Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

Important roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have recently been described in many plant developmental processes (Swanson and Gilroy 2010; Mittler et al. 2011) including guard cell physiology, flower development, root hair elongation, and most relevantly cellular differentiation in the root apex and modulation of auxin transport via action on PIN auxin efflux proteins (Bashandy et al. 2010; Tsukagoshi et al. 2010). ROS and RNS make excellent signaling molecules as their toxicity requires they be kept at low levels in cells, which allows subtle changes in their synthesis to lead to large magnitude differences in their levels, like other important signaling molecules, such as calcium and protons. As a result, there is a precise balance between synthesis and scavenging of these molecules that allows their level to be carefully modulated.

ROS include H2O2, O2 •−, and 1O2, which are by-products of aerobic cellular metabolism. Their accumulation is determined by the balance between production and detoxification by antioxidants. In both plants and animals, ROS can be generated through respiratory burst/NADPH oxidases (Suzuki et al. 2011; Marino et al. 2012), while ROS levels are reduced through antioxidant protein networks including thioredoxins, glutathione/glutaredoxins, and peroxidases (Mittler et al. 2011) or by chemical antioxidants, such as flavonoids or ascorbate (Hernandez et al. 2009; Pollastri and Tattini 2011). Plants with mutations in the genes encoding proteins that synthesize ROS or antioxidants have linked ROS to plant development (Mittler et al. 2011).

The most abundant RNS in plants is nitric oxide (NO), which is a gaseous free radical displaying a broad spectrum of regulatory functions involved in physiological processes (Delledonne 2005). In animals, nitric oxide synthase (NOS) defines NO levels, but no obvious plant NOS homolog has yet been identified. Some studies have shown the importance of a NOS-like pathway in mediating NO responses in plants, while other studies suggest that the enzyme nitrate reductase (NR) is more important (Besson-Bard et al. 2008a, b). The diverse enzymatic and nonenzymatic reactions in plant NO synthesis are illustrated in Fig. 1. NO levels and bioactivity are also controlled by scavengers, which include class 1 hemoglobin (Hb1) (Fig. 1; Dordas et al. 2004; Perazzolli et al. 2004; Igamberdiev and Hill 2004) in a reaction that is dependent on the presence of both NAD(P)H and AHb1 (Igamberdiev et al. 2005). Strong hypoxic induction of the AHb1 gene (At2g16060) occurs in Arabidopsis under conditions that also induce enzymes of nitrogen metabolism (Klok et al. 2002), suggesting a mechanism for a rapid and transient elevation of NO levels, followed by scavenging to prevent cellular damage.

Fig. 1
figure 00051

Formation of reactive nitrogen species (RNS) with emphasis on the diverse enzymatic or nonenzymatic reactions potentially involved in NO synthesis in plants. AtNOA Arabidopsis thaliana nitric oxide associated, RNOH substituted derivatives of hydroxylamine. NO nitric oxide, NO 3 − nitrate, NO 2 − nitrite, N 2 O 3 nitrogen trioxide, NO 2 • nitrogen dioxide radical, O 2 •− superoxide, ONOO − peroxynitrite. Adapted from Moreau et al. (2010)

Full size image

Both RNS and ROS directly modulate the activity of proteins through posttranslational modification (PTM). PTMs mediated by RNS, such as cysteine S-nitrosylation or tyrosine nitration (Moreau et al. 2010; Arc et al. 2011), can result in an alteration of diverse protein functions. Similarly, ROS leads to oxidation of specific cysteine residues, which control protein activity. The best described ROS-dependent regulation of a mammalian protein is tyrosine phosphatase 1B (PTP1B), where the activity of the protein has been shown to be regulated by a specific single cysteine oxidation which in turn regulates the insulin signaling pathway (Tonks 2006). Recent data suggest that protein nitration and S-nitrosylation could be more than a biological marker of nitrosative stress and could participate in protein turnover or signal transduction in plants (Corpas et al. 2007; Ischiropoulos 2009; Stamler et al. 1992; Jaffrey and Snyder 2001). The specificity of these modifications indicates that they may act as regulatory switches in signal transduction pathways (Hess et al. 2005), analogous to protein phosphorylation (Spickett et al. 2006). Recent evidence, using mutants or chemical treatments that raise or lower ROS and RNS, suggests that auxin transport proteins (or proteins that control their synthesis or targeting) may be targets of ROS and/or RNS regulation by oxidation, nitration, or S-nitrosylation.

