Apoplastic reactive oxygen species transiently decrease auxin signaling and cause stress-induced morphogenic response in Arabidopsis 1 .

Reactive oxygen species (ROS) are ubiquitous signaling molecules in plant stress and development. To gain further insight into the plant transcriptional response to apoplastic ROS, the phytotoxic atmospheric pollutant ozone (O 3 ) was used as a model ROS inducer in Arabidopsis ( Arabidopsis thaliana ) and gene expression was analyzed with microarrays. In contrast to the increase in signaling via stress hormones salicylic acid (SA), jasmonic acid (JA) and ethylene, ROS treatment caused auxin signaling to be transiently suppressed, which was confirmed with a DR5-uidA auxin reporter construct. Transcriptomic data revealed that various aspects of auxin homeostasis and signaling were modified by apoplastic ROS. Furthermore, a detailed analysis of auxin signaling showed that transcripts of several auxin receptors and Aux/IAA transcriptional repressors were reduced in response to apoplastic ROS. The ROS-derived changes in expression of auxin signaling genes partially overlapped with abiotic stress, pathogen responses and SA signaling. Several mechanisms known to suppress auxin signaling during biotic stress were excluded indicating that ROS regulated auxin responses via a novel mechanism. Using mutants defective in various auxin ( axr1, nit1, aux1, tir1 afb2, iaa28-1, iaa28-2 ) and JA responses ( axr1 , coi1-16 ), ROS induced cell death was found to be regulated by JA but not by auxin. Chronic ROS treatment resulted in altered leaf morphology, a stress response known as “stress-induced morphogenic responses” (SIMR). Altered leaf shape of tir1 afb2 suggests that auxin was a negative regulator of SIMR in the rosette. and analyzed with a linear mixed model having hierarchical random effects for plant and leaf within a plant. A logarithm of the data was taken before modeling to improve the linear model fit of the data.


INTRODUCTION
Environmental challenges activate a complex signaling network initiating processes that ultimately determine plant stress tolerance. Both abiotic and biotic stresses cause increased production of reactive oxygen species (ROS), such as superoxide (O . 2 -), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ) and hydroxyl radical (OH -). In addition to being hazardous by-products of metabolism, ROS are important ubiquitous signaling molecules with diverse roles depending on the specific ROS, their subcellular localization and the stress in question. Plants have evolved sophisticated antioxidant systems to cope with increased ROS concentrations, but interestingly, also possess enzymatic tools to themselves produce ROS for both intra-and inter-cellular signaling purposes (Mittler et al., 2011).
Apoplastic ROS can be produced by plasma membrane-localized NADPH oxidases (RESPIRATORY BURST OXIDASE HOMOLOGS; RBOHs) and by cell wall peroxidases in response to several pathogens (Torres 2010). ROS production by RBOHD is also induced also by heat, wounding, salt stress, high light and cold (Miller et al., 2009). The mechanisms by which cells sense extracellular ROS, leading to intracellular signaling, are not yet identified. The gaseous ROS, ozone (O 3 ), enters leaves through stomata, and degrades in the apoplast into O .

2
and H 2 O 2 , which also cause the activation of RBOHD and RBOHF (Joo et al., 2005;Vahisalu et al., 2010). Therefore, O 3 can be used to deliver a precise and controlled apoplastic ROS burst for the study signaling events shared by a multitude of stresses.
ROS-induced signaling is entwined with plant hormonal responses. Ethylene (ET) biosynthesis is an early O 3 -response, and later salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) are produced (Overmyer et al., 2005). ET and SA signaling promote enhanced ROS production and programmed cell death (PCD), which all together form a self amplifying loop. JA attenuates this cycle by reducing ROS production downstream of ET and cell death. This form of PCD has relevance to both abiotic stress symptom formation and resistance to biotic stress (Overmyer et al., 2000). ABA is important especially as the regulator of stomatal closure and O 3 entry (Vahisalu et al., 2008;Vahisalu et al., 2010;Brosché et al., 2010). Recently, also the connections between oxidative stress and the classical plant hormone auxin have gained attention. Defects in the antioxidative capacity of a thioredoxin and glutathione mutant resulted in altered auxin homeostasis and development (Bashandy et al., 2010). Iglesias et al. (2010) have shown that auxin receptor mutants were more tolerant to H 2 O 2 , methyl viologen (paraquat) and salinity stress. Suppression of auxin signaling mediates pathogen tolerance via SA-auxin antagonism (Wang et al., 2007) or pathogen-inducible microRNA393 (miR393), which targets several auxin receptors for degradation (Navarro et al., 2006). Expression of auxin responsive genes is decreased by H 2 O 2 treatment, via MAP (MITOGEN ACTIVATED PROTEIN) kinase activation (Kovtun et al., 2000). Ultimately, prolonged stress exposure leads to altered growth patterns including more compact growth, reduced cell division and increased lateral growth (Potters et al., 2007;2009). This response termed "stress-induced morphogenic response" 8 expression peaked late (8h) and remained slightly elevated at 24h. Characteristic to this profile were biological processes related to ubiquitin-dependent protein degradation and various abiotic stress responses, such as response to salt and osmotic stress traditionally associated with the stress hormone ABA. Profile IV included the most rapidly regulated genes and had 74 enriched biological processes, which included several types of protein modifications (lipidation, myristoylation and phosphorylation), signaling and response to stress, response to bacterium, and regulation of immune responses. One of the first responses detected after bacterial infection or treatment of plants with PAMPs, such as flg22, is an apoplastic ROS burst (Torres 2010). The biological processes represented in profile IV suggest that the early ROS burst in bacterial infection and O 3 -derived ROS formation have a similar signaling role. Profiles II and III had somewhat lower expression and later peaks compared to profile IV. They were enriched in biological processes for secondary metabolism. Profile V included biological processes related to pollen and cell recognition. Recently ROS have emerged as regulators of pollen tube growth (Potocký et al., 2007). Profile VI contained genes with rapidly decreased expression that at 8h had returned to basal level. The biological processes in this group of genes related to ion homeostasis, developmental growth and auxin stimulus. Profiles VII, VIII and IX had decreased expression at 4 to 24 hours and included photosynthesis and other chloroplast-related processes. Additionally, profile VIII was enriched for processes related to glucosinolate metabolism.
Genes belonging to each expression profile and the GO enrichment results are available in Supplemental Table S1.

