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Auxin is critical for plant growth and orchestrates almost all developmental processes (Woodward & Bartel, 2005a; Aloni et al., 2006; Teale et al., 2006). Auxin is a collective name for several molecules with a wide spectrum of growth-promoting activities. They are synthesized not only in plants (Woodward & Bartel, 2005a), but also in fungi (Ulrich, 1960; Splivallo et al., 2009; Rao et al., 2010) and bacteria (Bianco et al., 2006a,b; Spaepen et al., 2007; Bai et al., 2012), and can also be produced synthetically (e.g. 1-napthylacetic acid, NAA). In a narrow sense, the term auxin is often used for the most potent native compound, namely indole-3-acetic acid (IAA).
Nearly every developmental stage (embryonic and postembryonic) and every growth process (formation of lateral organs, growth of leaves) of a plant is affected by IAA directly (Heisler et al., 2005; Teale et al., 2005, 2006) or indirectly by secondary induced signaling molecules (Pagnussat et al., 2004). Furthermore, phototropism and gravitropism are closely connected to auxin transport and action (Friml et al., 2002; Li et al., 2005; Wan et al., 2012).
A central point in the understanding of all the facets of auxin activity is the identification of specific receptors and interaction partners at the cellular level. Around 20 yr ago, the first auxin binding protein (ABP1) was found (Hesse et al., 1989). Located at the cellular periphery and being responsible for fast reaction to exogenous auxin (Dahlke et al., 2010; Tromas et al., 2010), ABP1 participates in extracellular auxin-mediated cell elongation by regulating endocytosis (Robert et al., 2010) and activating ROP signaling (Xu et al., 2010), as well as activating ion channels and H+-ATPases at the plasma membrane (Felle et al., 1991; Rück et al., 1993; Maurel et al., 1994; Christian et al., 2006). The proton efflux acidifies and loosens the cell walls, thus enabling turgor-driven cell expansion (e.g. Christian et al., 2006). In addition, an intracellular auxin receptor has been found. This receptor controls the degradation of a subset of transcription factors and thereby modulates transcriptional auxin responses (Dharmasiri et al., 2005; Kepinski & Leyser, 2005). However, many aspects of auxin action are not understood. For instance, the additional dependence of auxin on downstream signals, such as reactive oxygen species (ROS) and nitric oxide (NO), in the regulation of root organogenesis and architecture is still puzzling (Joo et al., 2001, 2005; Pagnussat et al., 2003, 2004; Hu et al., 2005; Yadav et al., 2013).
NO is a well-known stress signaling molecule that plays a crucial role during plant defense against pathogens (e.g. Bolwell et al., 1999). Recently, a more fundamental role in basic growth processes has been discussed. Rather surprisingly, NO has been reported to function as a downstream signaling molecule of auxin-induced lateral and adventitious root formation (Pagnussat et al., 2003, 2004). Moreover, gravistimulation of roots not only induces auxin accumulation at the lower root flank, but also of NO (Joo et al., 2001, 2005; Hu et al., 2005), and a reduction in the NO level inhibits gravitropic bending of gravistimulated root apices (Joo et al., 2005).
An interesting avenue that may help to understand NO-dependent auxin signaling is to take a closer look at the effects of indole-3-butyric acid (IBA), which is a naturally occurring auxin known from several plant species (Epstein & Ludwig-Müller, 1993). Unlike IAA, it is believed that IBA has nearly no auxin-typical transcriptional capacity on its own (e.g. Oono et al., 1998). IBA is considered to serve mainly as a transport and storage form of IAA (Bartel, 1997; Zolman et al., 2000). IAA can be converted to IBA (Ludwig-Müller & Epstein, 1991) and IBA is converted back to IAA in a peroxisomal β-oxidation-like process (Zolman et al., 2000). In plants, β-oxidation is localized in the peroxisomes (Gerhardt, 1992; Kindl, 1993). Mutants with defects of peroxisomal biosynthesis or β-oxidation are resistant to externally applied IBA (Zolman et al., 2000, 2001a,b; Zolman & Bartel, 2004). Several mutants of peroxisomal enzymes appear to be solely linked to β-oxidation-like processing of IBA to IAA (Zolman et al., 2008; Strader et al., 2011). Moreover, mutants defective in β-oxidation are impaired in IBA to IAA conversion (Strader et al., 2010). These genetic approaches indicate that IBA to IAA conversion could be the explanation for the unique biological activity of IBA.
