Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens

FEBS Letters 580 (2006) 6175–6181

Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens Ken-ichiro Hayashia,1, Hiroshi Kawaideb,1, Miho Notomib, Yuka Sakigia, Akihiko Matsuoc, Hiroshi Nozakia,* a Department of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama City 700-0005, Japan Division of Agriscience and Bioscience, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo 183-8509, Japan Division of Environmental Biochemistry, Faculty of Social Information Science, Kure University, Gohara-cho, Kure city, Hiroshima 724-07, Japan b

c

Received 28 July 2006; accepted 5 October 2006 Available online 17 October 2006 Edited by Mark Stitt

Abstract ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/ KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16a-hydroxykaurene).  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ent-kaurene synthase; Moss; Gibberellin; Biosynthesis; Physcomitrella patens

1. Introduction ent-Kaurene is a tetracyclic diterpene hydrocarbon and one of the biosynthetic intermediates in the production of the gibberellins (GAs), plant hormones. It is synthesized successively by two-step cyclization from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP). In higher plants, the angiosperms, each cyclization is catalyzed by two distinct enzymes, copalyl diphosphate synthase (CPS, formerly entkaurene synthase A) and ent-kaurene synthase (KS, formerly ent-kaurene synthase B). Diterpene cyclases are classified into

* Corresponding author. Fax: +81 86 256 9559. E-mail address: [email protected] (H. Nozaki). 1

two groups (types A and B) on the basis of cyclization mechanisms and aspartate-rich motifs (Fig. 1) [1]. Plant CPS belongs to type B cyclases, and the second aspartate of the DXDD motif in CPS acts as the proton donor to initiate the cyclization of GGDP [2]. On the other hand, KS is a type A cyclase with a DDXD motif, and it initiates the cyclization of CDP by an elimination of the diphosphate group in CDP and a rearrangement of the carbon skeleton of CDP to form an ent-kaurene skeleton [3,4]. In contrast to the unifunctional plant enzymes CPS and KS, the gymnosperm (conifer)-derived enzymes abietadiene synthase (AS) and abietadiene/levopimaradiene synthase (LAS) are bifunctional diterpene cyclases [5,6]. AS and LAS have two cyclization domains (DXDD and DDXXD) in a single polypeptide, and they directly catalyze the formation of an abietadiene or levopimaradiene skeleton from GGDP via CDP as a reaction intermediate (Fig. 1) [7]. Furthermore, functional analyses of ent-kaurene synthases from Phaeosphaeria sp. L487 and Gibberella fujikuroi have revealed that fungal ent-kaurene synthases (CPS/KS) are bifunctional diterpene cyclases having the activities of both plant CPS and plant KS (Fig. 1) [8,9]. The fungal CPS/KS have both CPS and KS active domains and have a conserved DXDD and DEXXEA motif in the CPS and KS domain, respectively [10,11]. The moss Physcomitrella patens is a model organism for the study of the genetics and development of bryophytes, because of a significant full-length cDNA EST collection, accessible whole-genome sequences, and established reverse genetic approaches [12,13]. Therefore, its study provides new insight into the phylogeny and evolution of land plants, including the biosynthesis and signaling of plant hormones. P. patens is reported to produce ent-16a-hydroxykaurene as a major secondary metabolite [14]. However, no report on the biosynthesis and physiological role of gibberellins in mosses has been published. Therefore, a study of the ent-kaurene synthases of P. patens has the potential to throw light on the phylogeny and evolution of gibberellin biosynthesis and signaling in land plants. In this paper, we describe the identification of the P. patens bifunctional ent-kaurene synthase gene (PpCPS/KS), and the cell-free protein expression and functional analysis of two CPS and KS domains in PpCPS/KS. Additionally, this is the first report on a diterpene cyclase from a bryophyte.

Both authors equally contributed to this study.

