Phytochemistry xxx (2015) xxx–xxx

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Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha Yusuke Yamamoto a, Jun Ohshika a, Tomohiro Takahashi a, Kimitsune Ishizaki b, Takayuki Kohchi c, Hideyuki Matusuura a, Kosaku Takahashi a,⇑ a b c

Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Graduate School of Science, Kobe University, Kobe 657-8501, Japan Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

a r t i c l e

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Article history: Received 18 February 2014 Received in revised form 25 February 2015 Available online xxxx Keywords: Allene oxide cyclase Marchantia polymorpha Marchantiaceae Octadecanoid pathway 12-Oxo-phytodienoic acid Stress response

a b s t r a c t 12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JAdependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha. Ó 2015 Published by Elsevier Ltd.

1. Introduction Oxylipins are lipid-derived compounds, and some of them function as signals that mediate responses to abiotic and biotic stresses in plants (Mosblech et al., 2009). They involve jasmonates, jasmonic acid (JA, 7), and jasmonic acid-related compounds such as cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA, 4) and

Abbreviations: AOC, allene oxide cyclase; AOS, allene oxide synthase; 12,13-EOT, 12,13(S)-epoxy-(9Z,11E,15Z)-octadecatrienoic acid; GC–MS, gas chromatography mass spectrometry; 13-HPOT, 13(S)-hydroperoxylinolenic acid; JA, jasmonic acid; JA-Ile, jasmonoyl-L-isoleucine; JAR1, jasmonate:amino acid synthetase; 13-LOX, 13lipoxygenase; MRM, multiple reaction monitoring; OPDA, 12-oxo-phytodienoic acid; OPC-8, 3-oxo-2-[(Z)-pent-2-enyl]-cyclopentane-1-octanoic acid; OPR3, OPDA reductase 3; UPLC–MS/MS, ultra performance liquid chromatography tandem mass spectrometry. ⇑ Corresponding author at: Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan. Tel.: +81 11 706 3349; fax: +81 11 706 2505. E-mail address: [email protected] (K. Takahashi).

jasmonoyl-L-isoleucine (JA-Ile, 8) (Fig. 1). These compounds regulate various physiological and developmental processes such as growth, senescence, and stress adaption. The biosynthesis of 7 (Fig. 1) starts with the oxidation of a-linolenic acid (1) at position 13 by 13-lipoxygenase (LOX) (Schaller and Stintzi, 2009). The resulting hydroperoxide, 13(S)-hydroperoxylinolenic acid (13HPOT, 2), is converted into an unstable allene oxide, 12,13(S)epoxy-(9Z,11E,15Z)-octadecatrienoic acid (12,13-EOT, 3), by allene oxide synthase (AOS). Allene oxide cyclase (AOC) catalyzes the cyclization of 12,13-EOT to form 4, which has a cyclopentenone structure. This reaction step is crucial to construct the basic structure of JA (4). The biosynthetic reactions that generate 4 are performed in the chloroplasts of flowering plants. Compound 4 is translocated from chloroplasts to peroxisomes, where it is reduced by OPDA reductase 3 (OPR3) to yield 3-oxo-2-[(Z)-pent-2-enyl]cyclopentane-1-octanoic acid (OPC-8, 5). Three rounds of b-oxidation of 5 afford (+)-iso-JA (6), which is subsequently isomerized to (-)-JA (7). Jasmonate:amino acid synthetase, JAR1, generates 8, an active form of 7 (Fonseca et al., 2009).

http://dx.doi.org/10.1016/j.phytochem.2015.03.008 0031-9422/Ó 2015 Published by Elsevier Ltd.