Time point analysis of biological processes affected by O 3 treatment
To further investigate O 3 -induced changes in gene expression, we analyzed biological processes enriched at each time point (Supplemental Table S2). Altogether 502 biological processes were enriched among the genes showing increased expression in O 3 -exposed plants, whereas 301 biological processes were enriched among genes with decreased expression (Supplemental Table S2). Within the set of genes exhibiting increased transcript levels, enrichment was seen consistently across all time points for 81 biological processes assigned to biotic and abiotic stresses, such as defense response, response to osmotic stress, response to oxidative stress, response to temperature stimulus and response to chitin (Supplemental Table S2). In contrast, for genes with decreased transcript levels, there was no biological process enrichment spanning all time points and only photosynthesis-related processes were enriched in four out of the five time points (Supplemental Table S2). Response to auxin stimulus and the partially overlapping process of cell morphogenesis were identified as the only enriched biological processes among genes with decreased transcript levels at 1h (Table I), consistent with profile VI (Fig. 1C). Consequently, GO categories associated with plant hormones were studied in more detail. The variation in the number of altered transcripts for hormone-responsive genes 9 throughout the time course was hormone-specific (Table I). ABA responsive genes were the largest group of hormone-related genes with elevated expression levels. Timing and direction of ET, SA and JA response activation was consistent with previous work (Overmyer et al., 2003). Novel hormone responses were also detected; the induction of brassinolide (BR) and gibberellic acid (GA) responses by ROS have not been previously reported. Auxin responsive transcripts were the largest group of hormone-regulated genes with decreased expression (Table I).

Promoter elements mediating the transcriptional response to apoplastic ROS
Promoter analysis was used to explore the role of cis-elements in the regulation of apoplastic ROS mediated gene expression. A list of confirmed Arabidopsis promoter elements from the databases Agris (Yilmaz et al., 2011), PlantCARE (Lescot et al., 2002), and PLACE (Higo et al., 1999, together with a few added elements from the literature (see Materials and Methods for details) was used for the analysis of 500 bp promoter fragments of genes regulated at each individual time point (Supplemental Table S3). The most abundant enriched element was the W-box element TTGAC (Supplemental Table S3), a target for WRKY transcription factors, which are key regulators of abiotic and biotic stress responses (Rushton et al., 2010). Due to the large number of enriched elements (48) and the redundancy in their regulatory sequence, a subset of promoter elements was chosen for a more detailed analysis within 500bp promoters of genes in profiles I-IX (Fig. 1C). The W-box was enriched in the fast and highly induced expression profiles III-V, which are enriched for response to various biotic stresses ( Fig. 1C-D). O 3 -exposure leads to elevated ABA concentration, particularly at the late time point 8h (Overmyer et al., 2008). The role of this increase in ABA has remained obscure. The promoter analysis revealed that profiles with expression changes at late time points (both increased and decreased expression) were enriched for the ABA response element (ABRE) and ABRE-like element . Consistent with this, profile I had biological processes enriched for salt and osmotic stress, which are regulated through ABA signaling. Overall this suggests that late responses in gene expression to apoplastic ROS could be regulated through increased ABA concentration and ABA signaling.

