Oligogalacturonide-Auxin Antagonism Does Not Require Posttranscriptional Gene Silencing or Stabilization of Auxin Response Repressors in Arabidopsis1[W] Daniel V. Savatin, Simone Ferrari, Francesca Sicilia, and Giulia De Lorenzo* Dipartimento di Biologia e Biotecnologie “C. Darwin,” Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Universita` di Roma, 00185 Rome, Italy a-1-4-Linked oligogalacturonides (OGs) derived from plant cell walls are a class of damage-associated molecular patterns and well-known elicitors of the plant immune response. Early transcript changes induced by OGs largely overlap those induced by flg22, a peptide derived from bacterial flagellin, a well-characterized microbe-associated molecular pattern, although responses diverge over time. OGs also regulate growth and development of plant cells and organs, due to an auxin-antagonistic activity. The molecular basis of this antagonism is still unknown. Here we show that, in Arabidopsis (Arabidopsis thaliana), OGs inhibit adventitious root formation induced by auxin in leaf explants as well as the expression of several auxin-responsive genes. Genetic, biochemical, and pharmacological experiments indicate that inhibition of auxin responses by OGs does not require ethylene, jasmonic acid, and salicylic acid signaling and is independent of RESPIRATORY BURST OXIDASE HOMOLOGUE D-mediated reactive oxygen species production. Free indole-3-acetic acid levels are not noticeably altered by OGs. Notably, OG- as well as flg22-auxin antagonism does not involve any of the following mechanisms: (1) stabilization of auxin-response repressors; (2) decreased levels of auxin receptor transcripts through the action of microRNAs. Our results suggest that OGs and flg22 antagonize auxin responses independently of Aux/Indole-3-Acetic Acid repressor stabilization and of posttranscriptional gene silencing.
Signaling for defense is tightly interconnected with the hormone response pathways that regulate plant growth and development. While ethylene (ET) and jasmonic acid (JA) are clearly integral components of signal transduction cascades leading to resistance, other hormones such as auxin show a more complex interplay with defense signaling (Bari and Jones, 2009). The observations that defense responses induced by a Phytophthora glucan preparation are inhibited by auxin in protoplasts (Leguay and Jouanneau, 1987) and that auxin-induced growth is competitively inhibited by elicitor-active oligogalacturonides (OGs) in pea (Pisum sativum) stem segments (Branca et al., 1988) provided, to our knowledge, the first evidence of an antagonistic action between this hormone and elici1 This work was supported by the Ministero dell’Universita` e della Ricerca (Programmi di Ricerca Scientifica di Interesse Nazionale [PRIN] 2007), the European Research Area (ERA-NET) Plant Genomics (grant no. RBER063SN4), the European Research Council (ERC Advanced Grant no. 233083), the Ministero delle Politiche Agricole e Forestali (PROTEO-STRESS 2007–2009), Institute Pasteur—Fondazione Cenci Bolognetti (2008–2010), and Universita` di Roma La Sapienza (ATENEO, 2006–2009). * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Giulia De Lorenzo (
[email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.111.184663
tors. More recently, the microbe-associated molecular pattern (MAMP) flagellin and its derived active peptide flg22, as well as the defense-associated hormone salicylic acid (SA) were shown to possess auxinantagonistic activity (Navarro et al., 2006; Wang et al., 2007). Perception of flg22 has been associated with the expression of the microRNA393 (hereafter miR393), which negatively regulates the expression of genes encoding the auxin receptors TRANSPORT INHIBITOR RESPONSE1 (TIR1), AUXIN F-BOX2 (AFB2), and AFB3, therefore stabilizing the Auxin/ Indole-3-Acetic Acid (Aux/IAA) repressors of auxin response factors (Navarro et al., 2006). In contrast, SAmediated repression of auxin receptors appears to be independent of miR393 (Wang et al., 2007). The ability to antagonize auxin is particularly intriguing in the case of endogenous elicitors such as OGs, linear oligosaccharides of a-1,4-D-galactopyranosyluronic acid residues. These are released from the homogalacturonan of the plant cell wall by mechanical damage (wounding) or pathogen-secreted enzymes such as endo-polygalacturonases (PGs), and therefore are typical damage-associated molecular patterns (DAMPs; Ridley et al., 2001; Brutus et al., 2010). The highest elicitor activity is associated with OGs with a degree of polymerization (DP) of about 10 to 16 (Ridley et al., 2001), the formation of which is favored when microbial PGs interact with PG-inhibiting proteins, specific Leu-rich repeat recognition proteins located in the plant apoplast (De Lorenzo and Ferrari,
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2002; Casasoli et al., 2009). Recently, the Arabidopsis (Arabidopsis thaliana) WALL-ASSOCIATED KINASE1 has been described as a receptor for OGs (Brutus et al., 2010; De Lorenzo et al., 2011). The biological responses triggered by OGs are well documented and similar in many aspects to those of MAMPs (Galletti et al., 2009). For example, OGs and flg22 activate defense responses effective against the microbial pathogens Botrytis cinerea and Pseudomonas syringae, respectively, independently of SA, ET, and JA (Zipfel et al., 2004; Ferrari et al., 2007). Both elicitors trigger a fast and transient response characterized by activation of early stages of multiple defense signaling pathways. However, the response to flg22 is stronger in both the number of genes differentially expressed and the amplitude of change. Even at very high concentrations, OGs do not induce a response that is as comprehensive as that seen with flg22. For example, SA-dependent secretory pathway genes and PR1 expression are substantially induced only by flg22 (Denoux et al., 2008). The cell wall is a structure that is dynamically regulated and continually remodeled to regulate plant growth and development (Vorwerk et al., 2004). OGs are therefore likely released in muro also in the absence of pathogens or mechanical damage, and their capability of antagonizing auxin may play an important role during development. Indeed, OGs regulate several developmental-related processes such as elongation in pea stem segments (Branca et al., 1988), adventitious root formation (Bellincampi et al., 1993), and pericycle cell differentiation (Altamura et al., 1998) in tobacco (Nicotiana tabacum). In all these cases, their action is antagonistic to the effects of exogenous auxin. In this work we examined whether OGs have auxinantagonistic activity in the model plant Arabidopsis, which may allow a better dissection of their mechanism of action. Our results demonstrate that, in Arabidopsis, OGs inhibit adventitious root formation induced by exogenously added auxin in leaf explants as well as the expression of several early auxinup-regulated genes. The inhibition of early auxin responses exerted by OGs as well as that by flg22 is mediated neither by miR393 activity nor by stabilization of Aux/IAA repressors. Furthermore, it is not affected in mutants defective in microRNA biosynthesis and posttranscriptional gene silencing (PTGS). We conclude that inhibition of auxin-induced responses by OGs and flg22 occurs through a mechanism, not yet characterized and independent of PTGS.
