The Role of Reactive Oxygen Species in Hormonal Responses

Reactive oxygen species (ROS) are versatile molecules mediating a variety of cellular responses in plant cells, including programmed cell death (PCD), development, gravitropism, and hormone signaling. A picture showing how ROS function in signal transduction networks has started to emerge as the result of recent studies providing genetic, cell biological, and physiological evidence describing roles for ROS in signaling (Apel and Hirt, 2004; Laloi et al., 2004; Mittler et al., 2004; Mori and Schroeder, 2004). However, further efforts are necessary to characterize the targets and molecular functions of ROS, as well as the complex interplay of ROS-generating and ROS-scavenging mechanisms. Moreover, the interactions of nitric oxide with other ROS species in hormone signaling is a subject of interest (Desikan et al., 2004; Wendehenne et al., 2004; Guo and Crawford, 2005; Bright et al., 2006). Due to limited space, in this Update article we focus on recent progress made in understanding the roles of ROS in hormone signaling.

AUXIN, ETHYLENE, AND ROS

ROS have been implicated as second messengers in several plant hormone responses. Joo et al. (2001) showed that ROS are asymmetrically generated in roots by gravistimulation to regions of reduced growth. A function for ROS in root curvature was reported by inhibiting cell growth, thus contributing to tropisms. Auxin also induced ROS production in roots and the auxin transport inhibitor N-1-naphthylphthlamic acid did not inhibit hydrogen peroxide (H₂O₂)-induced root curvature, leading to the suggestion that ROS play a role downstream of transport in auxin signaling and gravitropism (Joo et al., 2001).

A pharmacological study suggested that ethylene and ROS are required for root nodule initiation and function as positive transducers downstream of the Nod factor response in a semiaquatic legume (D'Haeze et al., 2003). Additional studies have suggested roles of ethylene in either stomatal opening or closing, depending on the plant species (Giulivo, 1986). Recently, it was reported that H₂O₂-mediated stomatal closure is completely disrupted in the loss-of-function mutant of the ethylene receptor etr1-7, suggesting a role for ETR1 in guard cell ROS signaling and stomatal closure (Desikan et al., 2005). Interestingly, in another recent study, ethylene was proposed to counteract stomatal closure (Tanaka et al., 2005). Abscisic acid (ABA)-induced stomatal closure was inhibited by ethylene or the hormone precursor 1-aminocyclopropane-1-carboxylic acid and by the ethylene-overproducing mutation eto1-1 (Tanaka et al., 2005). Moreover, this ethylene-induced inhibition of stomatal closure was suppressed in two ethylene-insensitive mutants, the dominant etr1-1 allele, and ein3-1 (Tanaka et al., 2005). Reverse transcription-PCR analysis with guard cell-enriched epidermal strips showed that ETR1 is expressed in guard cells (Desikan et al., 2005). ATH1 and AG oligonucleotide-based (Affymetrix) microarray analyses of guard cell-expressed genes suggest that at least the ETR1, ERS1, and EIN4 ethylene receptor genes are expressed in guard cells, with ERS1 apparently showing the highest expression level in these experiments (Leonhardt et al., 2004; Heggie and Gray, 2005; J. Kwak, N. Leonhardt, Y. Yang, and J. Schroeder, unpublished data). As these receptors share overlapping redundant negative regulator functions (Hua and Meyerowitz, 1998), it would be interesting to analyze stomatal responses in etr1ein4, etr1ers1, and ers1ein4 double mutants in addition to the recessive loss-of-function etr1-7 line (Desikan et al., 2005). The dual functions of ethylene receptors proposed in these two studies—transduction of stomatal closure and inhibition of ABA-induced stomatal closure in Arabidopsis (Arabidopsis thaliana)—will require further analyses (Desikan et al., 2005; Tanaka et al., 2005).