2 ROS Regulation of Auxin Transport

ROS have been reported to modulate polar auxin transport by influencing auxin efflux and influx-dependent transport. Recent genetic analysis indicates that polar auxin transport is impaired in plants with altered ROS accumulation. Plants with defects in genes encoding thioredoxin reductase (ntra and ntrb) and an enzyme of glutathione biosynthesis (cad2) exhibit altered ROS homeostasis, due to the absence of these antioxidant proteins (Bashandy et al. 2010). The ntra ntrb cad2 triple mutant has impaired auxin transport and developmental phenotypes consistent with altered auxin transport including PIN-shaped inflorescences and reduced lateral root formation (Bashandy et al. 2010). In addition, atgrxs17 and ntra ntrb cad2 plants show auxin-related morphological phenotypes and reduced expression of the auxin-responsive reporter, DR5 pro :GUS indicating that they have deficiencies related to auxin action or auxin accumulation (Bashandy et al. 2010; Cheng et al. 2011). Consistent with this latter possibility, root growth defects of this triple mutant are rescued by auxin treatment, suggesting a defect in transport, rather than signaling (Bashandy et al. 2010). Decreased transcript levels for auxin efflux and influx carriers in response to elevated ROS suggest that ROS may modulate auxin polar transport at the level of synthesis of transport proteins (Blomster et al. 2011; Tognetti et al. 2012). Intriguingly, the auxin transport inhibitor TIBA and O3 treatment (used as a tool to produce an apoplastic ROS burst) exhibited similar transcriptional effects on auxin-responsive genes (Blomster et al. 2011). Similarities between the responses to oxidative stress and auxin transport inhibitors suggest ROS may act on plant morphology by inhibiting auxin transport either at the level of synthesis or turnover of auxin transport proteins.

Elevated ROS may also alter auxin transport by affecting the stability of auxin efflux carriers. The fluorescence of GFP fusions to PIN1, PIN2, PIN3, and PIN7 are decreased in the root tips of primary roots, but not adventitious roots, treated with BSO (Koprivova et al. 2010; Bashandy et al. 2010). One set of authors suggest that since BSO did not abolish transcription of PIN1, and the effect of BSO was complemented by dithiothreitol, the authors conclude that as yet an uncharacteristic post-transcriptional redox mechanism regulates the accumulation of PIN proteins, and thus auxin transport, in the root tips (Koprivova et al. 2010). Taken together, these data suggest the intriguing result that BSO treatment decreases PIN protein abundance with both transcriptional and post-translational role implicated.

Mutations in genes encoding proteins that are involved in the synthesis of chemical antioxidants, such as flavonols, suggest an additional link between ROS and auxin transport (Pollastri and Tattini 2011). Flavonols have been shown to regulate auxin transport and dependent physiological processes, including root elongation, gravitropism, and branching (Brown et al. 2001; Buer and Muday 2004; Peer et al. 2004; Buer and Djordjevic 2009; Lewis et al. 2011). Auxin transport is elevated in inflorescences, hypocotyls, and roots of plants with the tt4-2 mutation, which make no flavonoids (Murphy et al. 2000; Brown et al. 2001). A comparison of the root gravitropic responses of wild-type and several tt4 alleles identified a delay in root gravitropism when flavonoid synthesis is abolished, which is reversed by chemical complementation by naringenin (Buer and Muday 2004; Buer et al. 2006; Lewis et al. 2011). Flavonoids promote gravitropism presumably by regulating auxin movement in the root tip that modulates differential growth (Buer and Muday 2004). Finally, factors that regulate flavonoid biosynthesis also affect auxin transport, such as light levels (Jensen et al. 1998; Rashotte et al. 2003), wounding and pathogen attacks (Mathesius et al. 1998; Berleth and Sachs 2001), ethylene levels (Lewis et al. 2011), and gravity stimulation (Buer and Muday 2004; Buer et al. 2006).

