Plant Growth Regulation

, 65:239

Cloning and characterization of an allene oxide cyclase, PpAOC3, in Physcomitrella patens

Authors

  • Takahiro Hashimoto
    • Division of Applied Bioscience, Research Faculty of AgricultureHokkaido University
    • Division of Applied Bioscience, Research Faculty of AgricultureHokkaido University
  • Michio Sato
    • Division of Applied Bioscience, Research Faculty of AgricultureHokkaido University
  • P. K. G. S. S. Bandara
    • Division of Applied Bioscience, Research Faculty of AgricultureHokkaido University
  • Kensuke Nabeta
    • Division of Applied Bioscience, Research Faculty of AgricultureHokkaido University
Original paper

DOI: 10.1007/s10725-011-9592-z

Cite this article as:
Hashimoto, T., Takahashi, K., Sato, M. et al. Plant Growth Regul (2011) 65: 239. doi:10.1007/s10725-011-9592-z

Abstract

Allene oxide cyclase (AOC) is a key enzyme in the octadecanoid pathway of flowering plants that synthesizes 12-oxo-phytodienoic acid (OPDA), which is a biosynthetic precursor of the signal molecule jasmonic acid (JA). A database search of the Physcomitrella patens genome revealed the presence of an AOC gene unique from the two previously reported AOC genes, PpAOC1 and PpAOC2. After cloning the identified AOC gene, designated PpAOC3, the obtained cDNA sequence (897 bp) was larger than the predicted AOC gene (765 bp) in the database because a speculated intron was not fully deleted. Although PpAOC3 did not display high similarity with AOC proteins from other species, recombinant PpAOC3 exhibited AOC activity and translocated to chloroplasts, as is observed for other AOC proteins. Notably, the expression profile of PpAOC3 differed from the other PpAOCs, as its expression in protonemata was higher than that in gametophores. Although the function of oxylipins such as OPDA and JA remains elusive in P. patens, further characterization of the enzymes in the octadecanoid pathway and the role of oxylipin will aid in the elucidation of physiological processes in this model bryophyte.

Keywords

Physcomitrella patensAllene oxide cyclaseChloroplastOctadecanoid pathway

Abbreviations

AOC

Allene oxide cyclase

AOS

Allene oxide synthase

12,13-EOT

(12,13S)-epoxy-(9Z,11E,15Z)-octadecatrienoic acid

13-HPOT

13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid

IPTG

Isopropyl β-d-thiogalactopyranoside

JA

Jasmonic acid

LOX

Lipoxygenase

OPC-8:0

3-oxo-2[(Z)-pent-2-enyl]-cyclopentane-1-octanoic acid

OPDA

12-oxo-phytodienoic acid

OPR

OPDA reductase

Introduction

Jasmonic acid (JA) is a plant hormone that is involved in various physiological processes in flowering plants (Wasternack 2007). The biosynthesis of JA occurs in the octadecanoid pathway (Schaller, and Stinzi 2009), which is initiated by the oxygenation of α-linolenic acid at position 13 by 13-lipoxygenase (13-LOX). The resultant hydroperoxide, 13-HPOT, is converted into an unstable allene oxide, (12,13S)-epoxy-(9Z,11E,15Z)-octadecatrienoic acid (12,13-EOT), by allene oxide synthase (AOS). Allene oxide cyclase (AOC) catalyzes the cyclization of 12,13-EOT to produce 12-oxo-phytodienoic acid (OPDA), which has a cyclopentenone structure. This reaction step is important to construct the basic structure of JA. Following the reduction of OPDA by OPDA reductase (OPR) to yield 3-oxo-2[(Z)-pent-2-enyl]-cyclopentane-1-octanoic acid (OPC-8:0), three cycles of β-oxidation affords (+)-iso-JA, which is subsequently isomerized to (-)-JA.

The JA signaling pathway has been intensively studied, and the framework of the JA signal transduction mechanism has been determined. The binding of (+)-7-iso-jasmonoyl-l-isoleucine conjugate, which is an active form of JA, to COI1 facilitates the formation of complexes with JAZ proteins, which serve to regulate plant growth and development. COI1 was recently demonstrated to serve as a receptor of JA signal transduction (Katsir et al. 2008).

