Carotenoid Analysis of a Liverwort Marchantia polymorpha and Functional Identification of its Lycopene b- and e-Cyclase Genes 1
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa, 921-8836 Japan 2 Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto, 606-0805 Japan *Corresponding author: E-mail, [email protected]; Fax, +81-76-227-7557. (Received July 23, 2013; Accepted November 13, 2013)
Carotenoid biosynthesis in bryophytes has yet to be clarified. The liverwort Marchantia polymorpha L. is known to be an early land plant and is an emerging bryophyte model. In order to gain insight into the evolution of carotenoid biosynthesis in plants, we studied carotenoid biosynthesis in this liverwort. As is the case in higher plants, liverwort thalli contain lutein and b-carotene, as major carotenoids, as well as zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin. Based on liverwort expressed sequence tag (EST)/cDNA and genome sequences, we isolated two cyclase genes encoding lycopene b-cyclase (LCYb) and lycopene e-cyclase (LCYe), which were involved in the synthesis of b-carotene and a-carotene. These enzymes were phylogenetically positioned between corresponding proteins of a green alga (Chlorophyta) and higher plants. Functional analysis of the two genes was performed using a heterologous Escherichia coli expression system, in which the Pantoea ananatis lycopene biosynthesis genes were coexpressed. The results indicated liverwort LCYb activity for the synthesis of b-carotene from lycopene, which was the same as that of higher plants. On the other hand, liverwort LCYe was able to form two e-rings from lycopene to e-carotene via d-carotene, which was different from the Arabidopsis LCYe enzyme which generates only one e-ring from lycopene. Keywords: a-Carotene b-Carotene Liverwort Lycopene cyclase Marchantia polymorpha. Abbreviations: CD, circular dichroism; EST, expressed sequence tag; HR-ESI-MS, high resolution electrospray ionization mass spectrometry; LCYb, lycopene b-cyclase; LCYe, lycopene e-cyclase; NMR, nuclear magnetic resonance. The nucleotide sequences reported in this paper has been submitted to DDBJ under accession numbers AB794089 (MpLCYb) and AB794090 (MpLCYe).
Introduction Carotenoids are widely distributed groups of isoprenoid pigments. They are biosynthesized in all photosynthetic organisms as well as in some non-photosynthetic bacteria and fungi. Typical carotenoids (tetraterpenes) are derived from the first carotenoid phytoene, which is generated by condensation of two geranylgeranyl diphosphate (GGPP) molecules. The pathway of carotenoid biosynthesis has been studied extensively in higher plants, and nearly all of the genes involved have been isolated and characterized (Cunningham and Gantt 1998, Fraser and Bramley 2004, Chaundhary et al. 2010, Ruiz-Sola and Rodrı´guez-Concepcio´n 2012). In higher plants, some carotenoid biosynthesis genes are redundant in their chromosome genome, suggesting that these genes have been duplicated and have functionally diverged during plant evolution. In contrast to higher plants, few reports are available on carotenoid biosynthesis in bryophytes, which are thought to be early land plants. In order to clarify the evolution of carotenoid biosynthesis in plants, we conducted a detailed carotenoid analysis and identified carotenoid biosynthesis genes in a bryophyte. Lycopene cyclases catalyze cyclization reactions of lycopene (c,c-carotene), which is a key branch point in the carotenoid biosynthetic pathway. In general, higher plants have two branches that lead to b-carotene (b,b-carotene) and a-carotene [(60 R)-b,e-carotene]. Lycopene b-cyclase (LCYb) forms two b-rings from lycopene to b-carotene via g-carotene (b,c-carotene) (see Fig. 2). On the other hand, lycopene e-cyclase (LCYe) adds one e-ring to synthesize d-carotene [(6R)-e,c-carotene], which is converted to a-carotene that includes the e- and b-rings with LCYb. b-Carotene is one of the major carotenoids in the leaves of higher plants, and it is metabolized to zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin. Lutein is the predominant carotenoid in plant leaves, and is the hydroxylated product from a-carotene. In contrast, carotenoids with two e-rings are not ordinarily
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170, available online at www.pcp.oxfordjournals.org ! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
194
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Regular Paper
Miho Takemura1,*, Takashi Maoka1,2 and Norihiko Misawa1
Lycopene cyclase genes in liverwort
Results
Table 1 Carotenoid content in liverwort thallus 1
Carotenoid content (mg g
FW)
Male
Female
109.6
162.5
Carotenoid composition (%) a-Carotene
1.6
1.5
b-Carotene
30.9
29.2
Luteina
56.3
60.9
Lutein-5,6-epoxide
3.6
2.3
Zeaxanthin
1
1
Antheraxanthin
2.3
1.5
Violaxanthin
1.6
1.5
90 -cis-Neoxanthin
2.7
2.1
a
Lutein contains a small amount of cis-isomers such as 90 -cis lutein which might be artificially produced during carotenoid extraction.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
found in plants because most higher plant LCYe enzymes tend to form only one e-ring to the substrate lycopene. It has been reported, however, that romaine lettuce is an exception in that it accumulates substantial amounts of lactucaxanthin, a carotenoid with two e-rings and two hydroxyl groups (Phillip and Young 1995), and the LCYe from lettuce plants was found to convert lycopene efficiently to e-carotene [(6R)-e,e-carotene] that has two e-rings (Cunningham and Gantt 2001). LCYb and LCYe share significant similarities in their amino acid sequences, suggesting that they have originated from a common ancestor through gene duplication. Some higher plants, including tomato, possess two LCYb proteins, which are chloroplast specific and chromoplast specific, whereas Arabidopsis has only one LCYb (Lcy-b) gene (Cunningham et al. 1996, Ronen et al. 2000, Alque´zar et al. 2009). In contrast, the LCYe (Lcy-e) gene has been shown to be a single copy in all higher plants. The concentration and composition of carotenoids vary in plant tissues as a result of highly regulated processes. For example, lutein and b-carotene are the major carotenoids in tomato leaves, while violaxanthin and neoxanthin are the main carotenoids in tomato petals (Ronen et al. 1999). This result corresponded to the expression levels of LCYb and LCYe, i.e. the increased expression of LCYb and non-expression of LCYe were found in the petals (Ronen et al. 1999). In the present study, we focused on the LCYb and LCYe genes in a liverwort, Marchantia polymorpha L., which does not have flowers or fruit.
