthesis compensates for the reduction of auxin flow to the tip in the presence of cytokinin [177].
In general, easier-to-root varieties contain higher levels of free auxin whereas rooting-recalcitrant varieties have less auxin [179]. The positive correlation between auxin-levels and AR induction is further substantiated by spontaneous AR formation in auxin-overproduction mutants. Transgenic rice overexpressing YUCCA develop more crown roots [145]. The superroot mutants sur1 and sur2 are blocked in glucosinolated indole synthesis and thereby re-channel IAOx for IAA-synthesis, resulting in excessive AR formation [108,112,113,161,162]. The spontaneous rooting in sur1 and sur2 is lost in mutants with reduced Trp synthesis (e.g., mutants in ASA1, ASB1 and TSB1). However, other suppressors of sur2 are not controlling endogenous IAA-levels but are affected in auxin signaling [168]. A high auxin concentration in the plant is therefore as such not sufficient to induce AR.

Regulation of Auxin Homeostasis during Root Development by Other Hormones
The balance between auxin and cytokinin is of critical importance for determining the developmental fate of organs, with auxin promoting the root and cytokinin stimulating shoot development. At intermediate auxin/cytokinin ratios, callus, predominantly existing of actively dividing cells, is induced reflecting the stimulatory activity of both auxin and cytokinin [177,180]. The strong link between auxin and cytokinin (co)-activity is mediated by regulatory circuits that control each other's production and transport. Constitutive downregulation of cytokinin was reflected in a significant downregulation of auxin biosynthesis [178]. Furthermore, cytokinin strongly inhibits PIN-type auxin efflux carriers [181][182][183]. On the other hand, cytokinin-induced repression of the auxin transport machinery resulted in secondary stimulation of the auxin biosynthetic gene YUCCA6 [177,184]. The connectivity between auxin and cytokinin is not necessarily conserved across plant species but in for example poplar and Arabidopsis, auxin biosynthesis was induced by an increase in cytokinin [185,186]. Microarrays and q-RT-PCRs showed altered expression of metabolic genes from different auxin biosynthetic pathways upon cytokinin treatment, confirming that cytokinin interferes with auxin biosynthesis [187]. One of these genes is the Cytokinin-induced Root Curling 6 (CKRC6), which is allelic to ASA1. The ckrc6 mutant is insensitive to both cytokinin and ethylene while containing less IAA [188]. This results in a reduced gravitropic root growth phenotype, which can be compensated by exogenous application of auxin. The downstream Trp synthesis gene ASB1 is also regulated by cytokinin by means of the response regulator ARABIDOPSIS RESPONSE REGULATOR (ARR1). Cytokinin-treatment of the root system leads to an increase in auxin at the transition zone of the root meristem due to locally increased ASB1 expression [189].
The cross-talk between ethylene and auxin inhibits root elongation and regulates LR initiation and emergence [190,191]. Ethylene regulates the expression of two WEAK ETHYLENE INSENSITIVE (WEI2/ASA1 and WEI7/ASB1) genes, which encode subunits of anthranilate synthase, a rate-limiting enzyme in Trp biosynthesis [25,192]. These genes were identified in a screen for ethylene insensitivity. WEI2 is allelic to ASA1, involved in Trp biosynthesis and WEI8 to TAA1, an enzyme in the IPyA auxin biosynthetic pathway. These mutants underscore the link between ethylene signaling and auxin biosynthesis [25]. In ethylene-treated seedlings, an overall increase of the auxin response at the root tip was observed and this is also reflected in direct auxin measurements [191,193]. A member of ethylene-responsive AP2 transcription factors; ETHYLENE RESPONSE FACTOR1 (ERF1) was recently shown to bind the promoter of ASA1 in order to regulate auxin biosynthesis and ethylene-induced root growth inhibition [194]. Another interaction between ethylene and auxin biosynthesis was discovered in a chemical genetic strategy, using L-kynurenine, a chemical that represses the nuclear accumulation of the ethylene insensitive 3 (EIN3) transcription factor and TAA1/TAR were identified as a target for L-kyn [195]. Lastly, rice transgenic plants overexpressing OsEIL1, the closest homolog of EIN3, exhibited a short, coiled primary root and they had increased YUC8/REIN7 expression so auxin biosynthesis through the IPyA pathway is activated [196].