Bryophytes appear to possess characteristic signatures of early land plants and unique physiological processes from those of vascular plants. The genome of Physcomitrella patens, a model moss for plant evolutionary and developmental studies, has recently been elucidated (Lang et al. 2008). Although the function of oxylipins, such as OPDA and JA, are not clear in P. patens, a gene encoding AOS (PpAOS1) was previously cloned and confirmed to have AOS activity (Bandara et al. 2009). Moreover, two potential AOC genes, PpAOC1 and PpAOC2, were identified and analyzed, with both recombinant proteins exhibiting AOC activity. These AOC proteins were shown to localize in chloroplasts in a similar manner as in flowering plants. The phenotype analyses of disrupted mutants of PpAOC1 and PpAOC2 showed that these genes play a role in spore formation, respectively. Double knockout mutant was not obtained, indicating that both enzymes have overlapping functions in protoplast generation (Stumpe et al. 2010). The function of oxylipins such as OPDA and JA remains elusive in P. patens. We conducted a search of the DOE Joint Genome Institute database (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html) and identified a predicted AOC gene in P. patens that was distinct from the previously reported PpAOCs. We report here the cloning and functional analysis of the PpAOC3 gene.

Materials and methods

Plant material

Physcomitrella patens was propagated on either BCDATG or BCD agar media under continuous white fluorescence light at 25°C, as previously described (Nishiyama et al. 2000).

Cloning and overexpression of PpAOC3

Total RNA was extracted from P. patens grown on BCDATG agar medium under continuous white fluorescence light at 25°C for 3 weeks, and reverse transcription (M-MLV Reverse Transcriptase, Invitrogen, Carlsbad, CA USA) was performed according to the manufacturer’s instructions to yield cDNA. PCR was carried out with the generated cDNA using a specific primer set for PpAOC3, which were designed based on a sequence in the NCBI database (accession number XM_001771188). The confirmed coding sequence for PpAOC3 was then subcloned into the expression vector pQE30 (Qiagen, USA). The detailed cloning method is described in Supplementary material. The resultant expression vector, pQE30-PpAOC3 was transformed into E. coli M15 by standard procedures. After selection of transformants on LB agar medium containing 100 μg/ml ampicillin at 37°C, a single colony was grown overnight in 20 ml LB medium containing 100 μg/ml ampicillin at 37°C. A 10-ml aliquot of the culture was inoculated into 1 l of LB medium containing 100 μg/ml ampicillin and incubated at 22°C. Once the culture reached cell density at an OD600 of 0.6, recombinant protein synthesis was induced by the addition of 0.2 mM IPTG. After further incubation for 3 h, the cells were collected by centrifugation at 7,000g for 5 min, and were then washed once with a basal buffer (50 mM sodium phosphate buffer, pH 7.8). The cells were resuspended in 3 ml of basal buffer containing 0.3 M NaCl, 20 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and then disrupted by ultrasonication. After cell debris was removed by centrifugation at 14,000g for 20 min, 2 ml of the supernatant was subjected to Ni–NTA agarose column chromatography (2 ml, GE Healthcare, USA). The column was washed with 20 ml of basal buffer and PpAOC3 fused with a 6 × His-tag was then eluted with basal buffer containing 150 mM imidazole and 0.3 M NaCl. The eluate containing the recombinant protein was dialyzed against a buffer composed of 10 mM sodium phosphate (pH 7.0) and 1 mM DTT, and was then resolved by SDS–PAGE. The dialyzed His-tagged PpAOC3 protein was used for enzymatic activity analysis. Peptide mass fingerprinting analysis of PpAOC3 was performed according to the method of Sato et al. (2009).

GC–MS analysis of the PpAOC3 reaction product

An AOC reaction mixture (5 μg 13-HPOT, 5 μg PpAOS1, and 10 μg PpAOC3 in 1 ml of 10 mM sodium phosphate buffer, pH 7.0) was incubated for 15 min at 25°C. For termination of the AOC reaction, the reaction mixture was adjusted to approximately pH 3 by the addition of 1 M HCl. Conversion of cis-OPDA into trans-OPDA was done by alkaline treatment. The reaction mixture was extracted with the identical volume of ethyl acetate twice, evaporated, and the resulting residue was then methylated using ethereal diazomethane. The methylated residue was dissolved in 100 μl of CHCl3, and 1 μl of the solution was injected into a GC–MS spectrometer (1200L GC/MS/MS system, Varian, USA) equipped with a β-DEX fused silica capillary column (0.25 mm × 30 m, 0.25 µm film thickness, Supelco, USA). Helium was used as a carrier gas at a constant flow rate of 1.0 ml/min. The ion source and vaporizing chamber were heated to temperature of 200 and 220°C, respectively, and the ionization voltage was 70 eV. The column was initially heated to 50°C, held isothermal for 1 min, and then subsequently raised at 10°C/min to a final temperature of 190°C, which was held for 80 min. The retention times of the (+)- and (−)-trans-OPDA methyl esters were 86.8 and 88.4 min, respectively. Racemic trans-OPDA methyl ester was prepared according to the method of Bandara et al. (2009) to be used as a standard.