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.018

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K. Hayashi et al. / FEBS Letters 580 (2006) 6175–6181

Fig. 1. Biosynthesis of abietadiene and kaurene-type diterpenoids. Higher plants synthesize ent-kaurene with two different unifunctional enzymes, ent-copalyl diphosphate synthase (CPS) with DXDD motif and ent-kaurene synthase (KS) with DDXD motif. Fungal bifunctional ent-kaurene synthase [DXDD and DEXXE motifs] directly synthesize ent-kaurene from GGDP via carbocation intermediate. The moss P. patens ent-kaurene synthase, PpCPS/KS, [DXDD and DDXD motifs] directly synthesize two ent-kaurene diterpenes [ent-kaurene and ent-16-a-hydroxy-kaurene] from GGDP via carbocation intermediate. Abietadiene is directly synthesized from GGDP by a bifunctional abietadiene synthase (AS).

2. Materials and methods 2.1. Identification and analysis of PpCPS/KS The cDNA clone candidate for PpCPS/KS was searched in a BLAST search of the P. patens EST library generated by Nishiyama et al. [12], with the protein sequence of several plant CPSs, KSs and ASs as a query. The 3335-bp full-length PpCPS/KS cDNA clone (clone number: pdp33101, RIKEN Bio Resource Center, Japan) was sequenced on both strands. Homology genes of PpCPS/KS, CPS, and KS in the P. patens genome were searched against P. patens raw genome sequence data set in the PHYSCObase with PpCPS/KS and plant diterpene synthase genes as query. All genome clones with homology were assembled into contigs by a CAP-assembled program in PHYSCObase [http://moss.nibb.ac.jp/]. Multiple sequence alignments were generated with the CLUSTAL W program [15]. Phylogenetic analysis of deduced amino acid alignments used the neighbor-joining method [16] with the CLUSTAL W program. Prediction of the cleavage site of the plastidic transit peptide was performed with the ChloroP program [17]. 2.2. Expression constructs To analyze the enzyme function, the transcription product was expressed by an in vitro protein synthesis system. The ORF lacking the transit peptide region (D1–130) was amplified by PCR using Phusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland) and 5 0 -phosphorylated primers (ppdtc-forward primer, 5 0 ATGAACGAGTGGATTGAGG-3 0 and ppdtc-reverse primer, 5 0 CTATTCAGGTACTGGCTCG-3 0 ). The amplified fragment was ligated into downstream of polyhedrin 5 0 UTR in the pTD1 vector (Shimadzu, Kyoto, Japan) to construct a pTD-PpCPS/KS. Arabidopsis GA1 (At4g02780, AtCPS) and GA2 (At1g79460, AtKSB) ORFs were amplified by Phusion PCR from the cDNA library. The ORF of GA1 lacking the transit peptide region was amplified by PCR using the following primers: 5 0 -ATGGCATTCAAAGAAGCAGTGAAGAGTGTG-3 0 as a forward primer and 5 0 -CTAGACTTTTTGAAACAAGACTTTGGAGATGTG-3 0 as a reverse primer. The primers for amplification of GA2 ORF were 5 0 -ATGTTGGAGAAAGTGGAGCTTTCTGTTTCG-3 0 and 5 0 -TCAAGTTAAAGATTCTTCCTGTAAGCTAACAGG-3 0 . For production of GA1 and GA2 proteins, each truncated ORF was subcloned into the pTD1 vector. The pTD1 construct of PhCPS/KS–myc fusion protein, fungus Phaeosphaeria ent-kaurene synthase with c-myc at the C-terminus [8], was prepared as a positive control to verify in vitro expression of diterpene cyclase (data not shown). All DNA sequences of PCR-amplified ORFs were confirmed by ABI 3130xl Genetic Analyzer (Applied Biosystems).