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

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Y. Yamamoto et al. / Phytochemistry xxx (2015) xxx–xxx

COOH O

α-Linolenic acid (1) 13-LOX

COOH

OOH OPC-8 (5) COOH 13-HPOT (2)

β-oxidation ( x 3) O

O Isomerization

AOS O COOH 12,13-EOT (3)

COOH

COOH

(+)-iso-JA (6)

(+)-JA (7)

JAR1 O

AOC O COOH

O HOOC

NH

cis -(+)-OPDA (4) OPR3

JA-Ile (8)

Fig. 1. Biosynthesis of jasmonates from a-linolenic acid. AOC, allene oxide cyclase; AOS, allene oxide; 12,13-EOT, 12,13(S)-epoxy-(9Z,11E,15Z)-octadecatrienoic acid; 13HPOT, 13(S)-hydroperoxyoctadecatrienoic acid; JA, jasmonic acid; jasmonoyl-L-isoleucine, JA-Ile; JAR1, jasmonate:amino acid synthetase; 13-LOX, 13-lipoxygenase; OPDA, 12-oxo-phytodienoic acid; OPR3, OPDA reductase3; OPC-8, 3-oxo-2-[(Z)-pent-2-enyl]-cyclopentane-1-octanoic acid; OPDA; 12-oxo-phytodienoic acid.

The JA (7) signaling pathway has been intensively studied, and the framework of the JA (7) signal transduction mechanism has been elucidated (Wasternack and Hause, 2013). When the concentration of 8 is low, JAZ proteins suppress the activity of transcription factors such as MYC2, which binds to the promoter regions of jasmonate-responsive genes. Environmental stresses induce de novo synthesis of 7, which leads to 8. Binding of 8 to COI1, which is a part of the SCFCOI1 complex, facilitates the formation of complexes with JAZ proteins. After the ubiquitination of JAZ, this protein is degraded by the 26S proteasome. The release of the repression of MYC2 activity triggers JA-dependent gene expression. In addition, compound 4 has JA-dependent and JA-independent biological activities (Böttcher and Pollmann, 2009). A transcriptome analysis of Arabidopsis treated with 4 established that it specifically induces the expression of more than 150 genes (Taki et al., 2005). Recently, compound 4 was shown to play an important role in development in tomato (Goetz et al., 2012). However, the detailed function and signaling pathway of 4 remain to be elucidated. Bryophytes, including liverworts, mosses, and hornworts, comprise an early diverging lineage of land plants (Qiu et al., 2006) and have unique genetic and physiological properties that can be considered ancestral to those of vascular plants (Bowman et al., 2007). The genome of Physcomitrella patens, a model moss, has been sequenced (Rensing et al., 2008). The hormonal functions of auxin, cytokinin, and abscisic acid have been reported in P. patens (Prigge et al., 2010; von Schwartzenberg et al., 2007, Takezawa et al., 2011). In contrast to the plant hormones described above, the detailed functions of oxylipins are still unclear in P. patens. Nevertheless, in the octadecanoid pathway, recombinant AOS and AOC proteins have been shown to possess enzymatic activities (Bandara et al., 2009; Stumpe et al., 2010; Hashimoto et al., 2011). The disruption of PpAOC1 and PpAOC2 causes reduced fertility, aberrant sporophyte morphology, and interrupted sporogenesis in P. patens (Stumpe et al., 2010). The crystal structural analysis of PpAOC1 and PpAOC2 suggested that differential substrate specificities of the PpAOCs arise from a difference of active site conformations (Neumann et al., 2012). Bacterial infection induces AOS and OPR gene expression and the accumulation of 4.

Treatment of 4 reduced the growth of P. patens (Ponce de León et al., 2012). Compound 4 probably plays a role in physiological processes in P. patens. Recently positive feedback regulation of the octadecanoid pathway was shown in P. patens (Toshima et al., 2014). Marchantia polymorpha is distributed throughout the world and is a member of the liverworts, which are considered to be in the earliest diverging clade of land plants (Qiu et al., 2006). M. polymorpha has been established as a model organism because of its critical evolutionary position (Bowman et al., 2007) and the availability of its molecular genetic tools, including transformation techniques (Ishizaki et al., 2008; Kubota et al., 2013) and gene-targeting strategies (Ishizaki et al., 2013). There are several reports on the functions of plant hormones in M. polymorpha. Abscisic acid (ABA) inhibits M. polymorpha growth, and MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE (ABI1), functions as a negative regulator of ABA signaling; ABA signal transduction is probably present in M. polymorpha (Tougane et al., 2010). Auxin is important for both gametophyte and sporophyte development in M. polymorpha, and the mechanism of auxin-mediated transcriptional activation has already been established in M. polymorpha (Ishizaki et al., 2012). In contrast to the plant hormones described above, the function of jasmonates in M. polymorpha was not studied until recently. A database search identified the unique homolog of known plant AOC genes, which was designated MpAOC. In this study, the contents of 4 and 7 in M. polymorpha were investigated. Also examined was the role of MpAOC in OPDA (4) biosynthesis. This study demonstrated a possible role for 4, which is produced by MpAOC, as a signaling molecule that regulates growth and the response to wounding in the basal land plant, M. polymorpha.