Transcriptional regulation of the auxin signaling pathway by apoplastic ROS
The transient decrease in the expression of auxin-related genes in O 3 -treated plants ( Fig. 1C and Table   I) prompted a more detailed study of the auxin signaling pathway. Of the TIR1-AFB auxin receptor gene family (Dharmasiri et al., 2005) TIR1, AFB1, AFB3, AFB5 had decreased transcript levels in response to O 3 ( Fig. 2A). Transcript levels for Aux/IAA (Auxin/Indole-3-Acetic Acid; IAA;Liscum and 1 0 Reed, 2002) genes were mainly decreased with two exceptions being IAA10 and IAA28 whose transcript levels transiently increased at 2h (Fig. 2B). The expression of genes encoding ARF transcription factor proteins (Guifoyle and Hagen, 2007), which mediate auxin-responsive gene expression via the Auxin Responsive Element (AuxRE), were only marginally affected by apoplastic ROS, in both directions with no consistent trends (Fig. 2C). Signaling downstream of ARFs was studied using the auxin-responsive synthetic promoter DR5, which contains seven repeats of AuxRE, fused to the uidA reporter gene (Ulmasov et al., 1997). The uidA transcript levels were monitored with real time quantitative RT-PCR (qPCR). An early decrease in DR5-driven uidA transcript abundance was consistently detected in O 3 -treated plants, similar to the expression pattern of the auxinresponsive marker gene HAT2 (Fig. 2D). The DR5-uidA and HAT2 expression levels partially recovered already at 4h, during the O 3 treatment, and were indistinguishable from the controls by 8h ( Fig. 2D). This indicates that these AuxRE-dependent transcripts are transiently reduced in response to O 3 treatment. In 3-week-old rosettes the DR5-driven accumulation of the β -glucuronidase (GUS) activity encoded by DR5-uidA was distinctly localized in hydathodes and young leaves consistent with the localization of auxin biosynthesis in leaves (Supplemental Fig. S2;Teale et al., 2006). O 3 treatment caused no change in the staining pattern as detected by histochemical GUS staining (Supplemental Fig. S2).
To further characterize the connection between O 3 -altered gene expression and auxin signaling, we identified auxin regulated genes from several publicly available microarray data sets, which utilized different auxin concentrations and time points (see Material and Methods for the data sets used). One hundred seventy nine genes were identified as regulated by auxin, and of these, 60 genes were at least two-fold regulated by both auxin and apoplastic ROS. Thirty six genes were regulated by auxin and apoplastic ROS in the same direction (increased expression by both treatments or decreased expression by both treatments), whereas 24 genes showed an inverse regulatory pattern (increased expression by one treatment type and decreased expression by the other). Several Aux/IAA and SAUR genes, the HAT2 transcription factor and the auxin efflux carrier PIN3 were among the 22 transcripts belonging to the latter category.  , 2005;Schlereth et al., 2010). Surprisingly, 33% of auxin-responsive genes did not contain an AuxRE, not even the more general AuxRE sequence (TGTCnC), in their 1 kb promoter. Because this group of genes also included seven putative targets of transcriptional activator ARFs (Fig. 3A), the promoters of these genes were checked for the presence of AuxREs even further upstream. Indeed the 3 kb of all these genes contain at least one copy of the AuxRE (data not shown). Genes regulated by ARFs were predominantly found among the genes induced by auxin and repressed by apoplastic ROS (Cluster II), possibly due to enhanced stability of a negative regulator i.e. Aux/IAA in O 3 -treated plants. However, targets of these ARFs were to a lesser extent also found in the other clusters ( Fig.   3A). mutant. Expression of the auxin receptors, TIR1, AFB1, AFB3 and AFB5, and the auxin inducible genes SAUR6, SAUR68 and HAT2 was studied by qPCR in Col-0, npr1, sid2, NahG and ein2. All the genes studied exhibited decreased expression levels 2h after the start of the O 3 treatment in Col-0 similar to the microarray results obtained (Fig. 3B). This response was not changed in ein2, npr1 or sid2 mutants, which suggests that neither ET or SA signaling, nor O 3 -induced SA biosynthesis were involved in this decline in expression (Fig. 3B). The decreased expression of auxin receptors, especially TIR1 and AFB5, was slightly compromised but not absent in NahG plants (Fig. 3B). (SALK_129988C), of IAA28 exposed to O 3 (Supplemental Fig. S6). No differences were found between IAA28 mutants and their respective controls. In conclusion, SA, miR393, IAA28, MPK3 and MPK6 signaling do not appear to be involved in O 3 mediated reduction in expression of auxin related genes.