Root growth is also affected by the interplay between auxin and jasmonic acid (JA). Exogenous application of methyl jasmonate enhances LR formation but inhibits PR elongation [197,198]. Jasmonates reduce LR formation in the Arabidopsis jasmonate-induced defective lateral root1 (jdl1/asa1-1) mutant. Inactivation of JDL1/ASA1 suppressed LR formation and reduced auxin accumulation in the basal meristem, which indicates that jasmonates not only affect auxin biosynthesis but also affect auxin transport [199]. The molecular mechanisms modulating the crosstalk between jasmonate and auxin biosynthesis during LR formation act through the Ethylene responsive transcription factor 109 (ERF109), which binds specifically to the promoter elements of YUC2 and ASA1. The JA-signaling pathway is also linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 expression [200]. In conclusion, the auxin gradients needed for root development, secondary root initiation and emergence depend on synchronized interactions between auxin biosynthesis and interaction with other phytohormones.

Auxin Biosynthesis as an Integrator of Environmental Factors and Root Development
The primary function of the root system is to search for water and minerals and to provide physical support for the shoot to grow towards a light source. Hence light, water, minerals and gravitropic force influence root development and architecture. Light has been reported to regulate the endogenous level of auxins [201,202]. During AR formation, light influences the expression of the auxin responsive GH3 and ARF genes [203][204][205][206]. In Arabidopsis thaliana, the photomorphogenesis mutant red1 defective in SUR2/ATR4 (ALTERED TRYPTOPHAN REGULATION 4) failed to produce AR on the hypocotyl in red light [207]. Light is also shown to induce YUCCAs in the maize root [208]. De novo root organogenesis in Arabidopsis leaf explants involves different YUCCA genes depending on whether explants are incubated in the light or dark. The relevance of light as a factor regulating auxin homeostasis also follows from the identification of shade avoidance mutants some of which were affected in auxin biosynthesis genes. Tao et al. [67] identified TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1) in a shade avoidance mutant screen. TAA1 indeed contributes to local auxin biosynthesis during the elongation of the hypocotyl [209]. In low R:FR conditions, PHYTOCHROME INTERACTING FACTOR 7 (PIF7) is activated via dephosphorylation and binds to the YUCCA genes to regulate their expression [210]. This suggests a direct link between the light-mediated PHYTOCHROME B (PHYB) pathway and auxin accumulation.
In Arabidopsis seedlings, exogenous application of sugar enhanced the PR growth and increased LR density by activating auxin biosynthesis [211]. The enhanced root growth correlated with transcriptional activation of the auxin biosynthesis genes YUC2, YUC8, YUC9, CYP79B2, CYP79B3, transporters PIN1, PIN2 and signaling AUX/IAA genes [211,212]. Similarly, sucrose grown seedlings develop elongated roots and auxin accumulates in the shoots and roots [213].
Root branching is enhanced in nutrient rich-soils [214][215][216][217]. Nitrate is a growth-limiting nutrient and the major source of nitrogen (N) for plants [218]. High and low concentrations of N significantly inhibit respectively enhance LR development with no or limited effect on growth of the PR [219,220]. N-depletion suppresses auxin-levels in the shoot while it stimulates auxin accumulation in the root, in line with a reduction in TAA1 and TAR1 expression in the shoot and upregulation in the root [221][222][223]. Also in wheat, TAR transcription is affected by N-supply [224]. Not much is known about the mechanism by which N-availability regulates auxin biosynthesis genes. The overexpression of AGAMOUS-like 21 (AGL 21) has been shown to stimulate LR elongation as a function of N-availability in agreement with enhanced expression of YUC5, YUC8 and TAR3 [225]. An agl21 mutant had fewer and shorter LRs and exogenous auxin application rescued the root phenotype, pointing to a role in the regulation of auxin biosynthesis. In Arabidopsis, at low concentrations of phosphate, PR growth is impaired, while LR development and root hair formation are promoted [226]. The working hypothesis is that auxin accumulation is required to ameliorate root growth under phosphate-starving conditions and overexpression of YUC1 promotes root architecture changes in P-limiting conditions [227]. NFYA (NUCLEAR FACTOR Y A)-B1 responds to nitrate-and phosphate-starvation by modulating the expression of TAR2 [228]. Finally, sulphate-availability modulates transcriptional activation of nitrilase 3 (NIT3) and this leads to enhanced local auxin biosynthesis [229].
Gravitropism ensures roots to grow downward in their search of sufficient nutrients and water [230]. In the Arabidopsis root, an asymmetric auxin gradient between the lower and upper sides of roots is responsible for root curving in response to gravity [231,232]. Besides a differential auxin transport, differential TAA1 activation in the lower and upper epidermal cells, aids in the formation of an asymmetric auxin gradient and response to the gravity vector [26].