2. Results 2.1. Identification of OPDA in M. polymorpha Compound 4 has already been identified in not only flowering plants, but also the model moss P. patens (Oliver et al., 2009). Whether compound 4 exists in M. polymorpha was of interest,

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

Y. Yamamoto et al. / Phytochemistry xxx (2015) xxx–xxx

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and UPLC–MS/MS analysis clearly established overlapping peaks for it in a standard sample as well as in a methanol extract of M. polymorpha (Figs. 2A–C). Considering identical full mass scan spectral data corresponding to the two peaks (Fig. 2D and E), compound 4 was shown to be present in M. polymorpha. Also M. polymorpha was analyzed for 7; however, it was not detected (Fig. S1). In flowering plants, JA (7) biosynthesis is induced by environmental stresses, and the stress adaptation conferred by it is then enhanced via positive feedback regulation in the octadecanoid pathway (Wasternack and Hause, 2013). The amount of 4 was increased by pathogenic infection and mechanical wounding in P. patens (Ponce de León et al., 2012; Scholz et al., 2012). The endogenous concentration of 4 in M. polymorpha after wounding was analyzed using UPLC–MS/MS (Fig. 3). Its level was significantly increased 8 h after wounding, suggesting that wounding activated the biosynthesis of 4. 2.2. Effect of OPDA (4) on M. polymorpha growth Compounds 4 and 7 show growth inhibitory activities in flowering plants. Compound 4 also inhibits P. patens growth; however, 7 did not have any significant physiological effects on P. patens (Ponce de León et al., 2012). To examine the effects of 4 in M. polymorpha, M. polymorpha was grown on a medium supplemented with 4 for 20 days. Compound 4 suppressed growth in a dose-dependent manner when applied at concentrations ranging from 25 to 50 lM (Fig. 4). In contrast, compound 7 did not

Fig. 3. OPDA (4) accumulation in M. polymorpha subjected to wounding stress. M. polymorpha, which was grown on 0M51C agar medium was subjected to wounding stress and then harvested at the indicated times after wounding. The accumulation of OPDA (4) was analyzed by UPLC–MS/MS. The data represent the means ± SD (n = 5). The asterisks represent significant differences between the amount of OPDA (4) in wounded and control plants (Student’s t test, ⁄⁄p < 0.01).

significantly affect M. polymorpha growth at 50 lM (Fig. 4). Compound 4 has growth inhibitory activity in M. polymorpha, and compound 4-mediated signaling for growth inhibition probably exists in M. polymorpha.

Fig. 2. UPLC–MS/MS analysis of OPDA (4) in M. polymorpha. MRM mode analysis of a specific daughter peak at m/z 164.99, which was derived from the peak at m/z 291.37 [MH]. (A) Standard OPDA (4). (B) The M. polymorpha extract. (C) Co-injection of standard OPDA (4) and the M. polymorpha extract. Product ion mass spectra of m/z 291 [MH]. (D) Standard OPDA (4). (E) The M. polymorpha extract.

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

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2.3. Cloning and overexpressing MpAOC in Escherichia coli Given the presence of 4, the octadecanoid pathway was also hypothesized to exist in M. polymorpha. AOC is a critically important enzyme in the octadecanoid pathway, which constructs the basic jasmonate structure (Schaller and Stintzi, 2009). To identify AOC in M. polymorpha, the amino acid sequence of an AOC of P. patens (PpAOC1, CAD48752) was used for a tblastn search of the M. polymorpha genome database. A potential AOC gene (DDBJ accession number AB873099), which was designated MpAOC, was found in M. polymorpha. MpAOC was predicted to consist of 257 amino acids and have a molecular mass of 27.2 kDa. Alignments of the predicted amino acid sequences of MpAOC and several known plant AOCs indicated that MpAOC shares approximately