MpLCYb showed 61% and 17% amino acid identities with Arabidopsis thaliana LCYb (AtLCYb) and Pantoea ananatis CrtY, respectively (Supplementary Fig. S3). By the ChloroP program, 42 amino acid residues of MpLCYb at the N-terminus were predicted as the signal peptide to the chloroplast. MpLCYe showed 53% amino acid identity with A. thaliana LCYe (AtLCYe) (Supplementary Fig. S4). The ChloroP program predicted that 47 amino acid residues of MpLCYe at the N-terminus were the signal peptide to the chloroplast. A phylogenetic tree suggested that MpLCYb belonged to the LCYb1 (chloroplast-specific) family rather than to the LCYb2 (chromoplast-specific) family (Fig. 1).
Functional analysis of MpLCYb and MpLCYe
Carotenoids in M. polymorpha To clarify carotenoid biosynthesis in bryophytes, we carried out a detailed analysis of carotenoids in the liverwort M. polymorpha. The carotenoid content and composition of male and female thalli are shown in Table 1. No significant differences were found between males and females. Lutein and b-carotene were major carotenoids, and a-carotene, lutein-5,6-epoxide, zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin were also found in the thalli (Supplementary Figs. S1, S2). Their carotenoid compositions were similar to those in the leaves of higher plants (DemmigAdams et al. 1996, Fraser and Bramley 2004). These results suggested that liverworts have the same carotenoid biosynthetic pathway as higher plants.
Isolation of the lycopene b- and e-cyclase genes from M. polymorpha In this study, we focused on genes encoding LCYb and LCYe since the cyclization of lycopene is a key branch point in the carotenoid biosynthesis pathway. As a result of expressed sequence tag (EST) and genome sequence homology searches (T. Kohchi et al. personal communication), we found genes homologous to higher plant LCYb and LCYe genes. Both of these, namely MpLCYb and MpLCYe, were single-copy genes.
To identify the catalytic activity of the liverwort lycopene cyclases, we used a heterologous Escherichia coli expression system (Misawa et al. 1995). The E. coli carrying the plasmid pACCRT-EIB that contains the crtE, crtB and crtI genes from P. ananatis accumulates lycopene (Fig. 2). We introduced MpLCYb and/or MpLCYe into this E. coli. When pET-MpLCYb was introduced in the lycopene-accumulating E. coli, b-carotene was detected (Fig. 3b, e). On the other hand, the empty vector pET21a did not influence the composition of carotenoids (Fig. 3a, e). The introduction of pET-MpLCYe resulted in the formation of d-carotene and e-carotene (Fig. 3c, e; Supplementary Table S1). The chirality of (6R) for d-carotene and e-carotene was determined from circular dichroism (CD) data. When both MpLCYb and MpLCYe were simultaneously introduced in the lycopene-accumulating E. coli, a-carotene was predominantly produced (Fig. 3d, e; Supplementary Table S1). The chirality of (60 R) for a-carotene was determined from CD data. These results indicate that MpLCYb and MpLCYe activity generates b- and e-rings, respectively, from the c end of lycopene, g-carotene or d-carotene, as shown in Fig. 2.
Analysis of MpLCYb and MpLCYe expression To investigate the expression of MpLCYb and MpLCYe, we performed a semi-quantitative reverse transcription–PCR
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
195
M. Takemura et al.
(RT–PCR). Both MpLCYb and MpLCYe were actively expressed in the liverwort thalli (Supplementary Fig. S5). The levels of MpLCYb and MpLCYe expression were similar. No significant differences were found between males and females.