What has not yet been demonstrated is whether the role of flavonols is to alter ROS in the root and thereby regulate auxin transport through ROS signaling pathways or through more direct mechanisms (Pollastri and Tattini 2011). The levels of ROS species are elevated in plants with defects in flavonol synthesis, consistent with flavonols acting as antioxidants in vivo (Lewis and Muday, unpublished observation), but other mechanisms of flavonol regulation of auxin transport have been described. Quercetin has been shown to block auxin transport when ABCB proteins are expressed in heterologous systems (Fig. 2; Geisler et al. 2005; Bouchard et al. 2006). In addition, ABCB4 was shown to be epistatic to TT4 by double mutant analysis, indicating that flavonols act through ABCB4 to control basipetal auxin transport and gravitropism (Lewis et al. 2007). The inhibition of auxin transport includes disruptions of a complex between an ABCB protein and an immunophilin protein that is needed for maximal auxin transport (Bailly et al. 2008). Whether this protein complex is sensitive to oxidation state in the cell has not yet been reported. An additional intriguing possibility is that ROS and/or RNS control the activity of flavonols by converting them to a semiquinone state, which may have different inhibitory properties. In this scenario, the oxidized flavonol could then be reduced, restoring its capacity to inhibit protein complex formation. Resolving the role of flavonols in regulation of auxin transport via modulation of ROS levels awaits further experimentation.

Fig. 2
figure 00052

Role of nitric oxide and flavonols in auxin transport and direct effect of NO in the auxin signaling pathway. AUX1/LAX auxin uptake carrier, PIN1/ABCB auxin efflux carriers

Full size image

3 RNS Regulation of Auxin Transport

Auxin transport has a central role in auxin-regulated growth processes. Despite the effort to understand the mechanism of NO regulation of polar auxin transport, our knowledge is still limited. Hu et al. (2005) showed that gravistimulation of soybean primary roots induces asymmetric accumulation of NO, and this NO generation is stimulated by auxin since NPA treatments inhibit NO accumulation and gravitropic bending, suggesting that lateral auxin transport is essential for asymmetric NO generation.

Interestingly, high levels of endogenous NO in the cue1/nox1 background produce a drastic reduction in auxin movement from the root shoot junction to the root tip (acropetal or rootward auxin transport), through use of [3H]IAA radiotracer assays as described previously (Fig. 2; Lewis and Muday 2009; Fernández-Marcos et al. 2011). Additionally, high levels of endogenous or applied NO reduce the fluorescence of a PIN1:GFP fusion which participates in rootward auxin transport, without altering significantly PIN1 transcript levels (Fernández-Marcos et al. 2011). In contrast, the fluorescence of a GFP-fusion reporter for PIN2, which mediates basipetal or shootward IAA transport, was not altered significantly suggesting a specific effect of NO on rootward auxin transport in primary roots mediated by changes in PIN1 protein levels (Fernández-Marcos et al. 2011). Likewise, acropetal auxin transport is enhanced in mutants with lower levels of NO such as atnoa1, supporting the hypothesis that altered NO levels cause altered auxin transport capacity (Fernández-Marcos et al. 2011; unpublished data).

In a recent report, Bai et al. (2012) propose that treatment with 3-O-C10-HL (N-acyl-homoserine lactones, AHLs) promotes auxin-dependent adventitious root formation, possibly through H2O2- and NO-dependent cGMP signaling in mung bean (Vigna radiata) seedlings. This treatment is able to stimulate the generation of H2O2, NO, and the synthesis of cGMP to activate adventitious root formation. Treatment with 3-O-C10-HL enhances hypocotyl auxin basipetal transport and this effect can be reversed by scavenging H2O2 or NO, suggesting that these molecules act within a single pathway to promote hypocotyl basipetal auxin transport and adventitious root formation.