ROLES OF ROS IN GA₃ SIGNALING IN THE ALEURONE LAYER AND CELL DEATH

ROS have been shown to play a central role in PCD of barley (Hordeum vulgare) aleurone cells, which offer a well-developed system for studying cell biological and physiological functions of GA₃ responses. GA₃ initiates cell death of aleurone cells, whereas ABA inhibits cell death (Wang et al., 1996; Appleford and Lenton, 1997). H₂O₂ was shown to represent a major reactive oxygen leading to cell death in aleurone cells (Bethke and Jones, 2001). Furthermore, transcript levels and activities of ROS-scavenging enzymes, including catalase, ascorbate peroxidase, and superoxide dismutase, were significantly down-regulated in GA₃-treated aleurone cells, thus rendering these cells sensitive to oxidative damage and cell death, whereas ABA caused increases in catalase activity and CAT2 mRNA (Fath et al., 2001). Aleurone cells are devoid of functional chloroplasts (Jones, 1969), one of the ROS sources in plant cells. In aleurone cells, mitochondria are abundant and a cyanide-insensitive electron transport pathway switches to a cyanide-sensitive one in response to GA₃ that may contribute to ROS production (Fath et al., 2002). In addition, a reduction in the number of lipid-storing oleosomes correlates with increases in the enzyme activities of the glyoxylate cycle in aleurone cells, suggesting that mitochondria and glyoxysomes are major sources of ROS in aleurone cells (Fath et al., 2002).

ROS also play an important signaling role as regulators of PCD in response to pathogens (Levine et al., 1994). Two Arabidopsis NADPH oxidase genes, AtrbohD and AtrbohF, were reported to be a major source of pathogen-induced ROS generation (Torres et al., 2002). Interestingly, atrbohD/atrbohF double mutants showed reduced cell death in response to a bacterial pathogen, but enhanced cell death in response to a fungal pathogen. These opposite responses may derive from interaction with salicylic acid. Torres et al. (2005) reported that ROS produced by NADPH oxidases antagonize salicylic acid and suppress cell death in cells that are more distantly located from the cells at the site of infection. The cells at the site of infection undergo hypersensitive cell death. These results indicate that ROS play dual functions in both driving and suppressing PCD in different contexts in the pathogen response.

ROS AND ABA SIGNALING

Research on ABA signal transduction has characterized cell biological and genetic mechanisms upstream and downstream of ROS production, and we therefore describe these in further detail in this article. ROS were shown to induce increases in cytosolic Ca²⁺ and stomatal closure (McAinsh et al., 1996; Lee et al., 1999). Oligogalacturonic acid and chitosan treatments caused H₂O₂ production in guard cells, resulting in stomatal closure in tomato (Lycopersicon esculentum) and Commelina. These responses were suppressed by EGTA, catalase, and ascorbic acid (Lee et al., 1999). ABA activation of plasma membrane Ca²⁺-permeable channels contributes to ABA-induced cytosolic Ca²⁺ increases in guard cells of Vicia and Arabidopsis (Hamilton et al., 2000; Pei et al., 2000), together with organellar Ca²⁺ release and Ca²⁺-independent mechanisms (Schroeder et al., 2001; Hetherington and Woodward, 2003; Fan et al., 2004). Application of H₂O₂ to guard cells activates hyperpolarization-regulated Ca²⁺-permeable (I_Ca) channels and produces simultaneous cytosolic calcium elevations, and this activation is impaired in the ABA-insensitive gca2 mutant (Pei et al., 2000). ABA regulation of I_Ca channels requires cytoplasmic NADPH in whole-cell recordings of Arabidopsis guard cells (Murata et al., 2001). Furthermore, ABA enhances cellular ROS levels in Arabidopsis guard cells, in Vicia faba guard cells, and in maize (Zea mays) embryos (Guan et al., 2000; Pei et al., 2000; Zhang et al., 2001c). These studies define new ABA signal transduction events.

It was reported that H₂O₂ induces cytosolic alkalization in Vicia guard cells (Zhang et al., 2001a), whereas in another study, cytoplasmic alkalization preceded ROS production during stomatal responses to ABA and methyl jasmonate in Arabidopsis (Suhita et al., 2004). In V. faba guard cells, initial ABA-induced ROS increases were observed within 30 s of ABA application (Zhang et al., 2001c). The findings of cytosolic pH changes both before and after ROS may reflect the parallel and branched nature of the ABA-signaling network (Leung and Giraudat, 1998; Schroeder et al., 2001; Hetherington and Woodward, 2003; Fan et al., 2004). Nevertheless, further research into cellular ROS homeostasis and the timing of ROS production would be of interest and may require the development of time-resolved ratiometric reporters that show a high sensitivity to ROS, as have been developed for other second messengers (Allen et al., 1999; Zacharias et al., 2000; Zhang et al., 2002).