Discussion Based on the analysis of carotenoids in M. polymorpha thalli (Table 1), we proposed a carotenoid biosynthetic pathway in this bryophyte (Fig. 4). The carotenoid compositions of the liverwort thalli were similar to those of the leaves of higher plants. We isolated lycopene b- and e-cyclase genes from the liverwort, which we designated MpLCYb and MpLCYe, respectively. MpLCYb belonged to the LCYb1 (chloroplast-specific) subfamily and had activity to produce b-carotene from lycopene, as expected. A gene encoding the LCYb2 (chromoplast-specific) subfamily, which was mainly expressed in fruit (Alque´zar et al. 2009, Mendes et al. 2011), was not found in the liverwort. Because the liverwort has neither flowers nor fruit, the LCYb2 subfamily is likely to have evolved after the branch point of bryophytes. Two types of e-cyclase have been reported (Cunningham and Gantt 2001). One type forms only one e-ring to synthesize the monocyclic d-carotene from lycopene, e.g. the Arabidopsis
196
LCYe produced d-carotene exclusively (Cunningham and Gantt 2001). The other type generates two e-rings to form the bicyclic e-carotene. Lettuce LCYe primarily formed e-carotene from lycopene. Adonis and maize LCYe enzymes produced not only d-carotene but also e-carotene (Cunningham and Gantt 2001, Bai et al. 2009). MpLCYe was able to produce both d-carotene and e-carotene, while the former was more abundant than the latter. This ratio of d-carotene and e-carotene in the total carotenoids varied with E. coli culture temperature (data not shown). e-Carotene did not accumulate to detectable levels in the liverwort thalli, suggesting that the step from d-carotene to e-carotene occurred to a slight degree if at all. The amino acid residues of LCYe that influence the determination of e-ring number were reported (Cunningham and Gantt 2001, Bai et al. 2009) (Supplementary Fig. S3). The H residue in lettuce LCYe corresponding to the L448 in Arabidopsis LCYe confers bi-cyclase activity to the enzyme (Cunningham and Gantt 2001). However, Adonis and maize LCYes carry an L residue like Arabidopsis, but both are able to produce e-carotene (Bai et al. 2009). MpLCYe also has an L residue in this position, suggesting that the L residue is not sufficient to produce e-carotene. Bai et al. (2009) have also reported that S502 in maize LCYe is important for its specificity. Since MpLCYe has the same A residue as Arabidopsis and lettuce, it is unclear whether this A residue is essential for the bi-cyclase activity of MpLCYe. A mutational analysis would be useful in investigating the dual cyclase activity of MpLCYe in the future. When MpLCYb and MpLCYe were simultaneously expressed, a-carotene was detected as the predominant product, and mono-cyclic carotenes, g-carotene and d-carotene, were not detected (Fig. 3d). Moreover, b-carotene and e-carotene, which are bi-cyclic carotenes synthesized by each enzyme, were not found. Since MpLCYb and MpLCYe genes were on
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 1 Phylogenetic tree of the lycopene cyclases. GenBank accession numbers are as follows: tomato LCYb1 (SlLCYb1), EF650013; tomato LCYb2 (SlLCYb2), AF254793; grapefruit LCYb1 (CpLCYb1), AF152246; grapefruit LCYb2 (CpLCYb2), GQ214768; liverwort (M. polymorpha) LCYb (MpLCYb), AB794089; Arabidopsis LCYb (AtLCYb), U50739; Chlamydomonas LCYb (CrLCYb), AY860818; liverwort LCYe (MpLCYe), AB794090; Arabidopsis LCYe (AtLCYe), U50738; tomato LCYe (SlLCYe), Y14387; Chlamydomonas LCYe (CrLCYe), AY606130; Adonis LCYe (AaLCYe), AF321535; lettuce LCYe (LsLCYe), AF321538; maize LCYe (ZmLCYe), EU924262; Pantoea ananatis CrtY (CrtY), D90087.
Fig. 2 Pathway of carotenoid biosynthesis in transformed E. coli used in this study. The reactions indicated by gray arrows have not been examined directly. It is thus unclear via which route a-carotene is formed.
Lycopene cyclase genes in liverwort
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 3 HPLC analysis of products formed from lycopene in transformed E. coli. (a–d) HPLC chromatograms of the extracts from E. coli that carried (a) pACCRT-EIB plus pET21a vector; (b) pACCRT-EIB plus pET-MpLCYb; (c) pACCRT-EIB plus pET-MpLCYe; and (d) pACCRT-EIB plus pETDuetMpLCYb/MpLCYe. (e) Absorption spectra of individual peaks: 1, lycopene; 2, b-carotene; 3, d-carotene; 4, e-carotene; 5, a-carotene.
the same vector, their expression levels were thought to be almost equal. Our results support the hypothesis that cyclization of lycopene occurs in a stepwise fashion, with closure of a single e-ionone ring to synthesize d-carotene, followed by b-ring closure to synthesize a-carotene (Fig. 2), which is mediated by a b-cyclase–e-cyclase complex (Cunningham and Gantt 1998). In maize, a null mutant of LCYb results in the accumulation of e-carotene and lactucaxanthin that are the hydroxylated product of e-carotene in the endosperm, whereas these carotenoids are not present in wild-type endosperm (Bai et al. 2009). This suggests that the presence of b-cyclase limits e-cyclase activity to mono-cyclization. The authors also suggested that the carotenoid profiles may be regulated by the differential expression of LCYe and LCYb in embryo and
endosperm tissues. In wild-type endosperm, high levels of LCYe accumulate, favoring the conversion of lycopene to d-carotene by LCYe and the production of lutein via a-carotene through the action of LCYb. On the other hand, in wild-type embryo, LCYb predominates, resulting in the conversion of lycopene to b-carotene by LCYb activity and subsequent production of zeaxanthin. We found that the MpLCYe gene was expressed at the same level as MpLCYb in the liverwort thalli. This situation is similar to that in maize endosperm and is consistent with results showing that lutein is the predominant carotenoid in the thalli. In this study, we identified the LCYb and LCYe genes of liverwort to gain insight into the evolution of carotenoid biosynthesis in plants. As discussed above, we suggested that the
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
197
M. Takemura et al.
LCYb2 (chromoplast-type) subfamily evolved after the branch point of bryophytes. It was also found that the liverwort genes were located between green alga (Chlorophyta) and higher plants, as expected. Analysis of other carotenoid biosynthesis genes will reveal the genetic evolution of carotenoid biosynthesis in plants.
Materials and Methods Plant material and growth conditions The female and male of M. polymorpha used in this study have been described by Okada et al. (2000). The liverwort was grown
198
on M51C solid medium (Takenaka et al. 2000) and incubated at 25 C under conditions of 16 h light/8 h dark in dim light (approximately 350 lux).