Microscopic analysis of transiently expressed PpAOC3-GFP

For construction of a vector for transiently expressing of PpAOC3-GFP, the cloned PpAOC3 sequence was subcloned into the pENTR4 vector (Supplementary material). The obtained entry clone was combined with the pUGWnew5 destination vector and Gateway LR Clonase Mix (Invitrogen, USA) to generate a plasmid encoding 35S::PpAOC3-GFP (pUGWnew5-PpAOC3). Transformation of pUGWnew5-PpAOC3 into protoplasts prepared from P. patens, which were grown on BCDATG medium for 3 days, was performed by PEG-mediated transformation (Baur et al. 2005). The localization of PpAOC-GFP was analyzed at 200× magnification using a TCS-SP5 confocal laser scanning microscope (Leica, Germany).

Semi-quantitative RT-PCR of PpAOC3

Total RNA were extracted from protonemata of P. patens grown on BCDATG agar medium for 4 days, and gametophores of P. patens grown on BCD agar medium for 60 days, respectively. The detailed RT-PCR method for PpAOCs is described in Supplementary material. The obtained PCR products were analyzed by gel electrophoresis and visualized with ethidium bromide.

Results and discussion

Cloning and overexpression of PpAOC3 in E. coli

A search of NCBI database identified a potential AOC gene (accession number XM_001771188) that was distinct from the two previously reported PpAOC genes. To examine whether this gene was involved in OPDA production, we attempted to clone and overexpress this AOC gene in E. coli. Although the predicted DNA size was 765 bp based on the database sequence, a ca. 900 bp product was amplified by nested PCR using cDNA template. After the nested PCR product was ligated into pBluescript SK II (+) (Stratagene, USA) to generate plasmid pSK-PpAOC3, sequence analysis revealed that a predicted intron sequence was not fully deleted, with an additional 132 bp DNA remaining in exons 1 (123 bp) and 2 (9 bp) of the cloned gene. Therefore, the 897 bp DNA sequence obtained in this experiment for the unique AOC gene in P. patens was most likely the correct size, and was designated PpAOC3 (Fig. 1).
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Fig. 1

The gene structure of the predicted PpAOC3 gene and cloned PpAOC3 gene in P. patens. The predicted PpAOC3 gene is based on the mRNA sequence in the NCBI database (XM_00177188: 1–765 bp), and contains four exons (boxes) and three introns (lines). Arrows indicate the additional DNA sequences present in exons 1 and 2 of PpAOC3 compared with the predicted gene in P. patens

PpAOC3 was predicted to consist of 298 amino acids and have a molecular mass of 33 kDa. Among AOC proteins, PpAOC3 has a relatively high molecular mass, and is larger than the two other reported PpAOCs that have sizes of ca. 20 kDa (Stumpe et al. 2010). Alignments of the predicted amino acid sequences of PpAOC3 with several known plant AOCs indicated that PpAOC3 shares approximately 30% identity with AOCs of Arabidopsis thaliana, tomato (Solanum lycopersicum), rice (Oryza sativa), corn (Zea mays), and PpAOC1 and PpAOC2 (Fig. 2).
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Fig. 2

Amino acid sequence alignments of AOCs of several plant species with PpAOC3. The amino acids sequence of PpAOC3 was compared with AtAOC2 (Arabidopsis thaliana, accession number AJ308484), LeAOC (Lycopersicon esculentum, accession number AJ272026), ZmAOC (Zea mays, accession number AY488136), PpAOC1 (Physcomitrella patens, accession number XP_001783824), PpAOC2 (P. patens, accession number XP_001751254), and PpAOC3 (P. patens, the sequence shown by this report). Black and gray boxes indicate identical and similar amino acid residues, respectively, while arrows indicate the amino acids Glu113, Phe152, and Cys172, which are critical for AOC activity