2.3. Site-directed mutagenesis Site-directed mutagenesis was carried out by PCR using pTD-PpCPS/ KS as the template. The amino acid residues of N-terminal 417DVDD motif were changed into AVAD motif. Initially, two pairs of gene-specific primers, 5 0 -GCAGATTGTACTGAGAGTA-3 0 (pTD1-forward) and 5 0 -GTGTCGGCCACGGCCTGCACGG-3 0 (AVAD-reverse, mutation sites underlined), and 5 0 -CCGTGCAGGC- CGTGGCCGACAC-3 0 (AVAD-forward, mutation sites underlined) and 5 0 -ACAACGCACAGAATCTAGC-3 0 (pTD1-reverse), were used to amplify two ORF regions of PpCPS/KS. Then the AVAD mutant ORF was amplified by PCR with pTD1-forward and -reverse primers and the two ORF regions as templates. The amino acid residues of the C-terminal 635DDYFD motif were changed into AAYFD in the same manner as described above. Two gene-specific mutant primers, 5 0 -GATCGAAGTAAGCGGCTAGAACGG-3 0 (AAYFD forward, mutation sites underlined) and 5 0 -CCGTTCTAGCCGCTTACTTCGATC-3 0 (AAYFD reverse, mutation sites underlined) were used for PCR amplification of the mutant ORF. These mutant ORFs were subcloned into the pTD1 vector described above and the sequence of ORFs was confirmed. 2.4. Cell-free protein production and GC–MS analysis of enzymatic products All pTD1-related constructs were digested with NotI to linearize the double-stranded DNA templates. PpCPS/KS mRNA was prepared in vitro according to the manufacturer’s protocol (AmpliScribe T7Flash Transcription Kit, Epicentre, WI, USA). The mRNA synthesized was used in a cell-free protein synthesis system, Transdirect insect cell, which contains an extract prepared from Spodoptera frugiperda 21 insect cells (Shimadzu). Expression in vitro was obtained according to the manufacturer’s protocol. To verify the expression in vitro of fungus PhCPS/KS–myc protein, the fusion protein was analyzed by western blot using anti-c-myc-AP antibody (Invitrogen) and BCIP/NBT development (Western Blue stabilized substrate, Promega). The reaction mixture was centrifuged (16 000 · g, 4 C, 10 min) to remove insoluble debris, and the supernatant was used for enzyme assay. An enzyme reaction was started at the addition of 1 lg of GGDP as substrate and 5 mM of MgCl2 as a cofactor. After overnight incubation at 30 C, the enzymatic products were extracted three times with n-hexane and concentrated with a gentle stream of dry N2 gas. The sample was analyzed by the JEOL (Tokyo, Japan) JMS-BU25 GC–MS system (ionization energy at 70 eV, filament at 300 lA) coupled with a DB-5 capillary column (0.25 mm in diameter, 15 m long, 0.25 lm film thickness; J&W Scientific, Folson, CA, USA). After injection of the sample at 80 C for a 1-min isothermal hold, the column temperature was programmed at 30 C min1 intervals to 200 and at 5 C min1

K. Hayashi et al. / FEBS Letters 580 (2006) 6175–6181 intervals to 250 C. The flow rate of the helium carrier gas was 1 ml min1.

3. Results 3.1. Identification and sequence characterization of P. patens ent-kaurene synthase Plant diterpene synthases such as CPSs, KSs and ASs have high homology across different species. The P. patens fulllength cDNA EST database (PHYSCObase) was searched to isolate potential ent-kaurene synthase cDNA clones [12]. The EST database search revealed a unique cDNA clone (PpCPS/KS) with high sequence similarity to the C-terminus of Arabidopsis CPS. Sequencing of both strands in the PpCPS/KS cDNA clone resulted in a 3335-bp segment with one ORF of 2646-bp with an ATG start codon at position 403 and a TAG stop codon at position 3048. Comparison of the deduced 881-residue sequence of ORF (101 kDa) with known plant diterpene synthases (Fig. 2) indicated high similarity to conifer AS (45% identity, AAK83563), LAS (45% identity, AAS47691) and a number of plant CPSs (46–54%