Analysis of carotenoids in liverwort Analysis of carotenoids in liverwort was carried out as described previously (Maoka et al. 2007, Maoka et al. 2008). Sufficient amounts of acetone were added to the male thalli (38 g FW) and the female thalli (52 g FW) of M. polymorpha, and mixed for several hours at room temperature. Each acetone extract was partitioned after the addition of hexane : diethyl ether (Et2O) (1 : 1, v/v) and water. The upper solvent layer was evaporated, and saponified with methanol (MeOH) including 5% potassium
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 4 Proposed carotenoid biosynthetic pathway in M. polymorpha thalli. The names of carotenoids detected in M. polymorpha are shown with red letters.
Lycopene cyclase genes in liverwort
Cloning of the lycopene b- and e-cyclase genes from M. polymorpha To find sequences homologous to the known LCYb and LCYe genes, homology searches of the genome, cDNA and EST sequences of liverwort (T. Kohchi et al. personal communication) were performed. Based on the homologous sequences obtained, the primers were designed and the coding regions of each gene were amplified by PCR of liverwort cDNAs. The following primers were used: MpLCYbF, 50 -CATATGAGCTCGAC GAGGTATG-30 ; MpLCYbR: 50 -GGATCCTTATTGCCGCTTCAT CAATG-30 ; MpLCYeF, 50 -CATATGGGAACCATAGATCGG-30 ; and MpLCYeR, 50 -CTCGAGTCATCTCATCTCCTTCGC-30 (the underlined sequences were added for cloning). Then PCR products were cloned into the plasmid vector and sequenced.
Sequence analysis Homology search was performed by BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi). Amino acid alignments and a phylogenetic tree were constructed using CLUSTAL W (http://www.clus tal.org/). Transit peptides of the gene products were predicted by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/).
Expression of liverwort LCYb and LCYe genes in Escherichia coli The coding regions of the liverwort LCYb (MpLCYb) and LCYe (MpLCYe) were cloned into pET21a (Merck Millipore) independently. These plasmids were designated pET21-MpLCYb and pET21-MpLCYe. For complementary expression of the MpLCYb and MpLCYe genes, they were cloned simultaneously into pETDuet (Merck Millipore) and called pETDuet-MpLCYb/ MpLCYe. Each plasmid was introduced into the lycopeneproducing E. coli [BL21 (DE3)], which carried plasmid pACCRT-EIB for the expression of the crtE, crtB and crtI genes from the bacterium Pantoea ananatis (Misawa et al. 1995). Each transformed E. coli was grown in 2YT medium at 37 C until an optical density of 0.8–1.0 was achieved, induced with 0.05 mM of isopropyl-b-D-thiogalactopyranoside (IPTG) and further cultured at 21 C for 2 d.
Extraction and analysis of carotenoids from E. coli cells Extraction of carotenoids from recombinant E. coli was performed by the method described by Fraser et al. (2000). Escherichia coli cultures were centrifuged and cell pellets were extracted with MeOH using a mixer for 5 min. Tris–HCl (50 mM, pH 7.5) (containing 1 M NaCl) was added and mixed. Chloroform was then added to the mixture and incubated for 5 min. After centrifugation, the chloroform phase was collected and dried by centrifugal evaporation. Dried residues were re-suspended with ethyl acetate, and applied to HPLC with a Waters Alliance 2695–2996 (PDA) system. HPLC was carried out according to the method described (Yokoyama and Miki 1995) using TSKgel ODS-80Ts (4.6 150 mm, 5 mm; Tosoh). This was developed at a flow rate of 1.0 ml min1 at 25 C with solvent A (water : MeOH, 5 : 95, v/v) for 5 min, followed by a linear gradient from solvent A to solvent B (tetrahydrofuran : MeOH, 3 : 7, v/v) for 5 min, and solvent B alone for 8 min. Individual carotenoids were identified by comparing retention times and absorption spectra with those of the authentic standards. a-Carotene, d-carotene and e-carotene were purified from the recombinant E. coli cells according to a method similar to that of analysis of carotenoids in liverwort, and chemical structures were confirmed by UV-VIS, high resolution electrospray ionization MS (HR-ESI-MS), CD and 1H-NMR (500 MHz) (Maoka et al. 2007, Maoka et al. 2008).
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
hydroxide at room temperature. This was re-extracted with hexane : Et2O (1 : 1). Each carotenoid extract was then subjected to HPLC on silica gel (Cosmosil 5SL-II, 4.6 250 mm; Nacalai tesque Inc.), developed with acetone : hexane (2 : 8) at a flow rate of 1.0 ml min1, and monitored at 450 nm. Carotenes eluted from silica gel HPLC were further subjected to reversephase HPLC to separate b-carotene and a-carotene. The reverse-phase HPLC was carried out using a YMC-Pack ODS-A column (4.6 250 mm ID; YMC Co.), developed with chloroform : methanol (15 : 85) at a flow rate of 1.0 ml min1 and monitored at 450 nm. Carotenoid compositions were estimated by peak area in HPLC. Individual carotenoids were fractionated during HPLC, as needed, and analyzed by UV-VIS, fast atom bombardment mass spectrometry and 1H-nuclear magnetic resonance (NMR; 500 MHz, CDCl3).
Spectroscopic data a-Carotene [(60 R)-b,e-carotene]: UV-VIS (Et2O) 423 (shoulder), 445, 473 nm; 1H-NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4368; CD (Et2O) nm (e) 240 (+5.5), 260 (+4.5), 280 (0), 350 (+0.5). d-Carotene [(6R)-e,c-carotene]: UV-VIS (Et2O) 430, 454, 487 nm; 1H-NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4371; CD (Et2O) nm (e) 255 (0), 270 (+6.5), 290 (0), 303 (–15). e-Carotene [(6R)-e,e-carotene]: UV-VIS (Et2O) 417, 440, 468 nm; 1H NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4387; CD (Et2O) nm (e) 235 (0), 267 (+9), 285 (0), 300 (–1.8), 330 (–0.5).