RESULTS OGs Antagonize Auxin in Arabidopsis
Arabidopsis leaf explants cultured for 15 d on basal medium containing IAA developed adventitious roots, which emerged in a polar manner at the cut 1164
proximal edge of the explants. In particular, the number of roots per explant increased with increasing IAA concentration, reaching a maximum at 10 mM IAA (Fig. 1A). Inhibition of auxin-induced root formation was observed upon OG addition with a DP of 10 to 16 (30 mg mL21). Inhibition was nearly complete at 1 and 2.5 mM IAA, but was overcome by higher concentrations of IAA, reminiscent of the OG-IAA antagonism previously described in pea and tobacco (Branca et al., 1988; Bellincampi et al., 1993). OGs also inhibited the expression, induced by exogenous IAA, of GUS driven by the synthetic promoter DR5 (Ulmasov et al., 1997; Fig. 1B). Histochemical analysis showed that GUS activity was localized at the tip of the root apex in untreated seedlings and, upon treatment with IAA, extended to the entire root apical meristem and across the division zone, up to the differentiation zone; GUS activity also appeared at the sites of emergence of lateral root primordia. While the increase of GUS activity induced by exogenous IAA was inhibited by OGs, little effect was observed on the basal GUS activity present in the root apex, likely due to the long half-life of the preexisting enzyme (Fig. 1B). Next, we tested the effect of OGs on the expression of endogenous genes that are early up-regulated (within 1 h) by IAA (IAA5 [At1g15580], IAA19 [At3g15540], IAA20 [At2g46990], IAA22 [At1g19220], SAUR16 [At4g38860], SAUR-AC1 [At4g38850], and GH3.3 [At2g23170]; Goda et al., 2004) in both leaf explants and seedlings treated with IAA. OGs inhibited the accumulation of transcripts of all these genes in both types of plant material (Fig. 2; Supplemental Fig. S1). Inhibition by OGs was dose dependent (Supplemental Fig. S1). The genes examined can therefore be used as markers to study the auxin-OGs antagonism. We also analyzed whether, conversely, auxin is able to counteract the OG-induced protection against B. cinerea. Treatment of wild-type plants with auxin alone at concentrations higher than 5 mM increased resistance to B. cinerea (Fig. 3A), likely due to the induction of ET accumulation; indeed, ET is known to mediate basal resistance against B. cinerea (Thomma et al., 1999). Therefore, to test the effect of IAA on OG-induced resistance, we used the ET-insensitive 2-1 (ein2-1) mutant (Guzma´n and Ecker, 1990), and found that, in this genotype, IAA alone did not significantly affect susceptibility against the fungus but abolished the protection induced by OGs (Fig. 3B). Up-Regulation of IAA5 Expression by Cycloheximide Is Inhibited by OGs
To elucidate the basis of OG-IAA antagonism, we first examined whether OGs are capable of altering the levels of IAA in Arabidopsis. Endogenous levels of free IAA were unaffected in both Arabidopsis seedlings and adult leaves treated with OGs both in the absence and in the presence of auxin (Supplemental Fig. S2), suggesting that antagonism is likely played at Plant Physiol. Vol. 157, 2011
Oligogalacturonide-Auxin Antagonism in Arabidopsis
of protein biosynthesis in eukaryotic organisms (Schneider-Poetsch et al., 2010), on the expression of genes regulated by IAA. Because canonical Aux/IAA proteins have a similar and short turnover timing in vivo (5–15 min; Dreher et al., 2006), CHX treatment results in the rapid disappearance of these repressors and consequently in the up-regulation of the expression of target genes such as Aux/IAA, SAUR, and GH3 genes (Gil et al., 1994; Roux and Perrot-Rechenmann, 1997). We therefore analyzed the expression of IAA5 upon treatment with 10 mM CHX by quantitative reverse transcription (qRT)-PCR. As shown in Figure 4, within 15 min of treatment, the block of protein synthesis led to a rapid accumulation of IAA5 transcripts. OGs were then supplied at different times (0, 15, 30, 60, and 120 min) after CHX addition, and IAA5 transcripts were measured at a final time point of 150 min. Inhibition of IAA5 transcript accumulation was observed in all samples. Inhibition was nearly complete when OGs were added within 30 min of CHX treatment, and was still observed when OGs were added up to 2 h after treatment (Fig. 4). These data suggest that OGs affect the expression of IAA-regulated gene markers also in the absence of exogenous auxin and of Aux/IAA repressors, and that OG-IAA antagonism occurs also in the absence of de novo protein synthesis. Inhibition of Auxin-Regulated Gene Expression by OGs Is Independent of Stabilization of Aux/IAAs or miRNA Action
Figure 1. OGs inhibit auxin-induced formation of adventitious roots and expression of DR5::GUS in Arabidopsis. A, Arabidopsis Col-0 leaf explants were incubated in the presence of different concentrations of IAA, alone or in combination with OGs (30 mg mL21). After 15 d, the number of roots formed by each explant was determined. Each data point is the mean number of roots per explant (6SE, n = 9). Inset shows representative explants treated with 2 mM IAA alone (bottom) or in the presence of OGs (top). B, The graph at the top shows GUS activity (expressed as nmol 4-methylumbelliferone [MU] min21 mg21 protein) measured in 10-d-old DR5::GUS transgenic seedlings treated for 1 h with IAA (1.5 mM) in the presence or absence of OGs (100 mg mL21). Each bar represents the mean of three independent experiments (6SE, n = 9). The bottom section shows representative roots of seedlings treated as described above and stained for GUS activity.