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identities). Slightly higher similarity was found with plant KSs, (33–35% identities) and conifer E-a-bisabolene synthases (38% identities). This analysis together with signal peptide prediction suggested the existence of N-terminal putative transit peptide, indicating that the protein is transported into plastids just as are other plant diterpene synthases. Both the DVDDTA motif conserved in CPSs (type B: DXDDTA) and the DDYFD motif conserved in KS (type A: DDXXD) were found in the deduced amino-acid sequence, suggesting that PpCPS/KS would catalyze a sequential two-step cyclization as bifunctional cyclase, similar to AS and LAS [6,7]. Phylogenetic analysis also supported the similarity of PpCPS/KS to CPS and AS groups (Fig. 3). Phaeosphaeria sp. L487 and G. fujikuroi ent-kaurene synthases are bifunctional cyclases with a DXDD motif (in CPS domain) and DEXXE motif (in KS domain) [11]. PpCPS/KS as well as CPS and AS displayed moderate similarity (25–28% identities) to the CPS domain (from SAYDTA to DVDD motif) in the fungal CPS/KS, but not to the KS domain. Multiple sequence alignment analysis (Fig. 2) indicates that the N-terminal half sequence (SPYDTA to DVDD motif) has high similarity to AtCPS, and that the C-terminal half

Fig. 2. Comparison of plant diterpene synthases and PpCPS/KS. The deduced PpCPS/KS polypeptide was compared with Arabidopsis ent-coparyl diphosphate synthase (AtCPS), Arabidopsis ent-kaurene synthase B (AtKSB) and Abies grandis abietadiene synthase (AgAS). The dot represents identical amino acid to PpCPS/KS and dark gray shading indicates identical A.A. to each sequence. Light gray shading indicates A.A. with similar properties. Underline represents conserved motifs (SXYDTAWVA, DXDD, and DDXXD).

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Fig. 3. Phylogenetic analysis between plant diterpene synthases and PpCPS/KS. Plant origins of enzymes are as follows: AtCPS and AtKSB [Arabidopsis thaliana]; ZmCPS [Maize, Zea mays]; CmCPS [Pumpkin, Cucurbita maxima]; AgAS [Grand Fir, Abies grandis]; GbLS (levopimaradiene synthase) [Ginkgo biloba]; PaLS and PaLAS [Norway spruce, Picea abies]; TbTS (taxadiene synthase) [Taxus brevifolia]; SrKSB [Stevia rebaudiana]; CsKSB [Cucumber, Cucumis sativus]; and OsKSB, OsPS (syn-pimara-7,15-diene synthase) and OsCS (cassadiene synthase) [Rice, Oryza sativa].

sequence (570LKLAK to the end) resembles AtKSB. The BLAST search shows the highest similarity to CPS with N-terminal half sequence as query and to KS with C-terminal half sequence as query. Hence, PpCPS/KS seems to be a hybrid enzyme of CPS and KS, consisting of both plant CPS and KS active domains. Recently, the Joint Genome Institute achieved the wholegenome shotgun sequencing of the P. patens genome, and raw genome sequences are available in PHYSCObase. Therefore, the homology genes of PpCPS/KS, CPS, AS, LAS and KS were searched against the P. patens genome. However, no homology genes were found in the genome data set, except for pseudogenes. 3.2. Functional analysis of PpCPS/KS First of all, the in vitro protein expression system of a diterpene cyclase was verified by known fungal bifunctional ent-kaurene synthase using the Phaeosphaeria PhCPS/KS– myc fusion protein. Western blotting analysis of PhCPS/KS– myc with anti-c-myc antibody showed an expected band near 106 kDa (data not shown). In addition, PhCPS/KS–myc protein displayed the activity of the direct cyclization reaction from GGDP to ent-kaurene. A negative control of pTD1 plasmid without a cyclase showed no conversion of GGDP to diterpene products (data not shown). Thus, this in vitro protein expression system prepared from cell-free extracts of S. frugiperda insect cells is effective for the functional assay of terpene cyclases. The PpCPS/KS protein without tag peptide was also expressed and incubated in the presence of GGDP and magnesium ion. The products recovered with n-hexane were analyzed by GC–MS. Fig. 4A and Table 1 show the results of GC–MS analysis of the products converted from GGDP with PpCPS/ KS protein. Mass chromatogram (Fig. 4A) monitoring with ions at m/z 257 revealed at least two obvious diterpene products. A major molecular ion peak at m/z 290 [M]+ was identified as ent-16a-hydroxykaurene by a comparison with the NIST mass spectral database and by a direct comparison of