Apoplastic ROS regulation of genes with increased expression
The expression of DR5-uidA and HAT2 in response to apoplastic ROS indicated a role for AuxRE and auxin signaling in regulation of genes with transient decreased expression (Fig. 2D). HAT2 belongs to cluster II (Fig. 3A) characterized by expression increased by auxin and decreased by ROS. In contrast, cluster III genes were increased by both treatments as well as the SA-analog BTH. These treatments 1 3 all increase expression of the marker gene GST6 through ocs and TGACG elements (Chen et al., 1996;Chen and Singh, 1999). The TGACG element is a target for TGA type transcription factors and NPR1 (Zhou et al., 2000). To test if genes in cluster III where regulated via NPR1, SA or ET, four genes from this cluster (PBP1, WRKY40, ZAT10 and ACS6) and GST6 were tested for apoplastic ROS regulation in ein2, sid2, npr1 and NahG (Fig. 3B). In clean air control plants SA, ICS1 and NPR1 were positive regulators and EIN2 a negative regulator of PBP1 and WRKY40. In response to O 3 the expression of PBP1 and GST6 was increased in npr1 compared to Col-0, indicating a negative role for SA signaling during O 3 -consistent with our previous model for regulation of O 3 gene expression (Wrzaczek et al., 2010). Transgenic NahG plants, which cannot accumulate SA, did not show the same O 3 induction indicating that a fine tuned balance between signaling pathways will influence the outcome of apoplastic ROS regulated gene expression.

Auxin biosynthesis, conjugation and transport in O 3 -response
The concentration of biologically active free IAA is regulated by a multilevel network of biosynthesis,  Table S2). To investigate the role of auxin in ROS-triggered PCD, the O 3 sensitivity was assayed in several auxin-related mutants that do not have gross leaf abnormalities, which could obscure the analysis of stress responses. Furthermore, double mutants were constructed between rcd1 and mutants representing different aspects of auxin biology (synthesis, transport and signaling). AXR1 (AUXIN RESISTANT1) encodes a RUB conjugating enzyme regulating the SCF (Skp1-Cullin-F-box) activity necessary for auxin signaling (del Pozo et al., 2002). The F-box proteins TIR1 and COI1 are receptors for auxin and the bioactive JA conjugate JA-Ile, respectively, and both of their SCF complexes are targets for AXR1 regulation (Xu et al., 2002;Lorenzo and Solano, 2005). Since axr1 is defective in both auxin and JA responses (Tiryaki and Staswick, 2002), the coi1-16 mutant, which is specifically defective in only JA responses, was used to dissect auxin and JA signaling events. Both axr1 and coi1-16 were O 3 sensitive, but axr1 had slightly higher levels of cell death, quantified as ion leakage (Fig. 5A). The rcd1 axr1 and rcd1 coi1-16 double mutants had additive effects on O 3 sensitivity compared to the parental lines suggesting that axr1 and coi1-16 affect different regulatory processes in PCD than rcd1 (Fig. 5A). Loss of auxin biosynthesis and influx in the nit1-3 (nitrilase1-3) and aux1-7 mutants, respectively, did not alter the ROS-induced cell death as single mutants in the tolerant Col-0 background, or as double mutants in the sensitive rcd1 background (Fig. 5A). Furthermore, the auxin receptor double mutant tir1-1 afb2-3 and IAA28 mutants were not sensitive to an acute O 3 exposure (data not shown). In addition to their ROS phenotypes, both axr1 and rcd1 have altered leaf morphology; interestingly rcd1 axr1 plants were smaller than either of the parental lines whereas rcd1 coi1-16 had a habitus similar to rcd1 (Fig.   5B). Thus, cell death and leaf morphology experiments differentiated between axr1 and coi1-16, suggesting that JA signaling is predominately involved in the observed cell death responses, and auxin signaling in the developmental responses.