the level of perception or transduction of the IAA signal. Next, we tested whether OGs also inhibit the effects of cycloheximide (CHX), a well-known inhibitor Plant Physiol. Vol. 157, 2011
To assess whether inhibition of auxin-regulated responses by OGs also involves degradation of auxin receptor transcripts mediated by miRNA393 and stabilization of Aux/IAA repressors, as shown for flg22 by Navarro et al. (2006), we analyzed the expression of TIR1, AFB1, AFB2, and AFB3 genes by qRT-PCR in Columbia-0 (Col-0) seedlings treated with OGs for 30 min, and found no significant variation compared to the water treatment (Fig. 5A). We then determined mature miR393 levels by stem-loop qRT-PCR (Chen et al., 2005). OGs did not induce miR393 expression at 20 min (Fig. 5B) or at 1 h of treatment (Supplemental Fig. S3), although inhibition of auxin-induced gene expression was evident at this time point. We performed similar analyses upon treatment with flg22 (1 and 10 mM). Concentrations of 100 to 200 nM flg22 have been shown to be saturating for up-regulation of marker genes (Denoux et al., 2008) and inhibition of seedling growth (Galletti et al., 2011). We found that a concentration of 1 mM flg22 is effective for inhibiting auxin-regulated gene expression (Supplemental Fig. S4); the concentration of 10 mM is the same as that previously used by Navarro et al. (2006). As shown in Figure 5, we observed no significant variation of the expression of TIR1, AFB1, AFB2, and AFB3 and of miR393 in response to 1 mM flg22, compared to the water treatment. Decreased expression of the four receptor genes and up-regulation of miR393 was in1165
Savatin et al. Figure 2. Inhibition of auxin-regulated gene expression by OGs. Expression of the indicated genes was determined by qRT-PCR in 10-d-old Col-0 seedlings treated for 1 h with water, OGs (100 mg mL21), IAA (1.5 mM), or IAA + OGs. Expression of UBQ5 was used as a reference. Results are the mean of three independent experiments (6SE).
stead observed at 10 mM flg22, in agreement with previous data (Navarro et al., 2006). The inhibitory effect of OGs was also examined in a quadruple mutant defective in the TIR1, AFB1, AFB2, and AFB3 auxin receptors (Dharmasiri et al., 2005). In this mutant, IAA5 showed a slight induction in response to IAA; this induction was also inhibited by OGs (Supplemental Fig. S5). We then analyzed whether OGs determine the stabilization of Aux/IAA proteins by using a transgenic line expressing a heat-shock-inducible protein comprising the N-terminal domains I and II of AXR3/ IAA17 fused to GUS (HS::AXR3NT-GUS). This fusion protein shows ubiquitin-mediated degradation similar to that of the native protein (Gray et al., 2001). Transgenic seedlings were incubated at 37°C for 2 h, then treated at 25°C for different times (5, 15, 30, and 60 min) either with 1.5 mM IAA in the presence or absence of OGs (100 mg mL21) or flg22 (10 mM) or with elicitors alone. Quantification of GUS activity and immunoblot analysis using an anti-GUS antibody showed that AXR3NT-GUS was present in the untreated samples and in the samples treated with elicitors alone, whereas it disappeared, starting from 5 min, in the IAA-treated samples, either in the presence or in the absence of elicitors (Fig. 6). No significant stabilization of AXR3NT-GUS was observed in the seedlings treated for 1 h with OGs or 10 mM flg22 alone; the last result is 1166
in agreement with what observed at the same time point by Navarro et al. (2006). Thus, degradation of Aux/IAAs induced by exogenous auxin is not affected by OGs or flg22 in our experimental conditions. To provide genetic evidence of the lack of involvement of miRNAs in OG-mediated inhibition of auxin responses, we analyzed two different mutant lines previously described as defective in miRNA-mediated PTGS: argonauta1-27 (ago1-27), a hypomorphic mutant for the AGO1 gene (Morel et al., 2002), and an insertional KO line for HUA ENHANCER1 (hen1; Park et al., 2002). AGO1 is a PAZ PIWI DOMAIN protein that binds miRNAs to form effector complexes named RNA-induced silencing complexes and is involved in target mRNA cleavage based on sequence complementarity with miRNAs (Hannon, 2002). AGO1 binds miR393 and mediates its activity (Mi et al., 2008). HEN1 is involved in miRNA stabilization and is required for mature miRNA and siRNA accumulation (Park et al., 2002). In both ago1-27 and hen1 seedlings, treatment with either OGs or flg22 reduced IAA-induced accumulation of IAA5 and SAUR16 transcripts in a manner comparable with that occurring in wildtype seedlings (Fig. 7). Taken together, these results indicate that PTGS mediated by miRNAs, including miR393, is not involved in the inhibition of early auxin-induced gene expression exerted by OGs and flg22. Plant Physiol. Vol. 157, 2011