K. Hayashi et al. / FEBS Letters 580 (2006) 6175–6181

Fig. 4. GC–MS chromatogram of diterpene products of PpCPS/KS and its site-directed mutated proteins. Chromatogram monitored ions at m/z 257. Closed circle: ent-kaurene (Rt = 6.40 min). Closed triangle: ent-16a-hydroxykaurene (Rt = 8.07 min). Open triangle: copalol (Rt = 8.05 min). Underlines: cps or ks domains with mutated motifs in PpCPS/KS mutants.

authentic ent-16a-hydroxykaurene isolated from gametophores of the P. patens (Table 1). Another, minor, diterpene product was identified as ent-kaurene by direct comparison with an authentic sample (Table 1). The relative abundance of ent-16a-hydroxykaurene and ent-kaurene was about 86% and 14% in GC–MS estimates (total ion intensity), respectively. Thus, PpCPS/KS directly converts GGDP to ent-16ahydroxykaurene and ent-kaurene in the functional assay in vitro. On the other hand, no residual geranylgeraniol or ent-copalol, dephosphorylated alcohols from GGDP and CDP, were detected when the enzymatic reaction mixture was treated with excess bacterial alkaline phosphatase (data not shown). 3.3. Site-directed mutagenesis of PpKS The PpCPS/KS was confirmed to be a bryophytic bifunctional ent-kaurene synthase by functional assay in vitro. In this assay system, CDP, a possible intermediate in the sequential reaction, could not be detected (Fig. 4A). To confirm an intermediate through the conversion from GGDP to ent-kaurenes by PpCPS/KS, site-directed mutagenesis of two aspartate-rich motifs, DVDDTA (CPS domain) and DDYFD (KS domain), were carried out. Two aspartate residues of DVDDTA were substituted by alanine to prepare a mutant PpCPS/KS protein with AVADTA motif (Ppcps/KS-AVADTA). Another mutant was PpCPS/KS with AAYFD motif (PpCPS/ks-AAYFD). Results of the function of these mutant proteins are summarized in Table 1 and Fig. 4B–E. When these mutant proteins were produced by the in vitro protein expression system and incubated with GGDP, no ent-16a-hydroxykaurene and ent-kaurene were formed (data not shown). In the case of the PpCPS/ks-AAYFD mutant, CDP was detected as copalol after dephosphorylation by bacterial alkaline phosphatase and it was confirmed by a direct comparison with authentic CDP formed by AtCPS (Fig. 4B and D). On the other hand, no CDP was detected as a product of Ppcps/KS-AVAD protein

K. Hayashi et al. / FEBS Letters 580 (2006) 6175–6181

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Table 1 Mass spectral data for diterpene products of PpCPS/KS and its site-directed mutated proteins Expressed enzyme

Product

Observed ions, m/z (relative abundance, %)

PpCPS/KS

ent-kaurene ent-16a-hydroxykaurene

272 (M+, 61), 257 (100), 229 (69), 213 (38), 175 (35), 147 (73), 123 (86) 290 (M+, 7), 272 (44), 257 (36), 232 (30), 134 (51), 123 (76), 94 (100)

PpCPS/ks AtCPS

copalola copalola

290 (M+, 3), 275 (15), 257 (22), 177 (15), 137 (76), 95 (80), 81 (100) 290 (M+, 4), 275 (23), 257 (26), 177 (19), 137 (94), 95 (94), 81 (100)

PpCPS/ks+AtKSB

ent-kaurene copalol

272 (M+, 72), 257 (100), 229 (80), 213 (36), 175 (40), 147 (62), 123 (91) 290 (M+, 3), 275 (17), 257 (22), 177 (15), 137 (74), 95 (80), 81 (100)