45% identity with the AOCs of Arabidopsis thaliana, tomato (Solanum lycopersicum), rice (Oryza sativa), corn (Zea mays), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and P. patens (Fig. S2). To examine the evolutionary relationship between MpAOC and the other AOCs, A phylogenetic analysis was performed by the neighbor-joining method. This analysis showed that the position of MpAOC was different from that of all existing AOC clusters (Fig. S3). The ChloroP v 1.1 program predicted the existence of a chloroplast signal peptide composed of 62 amino acids at the Nterminus. To examine whether MpAOC was involved in the production of 4, MpAOC was cloned and the recombinant MpAOC fused with a His-tag at the N-terminus, instead of the chloroplast signal peptide, was overexpressed in E. coli M15 using the

Fig. 4. The growth inhibitory activity of OPDA (4) in M. polymorpha. M. polymorpha was grown at 22 °C on 1/2 Gamborg’s B5 agar medium supplemented with 4. OPDA and JA for 20 days under continuous white fluorescent light. (A) Photographs of M. polymorpha. (B) Fresh weight of M. polymorpha. The data represent the means ± SD (n = 6). The asterisks represent significant difference between the treated plants and control plants (Student’s t test, ⁄⁄p < 0.01).

Fig. 5. Chiral GC–MS analysis of the product generated from the MpAOC reaction. The product of the MpAOC enzymatic reaction was treated with an alkaline solution and then methylated by ethereal diazomethane. The methylated OPDA was analyzed by GC–MS (Varian 1200L GC/MS/MS system) using a b-DEX fused silica capillary column (0.25 mm  30 m, 0.25 lm film thickness, Supelco, USA). The molecular ion peak of the OPDA methyl ester at m/z 306 was monitored. The former and latter peaks represent the (+)- and ()-trans-OPDA methyl esters, respectively. (A) Racemic OPDA (4) standard. (B) Product of MpAOC enzymatic reaction.

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

Y. Yamamoto et al. / Phytochemistry xxx (2015) xxx–xxx

expression vector pQE30. After the purification of crude proteins by Ni–NTA agarose column chromatography, a single band representing recombinant MpAOC appeared in SDS–PAGE analysis (Fig. S4). Thus, MpAOC was successfully overexpressed in E. coli, and this protein was used for enzymatic analysis.

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was markedly induced by wounding (Fig. 7A). The application of 4 stimulated MpAOC expression moderately (Fig. 7B). These data indicated the presence of positive feedback regulation in the biosynthesis of 4 in M. polymorpha; positive feedback regulation in the octadecanoid pathway is indicated to be conserved in land plants.

2.4. Enzymatic activity of MpAOC The substrate of the AOC reaction, compound 3, which is the product of the AOS reaction in the octadecanoid pathway, is unstable and immediately converted to racemic OPDA (4), a-ketol, and c-ketol (Laudert et al., 1997). Accordingly, the enzymatic activity of MpAOC was examined in 1 ml of 50 mM phosphate buffer (pH 7.0) including recombinant MpAOC, recombinant PpAOS1 (Bandara et al., 2009), and compound 2 as a substrate. The product of the MpAOC reaction, cis-form of 4, was converted into transform of 4 by alkaline treatment, methylated by ethereal diazomethane, and analyzed by chiral GC–MS. To estimate the activity of MpAOC, the molecular ion peak of OPDA methyl ester at m/z 306 [M]+ was monitored (Laudert et al., 1997). The retention time of the molecular ion peak was identical to that of the peak of the prepared (+)-trans-OPDA methyl ester standard (Fig. 5) The synthesis of (+)-trans-OPDA indicated that MpAOC has AOC activity similar to that of other characterized AOCs.