ROS induced morphological responses
Chronic exposure to elevated O 3 causes biochemical changes, reduced growth and morphological changes in several plant species (Skärby et al., 2004;Li et al., 2006;Karnosky et al., 2007;Kontunen-Soppela et al., 2010;Street et al., 2011). Similarly, chronic exposure to several other stresses causes similar growth changes. These responses are adaptive, genetically programmed and have recently been referred to as the stress-induced morphological response (SIMR; Potters et al., 2007;2009 1 5 plants was monitored in chronic O 3 exposure to determine the role of auxin responses in ROS induced SIMR. Changes in leaf morphology and the relative growth rate were used as indexes of SIMR. The auxin insensitive receptor double mutant tir1-1 afb2-3 was used for these studies together with two mutant alleles of IAA28, an O 3 -inducible Aux/IAA gene (Figs. 2B, 3A and S6). All genotypes responded to prolonged O 3 -treatment with a significantly decreased growth rate compared to their respective clean air controls (Fig. 6A). The tir1 afb2 mutant exhibited a smaller rosette size and slower growth rate compared to Col-0 both under clean air control and O 3 -exposed conditions (Fig.   6A). In addition to decreased growth all genotypes exhibited changes in leaf morphology, which were documented photographically (Figs. 6 B-E) and quantified using the LAMINA leaf shape analysis software (Bylesjö et al., 2008;Figs 6 F-H). The typical O 3 response was epinastic curling in the leaf margin, as exemplified by Col-0 ( Fig. 6B and 6D) and quantified in the leaf area parameters (Fig. 6F).
This phenotype was a specific O 3 response, as it was not present in the clean air controls. Similar to the other genotypes, the tir1 afb2 mutant exhibited curling in the leaf margins(Figs. 6Eand 6F); additionally, epinastic curling on the leaf tip and curvature of the mid vein were seen (Fig. 6E). Tip curling was quantified as a reduced leaf length (Fig. 6G) . Furthermore, tir1 afb2 was also unique in that its leaf morphology phenotypes were subtly present already in the clean air controls (Fig. 6C), but to a much lesser extent than in O 3 -treated plants. This phenotype was quantified in the mean indent width parameter (Fig. 6H). The assay used here to quantify rosette size may not report a true rosette diameter for tir1 afb2 due to its leaf phenotype. The reduced rosette size and growth rates reported in figure 6A for tir1 afb2 are likely artificially underestimated due to leaf tip curling (Figs. 6E, G). In support of this view, end point rosette size determinations of tir1 afb2 were significantly smaller than Col-0 while fresh weight was not (data not shown). Therefore the tir1 afb2 growth rate was likely not different from Col-0.
We conclude that chronic O 3 decreased growth rate independent of auxin signaling. ROS dependent growth control in the root has been previously shown to be independent of auxin (Tsukagoshi et al., 2010). In contrast SIMR, measured as altered leaf morphology, was exaggerated in O 3 treated tir1 afb2 mutant plants, suggesting that O 3 -induced SIMR is auxin regulated. Supplemental Tables S1 and S2).
The early response to apoplastic ROS consisted mainly of genes with increased expression and belonged to GO categories which represent signaling and defense (Supplemental Table S2).
Interestingly, the GO category "regulation of transcription" was significant only at late time points, suggesting that the early changes in O 3 -induced gene expression were executed by preexisting components regulating the transcriptional response. Genes with a peak in expression at eight hours

Apoplastic ROS rapidly decreases auxin signaling
Several hormones are involved in regulation of apoplastic ROS responses (Overmyer et al., 2003).
Analysis of GO categories for each of the major plant hormones showed significant enrichment of genes related to SA, JA, ET, BR and ABA (Table I) in ROS induced genes. In contrast, the GO categories for GA and IAA (also BR) were enriched among genes with decreased expression (Table   I). This suppression of genes belonging to the GO category "response to auxin stimulus" together with recent findings showing that auxin and ROS are regulators of plant development during stress (Potters et al., 2007;2009;Tognetti et al., 2011) prompted us to study auxin signaling in more detail. The 1 7 highly sensitive auxin reporter construct DR5-uidA indicated a four-fold decrease in auxin response already at 1h of O 3 treatment (Fig. 2D). It is possible that this results from a decrease in auxin concentration due to decreased biosynthesis or increased inactivation via conjugation or even direct auxin oxidation during oxidative stress (Normanly, 2010). This was addressed by auxin measurements and the method used is mass specific, i.e. detects the free active form but not conjugated or oxidized auxins. The concentration of free active auxin did not change, suggesting auxin homeostasis is not involved. However, since only one form of auxin was measured from whole rosettes, the possibility of cell type specific changes and increased auxin flux, i.e. simultaneous increases in both biosynthesis and in activation, cannot be excluded. Alternatively, the rapid decrease in auxin response mentioned  1 8 would lead to decreased output from the auxin signaling pathway (Fig. 7). Furthermore, this mechanim would inhibit de novo synthesis of Aux/IAAs and consequently allow auxin signaling to return to pre-stress levels, as was observed with DR5-uidA and HAT2 expression (Figs. 2D and 7).
Apoplastic ROS could also directly regulate the activity or localization of ARFs via yet unknown mechanims.