In auxin bioassays, IBA often shows only weak activity (Woodward et al., 2005a), but there are exceptions. IBA, but not IAA, can efficiently induce adventitious roots in Arabidopsis (Ludwig-Müller et al., 2005). Moreover, it has emerged that IBA can promote lateral root formation (LRF) independently of IAA (Ludwig-Müller, 2000; Chhun et al., 2003; Strader et al., 2010, 2011). Altogether, several pieces of evidence indicate that the straightforward scenario of IBA being only a precursor of the active auxin IAA is not sufficient to explain its activity features. It has been shown that IBA, much like IAA, uses NO as a downstream signal to induce lateral roots (Kolbert & Erdei, 2008; Kolbert et al., 2008; Yadav et al., 2013). NO signal transduction cascades relevant for root primordial formation include cyclic GMP, phospholipase D, phosphatidic acid, ROS and PIN transporters of auxin (Pagnussat et al., 2003; Lanteri et al., 2008; Li et al., 2011; Bai et al., 2012; Li & Jia, 2013).
Here, we show the effects of IAA and IBA in different root development model systems using wild-type and mutant plant lines of maize and Arabidopsis. Particular emphasis is paid to NO-mediated IAA and IBA effects on root growth and development of root systems. We use the protein degradation inhibitor Terfestatin A as an additional experimental tool. This inhibitor prevents the auxin-specific changes of gene repression (Yamazoe et al., 2005) and enables discrimination between transcriptional and redox-based activities of auxin.
IBA shows paradoxical behavior relative to other auxins, such as IAA, NAA or 2,4-dichlorophenoxyacetic acid (2,4-D). It displays only weak auxin activity in root growth inhibition assays (Woodward & Bartel, 2005a), but its ability to induce lateral and adventitious roots is comparable with that of other auxins (Nordström et al., 1991; Zolman et al., 2000; Ludwig-Müller et al., 2005). The lateral rootless 1 (lrt1) Oryza sativa (rice) mutant is less sensitive to auxin (IAA, IBA and 2,4-D) with respect to root elongation. In addition, the only auxin that can restore lateral root initiation in the mutant is IBA (Chhun et al., 2003). Overall, this suggests that, at least in lateral root initiation, IBA performs an essential role, which cannot be replaced by IAA. Our present data show that the spatially and temporally coordinated release of IAA and NO via peroxisomes is the critical process, which makes IBA a unique player in lateral root initiation.
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IAA influences virtually every aspect of plant growth and development (Woodward & Bartel, 2005a; Aloni et al., 2006; Teale et al., 2006). In addition to the well-documented effect on the transcriptional level (Abel, 2007), other effects occur via signaling at the plasma membrane and endomembranes, via diverse downstream signals including NO (Mockaitis & Howell, 2000; Pagnussat et al., 2003, 2004; Lanteri et al., 2006, 2008; Bai et al., 2012; Chen et al., 2012; Li & Jia, 2013; Wang et al., 2013). Genetic analysis has uncovered many aspects of auxin biosynthesis and metabolism (reviewed in Woodward & Bartel, 2005a), and details of transcriptional regulation pathways of auxin-inducible genes (Sawa et al., 2002; Pufky et al., 2003; Cluis et al., 2004; Himanen et al., 2004). Studies to compare different auxins have shown fundamental differences in their function on plant growth, enabling a classification of active and inactive auxins. However, IBA shows conflicting results in different growth assays, making it difficult to classify it into one or the other group. In this work, we have conducted a set of experiments to examine the role of IBA, IAA and NO, and their connection within a possible signaling pathway in LRF.