4 Other ROS and RNS Connections to Auxin

ROS and RNS can also affect the dynamics of the actin cytoskeleton and may alter actin-dependent targeting of auxin transport proteins. At the level of subcellular dynamics and polar targeting, there is increasing evidence that auxin regulates polar auxin transport by inhibiting PIN endocytosis (Dhonukshe et al. 2008; Lin et al. 2012; Nagawa et al. 2012), which is actin dependent (Geldner et al. 2001). Whether ROS have an effect on auxin distribution as a result of their regulation of the cytoskeleton, vesicle trafficking, and membrane dynamics remains to be elucidated. However, it is already known that NO affects the functioning of the actin cytoskeleton. In response to NO levels, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin depleted/depolymerized (Kasprowicz et al. 2009). Additionally, actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly formed cell plates (Kasprowicz et al. 2009). Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signaling in planta (Kasprowicz et al. 2009).

4.1 Auxin Promotes ROS Accumulation

Auxin induces changes in redox status leading to a more oxidizing cellular environment (Takahama 1996; Joo et al. 2001; Jiang and Feldman 2003; Li et al. 2009; Wang et al. 2010; De Tullio et al. 2010). This change in redox status is mainly due to the generation of several ROS, such as hydrogen peroxide (H2O2) (Brightman et al. 1988; Joo et al. 2001) and superoxide ions (O2 •−) (Schopfer 2001). These ROS may be generated by oxidation of IAA (Kawano 2003) or, indirectly, as a consequence of auxin affecting the activities or synthesis of redox-associated systems (Takahama 1996; Kisu et al. 1997; Jiang and Feldman 2003; Pignocchi et al. 2003).

Redox processes are important for regulating root growth. This regulation may act through mechanisms dependent (Duan et al. 2010) or independent (Tsukagoshi et al. 2010) on the auxin signaling pathway. ROS accumulation in the quiescent center (QC) is an interesting example of how auxin induces changes in redox status. The redox status of the QC, where auxin is strongly accumulated, is different from that in adjacent rapidly dividing cells. The QC has a more oxidizing environment (Kerk and Feldman 1995; Sanchez-Fernandez et al. 1997; Kerk et al. 2000; Jiang and Feldman 2003; Liso et al. 2004) and a large group of transcripts associated with regulating redox status are localized to this tissue (Jiang et al. 2010).

Auxin also promotes ROS accumulation during gravitropic bending (Joo et al. 2001). Gravistimulation elicits a transient increase in intracellular ROS. The action of asymmetrically applied H2O2 in causing root curvature does not depend upon auxin redistribution, suggesting that ROS play a role as a downstream component in the auxin response pathway. Increased ROS concentrations may in turn trigger nitric oxide (NO) generation by nitrate reductase (NR) (Wang et al. 2010) and NO synthase (NOS)-like enzymes (Neill et al. 2008; Li et al. 2009). This probably occurs through the rapid phosphorylation of MAP kinase 6 (MAPK6) (Kovtun et al. 2000; Wang et al. 2010) and/or the action of the protein kinase OX1 and involves Ca2+ (Rentel et al. 2004). Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an inhibitor of nitrate reductase reduces gravitropic bending, indicating that NO synthesis is an important component of the gravitropic response (Hu et al. 2005).

Additional experiments have revealed possible mechanisms of auxin-induced ROS synthesis. The activation of PtdIns 3-kinase and NADPH oxidase is required for auxin-induced production of ROS, regulating plant cell expansion through the activation of Ca2+ channels (Joo et al. 2001; Foreman et al. 2003). Recent results suggest that NADPH oxidase may also be regulated by the Feronia (FER) receptor-like kinase (Duan et al. 2010) and RAC/ROP GTPases (Tao et al. 2002; Xu et al. 2010). Specifically, FERONIA acts as a surface regulator of the RAC/ROP signaling pathway which in turn regulates NADPH oxidase-dependent ROS production (Wu et al. 2011) (Fig. 3).