To determine the evolutionary relationship between PpAOC3 and the AOCs of flowering plants, we performed phylogenetic analysis by the neighbor-joining method. Our analysis revealed that PpAOC3 was grouped in a cluster with PpAOC1 and PpAOC2, which was clearly separated from the clusters composed of AOCs from flowering plant species. Moreover, PpAOC3 was significantly removed from the other PpAOCs (Fig. 3). Notably, although the critical amino acids for AOC activity, Glu113, Phe152, and Cys172, were conserved in PpAOC3 (Hofmann et al. 2006), the iPSORT program (http://hc.ims.u-tokyo.ac.jp/iPSORT/) predicted the absence of chloroplast-targeting signal peptides at the N-terminus.
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Fig. 3

Phylogenetic tree of AOCs from flowering plants and P. patens. Amino acid sequences were aligned using Clustal W and a phylogenetic tree was generated using Treeview software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Analysis was performed with AtAOC1, AtAOC2, AtAOC3, and AtAOC4 (Arabidopsis thaliana, accession numbers AJ308483, AJ308484, AJ308485, and AJ308486, respectively), BsAOC (Bruguiera sexangula, accession number AB037929), HlAOC (Humulus lupulus, accession number AY687339), HvAOC (Hordeum vulgare, accession number AJ308488), LeAOC (Lycopersicum esculentum, accession number AJ272026), PsAOC (Pisum sativum, accession number AB095986), ZmAOC (Zea mays, accession number AY488136), PpAOC1 (Physcomitrella patens, accession number XP_001783824), PpAOC2 (P. patens, accession number XP_001751254), and PpAOC3 (P. patens, the sequence shown by this report). The scale bar corresponds to 0.1 substitutions per nucleotide

To evaluate the localization and enzymatic activity of PpAOC3, we attempted to overexpress PpAOC3 with a His-tag at the N-terminus in E. coli M15 using the expression vector pQE30. After purification of crude proteins with Ni–NTA agarose column chromatography, two bands of approximately 33 and 30 kDa in size were appeared in SDS–PAGE analysis (Fig. 4a). The larger one had an expected molecular mass of PpAOC3 (33 kDa). The peptide mass fingerprinting analysis of the smaller protein showed that smaller size-band (30 kDa) was due to degradation of PpAOC3 during protein preparation (Fig. 4b). Thus PpAOC3 was successfully overexpressed in E. coli and then the mixture of the recombinant PpAOC3 proteins was subjected to enzymatic analysis.
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Fig. 4

Bacterial overexpression of PpAOC3 protein. a, SDS–PAGE analysis of PpAOC3 overexpressed in E. coli. M marker, P recombinant PpAOC3. b Alignment of peptide sequence of 30 kDa protein with PpAOC3. The peptide sequence of small size protein was determined by peptide mass fingerprinting analysis. The boxes show the peptide sequences identical to PpAOC3 sequence

Enzymatic activity of PpAOC3

The substrate of the AOC reaction, 12,13-EOT, which is the product of the AOS reaction in the octadecanoid pathway, is unstable and immediately converted to other compounds. Accordingly, the enzymatic activity of PpAOC3 was evaluated in 1 ml of phosphate buffer (pH 7.0) containing recombinant PpAOC3 (10 μg), recombinant PpAOS1 (5 μg), and 13-HPOT (5 μg) as a substrate. The product of the PpAOC3 reaction, cis-OPDA, was converted into trans-OPDA by alkaline treatment, methylated by ethereal diazomethane, and then analyzed by chiral GC–MS. To evaluate the activity of PpAOC3, the molecular ion peak of OPDA methyl ester at m/z 306 [M]+ was monitored (Laudert et al. 1996). The retention time of the molecular ion peak detected in this analysis was 86.8 min, which was identical to that of the peak of the prepared (+)-trans-OPDA methyl ester standard (Fig. 5). Additionally, the formation of greater than 95% of (+)-trans-OPDA indicated that PpAOC3 exhibits similar AOC activity as other characterized AOCs. The additional amino acids present in PpAOC3 compared with PpAOC1 and 2 did not appear to significantly affect AOC activity, suggesting that the spatial arrangement of the active site is conserved in PpAOC3.
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Fig. 5