IBA occurs naturally in different plant species and tissues (Epstein & Ludwig-Müller, 1993). It can be converted into IAA and vice versa, indicating a strong link between the metabolism of the two auxins (reviewed in Woodward & Bartel, 2005a). IBA shows the unique auxin ability of a polar cell-to-cell transport, but possibly by a different mechanism from IAA (Rashotte et al., 2003; Poupart et al., 2005). IBA and NO are known to promote the formation of lateral roots (Zolman et al., 2000; Correa-Aragunde et al., 2004) and adventitious roots (Nordström et al., 1991; Ludwig-Müller et al., 2005; Yadav et al., 2013). These are all typical auxin traits but, as we have shown here using β-oxidation-defective mutants of Arabidopsis, IBA does not show the transcriptional activity expected of an active auxin (Fig. 2).
The root growth of mutants with specific β-oxidation defects is insensitive to IBA, and our experiments show that IBA is unable to activate the transcription of Aux/IAA genes in such mutants (Fig. 1). Moreover, these mutants show very low IBA to IAA conversion rates (Table 2; Strader et al., 2010, 2011). All this suggests that IBA is converted to IAA in a process paralleling fatty acid β-oxidation (Zolman et al., 2000). β-Oxidation of fatty acids in plants resides solely in the peroxisomes (Gerhardt, 1992; Kindl, 1993). This supports the assumption that the conversion of IBA to IAA is indeed peroxisomal.
Terfestatin A is a root-specific auxin signaling inhibitor which disturbs the SCFTIR proteasome complex and disables the transcription of Aux/IAA response proteins and of the synthetic DR5-reporter (Yamazoe et al., 2005). The blocking of auxin-inducible gene transcription by Terfestatin A prevents the formation of lateral roots and neutralizes the positive LRF-promoting effect of IAA. However, the inhibition of SCFTIR-controlled gene expression has a weaker detrimental effect on IBA activity, pointing to the existence of a second LRF-promoting pathway in response to IBA (Fig. 2).
The fluorescent dye DAF-2 DA is often used as a marker of NO. However, its specificity is sometimes questioned because of the possibility that it detects not NO itself, but its oxidized products, such as N2O3 (Kojima et al., 1998). Indeed, the DAF–NO detection system seems to require oxygen to form fluorescent molecules (Arita et al., 2006). This may constrict the usage of the dye (not suitable under anoxia), but the generated fluorescence signal still reflects the level of NO produced after a stimulus. The fluorescence signal of DAF–NO reveals that IBA treatment generates a considerable amount of NO (Fig. 3). Importantly, this response was not found in the IBA-insensitive mutants used in this study, indicating that intact peroxisomal β-oxidation is necessary for this NO generation. This is consistent with the important finding of our present study that peroxisomes are a major NO source after IBA treatment (Fig. 4). We propose the scenario that the IBA to IAA conversion is responsible for the peroxisomal NO signal of IBA-exposed roots, which drives LRF. Consistent with this hypothesis, the impact of IBA on LRF is more susceptible to changes in the NO level than the impact elicited by IAA (Fig. 1). IBA is nearly inactive on LRF after inhibition of NO production by cPTIO, as well as in the Arabidopsis mutant noa1 with its diminished ability to produce NO (Guo et al., 2003; Guo & Crawford, 2005). Importantly, the IBA to IAA conversion is not significantly affected in noa1 (Table 2).