Auxin-responsive genes are targeted by several stresses
The mechanism by which apoplastic ROS affect auxin responses was further studied by identification of auxin-responsive genes from several publically available microarray experiments (Paponov et al., 2008) and comparing them to our set of O 3 -regulated genes by hierarchical clustering. Although some discrepancy might be brought into the comparison by the differences in plant age (i.e. auxin array experiments have been performed on seedlings vs. three-weeks-old plants used in the O 3 array experiment) and tissue types (whole plant vs. rosette only), it still gave an indication of which processes are regulated both by auxin and ROS. Approximately 30% of auxin-regulated genes were also regulated by O 3 and several Aux/IAA genes were coordinately regulated by several stresses together with apoplastic ROS (Fig. 3A). The commonly occurring AuxRE is found in approximately 25% of Arabidopsis 500 bp promoters (Keilwagen et al., 2011). Auxin-responsive genes have a variable number of AuxRE elements in their 2kb promoters (Lee et al., 2009), and in our analysis they were absent in the 1kb promoters of some auxin responsive genes (Fig. 3A). This may have complicated the promoter element analysis and therefore explain the lack of AuxRE enrichment in the expression profile VI containing genes classified as auxin responsive (Fig. 1C-D). However, due to the decrease in DR5-uidA expression (Fig. 2D), ARF function must at some point be altered by apoplastic ROS. ARFs can be classified into transcriptional activators and repressors according to their amino acid sequence, but the molecular function of individual ARFs is still largely unexplored.
No major changes in expression of 16 ARFs (including all the activator ARFs) were found (Fig. 2C  increased expression by auxin; and contained many genes with a role in auxin signaling, Aux/IAAs and SAURs. In contrast, cluster III included genes with increased expression by both auxin and apoplastic ROS; the annotation of these genes revealed no obvious link to auxin signaling and no enrichment of the AuxRE element. The stress responsive marker gene GST6 has increased expression by auxin, SA and H 2 O 2 , and is regulated through ocs and TGA elements (Chen et al., 1996;Chen and Singh, 1999;Zhou et al., 2000). The similarity between GST6 expression and cluster III prompted to us to study the expression of several genes in response to O 3 in SA and ET mutants (Fig. 3B).
Transcripts analyzed included transcription factors (ZAT10 and WRKY40), ethylene biosynthesis (ACS6), pinoid binding protein (PBP1) and GST6. Already in clean air some differences were observed between the mutants; decreased expression of WRKY40 and PBP1 in sid2, npr1 and NahG, and in contrast higher expression of these genes in ein2. This indicates that SA signaling is a positive regulator of these genes, and ET a negative regulator in non-stressed conditions. Interestingly, the role of SA appears to be reversed during O 3 treatment, and PBP1 had increased expression in npr1 relative

Hormone Interactions
So far, the discussion of auxin signaling has focused on mechanisms within the classic auxin signaling pathway (Fig. 7). These processes, of course, do not define a simple linear pathway, rather they are connected in a complex web of interacting hormone signaling pathways. In response to apoplastic ROS, there is clear evidence for the involvement of phytohormones other than auxin (Table I) which might lead to long term developmental alterations; consequently we studied the SIMR of plants chronically exposed to O 3 . The chronic exposure to apoplastic ROS altered plant morphology which was enhanced in the auxin receptor tir1 afb2 double mutant (Fig. 6). This is in contrast to previous studies that report an attenuated SIMR response in auxin receptor mutants (Iglesias et al., 2010;Zolla et al., 2010). However, previous SIMR studies have been performed in the plant root, a tissue in which many auxin effects are the opposite of those seen in the shoot. Thus, auxin appears to act as a negative regulator of SIMR in the shoot (Fig. 7). While auxin is involved in the regulation of stress morphology, it had no effect (Fig. 6A)  Collectively, ROS-auxin interactions are observed at two biological processes, gene expression and SIMR, which may be connected to each other (Fig. 7). Apoplastic ROS led to a rapid transient decrease in AuxRE driven expression as exemplified by DR5-uidA and HAT2. We propose that the entry point for ROS in the auxin signaling pathway is through stabilization and/or degradation of Aux/IAAs (Fig. 7). In particular, two Aux/IAA genes, IAA10 and IAA28, were transcriptionally induced by O 3 and they could mediate decreased expression of AuxRE containing genes by specific interactions with repressor ARFs. Mutant plants with altered IAA28 protein function did not exhibit any change in their SIMR or HAT2 gene expression response, suggesting that IAA28 is not involved in regulating these processes (Figs. 6A and S6). However, IAA10 may be functionally redundant with IAA28 and studies with iaa28 iaa10 double mutants will be required to fully address the involvement of these proteins. What other support exists for this model of ROS-auxin interaction? The auxin transport inhibitor TIBA gave a strikingly similar result to O 3 in regulation of auxin responsive genes ( Fig. 3A), indicating that re-distribution of auxin is a regulator for this set of genes. This similarity between ROS and TIBA is not restricted to gene expression as it is also observed in SIMR (Pasternak et al., 2005).
Plant survival in a changing environment requires adaptation to the prevailing conditions. The use of ROS in combination with auxin may provide plants with an elegant mechanism to optimize plant performance during acute and chronic stresses.
www.plantphysiol.org on August 23, 2017 -Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.

Plant material and O 3 treatment
For microarray experiments, wild-type Columbia (Col-0) and rcd1-1 seeds were sown on 1:1 peatvermiculite mixture, stratified for 2 days and grown in controlled environment chambers (Weiss were taken against a dark background, which was digitally made uniformly black. The axr1, aux1-7, nit1-3 and iaa28-2 (SALK_129988C) mutants were obtained from the European Arabidopsis Stock Centre (http://arabidopsis.info/) and the coi1-16, tir1-1 afb2-3 and iaa28-1 mutants were kind gifts from John Turner, Mark Estelle and Bonnie Bartel, respectively. Double mutants were constructed with rcd1 or coi1-16 as the pollen acceptor. Double mutants were initially screened for the visible rcd1 phenotype (curly leaves and compact rosette) or methyl-jasmonate insensitive root growth of

