Cloning and characterization of an allene oxide cyclase, PpAOC3, in Physcomitrella patens
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- Hashimoto, T., Takahashi, K., Sato, M. et al. Plant Growth Regul (2011) 65: 239. doi:10.1007/s10725-011-9592-z
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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.
KeywordsPhyscomitrella patensAllene oxide cyclaseChloroplastOctadecanoid pathway
Allene oxide cyclase
Allene oxide synthase
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
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
Enzymatic activity of PpAOC3
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.
Semi-quantitative PCR analysis of PpAOCs
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.
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.