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play partially overlapping but essential functions (Krysan et al., 2002). IAA-inducible gene expression in the presence or absence of OGs was analyzed by qRT-PCR in the double anp1 anp2, anp1 anp3, and anp2 anp3 mutants. As shown in Figure 8, inhibition of gene expression by OGs occurred in all double mutants, suggesting that none of the three ANPs plays a predominant role in this process. AtrbohD Function and ET-, SA-, and JA-Mediated Signaling Pathways Are Not Required for OG-Auxin Antagonism
Figure 3. Auxin inhibits OG-triggered protection against B. cinerea. Leaves from 3-week-old Arabidopsis plants were syringe infiltrated with IAA or OGs (200 mg mL21), as indicated. Twenty-four hours after infiltration, leaves were inoculated with B. cinerea, and lesion area was measured 48 h postinoculation. A, Arabidopsis Col-0 leaves were infiltrated with water (control) or IAA at the indicated concentrations. B, Arabidopsis ein2-1 leaves were infiltrated with water (control), IAA (20 mM), OGs (200 mg mL21), or IAA + OGs. Values are means (6SE) of at least 12 lesions. Different letters indicate statistically significant differences according to ANOVA followed by Tukey’s honestly significant difference test (P , 0.05). Experiments were repeated three times with similar results.
Although hydrogen peroxide (H2O2) negatively controls auxin-regulated gene expression (Kovtun et al., 2000), extracellular H2O2 accumulation induced by OGs does not mediate their auxin-antagonistic activity in tobacco leaf explants (Bellincampi et al., 2000). OGinduced reactive oxygen species (ROS) formation in Arabidopsis is mainly provided by the RESPIRATORY BURST OXIDASE HOMOLOGUE D (AtrbohD), a plasma membrane-associated NADPH oxidase (Torres et al., 2002; Galletti et al., 2008). We therefore determined whether in atrbohD mutant plants OG-mediated inhibition of auxin-regulated gene marker expression is affected. atrbohD seedlings showed a normal induction of marker genes in response to IAA and their expression was inhibited by OGs at the same extent as in wild-type seedlings (Fig. 9). AtrbohD-mediated
Double KO Mutants Lacking the MEK Kinases Arabidopsis NPK1-Related Protein Kinases Are Not Affected in OG-Auxin Antagonism
Because it has been shown that activity of GH3 promoter induced by IAA is negatively regulated by Arabidopsis NPK1-Related Protein Kinases (ANPs) in protoplasts (Kovtun et al., 2000), we assessed the possible involvement of these proteins in the OGauxin antagonism. The ANP family comprises three genes (ANP1, ANP2, and ANP3) that represent a distinct branch of MEK kinases (MAPKKKs) likely involved in cytokinesis (Krysan et al., 2002). Single anp mutants and double anp1 anp2 mutants have no growth or developmental defects; in contrast, anp1 anp3 plants show defects in their floral organs and anp2 anp3 plants show defects at all developmental stages analyzed (Krysan et al., 2002). The triple KO mutant could not be obtained, suggesting that ANPs Plant Physiol. Vol. 157, 2011
Figure 4. Up-regulation of IAA5 expression by CHX is inhibited by OGs. Ten-day-old seedlings were treated with CHX (10 mM) alone (solid line) or in the presence of OGs (100 mg mL21, dashed lines) added at different times (0, 15, 30, 60, and 120 min, as indicated by arrows). Accumulation of IAA5 transcripts was determined 150 min after the beginning of CHX treatment by qRT-PCR using UBQ5 as a reference gene. Results are the mean of three independent experiments (6SE). 1167
Savatin et al. Figure 5. Levels of auxin receptor transcripts and miR393 are not significantly altered by OG treatment. A, Levels of TIR1, AFB1, AFB2, and AFB3 transcripts 30 min after treatment with water (H), OGs (100 mg mL21), and flg22 (1 and 10 mM) were determined in 10-d-old Col-0 seedlings by qRT-PCR, using UBQ5 as a reference gene. B, Levels of mature miR393 were determined 20 min after treatment by stem-loop qRT-PCR, using rRNA 5S as a reference. Results are the mean of three independent experiments (6SE).
accumulation of extracellular H2O2 therefore does not play a major role in auxin-OG antagonism. Finally, we assessed whether OG-mediated inhibition of auxin responses is independent of the signaling pathways mediated by SA, JA, and ET, as in the case of the induction of defense-related responses by OGs (Ferrari et al., 2007; Galletti et al., 2008). To this aim, we analyzed the Arabidopsis npr1 ein2 jar1 (nej) triple mutant, which is impaired in responses mediated by SA, ET, and JA, due to mutations in the genes NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1, EIN2, and JASMONATE RESISTANT1 (Clarke et al., 2000). nej seedlings showed a normal induction of IAA5 and SAUR16 expression in response to IAA and their expression was inhibited by OGs at the same extent as in the wild-type seedlings (Fig. 9).