Expression analysis of the MpLCyb and MpLCYe genes Total RNA of liverwort thalli was extracted using an RNeasy Plant mini kit (Qiagen) and treated with DNase I. A 1 mg aliquot of total RNA was reverse-transcribed with oligo(dT) primer using ReverTra Ace (TOYOBO). PCR was performed using the following primers: MpLCYbF3, 50 -CGAGGTTGACTGCCATCC30 ; MpLCYbR1, 50 -GGATCCTTATTGCCGCTTCATCAATG-30 ; MpLCYeF3, 50 -GTTGAGTGCGAGAACTTC-30 ; MpLCYeR1,
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
199
M. Takemura et al.
50 -CTCGAGTCATCTCATCTCCTTCGC-30 ; MpACT1F, 50 -TTCA ACGTTCCTGCCATGTA-30 ; and MpACT1R, 50 -GATCTCCCTT GTCATACGGT-30 MpACT (Marchantia polymorpha ACTIN) was used as an internal control.
Supplementary data Supplementary data are available at PCP online.
Acknowledgments
Disclosures The authors have no conflicts of interest to declare.
References Alque´zar, B., Zacarı´as, L. and Rodrigo, M.J. (2009) Molecular and functional characterization of a novel chromoplast-specific lycopene b-cyclase from Citrus and its relation to lycopene accumulation. J. Exp. Bot. 60: 1783–1797. Bai, L., Kim, E., DellaPenna, D. and Brutnell, T.P. (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J. 59: 588–599. Chaundhary, N., Nijhawan, A., Khurana, J.P. and Khurana, P. (2010) Carotenoid biosynthesis genes in rice: structural analysis, genomewide expression profiling and phylogenetic analysis. Mol. Genet. Genomics 283: 13–33. Cunningham, F.X. Jr. and Gantt, E. (1998) Gene and enzymes of carotenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 557–583. Cunningham, F.X. Jr. and Gantt, E. (2001) One ring or two? Determination of ring number in carotenoids by lycopene e-cyclases. Proc. Natl Acad. Sci. USA 98: 2905–2910. Cunningham, F.X. Jr., Pogson, B., Sun, Z., McDonald, K.A., DellaPenna, D. and Gantt, E. (1996) Functional analysis of the b and e lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8: 1613–1626. Demmig-Adams, B., Gilmore, A.M. and Adams, W.W. III (1996) In vivo functions of carotenoids in higher plants. FASEB J. 10: 403–412.
200
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
We thank Dr. Takayuki Kohchi, Dr. Katsuyuki T. Yamato and Dr. Kimitsune Ishizaki for their assistance in searching liverwort sequences.
Fraser, P.D. and Bramley, P.M. (2004) The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43: 228–265. Fraser, P.D., Elisabete, M., Pinto, S., Holloway, D.E. and Bramley, P.M. (2000) Application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J. 24: 551–558. Maoka, T., Akimoto, N., Ishiguro, K., Yoshinaga, M. and Yoshimoto, M. (2007) Carotenoids with a 5,6-dihydro-5,6-dihydroxy-b-end group, from yellow sweet potato ‘Benimasari’, Ipomoea batatas LAM. Phytochemistry 68: 1740–1745. Maoka, T., Akimoto, N., Kuroda, Y., Hashimoto, K. and Fujiwara, Y. (2008) Pittosporumxanthins, cycloaddition products of carotenoids with a-tocopherol from seeds of Pittosporum tobia. J. Nat. Prod. 71: 622–627. Mendes, A.F.S., Chen, C., Gmitter, F.G. Jr., Moore, G.A. and Costa, M.G.C. (2011) Expression and phylogenetic analysis of two new lycopene b-cyclases from Citrus paradisi. Physiol. Plant 141: 1–10. Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T. et al. (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177: 6575–6584. Okada, S., Fujisawa, M., Sone, T., Nakayama, S., Nishiyama, R., Takenaka, M. et al. (2000) Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha. Plant J. 24: 421–428. Phillip, D. and Young, A.J. (1995) Occurrence of the carotenoid lactucaxanthin in higher plant LHC II. Photosynth. Res. 43: 273–282. Ronen, G., Carmel-Goren, L., Zamir, D. and Hirschberg, J. (2000) An alternative pathway to b-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc. Natl Acad. Sci. USA 97: 11102–11107. Ronen, G., Cohen, M., Zamir, D. and Hirschberg, J. (1999) Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant. Delta Plant J. 17: 341–351. ´ . and Rodrı´guez-Concepcio´n, M. (2012) Carotenoid bioRuiz-Sola, M.A synthesis in Arabidopsis: a colorful pathway. The Arabidopsis Book e0158. doi: 10.1199/tab.0158. Takenaka, M., Yamaoka, S., Hanajiri, T., Shimizu-Ueda, Y., Yamato, K.T., Fukuzawa, H. et al. (2000) Direct transformation and plant regeneration of the haploid liverwort Marchantia plymorpha L. Transgenic Res. 9: 179–185. Yokoyama, A. and Miki, W. (1995) Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin-producing bacterium Agrobacterium aurantiacum. FEMS Microbiol. Lett. 128: 139–144.