Ca²⁺-permeable channels are activated by hyperpolarization in guard cells, root hair cells, Fucus rhizoids, root epidermal cells, and mesophyll cells (Gelli and Blumwald, 1997; Hamilton et al., 2000; Kiegle et al., 2000; Pei et al., 2000; Véry and Davies, 2000; Coelho et al., 2002; Demidchik et al., 2003; Foreman et al., 2003; Stoelzle et al., 2003). ROS regulation of these channels has been found in several systems (Pei et al., 2000; Coelho et al., 2002; Demidchik et al., 2003; Foreman et al., 2003; for review, see Mori and Schroeder, 2004). An open question is whether ROS directly regulate Ca²⁺-permeable channels and/or upstream modulators (Mori and Schroeder, 2004). In vitro biochemical studies revealed that H₂O₂ directly inactivates the ABI1 and ABI2 type 2C protein phosphatase (PP2C) enzymes that function in ABA signaling (Meinhard and Grill, 2001; Meinhard et al., 2002). ABI1 and ABI2 function as negative regulators of ABA signaling (Merlot et al., 2001; Yoshida et al., 2006a). Knockout mutants for two other PP2Cs, PP2C-HAB and PP2CA, show strong hypersensitivity to ABA, showing that PP2C-HAB and PP2CA also negatively regulate ABA signaling (Leonhardt et al., 2004; Saez et al., 2004; Kuhn et al., 2006; Yoshida et al., 2006b). Thus, all four of these PP2Cs may be prime candidates as targets for ROS in mediating ABA responses.

ABA activation of Ca²⁺-permeable channels is disrupted in the two dominant PP2C mutants, abi1-1 and abi2-1 (Murata et al., 2001). ABA-induced ROS production and ABA activation of I_Ca channels is impaired in the abi1-1 mutant. The dominant abi2-1 mutation causes disruption of both H₂O₂ activation of Ca²⁺-permeable channels and H₂O₂-induced stomatal closing (Murata et al., 2001; Fig. 1). The ABA-activated protein kinase OST1 was identified to function upstream of ROS production in ABA signal transduction because the ost1 mutation disrupted ABA-induced ROS generation in guard cells (Mustilli et al., 2002; Fig. 1). OST1 is the Arabidopsis homolog of the Vicia ABA-activated protein kinase, AAPK (Li et al., 2000; Mustilli et al., 2002). Consistent with the disruption of ABA-induced ROS, increases in abi1-1 and ost1, but not abi2-1 (Murata et al., 2001; Mustilli et al., 2002), a recent study revealed that the ABI1 protein interacts with OST1 and the abi1-1 mutation, but not abi2-1 mutation, disrupts ABA activation of the OST1 protein kinase activity (Yoshida et al., 2006a). These data and the relatively strong ABA insensitivity of abi1-1 and ost1 mutants indicate that abi1-1 and OST1 may function upstream in the ABA-signaling network before the proposed branch points (Fig. 1).

Figure 1.

Simplified working model showing genetic interactions and putative functions and putative locations within the ROS-mediated ABA-signaling network in guard cells. Positive and negative regulators are shaded in green and red, respectively. PI3K, Phosphatidylinositol-3 kinase; PI4K, phosphatidylinositol-4 kinase.

An electrophysiological study provides evidence for a protein kinase-dependent and ROS-independent activation of I_Ca channels in V. faba guard cells (Fig. 1), showing that the PP1/PP2A protein phosphatase inhibitors, okadaic acid and calyculin A, enhanced I_Ca channel activity (Köhler and Blatt, 2002). In addition, phospholipids have been implicated in ABA-triggered cytosolic Ca²⁺ increases and stomatal closure (Staxen et al., 1999; Lemtiri-Chlieh et al., 2000). Treatment of epidermal strips with the phosphatidylinositol (PI)-3-kinase and PI-4-kinase inhibitors wortmannin and LY294002 inhibited PI kinase enzyme activities, enhanced stomatal opening, and impaired ABA-induced cytosolic Ca²⁺ increases, indicating that PI-3-P and PI-4-P modulate ABA signaling in guard cells (Jung et al., 2002). Furthermore, wortmannin and LY294002 inhibited ABA-triggered ROS production and stomatal closure, suggesting that PI-3-P (or products thereof) may function in the ABA-ROS-signaling branch in guard cells (Park et al., 2003).