Chiral GC–MS analysis of the product generated from the PpAOC3 reaction. The product of the PpAOC3 enzymatic reaction was methylated by ethereal diazomethane and analyzed by GC–MS spectrometry, and the molecular ion peak of OPDA methyl ester at m/z 306 was monitored. a Prepared methylated OPDA standard. b Product of the PpAOC3 enzymatic reaction. The peaks with retention times of 86.8 and 88.4 min represent (+)- and (−)-trans-OPDA methyl ester, respectively

Cellular localization of PpAOC3

In flowering plants, AOC proteins are localized in chloroplasts where they participate in JA biosynthesis. PpAOC1 and PpAOC2 have also been shown to localize in chloroplasts, even though these proteins are predicted to lack chloroplast-targeting signal peptides at the N-terminal region by the ChloroP v 1.1 program (Stumpe et al. 2010). Using the iPSORT program, we also failed to identify a chloroplast-targeting signal peptide in PpAOC3. This finding is supported by a previous suggestion that the transportation mechanisms of proteins from the cytosol to chloroplasts in P. patens differs from those in flowering plants.

To examine whether PpAOC3 is localized to chloroplasts in P. patens, a 35S::PpAOC3-GFP plasmid was constructed and transformed into P. patens protoplasts, which were then observed by confocal laser scanning microscopy. The green fluorescence signal of GFP was clearly detected in chloroplasts, which were identified by the red fluorescence of chlorophyll (Fig. 6). These data indicated that PpAOC3 is localized in chloroplasts in an identical manner as is observed for other AOC proteins. Considering that the structure of the N-terminal region in PpAOC3 is distinct from those of PpAOC1 and PpAOC2, a protein transport mechanism into chloroplasts that is independent of the N-terminus is suggested to exist in P. patens.
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Fig. 6

Expression of PpAOC3-GFP fusion protein in chloroplasts of protoplasts of P. patens. A PpAOC3-GFP fusion construct was introduced into protoplasts by PEG-mediated transformation. Images were obtained by confocal laser scanning microscopy with excitation at 488 nm and emission at 530 nm for detection of the GFP signal, and emission over 655 nm for detection of autofluorescence from chloroplasts. Scale bar 10 μm

Semi-quantitative PCR analysis of PpAOCs

In P. patens, spores germinate to form a filamentous tissue called protonema. Protonemata continue to develop and eventually differentiate into gametophores, which have a similar morphology as seed plants. As the morphological structures of these two developing stages are significantly different, we were interested in determining the expression levels of PpAOCs in protonemata and gametophores. Semi-quantitative RT-PCR was performed to evaluate mRNA expression of PpAOCs in each of these structures (Fig. 7). Our analysis revealed that PpAOC1 was the most highly expressed of the three PpAOCs genes, suggesting that PpAOC1 might play a major role for OPDA production in P. patens. Although the expression levels of PpAOC1 and PpAOC2 in protonemata were nearly identical as those in gametophores, PpAOC3 was preferentially expressed in protonemata. Accordingly, the differential expression patterns of these PpAOCs might influence the development of P. patens.
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Fig. 7

Semi-quantitative PCR analysis of PpAOCs in the protonemata and gametophores of P. patens. The RT-PCR products of the PpAOC1, PpAOC2, and PpAOC3 genes amplified by the indicated number of cycles from protonemata (P) and gametophores (G) were analyzed by gel electrophoresis and visualized by ethidium bromide

Conclusions

We have identified a gene in P. patens encoding an AOC protein, designated PpAOC3, which was shown to be 132 bp larger than the predicted gene (765 bp) in the database. PpAOC3 did not display high similarity with other characterized AOC proteins, but did exhibit AOC activity and was localized to chloroplasts, similar manner as occurs in flowering plants. Notably, PpAOC3 was preferentially expressed in protonemata, unlike the two other PpAOCs. Although the function of oxylipins such as OPDA and JA remains elusive in P. patens, further characterization of the enzymes in the octadecanoid pathway and the role of oxylipin will aid in the elucidation of physiological processes in this model bryophyte.

Acknowledgments

We are grateful to Prof. Y. Hasebe at the National Institute for Basic Biology for supplying the vector pUGWnew5. We also thank Dr. T. Fujita at Hokkaido University for his valuable advice. This research was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) to K.T.

Supplementary material

10725_2011_9592_MOESM1_ESM.doc (56 kb)
Supplementary material 1 (DOC 56 kb)

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