The PR elongates under mild drought conditions, allowing access to deeper water reserves and this growth is supported by modulating auxin activity in the PR meristem. Transgenic plants (Arabidopsis, potato and poplar) overexpressing iaaM, YUC6 or YUC7, produce more IAA and show enhanced drought resistance, while the yuc1/yuc2/yuc6 triple mutant is more sensitive to drought stress [233][234][235][236][237]. However, co-overexpression of iaaL, an IAA-conjugating enzyme and YUC6, reduces the endogenous IAA-levels but drought stress tolerance remained unaffected, which indicated that the tolerance is not solely based on IAA-overproduction or -levels [233]. Also, IAA-Ala Resistant3 (IAR3) plays a role in modulating root architecture during osmotic stress. This hydrolase is capable of generating free IAA from IAA-amino acid conjugates under drought stress. The locally generated auxin then stimulates LR development to promote plant survival in dry conditions [238]. Water availability stimulates auxin biosynthesis and -transport via the regulation of TAA1 and PIN3. Newly initiated LR are preferentially initiated towards the water gradient and this as well might be mediated by local auxin-biosynthesis [239]. This hydro-patterning is reduced in wei8 and sav3 but can be rescued by constitutive expression of TAA1 in the root epidermis [240].
Aluminum (Al), when present as Al 3+ ion, constitutes a major treat to the survival of plants in acidic soils (pH < 5) [241,242]. Al-induced inhibition of root growth is mediated by auxin [243][244][245]. In Arabidopsis, Al induced TAA1 directly or through the intervention of ethylene and as a consequence inhibited root growth [245,246]. Similarly, auxin-overproducing yucca mutants had an increased Al-sensitivity and contributed to the root growth inhibition in response to Al-stress [247,248]. Besides an effect on auxin biosynthesis, a reduction of PIN2 expression and localization contributed to the Al-induced root inhibition [249,250]. The metal ion of manganese (Mn) also inhibited PR growth and LR development by reducing the expression of YUC2, YUC3, SUR1, ASA1, PIN4 and PIN7 [251]. Boron-deprivation led to a significant increase of the endogenous auxin content thanks to the induction of TAA1, TAR2, YUC3 and YUC8 together with a decrease of the rootward IAA-transport (mediated by PIN1 and PIN4) [252].
In addition to abiotic environmental factors, root growth is influenced by beneficial growth-promoting rhizobacteria (PGPR) or fungi (PGPF) growing in the vicinity, on or in root tissue and pathogens [253]. The growth hormone auxin plays an important role in the communication between these microbes and the plant. Many plant-pathogenic microorganisms produce indole-3-acetic acid (IAA), which often determines their pathogenicity or influence endogenous auxin biosynthesis [254][255][256]. In general, both abiotic and biotic stresses and environmental conditions affect root development through the modulation of auxin biosynthesis.

Meta-and Catabolic Processes Controlling Auxin Levels
For auxin to display its crucial role during plant development, the endogenous concentration has to be spatio-temporally regulated. The role of biosynthesis has been discussed above but also inactivation, metabolism and storage contribute to the establishment of local levels of active auxin. IAA is known to act as a free acid but also conjugated forms, with a wide variety of sugars, peptides and amino acids are found in plants [17,18,65,257].
IBA can be considered a precursor of IAA. IBA is metabolized slower than other conjugates and less prone to non-enzymatic degradation. Furthermore, IBA does not undergo oxidation [258]. IBA is transported in the root, probably by the carrier proteins PLEIOTROPIC DRUG RESISTANCE 8 (PDR8) and PDR9 [6,58]. IBA-activity depends on peroxisomal import through PEROXISOMAL ABC TRANSPORTER1 (PXA1) and subsequent β-oxidation to IAA. The peroxisomal enzyme 3-ketoacyl-CoA thiolase PED, contributes to both fatty acid and IBA β-oxidation. Despite ample reports on bioactivity of IBA, its biological role is still controversial [273,274] (Purple pathway Figure 1).