DISCUSSION
The activity of MAMPs and SA against pathogens has been linked also to their ability to antagonize auxin
(Navarro et al., 2006; Wang et al., 2007); therefore, it is likely that this ability is also important for DAMPs, that, being endogenous signals, may control hormone response pathways in growth and developmental processes. Indeed, OGs negatively regulate a developmental response such as auxin-induced formation of adventitious roots in Arabidopsis (this work) and tobacco leaf explants (Bellincampi et al., 1993). Adventitious root formation is a complex process affected by multiple factors, including phytohormones; among them auxin plays a central role. Inhibition of root formation is observed when OGs are supplied together with up to 2.5 mM IAA, and is overcome by higher amounts of auxin, reminiscent of the OG-IAA antagonism previously described in other plant species (Branca et al., 1988; Bellincampi et al., 1996). Conversely, IAA is capable of counteracting defense responses triggered by OGs, as indicated by the reduction of OG-induced protection against B. cinerea observed in both Arabidopsis (this work) and tobacco (Ferrari et al., 2008). Interestingly, in Arabidopsis this effect is evident in the ET-insensitive ein2 mutant but
Figure 6. AXR3NT is not stabilized by elicitors in the presence of exogenous auxin. A, Ten-day-old HS::AXR3NT-GUS transgenic seedlings were incubated for 2 h at 37°C and then treated with water, OGs (100 mg mL21), IAA (1.5 mM), flg22 (10 mM), or IAA + elicitors for 5, 15, 30, and 60 min. Graphs show GUS activity measured at the indicated times and expressed as nmol 3 1022 MU min21 mg21 protein. Results are the mean of three independent experiments (6SE). B, Levels of AXR3NT-GUS (AXR3) are shown, determined by western-blot analysis using a GUS-specific antibody. Levels of actin were determined using a specific antibody to asses equal loading. 1168
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Oligogalacturonide-Auxin Antagonism in Arabidopsis Figure 7. Inhibition of auxin-regulated gene expression by OGs and flg22 in mutants defective in PTGS. Expression of the auxin-regulated genes IAA5 (top) and SAUR16 (bottom) was determined by qRT-PCR in 10-d-old Col-0, ago1-27, and hen1 seedlings treated for 1 h with water, OGs (100 mg mL21), flg22 (1 mM), IAA (1.5 mM), or IAA + elicitors. Expression of UBQ5 was used as a reference. Results are the mean (6SE) of three biological replicates. Experiments were repeated at least two times with similar results.
not in wild-type plants, likely because IAA induces the production of ET, which plays an important role in resistance against B. cinerea (Thomma et al., 1999). Auxin-induced production of ET may confer protection against this pathogen in spite of the downregulation of other defense responses by IAA. Induction of ET accumulation may explain why an intact auxin response pathway is important for resistance to this pathogen (Llorente et al., 2008). The activity of OGs not only affects long-term responses to auxin such as adventitious root formation, but also early responses such as the up-regulation of IAA5, SAUR16, and SAUR-AC1. Inhibition of the expression of these genes by OGs is already observed within 30 min, suggesting that the OG-triggered cascade that affects auxin signaling occurs rapidly and leads to the subsequent activation of long-term effects. Inhibition of auxininduced gene expression by OGs is confirmed by the ability of OGs to prevent IAA-induced expression of GUS driven by the auxin-inducible promoter DR5. Notably, the effect of OGs on early IAA-regulated gene expression is not mediated by an alteration of auxin homeostasis, as free IAA levels are not significantly changed within 1 h after treatment. This is in agreement with previous observations showing that OGs do not cause a faster depletion (uptake, modification, or breakdown) of IAA in tobacco leaf explants (Bellincampi et al., 1996). Plant Physiol. Vol. 157, 2011
In this work we focused on the early effects of OGs, compared to flg22, by monitoring the expression of the auxin-regulated genes. We found that miR393 does not mediate the inhibitory effects of both elicitors, in contrast with previous reports indicating that miRNA393 is responsible for the flg22-induced repression of early auxin-regulated gene expression (Navarro et al., 2006). The inconsistency may be explained by the fact that flg22, at very high concentration, triggers the induction of the miRNA393 and a decrease of auxin receptor transcripts, which apparently correlated with the inhibition of auxin responses. Our results show that in response to OGs and flg22, when this is not used at oversaturating concentrations, (1) miR393 expression is not altered up to 1 h after elicitor treatment; (2) transcripts of the genes encoding the auxin receptors TIR1 and AFBs are not decreased; and (3) AXR3NT is not stabilized in elicitor-IAA cotreatments. Furthermore, in auxin-OG and auxinflg22 antagonism experiments, ago1-27 (Morel et al., 2002) and hen1 (Park et al., 2002), two mutants lacking microRNA-mediated PTGS, show a behavior similar to that of the wild type. Searching for elements involved in auxin-OG antagonism, ANPs (ANP1, ANP2, ANP3) emerged as interesting candidates. In fact, constitutively active forms of these MAPKKKs, generated through deletion 1169
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ANP function is essential for plant development and growth, it has been so far impossible to obtain a triple KO mutant (Krysan et al., 2002); therefore, whether these MAPKKKs play an essential but redundant role
Figure 8. Inhibition of auxin-regulated gene expression by OGs in Arabidopsis anp double KO mutants. Expression of auxin-regulated gene markers was determined by qRT-PCR in 10-d-old Ws and anp double-mutant seedlings treated for 1 h with water, OGs (100 mg mL21), IAA (1.5 mM), or IAA + OGs. Expression of UBQ5 was used as a reference. Results are the mean of three independent experiments (6SE).