AtCPS+Ppcps/KS

ent-kaurene ent-16a-hydroxykaurene

272 (M+, 76), 257 (100), 229 (71), 213 (34), 175 (29), 147 (62), 123 (76) 290 (M+, 7), 272 (53), 257 (48), 232 (33), 134 (51), 123 (78), 94 (100)

ent-16a-hydroxykaureneb ent-kaureneb

290 (M+, 7), 272 (53), 257 (45), 232 (28), 134 (45), 123 (69), 94 (100) 272 (M+, 70), 257 (100), 229 (67), 213 (36), 175 (26), 147 (62), 123 (74)

Underlines represent cps or ks domains with mutated motifs in PpCPS/KS mutants. a CDP was treated with excess amount of bacterial alkaline phosphatase to detect as copalol. b Mass spectral data of authentic samples under same measurement condition.

(data not shown). When AtCPS was added to the reaction mixture containing Ppcps/KS-AVAD protein, ent-16a-hydroxykaurene and ent-kaurene were detected, a product profile the same as that of wild-type protein (Fig. 4C). When AtKSB was added to the reaction mixture of PpCPS/ks-AAYFD protein, ent-kaurene and residual CDP were formed, but not ent16a-hydroxykaurene (Fig. 4E). When the two mutant proteins, Ppcps/KS-AVAD and PpCPS/ks-AAYFD, were incubated in the same reaction mixture, the product profile was the same as that of wild type protein (data not shown). These findings clearly demonstrate that the DVDDTA and DDYFD motifs, respectively, are responsible for CPS and KS activity in PpCPS/KS.

4. Discussion We demonstrate here that PpCPS/KS is a bifunctional diterpene cyclase catalyzing the cyclization reaction of GGDP to ent-kaurene and ent-16a-hydroxykaurene. Heterologous expression by Escherichia coli has been used for functional assay on many diterpene cyclase genes fused with glutathionS-transferase [8,18]. This procedure, however, has often encountered a problem such as lack of expression or extremely low expression of active protein and the formation of inclusion bodies. Indeed, active PpCPS/KS protein was not expressed in E. coli. In the present study we have used an in vitro protein expression system derived from insect cells to produce diterpene cyclase protein without a large tag such as glutathionS-transferase. The results, including experiments on mutated proteins, were successfully obtained in a shorter time than that required by the E. coli system. During the functional assay, no residual substrate GGDP was detected by GC–MS analysis of the reaction mixture. It has been reported that many cell-free protein systems contain prenyl transferases catalyzing the prenylation of proteins with farnesyl diphosphate and GGDP [19]. Hence, residual GGDP might be completely consumed by prenylation in the reaction mixture. If the insect cell extract contains a terpene cyclase, GGDP might be converted to a diterpene hydrocarbon. However, no diterpene products were detected in the negative control, and this indicates that no diterpene cyclase was contained in the cell-free extract. Thus,