3
repeats, data not shown). GUS staining was performed according to Weigel and Glazebrook (2002).
IAA measurements of O 3 -treated Col-0 samples harvested in liquid nitrogen 0, 2, 4 and 8h after start of O 3 -exposure were repeated twice with similar results. Hormones were extracted and quantified with the vapor-phase extraction method described by Schmelz et al. (2004). GC-MS analysis was performed on a Agilent 6890N/5973N with G1088B electronics upgrade as a splitless injection in a single ion monitoring mode as in Montesano et al. (2005). Ions 130, 135, 189, and 194 et al., 2008). A matching gene was defined to be either an ungapped perfect match of >37 nucleotides or a perfect match of >44 nucleotides with one gap. If several genes fulfilled the criterion, the oligo was termed "ambiguous", if no matches were found the oligo was labelled "nomatch". Altogether the remapping resulted in 21071 genes, 2634 ambiguous oligos and 738 nomatch oligos. Only oligos binding specifically to a single gene were used in the further analysis.

Microarray data analysis
Microarray data preprocessing and analysis was carried out using scripts in R, version 2.12.1. For clustering of expression profiles, pairwise distances between the time series of differentially expressed genes were computed as in Toh and Horimoto (2002). The pairwise distance matrix was then clustered using affinity propagation (Frey and Dueck, 2007), implemented in R package apcluster. The algorithm was initialized with 10 random initializations with self similarity of -21.10333. The solution giving the best net similarity was selected.

Preprocessing of the microarrays was carried out as in
Gene ontology (GO) enrichment analysis was carried out for the differentially expressed genes clustered in each of the expression profiles using annotation of TAIR10 (ftp://ftp.arabidopsis.org/home/tair/Ontologies/) and scripts in R. The enrichment was analyzed using the Fisher Exact test. A Benjamini-Hochberg false discovery rate correction of the p-values was applied for each GO category. GO enrichment was performed separately for differentially expressed genes at each time point.

Real-time quantitative RT-PCR
Verification of the microarray results and additional gene expression analysis was performed with qRT-PCR. RNA was isolated and treated with DNaseI as in Jaspers et al. (2009). Reverse transcription was performed with 5 µg of RNA with RevertAid Premium RT and Ribolock Rnase inhibitor (Fermentas) and the reaction diluted to the final volume of 200 µl. qRT-PCR was performed in triplicate using 1µl cDNA template per reaction with primers, iQ TM SYBR GREEN supermix (Bio-Rad) and water. The cycle conditions with Bio-Rad CFX were: 1 cycle initiating with 95°C 10 min, 39 cycles with 95°C 15s, 60°C 30s, 72°C 30s and ending with melting curve analysis. Primer sequences and amplification efficiencies determined with the Bio-Rad CFX Manager program from a cDNA dilution series are given in Supplemental Table S4. The raw C(t) values were normalized to ACTIN2 (At3g18780). Fold changes and p-values were computed with scripts in R using linear mixed models. Based on likelihood ratio test statistic, a random effect for each biological repeat was incorporated (a p-value <0.05 was considered significant), otherwise a standard linear model was used. Contrasts were computed with multcomp package (Bretz et al., 2010), with single-step p-value correction.
www.plantphysiol.org on August 23, 2017 -Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Undiluted cDNA was used for miR393 quantification with primers in Supplemental Table S4 and miR393 expression was normalized to ACTIN2.

Promoter analysis
For promoter analysis, the 500 bp, 1000 bp and 3000 bp promoter sequences of TAIR10 were Enrichment of motifs was determined with Fisher exact test.