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa, 921-8836 Japan 2 Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto, 606-0805 Japan *Corresponding author: E-mail, [email protected]; Fax, +81-76-227-7557. (Received July 23, 2013; Accepted November 13, 2013)
Carotenoid biosynthesis in bryophytes has yet to be clarified. The liverwort Marchantia polymorpha L. is known to be an early land plant and is an emerging bryophyte model. In order to gain insight into the evolution of carotenoid biosynthesis in plants, we studied carotenoid biosynthesis in this liverwort. As is the case in higher plants, liverwort thalli contain lutein and b-carotene, as major carotenoids, as well as zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin. Based on liverwort expressed sequence tag (EST)/cDNA and genome sequences, we isolated two cyclase genes encoding lycopene b-cyclase (LCYb) and lycopene e-cyclase (LCYe), which were involved in the synthesis of b-carotene and a-carotene. These enzymes were phylogenetically positioned between corresponding proteins of a green alga (Chlorophyta) and higher plants. Functional analysis of the two genes was performed using a heterologous Escherichia coli expression system, in which the Pantoea ananatis lycopene biosynthesis genes were coexpressed. The results indicated liverwort LCYb activity for the synthesis of b-carotene from lycopene, which was the same as that of higher plants. On the other hand, liverwort LCYe was able to form two e-rings from lycopene to e-carotene via d-carotene, which was different from the Arabidopsis LCYe enzyme which generates only one e-ring from lycopene. Keywords: a-Carotene b-Carotene Liverwort Lycopene cyclase Marchantia polymorpha. Abbreviations: CD, circular dichroism; EST, expressed sequence tag; HR-ESI-MS, high resolution electrospray ionization mass spectrometry; LCYb, lycopene b-cyclase; LCYe, lycopene e-cyclase; NMR, nuclear magnetic resonance. The nucleotide sequences reported in this paper has been submitted to DDBJ under accession numbers AB794089 (MpLCYb) and AB794090 (MpLCYe).
Introduction Carotenoids are widely distributed groups of isoprenoid pigments. They are biosynthesized in all photosynthetic organisms as well as in some non-photosynthetic bacteria and fungi. Typical carotenoids (tetraterpenes) are derived from the first carotenoid phytoene, which is generated by condensation of two geranylgeranyl diphosphate (GGPP) molecules. The pathway of carotenoid biosynthesis has been studied extensively in higher plants, and nearly all of the genes involved have been isolated and characterized (Cunningham and Gantt 1998, Fraser and Bramley 2004, Chaundhary et al. 2010, Ruiz-Sola and Rodrı´guez-Concepcio´n 2012). In higher plants, some carotenoid biosynthesis genes are redundant in their chromosome genome, suggesting that these genes have been duplicated and have functionally diverged during plant evolution. In contrast to higher plants, few reports are available on carotenoid biosynthesis in bryophytes, which are thought to be early land plants. In order to clarify the evolution of carotenoid biosynthesis in plants, we conducted a detailed carotenoid analysis and identified carotenoid biosynthesis genes in a bryophyte. Lycopene cyclases catalyze cyclization reactions of lycopene (c,c-carotene), which is a key branch point in the carotenoid biosynthetic pathway. In general, higher plants have two branches that lead to b-carotene (b,b-carotene) and a-carotene [(60 R)-b,e-carotene]. Lycopene b-cyclase (LCYb) forms two b-rings from lycopene to b-carotene via g-carotene (b,c-carotene) (see Fig. 2). On the other hand, lycopene e-cyclase (LCYe) adds one e-ring to synthesize d-carotene [(6R)-e,c-carotene], which is converted to a-carotene that includes the e- and b-rings with LCYb. b-Carotene is one of the major carotenoids in the leaves of higher plants, and it is metabolized to zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin. Lutein is the predominant carotenoid in plant leaves, and is the hydroxylated product from a-carotene. In contrast, carotenoids with two e-rings are not ordinarily
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170, available online at www.pcp.oxfordjournals.org ! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
194
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Regular Paper
Miho Takemura1,*, Takashi Maoka1,2 and Norihiko Misawa1
Lycopene cyclase genes in liverwort
Results
Table 1 Carotenoid content in liverwort thallus 1
Carotenoid content (mg g
FW)
Male
Female
109.6
162.5
Carotenoid composition (%) a-Carotene
1.6
1.5
b-Carotene
30.9
29.2
Luteina
56.3
60.9
Lutein-5,6-epoxide
3.6
2.3
Zeaxanthin
1
1
Antheraxanthin
2.3
1.5
Violaxanthin
1.6
1.5
90 -cis-Neoxanthin
2.7
2.1
a
Lutein contains a small amount of cis-isomers such as 90 -cis lutein which might be artificially produced during carotenoid extraction.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
found in plants because most higher plant LCYe enzymes tend to form only one e-ring to the substrate lycopene. It has been reported, however, that romaine lettuce is an exception in that it accumulates substantial amounts of lactucaxanthin, a carotenoid with two e-rings and two hydroxyl groups (Phillip and Young 1995), and the LCYe from lettuce plants was found to convert lycopene efficiently to e-carotene [(6R)-e,e-carotene] that has two e-rings (Cunningham and Gantt 2001). LCYb and LCYe share significant similarities in their amino acid sequences, suggesting that they have originated from a common ancestor through gene duplication. Some higher plants, including tomato, possess two LCYb proteins, which are chloroplast specific and chromoplast specific, whereas Arabidopsis has only one LCYb (Lcy-b) gene (Cunningham et al. 1996, Ronen et al. 2000, Alque´zar et al. 2009). In contrast, the LCYe (Lcy-e) gene has been shown to be a single copy in all higher plants. The concentration and composition of carotenoids vary in plant tissues as a result of highly regulated processes. For example, lutein and b-carotene are the major carotenoids in tomato leaves, while violaxanthin and neoxanthin are the main carotenoids in tomato petals (Ronen et al. 1999). This result corresponded to the expression levels of LCYb and LCYe, i.e. the increased expression of LCYb and non-expression of LCYe were found in the petals (Ronen et al. 1999). In the present study, we focused on the LCYb and LCYe genes in a liverwort, Marchantia polymorpha L., which does not have flowers or fruit.