PpCPS/KS proteins including site-directed mutants were effectively synthesized by the in vitro expression system without laborious investigation of the expression condition as in the bacterial system. Our expression strategy using an in vitro system will be a powerful tool for high-throughput analysis of the function of (di)terpene cyclases. Even though diterpene cyclases responsible for the biosynthesis of antheridiogen (gibberellin derivatives regulating antheridium development in ferns) have been unknown [20], ent-kaurene biosynthesis in bryophytes is different from that in higher plants. This study showed that PpCPS/KS produced ent-CDP the same as plant CPS and consecutively converted CDP to ent-kaurene. Fungal ent-kaurene biosynthetic enzymes also catalyze sequential conversion of CDP to ent-kaurene as does PpCPS/KS [8,11]. The C-terminal region of PpCPS/KS showed homology with plant KS, producing ent-kaurene alone as a product. In PpCPS/KS, this region is responsible for both ent-16a-hydroxykaurene and ent-kaurene formation. At the final step in formation of the ent-kaurene skeleton, the carbocation intermediate would be quenched by H2O or double bond formation to yield ent-16a-hydroxykaurene or ent-kaurene as final products, respectively. Fungus aphidicolan-16bol synthase has a comparable quenching mechanism to that in PpCPS/KS, although no similarity of C-terminal regions is found among known terpene cyclases [18]. The PpCPS/KS product, ent-16a-hydroxykaurene, has been reported to be accumulated in P. patens tissue at a concentration of nearly 500–1000 lM [14]. Taken together with a single PpCPS/KS gene in the genome in P. patens, PpCPS/KS would contribute to the entire production of ent-16a-hydroxykaurene. On the other hand, the fate of ent-kaurene is unclear at present. Our product profile of PpCPS/KS implies that PpCPS/KS would synthesize considerable amounts of ent-kaurene in P. patens. Further investigation might detect the accumulation of entkaurene in P. patens tissue although low accumulation might be observed, because this hydrocarbon is volatile [21]. The moss P. patens represents an early stage in the evolutionary history of land plants, and its morphology and stages of differentiation are simpler than in seed plants [22]. It has been demonstrated that some plant hormones, auxin, abscisic acid (ABA) and cytokinin, are biosynthesized and act as plant hormones in P. patens [23–27]. Genes homologous

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to Arabidopsis auxin-responsive Aux/IAA and ABA-responsive LEA genes are present in P. patens and the gene expression is also regulated by auxin and ABA in P. patens [28,29], suggesting these hormone signaling mechanisms to be evolutionally conserved. In contrast, there is no clear evidence for the presence of endogenous GA and the common physiological GA response in moss. In higher plants, cell elongation, such as in the stem, is known to be a characteristic physiological response to GA; however, no obvious morphological changes in response to exogenously applied excess GA3 (100 lM) were observed during P. patens development (data not shown). On the other hand, the P. patens genome and EST search revealed the conservation of homology genes of GA biosynthesis genes, such as GA 20-oxidase and GA 3-oxidase, and GA signaling components, such as GAI, RGA, and GID; this implies that these conserved components might function in GA biosynthesis and the signaling pathway of P. patens. These findings prompt the following question: why does exogenous GA, unlike auxin and ABA, not exert profound physiological effects on moss development? There are several possibilities. One explanation is that these conserved GA-signaling genes play a distinct physiological role in higher plants. In flowering plants, LFY plays a crucial role in integrating the signals controlling flower development. Tanahashi et al. demonstrated that LFY homology genes, PpLFYs, were conserved in P. patens and that they regulate the first cell division after formation of the zygote [30]. These conserved proteins have a distinct physiological role at the same reproductive phase. A second possible explanation is that physiologically active GA differs between the moss and the seed plant. At present, GA1 and GA4 are recognized as biologically active GAs in higher plants. On the other hand, ferns of the Schizaeaceae reorganize GA methyl ester as active GA for the development of the antheridium, whereas this GA is inactive in higher plants [31]. Lastly, it is possible that physiological responses to GA are already saturated by endogenous GA levels at the site of action, and that therefore the cells would not further respond to exogenous GA. Exogenous brassinosteroid, a plant hormone, also showed no obvious profound effects on the intact Arabidopsis plant, but defects in brassinosteroid biosynthesis can lead to obvious phenotypic defects, such as dwarfism, observed in brassinosteroid biosynthesis mutants [32]. To assess the biological role of GA in mosses, especially in P. patens, the disruption of GA biosynthesis would be a straightforward approach. The roles of GA biosynthesis and physiological functions of PpCPS/KS are now under investigation. Acknowledgements: We thank Drs. Tomomichi Fujita (Hokkaido University), Takashi Murata and Mitsuyasu Hasebe (NIBB, Japan) for useful suggestions regarding P. patens EST and genome analysis, and also thank Drs. Mitsui Ryoji and Yoshiko Minami (Okayama University of Science, Japan) for help with the molecular cloning and Dr. Masaaki Ito (Shimadzu Biotech) for helpful suggestions on cell-free protein production. This study was partially supported by Grants (16208012) to H.K. from JSPS.

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