of the regulatory domain, inhibit auxin-induced activation of the GH3 promoter in Arabidopsis protoplasts (Kovtun et al., 2000). However, the three possible combinations of double KO mutants (anp1 anp2, anp1 anp3, and anp2 anp3) showed an unaltered OG-induced inhibition of auxin-regulated gene expression. Because 1170
Figure 9. Inhibition of auxin-regulated gene expression by OGs in atrbohD and nej mutants. Expression of auxin-regulated genes was determined by qRT-PCR in 10-d-old Col-0, atrbohD, and nej seedlings treated for 1 h with water, OGs (100 mg mL21), IAA (1.5 mM), or IAA + OGs. Expression of UBQ5 was used as a reference. Results are the mean of three independent experiments (6SE). Plant Physiol. Vol. 157, 2011
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could not be assessed. Our results however suggest that none of the three family members plays a major role in OG-IAA antagonism. ROS play important roles in both developmental processes and resistance against biotic and abiotic stresses (for review, see Carol and Dolan, 2006; Kotchoni and Gachomo, 2006), and negatively control auxinregulated gene expression (Kovtun et al., 2000). However, the extracellular oxidative burst generated in response to OGs by the NADPH oxidase AtrbohD (Galletti et al., 2008) is not required for OG-auxin antagonism. This result is in agreement with previous data showing that extracellular ROS production induced by OGs is not involved in the inhibition of the auxin-induced expression of the rolB gene in tobacco (Bellincampi et al., 2000). The analysis of the nej triple mutant defective in SA, ET, and JA signal transduction pathways shows that also the pathways mediated by these signal molecules do not play a major role in the auxin-OGs antagonism, consistently with previous results showing that the expression of several OG-responsive genes, as well as the OG-induced protection against B. cinerea, are independent of SA, ET, and JA (Ferrari et al., 2003; Galletti et al., 2008). In fact, treatment with OGs neither induces ET production in tobacco or in Arabidopsis (Ferrari et al., 2008; Brutus et al., 2010) nor the expression of several SA-dependent genes, which are instead up-regulated by flg22 (Denoux et al., 2008). The pathway activated by OGs, however, may partially overlap with that activated by SA, which also represses auxin signaling; in this case, stabilization of Aux/IAA repressors has been proposed to explain this effect (Wang et al., 2007). Notably, CHX experiments show that OG-IAA antagonism occurs not only in the absence of de novo protein synthesis, but also when repressors such as Aux/IAAs have been degraded. We observed a very limited increase of IAA5 expression when OGs were added at late time points, suggesting that the time elapsing between OG perception and the inhibition of the auxin-induced expression genes is relatively short and that inhibition occurs also after activation of auxin-regulated promoters has initiated. Similarly, previous analyses had shown that OGs very rapidly inhibit auxin-induced processes necessary not only to activate but also to maintain active the promoter of Agrobacterium rhizogenes rolB, a late auxin-responsive gene (Bellincampi et al., 2000). It is possible that the inhibitory effects of OGs may take place downstream in the auxin-regulated signaling cascade, perhaps through posttranslational regulation of elements other than Aux/IAA proteins or inactivation of auxin response factors. The fact that different defense-related pathways, i.e. those triggered by MAMPs, DAMPs, and SA, lead to the repression of responses mediated by auxin strongly suggests the importance of the down-regulation of the signaling pathway regulated by this hormone for counteracting pathogen infections. Plant Physiol. Vol. 157, 2011
MATERIALS AND METHODS Plant Material Arabidopsis (Arabidopsis thaliana) Col-0 and Wassilewskija (Ws) wild-type seeds were purchased from Lehle Seeds. The DR5-GUS transgenic line has been described by Ulmasov et al. (1997). The atrbohD KO line and the tir1 afb1 afb2 afb3 quadruple mutant were kindly provided by Jonathan G.D. Jones (Sainsbury Laboratory, John Innes Centre). The triple mutant nej was a kind gift from Xinnian Dong (Duke University). Heterozygous ago1-27 mutant seeds were a kind gift from Herve´ Vaucheret (Laboratoire de Biologie Cellulaire Institut National de la Recherche Agronomique, Centre de Versailles). The HS::AXR3NT-GUS transgenic line was previously described (Gray et al., 2001) and was kindly provided by Lionel Navarro (Centre National de la Recherche Scientifique, Institut de Biologie de l’Ecole Normale Supe´rieure-Paris). Seeds of the T-DNA insertional mutant hen1 (SALK_ 090960) were obtained from the Nottingham Arabidopsis Stock Centre (School of Biosciences, University of Nottingham, United Kingdom). Seeds of ein2-1 mutant were obtained from the Arabidopsis Biological Resource Center. The anp1 anp2, anp1 anp3, and anp2 anp3 double mutants (in the Ws background) were kindly provided by Patrick J. Krysan (Department of Horticulture, University of Wisconsin).