MpLCYb showed 61% and 17% amino acid identities with Arabidopsis thaliana LCYb (AtLCYb) and Pantoea ananatis CrtY, respectively (Supplementary Fig. S3). By the ChloroP program, 42 amino acid residues of MpLCYb at the N-terminus were predicted as the signal peptide to the chloroplast. MpLCYe showed 53% amino acid identity with A. thaliana LCYe (AtLCYe) (Supplementary Fig. S4). The ChloroP program predicted that 47 amino acid residues of MpLCYe at the N-terminus were the signal peptide to the chloroplast. A phylogenetic tree suggested that MpLCYb belonged to the LCYb1 (chloroplast-specific) family rather than to the LCYb2 (chromoplast-specific) family (Fig. 1).
Functional analysis of MpLCYb and MpLCYe
Carotenoids in M. polymorpha To clarify carotenoid biosynthesis in bryophytes, we carried out a detailed analysis of carotenoids in the liverwort M. polymorpha. The carotenoid content and composition of male and female thalli are shown in Table 1. No significant differences were found between males and females. Lutein and b-carotene were major carotenoids, and a-carotene, lutein-5,6-epoxide, zeaxanthin, antheraxanthin, violaxanthin and 90 -cis-neoxanthin were also found in the thalli (Supplementary Figs. S1, S2). Their carotenoid compositions were similar to those in the leaves of higher plants (DemmigAdams et al. 1996, Fraser and Bramley 2004). These results suggested that liverworts have the same carotenoid biosynthetic pathway as higher plants.
Isolation of the lycopene b- and e-cyclase genes from M. polymorpha In this study, we focused on genes encoding LCYb and LCYe since the cyclization of lycopene is a key branch point in the carotenoid biosynthesis pathway. As a result of expressed sequence tag (EST) and genome sequence homology searches (T. Kohchi et al. personal communication), we found genes homologous to higher plant LCYb and LCYe genes. Both of these, namely MpLCYb and MpLCYe, were single-copy genes.
To identify the catalytic activity of the liverwort lycopene cyclases, we used a heterologous Escherichia coli expression system (Misawa et al. 1995). The E. coli carrying the plasmid pACCRT-EIB that contains the crtE, crtB and crtI genes from P. ananatis accumulates lycopene (Fig. 2). We introduced MpLCYb and/or MpLCYe into this E. coli. When pET-MpLCYb was introduced in the lycopene-accumulating E. coli, b-carotene was detected (Fig. 3b, e). On the other hand, the empty vector pET21a did not influence the composition of carotenoids (Fig. 3a, e). The introduction of pET-MpLCYe resulted in the formation of d-carotene and e-carotene (Fig. 3c, e; Supplementary Table S1). The chirality of (6R) for d-carotene and e-carotene was determined from circular dichroism (CD) data. When both MpLCYb and MpLCYe were simultaneously introduced in the lycopene-accumulating E. coli, a-carotene was predominantly produced (Fig. 3d, e; Supplementary Table S1). The chirality of (60 R) for a-carotene was determined from CD data. These results indicate that MpLCYb and MpLCYe activity generates b- and e-rings, respectively, from the c end of lycopene, g-carotene or d-carotene, as shown in Fig. 2.
Analysis of MpLCYb and MpLCYe expression To investigate the expression of MpLCYb and MpLCYe, we performed a semi-quantitative reverse transcription–PCR
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
195
M. Takemura et al.
(RT–PCR). Both MpLCYb and MpLCYe were actively expressed in the liverwort thalli (Supplementary Fig. S5). The levels of MpLCYb and MpLCYe expression were similar. No significant differences were found between males and females.