The inactivation of auxin by oxidation resulting in the formation of the catabolite 2-oxindole-3-acetic acid (oxIAA) has only recently been studied in more detail but was already longtime considered a main auxin catabolism pathway [275,276]. oxIAA is an irreversible IAA catabolite with little bio-activity and it is not transported via the canonical polar auxin transport system [277]. Recently, three groups independently published back to back on the role of the Arabidopsis gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1) to catalyze the formation of oxIAA both in vitro and in vivo [278][279][280] (Purple pathway Figure 1). DAO was previously characterized in rice to have a role in auxin metabolism and reproductive development [281]. Arabidopsis mutants lacking this enzyme contain 50-95% less oxIAA (resp. data from [20,279]) and have phenotypes reminiscent of elevated auxin levels [279,280]. A second isoform, AtDAO2, is only expressed in the root cap and is also involved in the formation of oxIAA. Both genes are closely related to the apple Adventitious Rooting Related Oxygenase 1 (ARRO1) and rice DAO [280,282,283]. Temporal and tissue-specific inactivation of auxin by AtDAO1 is essential to regulate the plant's endogenous auxin levels. The localization of AtDAO1 in the border cells flanking the lateral root primordia (LRP), suggests that this enzyme is important to create an auxin maximum at the center of the root primordium [279]. In mutants lacking AtDAO1, the expression of GH3s as well as the synthesis of IAA-Asp and IAA-Glu is upregulated. This suggests that there is a compensation feedback between the different redundant IAA-regulating pathways [20,279,280]. In plants, there is a fast auxin conjugation response through the formation of conjugates such as IAA-Asp, which is more active at higher auxin concentrations and a constitutive slow conjugation response through oxidation and oxIAA formation, which acts at lower auxin concentrations [20,279]. The increase in auxin production in the dao1 mutant is mainly mediated via the IPyA pathway [20,280]. AtDAO2 was shown to follow a circadian rhythm-based oscillation in the root cap, which is a trigger for the activation of lateral root initials [58,59,281].
The generation of oxIAA is followed by a subsequent glycosylation to oxIAA-glucoside (oxIAA-Glc), which is very efficiently executed by UGT74D1 [284] (Purple pathway Figure 1). UDPglycosyltransferases (UGTs) are one of the largest families of glycosyltransferases in plants [281,283] and some of them act in the conversion of IAA to glucose-conjugates. UGT84B1 catalyzes the conversion of IAA to 1-O-(indol-3-ylacetyl)-β-D-glucose (IAA-Glc), while UGT74E2 catalyzes the formation of 1-O-(indol-3-ylbutanoyl)-β-D-glucose (IBA-Glc) from indole-3-butylic acid (IBA) and UGT74D1 converts both IAA and IBA but also NAA, 2,4-D and ICA to their corresponding glucosides [285][286][287]. The iaglu gene encodes IAA glucosyltransferase in Zea mays [288]. In Arabidopsis over 100 uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been identified that are classified into 14 subgroups [289,290]. Members of the L subgroup are potentially involved in the formation of IAA-glucose esters. Overexpression of UGT74D1 results in the loss of root gravitropism presumably due to increased glycosylation of ox-IAA and reduced IAA level at the root tip [284]. At the whole plant level, no decrease in IAA was observed, which suggest a compensating feedback mechanism [284]. A similar phenomenon was observed in plants overexpressing UGT84B1 in which an increase of IAA was measured [291]. The overexpression of UGT74E2 on the other hand led to an increase in IBA [287]. To allow for a temporal-and concentration-dependent regulation of auxin homeostasis, it was shown that GH3s act fast while UGT74D1 was slower upon auxin treatment. These results suggest that the OxIAA and GH3-pathways have distinct roles in IAA-homeostasis. The OxIAA pathway may function constitutively to maintain the basal levels of IAA in plants while the GH3 pathway may play a role in cases where plant cells have to rapidly reduce the relative amount of IAA in response to developmental and environmental changes [284].
Last but not lease, conjugation compartmentalization of IAA in the endoplasmic reticulum has been shown to regulate the availability of IAA to cellular conjugation mechanisms [292].

Conclusions
What have we learned so far from auxin-related studies is that the combined action of auxin biosynthesis and transport set the stage for adaptive developmental processes. The root system in particular is sensitive to auxin. Whereas the last decade's emphasis was more on auxin redistribution by means of polar and local transport, current research progress underscores the importance of local auxin biosynthesis. Auxin biosynthesis genes have been found to play a role in every aspect of root development translating environmental cues into adaptive responses (see Table 1). As auxin, derived from distantly located apical origins as well as locally synthesized auxin equally have impact on root architecture and development, the mechanism by which auxin is perceived and possibly how it enters the cells may hold specific information relevant for the adaptive growth responses. Future research should focus on local auxin signaling and spatial and temporal perception mechanisms controlling auxin-mediated establishment of root meristems.