Growth Conditions and Plant Treatments Leaf explants were obtained from 4-week-old Arabidopsis Col-0 plants grown in sterility (Phytatray, Sigma-Aldrich) on 0.53 Murashige and Skoog medium (Sigma-Aldrich) supplemented with 1% Suc and 0.8% plant agar at 22°C and under a 16-h light/8-h dark cycle (approximately 120 mmol m22 s21). Leaf explants (approximately 5 3 15 mm), consisting of the central part of the lamina including the midrib, were incubated in 0.53 Murashige and Skoog medium supplemented with 0.5% Suc. The medium was refreshed for three times every 30 min and then explants were incubated in the same medium in the presence of different concentrations of IAA (Sigma-Aldrich) alone or in combination with OGs in semidark conditions. After 15 d the number of roots formed by each explant was determined. For transcript analyses, leaf explants, prepared as described above, were incubated on 0.53 Murashige and Skoog medium supplemented with 0.5% Suc (0.53 Murashige and Skoog-Suc medium) in multiwell plates (approximately 7–8 explant/well). The medium was refreshed for three times every 30 min and then explants were incubated in an overnight recovery at 22°C with a 16-h light/8-h dark cycle and a light intensity of 120 mmol m22 s21, before treatments with 1 mM IAA alone or in combination with 10 mg mL21 or 30 mg mL21 OGs for 30 min. For seedling treatments, seeds were surface sterilized and germinated in multiwell plates (approximately 10 seeds/well) containing 2 mL per well of 0.53 Murashige and Skoog-Suc medium. Plates were incubated at 22°C with a 16-h light/8-h dark cycle at a light intensity of 120 mmol m22 s21. After 9 d, the medium was replaced and treatments were performed after an additional day. For HS::AXR3NT-GUS transgenic line, seedlings were treated at 37°C for 2 h in liquid 0.53 Murashige and Skoog-Suc medium and then incubated for 1 h, at 25°C, either with 1.5 mM IAA in the presence or absence of OGs (100 mg mL21) or flg22 (1 and 10 mM), or with elicitors alone. OGs with an average DP of 9 to 16 were prepared as previously described (Bellincampi et al., 2000). Matrix-assisted laser desorption/ionization time-offlight mass spectrometry was used to verify the DP of OG preparations. The flg22 peptide was synthesized by Prof. Maria Eugenia Schinina` (Sapienza Universita` di Roma). The protein synthesis inhibitor CHX (Sigma-Aldrich) was used for treatment of Col-0 seedling alone (at a concentration of 10 mM) or in the presence of OGs (100 mg mL21) added at different time points (T0, 15 min, 30 min, 1 h, and 2 h). Seedlings were harvested and immediately frozen in liquid nitrogen. Accumulation of IAA5 transcripts was determined by qRT-PCR 150 min after the beginning of CHX treatment.
Gene Expression Analysis Seedlings or leaf explants were frozen in liquid nitrogen, homogenized with a MM301 ball mill (Retsch), and total RNA was extracted with Isol-RNA lysis reagent (5 prime) according to the manufacturer’s protocol. RNA was treated with RQ1 DNase (Promega) and first-strand cDNA was synthesized using ImProm-II reverse transcriptase (Promega) according to the manufacturer’s 1171
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instructions. Real-time qPCR analysis was performed as previously described (Galletti et al., 2011) using a CFX96 real-time system (Bio-Rad). One microliter of cDNA (corresponding to 50 ng of total RNA) was amplified in a 30 mL reaction mix containing 13 GoTaq real-time PCR system (Promega) and 0.4 mM of each primer. Expression levels of each gene, relative to UBQ5, were determined using a modification of the Pfaffl method (Pfaffl, 2001) as previously described (Ferrari et al., 2006). Primer sequences are shown in Supplemental Table S1. The microRNA393 quantification by real-time stem-loop RT-PCR was performed as previously described by Chen et al. (2005) using the rRNA 5S as a standard. Small RNAs were purified from 10 mg of total RNA by high pure miRNA isolation kit (Roche) and used in a pulsed RT reaction performed in a final volume of 20 mL, as previously described (Varkonyi-Gasic et al., 2007). Three microliters of RT product were used as template for real-time PCR. Sequences of miR393 and 5S stem-loop primers are listed in Supplemental Table S1.
Histochemical Localization of GUS Activity The DR5-GUS construct and transgenic plants were previously described (Ulmasov et al., 1997). Histochemical GUS staining was performed by incubating whole seedlings in GUS staining buffer containing 50 mM sodium phosphate (pH 7.0), 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.1% (v/v) Triton X-100, 2% (v/v) dimethyl sulfoxide, and 1 mM 5-bromo-4-chloro-3indolyl-D-glucuronide at 37°C for 24 h (Jefferson, 1987).
GUS Activity Assay Seedlings were ground in liquid nitrogen and homogenized in 100 mL of extraction buffer containing 100 mM potassium phosphate (pH 7.8), 1 mM EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, and 1 mM dithiothreitol. To assay GUS activity, extracts (2.5 mL) were added to 197.5 mL of 2 mM 4-methylumbelliferyl-D-glucuronide in extraction buffer prewarmed to 37°C. The mixture was incubated at 37°C and the resulting fluorescence was measured with a Glomax Multi+ detection system (Promega) at 365 (excitation) and 455 (emission) nm after 20, 30, and 40 min. Protein content was determined according to the Bradford method (Bradford, 1976) to normalize GUS activity.