8
IPA=indole-3-pyruvic acid; IAAld=indole-3-acetaldehyde; IG=indole-3-methylglucosinolate; IAOx=indole-3-acetaldoxime; IAM=indole-3-acetamide; IAA=indole-3-acetic acid; IAN=indole-3acetonitrile. All the verified (after Normanly 2010) and putative genes involved in the IAA conjugation (GH3 family) and hydrolysis are shown. B) The concentration of free IAA was quantified from Col-0 plants at 0, 2, 4 and 8 hours after the onset of O 3 exposure (350 nL L -1 , 6 hrs). The experiment was repeated twice, one representative experiment is shown. Error bars depict standard deviation, n=4-5. C) Expression of auxin efflux carriers is reduced by apoplastic ROS. D) Auxin influx carriers show a slight decrease in response to apolastic ROS.  iaa28-1 plants were exposed to O 3 daily (6h, 350 nL L -1 ). For growth rate analysis rosette diameter was determined by finding the minimal circle that contained all leaves using ImageJ image analysis software (http://rsbweb.nih.gov/ij/) before and after 7 days of O 3 exposure. Relative growth rate was calculated by fitting a linear model to plant size (see Material and methods for details). B-E) 4-weeksold control clean air and ozone exposed plants: Under control clean air conditions Col-0 (B) are larger than tir1 afb2 plants (C) which have constitutively a smaller size and slightly curled leaves. After 2weeks of O 3 exposure, Col-0 exhibits stunted growth and curled leaves (D). Leaf curling in response to O 3 is more prominent in tir1 afb2 plants (E). Scale bars = 1cm. Leaf shape parameters, area (F), length (G), and mean indent width (H) were quantified using the LAMINA software (Bylesjö et al., 2008). All experiments were repeated twice with similar results, and the data analyzed with linear models. Bars = mean relative growth rate, error bars = standard deviation of the linear model. n=6. Supplemental Figure S3. Expression of pre-mir393a (left) and pre-miR393b (right) in response to apoplastic ROS was determined with qPCR. Pre-mir393a was not O 3 -responsive, whereas pre-miR393b expression was decreased both 1h and 2h after the start of O 3 exposure (350 nL L -1 ). In NahG plants this decrease was slightly lower than in Col-0. Averages (log 2 ratios) of 4 biological repeats are shown, error bars depict standard deviation.
Supplemental Figure S4. Expression of miR393 was quantified with qPCR after RT reaction with a miR393-specific stem-loop primer. cDNA was synthesized from the same RNA samples also with the standard oligo-dT and used for TIR1 expression analysis. Expression levels of miR393 and TIR1 were normalized to ACTIN2 from the oligo-dT-primed cDNA. RNA was isolated with TriReagent following the manufacture´s protocol. Figure S5. Expression of HAT2, SAUR68 and TIR1 in Col-0, mpk3 and mpk6 plants after 2h O 3 -treatment (350 nL L -1 ) was determined with qPCR. O 3 -treatment decreased TIR1, HAT2

Supplemental
and SAUR68 expression levels in Col-0, mpk3 and mpk6 in a similar way. There were no differences between the genotypes in clean air (data not shown). The experiment was repeated twice with similar results, a single representative experiment is shown.  Table I. Plant hormone responses are elicited by apoplastic ROS. The number of apoplastic ROS regulated genes belonging to plant hormone response categories (ABA, abscisic acid; BR, brassinolide; CK, cytokinin; ET, ethylene; GA, gibberellic acids; IAA, indole acetic acid (auxin); JA, jasmonic acid; and SA, salicylic acid). Gene ontology (GO) enrichment analysis was performed for genes with increased (log 2 ratio 1; q<0.05) or decreased expression (log 2 ratio -1; q<0.05) in O 3treated Col-0. The enrichment was calculated separately for each time point (0, 1, 2, 4, 8 and 24 hours), and no enriched processes were present in 0h samples (data not shown). Statistically significant (Q< 0.05) number of O 3 -responsive genes annotated to a given hormone response category is depicted with asterisk (*).  Table S1. Genes with statistically significant change of expression (q<0.05) in O 3 -treated plants. At least two-fold regulated genes were analyzed for overlap between time points, expression profiles (I-IX) and GO enrichment.               Figure 6. Chronic reduces plant growth. A) 2-weeks-old Col-0, iaa28-2, tir1 afb2, Ws-0 and iaa28-1 plants were exposed to daily ( ). For growth rate analysis rosette diameter was determined by finding the minimal circle that contained all leaves using ImageJ image analysis software (http://rsbweb.nih.gov/ij/) before and after 7 days of exposure. Relative growth rate was calculated by fitting a linear model to plant size (see Material and methods for details). B-E) 4-weeks-old control clean air and ozone exposed plants: Under control clean air conditions Col-0 (B) are larger than tir1 afb2 plants (C) which have constitutively a smaller size and slightly curled leaves. After 2-weeks of exposure, Col-0 exhibits stunted growth and curled leaves (D). Leaf curling in response to is more prominent in tir1 afb2 plants (E). Scale bars = 1cm. Leaf shape parameters, area (F), length (G), and mean indent width (H) were quantified using the LAMINA software All experiments were repeated twice with similar results, and the data analyzed with linear models. Bars = mean relative growth rate, error bars = standard deviation of the linear model. n=6.  . Auxin signaling pathway is modulated by apoplastic ROS. The binding of auxin to TIR/AFB receptors leads to degradation of AUX/IAA repressors via the 26S proteasome allowing activation of ARF transcription factors and changes in gene expression. O treatment leads to production of apoplastic ROS which could 3 suppress the auxin pathway by decreasing expression of TIR1/AFBs independently from miR393 and SA. O 3 may affect stability of Aux/IAAs (for example IAA10 and IAA28) or ARF activity could be directly modulated independently from auxin F-box proteins. ARFs regulate auxin-dependent gene expression, which includes Aux/IAA transcripts. Decreased levels of Aux/IAA transcripts provide a feed-back mechanism counteracting the increased Aux/IAA stability. In addition, apoplastic ROS modulated expression of several genes involved in auxin homeostasis (auxin signaling, polar transport and biosynthesis). Chronic O exposure leads to SIMR 3 which is under negative regulation of the auxin signaling pathway.