2.7. Phenotypic analysis of transgenic M. polymorpha overexpressing MpAOC The exogenous application of 4 inhibited M. polymorpha growth. The enzymatic activity of MpAOC indicated that compound 4 is

2.5. Subcellular localization of MpAOC The enzymes in the first half of the octadecanoid pathway, LOX, AOS, and AOC, are localized in chloroplasts where they participate in OPDA biosynthesis in flowering plants (Schaller and Stintzi, 2009). To examine whether MpAOC localizes to chloroplasts, a 35S::MpAOC-GFP plasmid was constructed and introduced into P. patens protoplasts, which were then observed by confocal laser scanning microscopy. The green fluorescent signal of GFP was clearly detected in chloroplasts, which were identified by the red autofluorescence of chlorophyll (Fig. 6). These results strongly suggested that MpAOC localizes to and functions in chloroplasts in a manner similar to the AOC proteins reported in other land plants. Compound 4 is therefore probably synthesized in M. polymorpha chloroplasts. 2.6. Effects of wounding and OPDA (4) treatment on MpAOC expression In flowering plants, the expression of genes encoding enzymes in the octadecanoid pathway is induced by environmental stresses and the treatment of 7. It was demonstrated that compound 4 also accumulates upon wounding in M. polymorpha (Fig. 3). To examine whether wounding and the treatment of 4 induce MpAOC expression, quantitative RT-PCR was performed to evaluate the level of mRNA accumulation for MpAOC, and the expression of MpAOC

Fig. 7. Quantitative RT-PCR analysis of MpAOC after wounding and OPDA treatment. (A) MpAOC expression in M. polymorpha, which was grown on 1/2 Gamborg’s B5, subjected to wounding. (B) MpAOC expression in M. polymorpha, which was grown on 1/2 Gamborg’s B5, treated with 25 lM OPDA. The data represent the means ± SD (n = 4). The asterisks represent significant difference between the treated plants and control plants (Student’s t test, ⁄⁄p < 0.01, ⁄p < 0.05).

Fig. 6. Expression of the MpAOC-GFP fusion protein in the chloroplasts of P. patens protoplasts. An MpAOC-GFP fusion construct was introduced into P. patens protoplasts by PEG-mediated transformation. Images were taken with a confocal laser-scanning microscope with excitation at 488 nm and emission at 530 nm for the detection of the GFP signal and an emission above 655 nm for the detection of autofluorescence from chlorophyll. Scale bar: 10 lm.

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

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synthesized through the octadecanoid pathway. A previous study in P. patens showed that sporophyte formation was impaired when either PpAOC1 or PpAOC2 was disrupted (Stumpe et al., 2010). The disruption of PpAOS1, which encodes a bottleneck enzyme in the octadecanoid pathway, caused a decrease in the endogenous level of 4 but did not cause phenotypic alterations (Scholz et al., 2012). To elucidate the endogenous function of MpAOC in M. polymorpha, transgenic M. polymorpha overexpressing MpAOC was constructed using Agrobacterium (Ishizaki et al., 2008). The transgenic lines were screened by RT-PCR to identify those overexpressing MpAOC. The phenotype comparison between the MpAOC overexpression lines and a vector control showed that the overexpression of MpAOC suppressed M. polymorpha growth (Fig. 8). This result was consistent with the effects of the exogenous application of 4. The analytical data for 4 established that its levels in the overexpression lines were higher than that in M. polymorpha containing the empty vector (Fig. 8). The MpAOC protein was shown to be functional in vivo. Taken together, these results suggest that compound 4 produced by MpAOC affects M. polymorpha growth. 3. Discussion Compound 4 was previously shown to be biosynthesized and to demonstrate growth inhibitory activity in the model moss P. patens. Compound 4 may therefore be a signaling molecule in P. patens (Ponce de León et al., 2012). M. polymorpha is a model