Immunoblot Assay Extraction of seedlings total proteins was performed in 50 mM Tris pH 7.5, 200 mM NaCl, EDTA 1 mM, 10% glycerol, 200 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (P8849, Sigma-Aldrich). Thirty micrograms of total protein extract were separated on a 7.5% acrylamide (30% acrylamide/Bis solution, 29:1, Bio-Rad) SDS-gel and transferred on nitrocellulose membranes (Hybond-C, Amersham) in 25 mM Tris, 192 mM Gly, pH 8.3, 20% methanol at 4°C for 1 h. The filter was stained with Ponceau Red and then blocked with 5% albumin from bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline (Bio-Rad) containing 0.2% (v/v) Tween 20 for 1 h prior to incubation for 1 h with primary antibody antib-glucuronidase or antiactin (Sigma-Aldrich). After extensive washes in phosphate-buffered saline containing 0.2% Tween 20, membranes were incubated with anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham). Membranes were washed and incubated for two minutes with ECL western-blotting substrate (Promega) prior to detection using ChemiDoc XRS+ (Bio-Rad).
Botrytis cinerea Growth and Plant Inoculation Botrytis cinerea was grown on 20 g L21 malt extract, 10 g L21 proteose peptone n. 3 (Difco), and 15 g L21 agar for 7 to 10 d at +24°C with a 12-h photoperiod before collection of spores. Rosette leaves from 4-week-old soilgrown Arabidopsis plants were syringe infiltrated with water solutions containing OGs, IAA, or both 24 h before being placed in petri dishes containing 0.8% agar, with the petiole embedded in the medium. Inoculation was performed by placing 5 mL of a suspension of 5 3 105 conidiospores mL21 in 24 g L21 potato dextrose broth (Difco) on each side of the middle vein. The plates were incubated at +22°C with a 12-h photoperiod. High humidity was maintained by covering the plates with a clear plastic lid. Under these experimental conditions, most inoculations resulted in rapidly expanding water-soaked lesions of comparable diameter. Lesion area was determined at 48 h after inoculation. 1172
Gas Chromatography-Mass Spectrometry Analyses for Free IAA Quantification Analysis of free IAA levels was performed in 10-d-old Col-0 seedlings or 3week-old Col-0 leaves (100 mg fresh weight) incubated with water, OGs (100 mg mL21), IAA (1.5 mM), or IAA + OGs for 30 or 60 min. As a positive control for the analysis, 5-d-old Arabidopsis ecotype Col glabra1-1 seedlings, kindly provided by Pier Domenico Perata (Scuola Superiore Sant’ Anna, Pisa), grown in water or 90 mM turanose as described by Gonzali et al. (2005) were analyzed. Analyses were performed according to Fujino et al. (1988). Briefly, after homogenization in liquid N2, samples were suspended in 1 mL dichlorometane, added with 5 ng of [13C6]-IAA (99 atom %, Cambridge Isotope Laboratories) as an internal standard, left at 4°C for 30 min, and centrifuged at 12,000g for 5 min. The organic fraction was dried under N2, derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS; Sigma; 70°C, 30 min), and redissolved in 50 mL dichlorometane. An aliquot (1 mL) of the derivatized sample was analyzed by gas chromatorgraphy-mass spectrometry using an Agilent 6850A gas chromatographer coupled to a 5973N quadrupole mass selective detector (Agilent Technologies). Chromatographic separations were carried out on a 30 m 3 0.25 mm i.d. fused-silica capillary column coated with cross-linked 5% phenylmethyl siloxane (film thickness 0.25 mm; Hewlett-Packard), as stationary phase. Injection mode: splitless at a temperature of 260°C. Column temperature program: 80°C (1 min), then to 280°C at a rate of 15°C/min, and held for 5 min. The carrier gas was helium at a constant flow of 1 mL min21. The spectra were obtained in the electron impact mode at 70 eV ionization energy, ion source 280°C, ion source vacuum 1025 Torr. Mass spectrometry analysis was performed using Agilent MSD productivity Chemstation that enables collection of both selective ion monitoring data and scan data (mass range scan from mass-to-charge ratio [m/z] 50–500 at a rate of 0.42 scans s21) in a single run. IAA quantitative analysis was performed in selective ion monitoring mode following three characteristic ions for both IAA-TBDMS derivative (m/z 130, m/z 232, m/z 289) and [13C]6-IAA TBDMS derivative (m/z 136, m/z 238, m/z 295). The obtained values were normalized to fresh weight of original sample.
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Inhibition of auxin-regulated gene expression by OGs occurs also in Arabidopsis leaf explants and is dose dependent. Supplemental Figure S2. Endogenous levels of free IAA are not significantly reduced by OGs in seedlings and adult leaves. Supplemental Figure S3. miR393 is not significantly altered by OG treatment. Supplemental Figure S4. Inhibition of auxin-regulated gene expression by 1 mM flagellin. Supplemental Figure S5. Inhibition of auxin-induced IAA5 gene expression by OGs in the quadruple mutant tir1 afb1 afb2 afb3. Supplemental Table S1. Primers used in this work.
ACKNOWLEDGMENTS We thank Gianni Salvi and Daniela Pontiggia (Dipartimento di Biologia e Biotecnologie “C. Darwin,” Sapienza Universita` di Roma) for the OG preparation and Dr. Alberto Macone (Department of Biochemistry R. Fanelli, Universita` di Roma La Sapienza) for gas chromatography-mass spectrometry technical support. We also thank Felice Cervone (Dipartimento di Biologia e Biotecnologie “C. Darwin,” Sapienza Universita` di Roma) for helpful discussions. Received August 5, 2011; accepted August 29, 2011; published August 31, 2011.
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