Interestingly, outward K⁺ channels are inhibited by H₂O₂, which could cause inhibition of ABA and/or ROS-induced stomatal closure (Torsethaugen et al., 1999; Zhang et al., 2001b; Köhler et al., 2003). Similarly, ozone causes secondary ROS production in guard cells and inhibits outward and inward K⁺ channels (Torsethaugen et al., 1999; Joo et al., 2005). Ozone inhibits stomatal opening, but does not inhibit stomatal closing (Torsethaugen et al., 1999). The residual outward K⁺ currents after ROS application may be sufficient to function in K⁺ efflux during stomatal closing, as previous analyses have shown that roughly 25% of K⁺ channel activities are sufficient for physiological K⁺ fluxes during stomatal movements (Schroeder et al., 1984; Kelly et al., 1995; Kwak et al., 2001). In addition to inhibiting outward K⁺ channels, H₂O₂ and ozone inhibited inward K⁺ channels in guard cells, which correlates with ABA maintenance of stomatal closure (Torsethaugen et al., 1999; Zhang et al., 2001b; Köhler et al., 2003). Nevertheless, the dual down-regulation of both outward and inward K⁺ channels by ROS either implicates localized oxidative bursts in guard cells or reversible regulation of ROS-dependent protein modifications during signaling.

NADPH OXIDASES AND CELL CALCIUM SIGNALING

Plant cells have a diverse array of enzymatic mechanisms responsible for ROS production (Apel and Hirt, 2004; Mittler et al., 2004). Among those possible cellular mechanisms, NADPH oxidases have been suggested to function in ABA signaling (Pei et al., 2000; Murata et al., 2001; Jiang and Zhang, 2002, 2003). Genetic evidence for an important source of ABA-triggered ROS production in guard cells came from the analysis of two guard cell-expressed transmembrane NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF (Kwak et al., 2003). Analyses of stomatal movement responses show that ABA-induced stomatal closure is partially impaired in two independent alleles of the atrbohD/atrbohF double mutant (Fig. 2). Cellular events were impaired in atrbohD/atrbohF double-mutant guard cells, including ABA-induced ROS increases, ABA activation of I_Ca channels, and ABA-induced cytosolic Ca²⁺ increases (Kwak et al., 2003; Fig. 1). Exogenous application of ROS recovered I_Ca channel activation and stomatal closing in atrbohD/atrbohF double-mutant guard cells. The partial impairment in ABA-induced stomatal closing of atrbohD/atrbohF double mutants is consistent with models in which parallel pathways function in the early ABA-signaling network (Leung and Giraudat, 1998; Schroeder et al., 2001; Hetherington and Woodward, 2003; Fan et al., 2004; Figs. 1 and 2). These findings on atrbohD/atrbohF double mutants provide molecular genetic evidence for a function of ROS as a second messenger in ABA signaling in guard cells.

Figure 2.

ABA-induced stomatal closure in two double-mutant alleles of the NADPH oxidases atrbohD and atrbohF. ABA-induced stomatal closure is partially impaired in the two independent alleles of atrbohD/atrbohF double mutants. atrbohD/atrbohF-1 carries a dSpm transposon insertion in the fifth exon of AtrbohD (insertion D3) and a dSpm insertion in the first exon of AtrbohF (insertion F3; Torres et al., 2002). atrbohD/atrbohF-2 carries a dSpm insertion in the second intron of AtrbohF (insertion F5; Torres et al., 2002) and a T-DNA insertion in the fourth intron of AtrbohD (Salk JP65_4B03L). Double-blind experiments were performed in which the ABA concentration and the genotype were unknown. Error bars represent se of n = 3 experiments, 60 stomata per bar. Error bars are not visible when they are too small.

The AtrbohC NADPH oxidase was demonstrated to function in root hair growth and plays an important role in mediating the tip-focused Ca²⁺ gradient in Arabidopsis root hair cells (Foreman et al., 2003). Reactive oxygen also activated I_Ca-like channels in root hairs and mediated Ca²⁺ influx in the Fucus rhizoid, suggesting that ROS activation of Ca²⁺-permeable channels is a more general signaling mechanism in plants (Coelho et al., 2002; Foreman et al., 2003). In animal cells, NADPH oxidases are regulated by cytosolic components, including Rho small G proteins (for review, see Bokoch and Knaus, 2003). Recent studies identified Rho small G proteins as regulators of ROS-generating enzymes in plants too. Carol et al. (2005) demonstrated that a RhoGTPase GDP dissociation inhibitor functions upstream of ROS produced by AtRbohC NADPH oxidase during root hair growth. Dominant mutations in the small G-protein ROP2 were shown to regulate ROS generation and phosphatidic acid-induced cell death (Park et al., 2004). Moreover, Joo et al. (2005) showed that heterotrimeric G-protein α- and β-subunits are differentially involved in ozone-triggered oxidative stress responses.