liverwort, which is classified into the Bryophytes. Therefore, compound 4 was also presumed to exist in M. polymorpha. The analytical data on 4 showed that it is present in M. polymorpha (Fig. 2). To the best of our knowledge, this result was the first evidence that compound 4 is present M. polymorpha. Moreover, wounding caused an increase in the concentration of 4 (Fig. 3). These results suggest that compound 4 is involved in the response to wounding stress in M. polymorpha as in flowering plants; however, the response to induction of formation of 4 in M. polymorpha was slower than that in flowering plants. M. polymorpha growth was suppressed by 4 at concentrations ranging from 25 to 50 lM (Fig. 4). Liverworts are considered to represent the earliest divergence of land plants, and compound 4 is involved in responses to adverse environmental conditions and growth in land plants. However, 7 was not detected, and 7 did not affect the M. polymorpha phenotype; in contrast, compound 4 suppressed growth in a dose-dependent manner. Stumpe et al. (2010) concluded that compound 7 is not present in the moss P. patens. Furthermore, treatment of 7 did not confer any abnormal biological phenotype in P. patens (Ponce de León et al., 2012). Accordingly, compound 7 likely does not have any significant physiological function in bryophytes. AOC catalyzes the formation of cyclopentenone, which is the basic structure of jasmonates (Schaller et al., 2008; Wasternack and Hause, 2013). The AOC enzymatic reaction establishes the enantiomeric structure of OPDA, the naturally occurring form. In this study, a gene encoding a functional AOC, MpAOC, in

Fig. 8. The phenotypes of the transgenic M. polymorpha overexpressing MpAOC. (A) The transgenic M. polymorpha overexpressing MpAOC (OX1 and OX2) and the transgenic M. polymorpha carrying an empty pMpGWB103 vector (VC) were grown on 1/2 Gamborg’s B5 agar medium at 22 °C for 20 days under continuous white fluorescent light. (B) The fresh weight of the OX1, OX2 and VC plants. (C) The endogenous concentration of OPDA (4) in the OX1, OX2 and VC plants. The concentrations of OPDA (4) were determined by UPLC–MS/MS. The data represent the means ± SD (n = 6). The asterisks represent significant difference between the transgenic lines (OX1 and OX2) and control plants (VC) (Student’s t test, ⁄⁄p < 0.01).

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

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M. polymorpha was successfully identified. The enzymatic activity of MpAOC required to synthesize the naturally occurring form of 4 was also demonstrated (Fig. 5), and microscopic observations using GFP fused with MpAOC suggested its localization to chloroplasts (Fig. 6). The overexpression of MpAOC increased the OPDA contents (Fig. 8); this result strongly suggested the presence of the first half of the octadecanoid pathway that synthesizes 4 in M. polymorpha. Considering the chloroplast localization of MpAOC, the first half of the octadecanoid pathway in chloroplasts could have been conserved during land plant evolution. Environmental stresses were shown to stimulate production of 4 in M. polymorpha in a manner similar to that in flowering plants and P. patens. As predicted, wounding induced MpAOC expression. Moreover, addition of exogenous 4 moderately provoked MpAOC expression, indicating that positive feedback regulation of synthesis of 4 occurs (Fig. 7). The slow response to induce MpAOC by wounding was almost consistent with the slow increase in the level of 4 caused by wounding (Fig. 3). The difference in time to induce MpAOC expression between wounding and treatment of 4 was observed (Fig. 7). These results suggested that it required time to initiate synthesis of 4 in M. polymorpha. Although the details have not been established, the slow induction of synthesis of 4 caused by wounding might be unique to liverworts including M. polymorpha. Analysis of 4 in several species of liverworts is needed for understanding the different response of wounding between liverworts and flowering plants (Agrawal et al., 2003; Sato et al., 2009). In the flowering plant A. thaliana, the expression of AOC genes in wounded leaves of Arabidopsis opr3 mutant (OPR3 is the enzyme immediately after AOC in the octadecanoid pathway) was lower than that in wild-type leaves (Stenzel et al., 2003). Compound 7 functions as the major signal of positive feedback regulation in the octadecanoid pathway (Laudert et al., 2000). Considering that 7 was suggested to be absent in M. polymorpha, it seems that M. polymorpha has a characteristic positive feedback mechanism in the octadecanoid pathway. Overexpression of MpAOC resulted in the accumulation of 4 in M. polymorpha (Fig. 8). In contrast, previous reports showed that flowering plants overexpressing genes encoding enzymes in the biosynthetic pathway of 4 failed to increase the levels of 7. Overexpression of AOS did not raise the endogenous level of either 4 or 7 in tobacco and Arabidopsis (Laudert et al., 2000). In flowering plants, the biosynthetic pathway is considered to be limited by substrate availability, and the biosynthesis and consumption of 7 achieves dynamic equilibrium (Stenzel et al., 2003). The regulatory mechanism of the octadecanoid pathway in M. polymorpha may be different from that in flowering plants, although the detailed mechanism has not been explored. Alternatively, compound 4 could stimulate the release of 1 in chloroplast lipid membrane, the starting material in biosynthesis of 4, and contribute to the positive feedback regulation of 4. The growth of transgenic M. polymorpha overexpressing MpAOC was suppressed compared with control plants (Fig. 8). This result suggested that the accumulation of 4 suppressed growth. To the best of our knowledge, this study is the first to report that the overexpression of a gene involved in the octadecanoid pathway retards plant growth.