Fig. 3
figure 00053

A model for the putative pathway of ROS and NO biosynthesis and signal transduction mediated by auxin-activated FER–RAC/ROP complex (modified from Duan et al. 2010 and Wang et al. 2010)

Full size image

4.2 ROS Represses Auxin-Inducible Promoters

Several lines of evidence suggest that ROS may modulate auxin sensitivity by repressing auxin-inducible gene expression (Navarro et al. 2006; Wang et al. 2007; Ludwikow and Sadowski 2008; Bashandy et al. 2010; Iglesias et al. 2010; Blomster et al. 2011; Cheng et al. 2011). Indeed, auxin-resistant mutants axr1 and axr3 are less sensitive to ROS than wild-type plants (Koprivova et al. 2010). This process seems to involve changes in MAPK activity. Specifically, ROS can activate an Arabidopsis MAPK, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6 (Kovtun et al. 2000). The activated MAPK cascade plays a dual role in regulation of gene expression activating stress-response genes that protect plants from diverse environmental stresses and repressing auxin-inducible promoters (Kovtun et al. 2000). Thus, the ANP-mediated MAPK cascade represents a molecular link between oxidative stress and the plant growth hormone auxin (Kovtun et al. 2000). In this scenario, NO may also collaborate with ROS to repress auxin-inducible promoters. Increased NO accumulation in cue1/nox1 mutant, where endogenous NO levels are enhanced, depletes auxin-dependent reporter expression in the apical auxin maximum (Fernández-Marcos et al. 2011).

4.3 RNS Regulation of Auxin Signaling

The synergistic effects of auxin and NO have been well characterized in the regulation of a variety of physiological processes of plants. One of the best described NO functions in plants is their involvement in the auxin-regulated signaling cascades determining root growth and morphology. During the last decade it has been reported that NO is involved in the promotion of adventitious roots (Pagnussat et al. 2002), in primary root growth and lateral root formation (Correa-Aragunde et al. 2004; Fernández-Marcos et al. 2011), in root hair development (Lombardo et al. 2006), and in gravitropic responses (Hu et al. 2005).

The role of NO in root development and the cross talk with hormones such as auxin is an emerging area of study. We found that high levels of NO, released by NO donors or using NO overaccumulating mutants (cue1/nox1), produced a decrease in the primary root length by reducing root meristem size and cell division rates (Fernández-Marcos et al. 2011). As auxin gradients are important factors in the regulation of these processes, the spatial pattern of the auxin response reporter DR5 pro :GUS/GFP after NO treatment and in the cue1/nox1 background was analyzed, showing an alteration in the root apical auxin maximum. NO also reduces elongation of root cells (Fernández-Marcos et al. 2012). It has been reported that attenuation of auxin transport and signaling delayed gibberellin (GA)-induced RGA (a DELLA protein) degradation (Fu and Harberd 2003), and as a consequence the NO-inhibition of elongation in the elongation-differentiation zone (EDZ) could be due to the promotion of DELLA activity and, consequently, PIN1 degradation in the presence of high levels of NO (Fernández-Marcos et al. 2012).

Consistent with robust changes in signaling and transcription, numerous NO-regulated genes have been identified. These genes are involved in different functional and biological processes (Huang et al. 2002; Polverari et al. 2003; Parani et al. 2004; Palmieri et al. 2008). However, the direct molecular targets of NO remain poorly documented in plants. Only a few intracellular S-nitrosylated proteins have been identified in plants (Astier et al. 2011; Lindermayr et al. 2005; Tanou et al. 2009). A recent and promising example is the NO-mediated modulation of auxin signaling through posttranslational modification of the TIR1 auxin receptor. S-nitrosylation of TIR1 promotes its interaction with Aux/IAA repressors, thereby facilitating their degradation (Fig. 2; Terrile et al. 2012).

5 Conclusions

In summary, ROS/RNS are important regulators of auxin-dependent growth and development through their effects on several distinct aspects of auxin biology. Recent reports indicate that auxin transport is perturbed in mutants affected in ROS/RNS homeostasis and/or under treatments to produce a burst of ROS/RNS. These results collectively suggest the existence of a regulatory loop between ROS and auxin transport with profound implications for a broad array of signaling processes (Fig. 4).

Fig. 4
figure 00054

The schematic diagram shows how ROS and NO impact auxin signaling and response by affecting its biosynthesis and distribution. (3) Joo et al. (2001); (5) Kawano (2003); (1) Hu et al. (2005); (4) Wang et al. (2010); (7) Astier et al. (2011); (6) Blomster et al. (2011); (8) Cheng et al. (2011); (2) Fernández-Marcos et al. (2011); (9) Terrile et al. (2012)

Full size image