The gene NOA1 has been suggested to encode a plant NO-synthase (Guo et al., 2003; Guo & Crawford, 2005), even though it shares no sequence homologies to animal NO-synthases, making a role as an NO-synthase in plants implausible. However, the noa1 protein participates in NO production from l-arginine, which is a typical NO-synthase feature. Published results indicate that NOA1 is a plastidic GTPase, and the decreased NO production in the noa1 mutant is an indirect consequence of the mutation (Moreau et al., 2008; Gas et al., 2009). Nevertheless, the mutant shows disturbed NO formation and is, as such, a valid candidate for our purposes. A similar approach using nitrate reductase (NR) mutants (nia1nia2) has suggested that NO formation, which is required for IBA action, is produced by NR (Kolbert & Erdei, 2008; Kolbert et al., 2008). However, these studies failed to provide convincing results to link NR activity with IBA activity, apart from an IBA insensitivity of the NR mutant. Strikingly, the inhibition of NR prevents not only IBA-induced, but also IAA-induced, NO formation (Fig. 3; see also Hu et al., 2005). Both noa1 and nia1nia2 showed only weak NO formation after IBA treatment, but both mutants have, in addition to their reduced capability for constitutive NO formation, pleiotropic phenotypes in metabolism, growth, development and stress responses (Modolo et al., 2005; Moreau et al., 2008; Gas et al., 2009). This makes it difficult, in general, to distinguish between effects caused by impaired NO biosynthesis from those caused by metabolic alterations.
We have shown that peroxisomes are a major cellular site of NO production during the IBA to IAA conversion. Interestingly, other reports have also shown that peroxisomes act as potential NO sources in plants. For example, NO has been detected in peroxisomes of pollen tubes, in which the NO level at the pollen tube tip determines the rate and orientation of pollen tube growth (Prado et al., 2004). This suggests that one mode of the peroxisome signal transduction mechanism is via the ability to act as an NO source. In vitro and in vivo studies have proposed NO-synthase-like activities in peroxisomes from pea leaf (Barroso et al., 1999; del Rio et al., 2004) and Arabidopsis roots (Corpas et al., 2009). An additional nitrite-dependent pathway for NO production, involving xanthine oxidase, has been identified (Zhang et al., 1998). Under anaerobic conditions, when NO is important for the hypoxic acclimation of roots (Mugnai et al., 2012), nitrite can be reduced to NO by the purified peroxisomal enzyme xanthine oxidoreductase (XOR) (Godber et al., 2000). Moreover, a role for XOR in the production of NO was proposed after expression and inhibition studies on phosphate deficiency in cluster roots of white lupin (Wang et al., 2010).
These examples highlight that our knowledge about NO signal transduction pathways is still fragmentary. All the conclusions and most of our knowledge about NO signaling and the genetic modulation of endogenous NO levels in plants are of a correlative nature. Despite the as yet unresolved questions concerning the NO-producing enzyme systems, an additional line of evidence shows the involvement of NO in IBA-induced LRF. The lrt1 maize mutant is insensitive to the auxins IAA, NAA and 2,4-D in terms of lateral root initiation (Hochholdinger & Feix, 1998), but IBA, as well as NO, can induce lateral roots in this mutant, resulting in a qualitative rescue of the phenotype. The primary root shows local induction of stunted lateral roots. Both treatments are amplified if applied together, resulting in a wild-type-like phenotype. An inhibition of NO production by cPTIO abolishes the ability of IBA to induce lateral roots completely, indicating the NO dependence of IBA activity in maize, as in Arabidopsis.
A question which still remains to be answered is the role of endogenous IBA. Recent studies have indicated that the IBA to IAA conversion is relevant for undisturbed seedling development by feeding into internal active auxin pools (Strader et al., 2011). One phenotypic aspect of many mutants that cannot utilize IBA is a shortage of lateral roots (e.g. Zolman et al., 2001a,b; Wiszniewski et al., 2008; Strader et al., 2010, 2011). This could be an indicator that IBA to IAA conversion is necessary for LRF. Our present data show that the spatially and temporally coordinated release of NO and IAA is needed for IBA bioactivity. IBA to IAA peroxisomal conversion not only results in tightly regulated IAA synthesis, but also promotes concomitant NO formation. It can be expected that other processes also requiring IBA, and also involving NO, will be shown to rely on peroxisomal IAA–NO generation. Here, we can mention polarized tip growth of root hairs and root adaptation to salt and water stresses (Lombardo et al., 2006; Strader et al., 2010, 2011; Tognetti et al., 2010; Strader & Bartel, 2011). Future work is necessary to elaborate what kind of NO-producing enzyme systems are responsible for the NO formation induced by IBA, and whether NO-mediated signaling is also utilized in other processes in which IBA is active or essential.