4. Concluding remarks This study indicates that compound 4 is a signaling molecule in the liverwort M. polymorpha. Thus, compound 4 has biological activities in not only flowering plants but also liverworts. Given that liverworts represent the most basal lineage in extant land plants, the function of 4 might have been inherited through the evolution of land plants with some alterations. A signaling

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mechanism of 4 has not yet been clarified in either bryophytes or flowering plants. Comparing the fundamental mechanisms, and functional evolution of 4 between bryophytes and flowering plants is an important challenge in understanding the physiological functions of jasmonates. 5. Experimental 5.1. Chemicals (+)-cis-OPDA (4) was prepared according to the method of Kajiwara et al. (2012). (±)-JA (7) was purchased from Sigma– Aldrich Co. (USA). 5.2. Plant materials Male and female accessions of M. polymorpha, Takaragaike-1 and Takaragaike-2, were maintained asexually (Ishizaki et al., 2008). F1 spores, which were produced by crossing Takaragaike1 and Takaragaike-2, were employed for transformation. The formation of sexual organs was elicited by far-red irradiation as described previously (Chiyoda et al., 2008). Mature sporangia were collected 3–4 weeks after crossing, air-dried for approximately 10 days, and stored at 80 °C until use. The plants were propagated on 0M51C medium containing 1.4% agar or 1/2 Gamborg’s B5 medium containing 1.4% agar under 50–60 lmol m2s1 continuous fluorescent light at 22 °C unless otherwise stated. 5.3. Analysis of OPDA (4) in M. polymorpha M. polymorpha, which was grown on 0M51C medium for 30 days, was used for the analysis of the endogenous concentrations of 4 and 7. For stress treatments, approximately 1 cm2 of a plant body was wounded using forceps. Unwounded plant tissue and tissue from plants subjected to wounding stress (approximately 1 g) were frozen with liquid N2 and extracted with EtOH (10 ml). Concentrations of 4 and 7 were analyzed by UPLC–MS/ MS according to the method of Sato et al. (2009) with some modifications (Supplementary data). 5.4. Cloning and overexpression of MpAOC Cloning and overexpression of MpAOC were performed according to a conventional method. The detailed experimental procedures are described in Supplementary data. 5.5. Analysis of the stereoselectivity of the MpAOC reaction Analysis of the stereoselectivity of the MpAOC reaction was conducted using chiral GC–MS according to the method of Hashimoto et al. (2011), with the detailed experimental procedure described in Supplementary data. 5.6. Microscopic analysis of transiently expressed MpAOC-GFP in P. patens protoplasts PCR was performed using the cDNA as a template and a forward primer (MpAOC-SLF, 50 -AAAGCAGGCTCCACCATGGCCGCCTCCATT30 ) and a reverse primer (MpAOCoe-R, 50 -AGCTGGGTCTAGATCT CAAGCCTTGACTTT-30 ). The PCR reaction was optimized for a 50-ll reaction mixture containing 5 ll of a dNTP mixture (2.0 mM each dNTP), 3 ll of 25 mM MgSO4, 1 ll of each primer (10 lM), 1 ll of pSK-MpAOC encoding the putative ORF of MpAOC (100 ng/ll), 5 ll of PCR buffer (10), 1 ll of KOD plus neo DNA polymerase (Toyobo, Japan), and 33 ll of Milli-Q H2O.

Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008

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Y. Yamamoto et al. / Phytochemistry xxx (2015) xxx–xxx

The reaction conditions were 94 °C for 2 min (initial denaturation); 30 cycles of 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min; and a final extension of 68 °C for 10 min. The PCR product was inserted into the pENTR4 entry vector (Invitrogen, USA) using the GeneArt seamless cloning and assembly kit (Invitrogen, USA). The resulting pENTR4-MpAOC entry clone was combined with the pUGWnew5 destination vector and Gateway LR Clonase Mix (Invitrogen, USA) to generate a plasmid encoding 35S::MpAOC-GFP (pUGWnew5MpAOC). The plasmid DNA was sequenced to confirm that the target gene was correctly inserted and free of sequencing errors. The transformation of pUGWnew5-MpAOC into protoplasts prepared from P. patens, which were grown on BCDATG medium for 3 days, was performed by PEG-mediated transformation. The localization of MpAOC-GFP was observed using a TCS-SP5 confocal laser scanning microscope (Leica, Germany). 5.7. Quantitative RT-PCR of MpAOC M. polymorpha, which was grown on 1/2 Gamborg’s B5 agar medium for 20 days, was either sprayed with 25 lM compound 4 or wounded. After 0, 2, 4, 6 and 8 h, total RNA was isolated by innuPREP Plant RNA kit (Analytik Jena AG, Germany). For real-time PCR, the total RNA (2 lg) was used as template for cDNA synthesis. First strand of cDNA was synthesized by M-MLV reverse transcriptase (Invitrogen, USA) with oligo (dT) primer according to manufacturer’s instructions. Real-time RT-PCR of the MpAOC was carried out using a 1-ll aliquot of the reaction mixture and SYBR green mixture (Takara, Japan). The PCR cycle was 95 °C for 5 s and 60 °C for 30 s. This cycle was repeated 40 times. The primer pairs were MpAOC-qPCR-F (50 -TCGTCAGCGCATCCTTCTTT-30 ) and MpAOC-qPCR-R (50 -TGTCACCCTCGTTGAACTCG-30 ) for MpAOC, and EF1a-qPCR-F (50 -AAGCCGTCGAAAAGAAGGAG-30 ) and EF1aqPCR-R (50 -TTCAGGATCGTCCGTTATCC-30 ) for EF1a. The EF1a gene was also amplified as an internal control. Real-time PCR was performed using Thermal Cycler Dice Real Time System (Takara, Japan). 5.8. Construction of M. polymorpha plants overexpressing MpAOC PCR was performed with the cDNA using a forward primer (MpAOC-SLF, 50 -AAAGCAGGCTCCACCATGGCCGCCTCCATT-30 ) and reverse primer (MpAOCoe-R, 50 -AGCTGGGTCTAGATCTCAAGCCT TGACTTT-30 ). The PCR reaction was optimized for a 50-ll reaction mixture containing 5 ll of a dNTP mixture (2.0 mM each dNTP), 3 ll of 25 mM MgSO4, 3 ll of each primer (5 lM), 1 ll of pSKMpAOC encoding the putative ORF of MpAOC (100 ng/ll), 5 ll of PCR buffer (10), 1 ll of KOD plus neo DNA polymerase (Toyobo, Japan), and 29 ll of Milli-Q H2O. The reaction conditions were 94 °C for 2 min (initial denaturation); 30 cycles of 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min; and a final extension of 68 °C for 10 min. The PCR product was inserted into the pENTR4 entry vector (Invitrogen, USA) using the GeneArt seamless cloning and assembly kit (Invitrogen, USA). The resulting pENTR4-MpAOC entry clone was combined with the pMpGWB103 destination vector and Gateway LR clonase mix (Invitrogen, USA) to generate a plasmid encoding EF::MpAOC (pMpGWB103-MpAOC). The construction of transgenic M. polymorpha overexpressing MpAOC using Agrobacterium was carried out according to the method of Ishizaki et al. (2008). Expression of MpAOC in the transgenic M. polymorpha overexpressing MpAOC was confirmed by semi-quantitative RT-PCR (Fig. S5). Acknowledgment We thank Dr. Hasebe (National Institute for Basic Biology) for kindly providing the plasmid, pUGWnew5.

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Please cite this article in press as: Yamamoto, Y., et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.03.008