Two plant enzymes, xanthine dehydrogenase and aldehyde oxidase, could provide sources for ROS production during water stress and/or ABA signaling (Yesbergenova et al., 2005). Interestingly, in contrast to their animal counterparts, xanthine dehydrogenase is capable of producing only O₂⁻, whereas aldehyde oxidase produced only H₂O₂ (Yesbergenova et al., 2005). A T-DNA insertional mutation in Arabidopsis xanthine dehydrogenase 1 showed loss of O₂⁻-producing activity. Furthermore, transcript levels of these enzymes were up-regulated upon ABA treatment and water stress (Yesbergenova et al., 2005). Additional ROS scavengers and/or ROS-producing enzymes are likely to function in ROS-signaling networks to ensure that a transient ROS burst does not trigger events that lead to cell damage and death.

ROS SIGNALING REQUIRES REGULATION OF ROS-SCAVENGING MECHANISMS

The findings that oxidative bursts function in various cellular signaling responses in plants, highlighted in this issue of Plant Physiology, suggest that ROS-scavenging mechanisms will be important for mediating and controlling these responses (Mittler et al., 2004), as illustrated in barley aleurone cells (Fath et al., 2001). For example, a strong oxidative burst will cause cellular damage and death (Mittler, 2002; Apel and Hirt, 2004). Furthermore, constitutive ROS elevations, even if not very high, could cause malfunction or desensitization of ROS-dependent signaling responses. Several studies suggest that ROS scavenger proteins play central roles in ABA signaling. Several ROS scavenger mRNAs are regulated in response to GA₃, ABA, and oxidative stress (Desikan et al., 2001; Fath et al., 2001; Vranova et al., 2002; Vandenabeele et al., 2003).

The cellular redox state influences diverse cellular functions and enzyme activities. An antisense suppression of catalase activity in transgenic tobacco (Nicotiana tabacum) plants resulted in increased ascorbate peroxidase and glutathione peroxidase levels and a 4-fold decrease in ascorbate (Willekens et al., 1997), a major H₂O₂-scavenging antioxidant in plant cells. Interestingly, transgenic tobacco plants in which both ascorbate peroxidase and catalase are suppressed were shown to be less sensitive to oxidative stress compared to single antisense plants suppressing either catalase or ascorbate peroxidase alone, suggesting that lack of H₂O₂-scavenging mechanisms might have turned on an alternative mechanism by which cells are protected (Rizhsky et al., 2002). A knockout mutation in ascorbate peroxidase 1 (APX1) caused the accumulation of H₂O₂ in Arabidopsis and decreases in the transcript levels of catalase and chloroplast superoxide dismutase and no significant changes in glutathione peroxidase transcript levels (Pnueli et al., 2003). Furthermore, stomates of the apx1 knockout plants neither closed in response to darkness nor opened in response to light (Pnueli et al., 2003). APX1 was also shown to play a role in protecting the chloroplast from oxidative damage (Davletova et al., 2005). Arabidopsis plants with increased dehydroascorbate reductase, which generates ascorbate from dehydroascorbate, showed an increased ascorbate redox state and a reduction in H₂O₂ levels in guard cells. Consistent with guard cell-signaling analyses, this resulted in increased stomatal conductance and reduced sensitivity to H₂O₂ and ABA (Chen and Gallie, 2004). This result suggests that ABA/ROS signaling in guard cells is controlled by the ascorbic acid redox state.

FUTURE PERSPECTIVES

ROS mediate diverse functions in a variety of cellular processes. Many exciting findings have revealed roles of ROS in hormonal responses in the past few years and many new questions arise. What are the downstream protein targets that are modified by ROS during signal transduction, enabling stimulus-specific cellular responses? Among those mechanisms responsible for ROS generation in plant cells, which combination of cellular mechanisms mediates ROS production for specific signaling cascades, and how do ROS producers and scavengers interact with each other to regulate cellular ROS levels? What are the roles for the other seven Atrboh genes whose functions are waiting to be unveiled? How is the enzyme activity of NADPH oxidases regulated in plants? Surely there is a lot more to come in this stimulating field.