Discussion Based on the analysis of carotenoids in M. polymorpha thalli (Table 1), we proposed a carotenoid biosynthetic pathway in this bryophyte (Fig. 4). The carotenoid compositions of the liverwort thalli were similar to those of the leaves of higher plants. We isolated lycopene b- and e-cyclase genes from the liverwort, which we designated MpLCYb and MpLCYe, respectively. MpLCYb belonged to the LCYb1 (chloroplast-specific) subfamily and had activity to produce b-carotene from lycopene, as expected. A gene encoding the LCYb2 (chromoplast-specific) subfamily, which was mainly expressed in fruit (Alque´zar et al. 2009, Mendes et al. 2011), was not found in the liverwort. Because the liverwort has neither flowers nor fruit, the LCYb2 subfamily is likely to have evolved after the branch point of bryophytes. Two types of e-cyclase have been reported (Cunningham and Gantt 2001). One type forms only one e-ring to synthesize the monocyclic d-carotene from lycopene, e.g. the Arabidopsis
196
LCYe produced d-carotene exclusively (Cunningham and Gantt 2001). The other type generates two e-rings to form the bicyclic e-carotene. Lettuce LCYe primarily formed e-carotene from lycopene. Adonis and maize LCYe enzymes produced not only d-carotene but also e-carotene (Cunningham and Gantt 2001, Bai et al. 2009). MpLCYe was able to produce both d-carotene and e-carotene, while the former was more abundant than the latter. This ratio of d-carotene and e-carotene in the total carotenoids varied with E. coli culture temperature (data not shown). e-Carotene did not accumulate to detectable levels in the liverwort thalli, suggesting that the step from d-carotene to e-carotene occurred to a slight degree if at all. The amino acid residues of LCYe that influence the determination of e-ring number were reported (Cunningham and Gantt 2001, Bai et al. 2009) (Supplementary Fig. S3). The H residue in lettuce LCYe corresponding to the L448 in Arabidopsis LCYe confers bi-cyclase activity to the enzyme (Cunningham and Gantt 2001). However, Adonis and maize LCYes carry an L residue like Arabidopsis, but both are able to produce e-carotene (Bai et al. 2009). MpLCYe also has an L residue in this position, suggesting that the L residue is not sufficient to produce e-carotene. Bai et al. (2009) have also reported that S502 in maize LCYe is important for its specificity. Since MpLCYe has the same A residue as Arabidopsis and lettuce, it is unclear whether this A residue is essential for the bi-cyclase activity of MpLCYe. A mutational analysis would be useful in investigating the dual cyclase activity of MpLCYe in the future. When MpLCYb and MpLCYe were simultaneously expressed, a-carotene was detected as the predominant product, and mono-cyclic carotenes, g-carotene and d-carotene, were not detected (Fig. 3d). Moreover, b-carotene and e-carotene, which are bi-cyclic carotenes synthesized by each enzyme, were not found. Since MpLCYb and MpLCYe genes were on
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 1 Phylogenetic tree of the lycopene cyclases. GenBank accession numbers are as follows: tomato LCYb1 (SlLCYb1), EF650013; tomato LCYb2 (SlLCYb2), AF254793; grapefruit LCYb1 (CpLCYb1), AF152246; grapefruit LCYb2 (CpLCYb2), GQ214768; liverwort (M. polymorpha) LCYb (MpLCYb), AB794089; Arabidopsis LCYb (AtLCYb), U50739; Chlamydomonas LCYb (CrLCYb), AY860818; liverwort LCYe (MpLCYe), AB794090; Arabidopsis LCYe (AtLCYe), U50738; tomato LCYe (SlLCYe), Y14387; Chlamydomonas LCYe (CrLCYe), AY606130; Adonis LCYe (AaLCYe), AF321535; lettuce LCYe (LsLCYe), AF321538; maize LCYe (ZmLCYe), EU924262; Pantoea ananatis CrtY (CrtY), D90087.
Fig. 2 Pathway of carotenoid biosynthesis in transformed E. coli used in this study. The reactions indicated by gray arrows have not been examined directly. It is thus unclear via which route a-carotene is formed.
Lycopene cyclase genes in liverwort
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 3 HPLC analysis of products formed from lycopene in transformed E. coli. (a–d) HPLC chromatograms of the extracts from E. coli that carried (a) pACCRT-EIB plus pET21a vector; (b) pACCRT-EIB plus pET-MpLCYb; (c) pACCRT-EIB plus pET-MpLCYe; and (d) pACCRT-EIB plus pETDuetMpLCYb/MpLCYe. (e) Absorption spectra of individual peaks: 1, lycopene; 2, b-carotene; 3, d-carotene; 4, e-carotene; 5, a-carotene.
the same vector, their expression levels were thought to be almost equal. Our results support the hypothesis that cyclization of lycopene occurs in a stepwise fashion, with closure of a single e-ionone ring to synthesize d-carotene, followed by b-ring closure to synthesize a-carotene (Fig. 2), which is mediated by a b-cyclase–e-cyclase complex (Cunningham and Gantt 1998). In maize, a null mutant of LCYb results in the accumulation of e-carotene and lactucaxanthin that are the hydroxylated product of e-carotene in the endosperm, whereas these carotenoids are not present in wild-type endosperm (Bai et al. 2009). This suggests that the presence of b-cyclase limits e-cyclase activity to mono-cyclization. The authors also suggested that the carotenoid profiles may be regulated by the differential expression of LCYe and LCYb in embryo and
endosperm tissues. In wild-type endosperm, high levels of LCYe accumulate, favoring the conversion of lycopene to d-carotene by LCYe and the production of lutein via a-carotene through the action of LCYb. On the other hand, in wild-type embryo, LCYb predominates, resulting in the conversion of lycopene to b-carotene by LCYb activity and subsequent production of zeaxanthin. We found that the MpLCYe gene was expressed at the same level as MpLCYb in the liverwort thalli. This situation is similar to that in maize endosperm and is consistent with results showing that lutein is the predominant carotenoid in the thalli. In this study, we identified the LCYb and LCYe genes of liverwort to gain insight into the evolution of carotenoid biosynthesis in plants. As discussed above, we suggested that the
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
197
M. Takemura et al.
LCYb2 (chromoplast-type) subfamily evolved after the branch point of bryophytes. It was also found that the liverwort genes were located between green alga (Chlorophyta) and higher plants, as expected. Analysis of other carotenoid biosynthesis genes will reveal the genetic evolution of carotenoid biosynthesis in plants.
Materials and Methods Plant material and growth conditions The female and male of M. polymorpha used in this study have been described by Okada et al. (2000). The liverwort was grown
198
on M51C solid medium (Takenaka et al. 2000) and incubated at 25 C under conditions of 16 h light/8 h dark in dim light (approximately 350 lux).
Analysis of carotenoids in liverwort Analysis of carotenoids in liverwort was carried out as described previously (Maoka et al. 2007, Maoka et al. 2008). Sufficient amounts of acetone were added to the male thalli (38 g FW) and the female thalli (52 g FW) of M. polymorpha, and mixed for several hours at room temperature. Each acetone extract was partitioned after the addition of hexane : diethyl ether (Et2O) (1 : 1, v/v) and water. The upper solvent layer was evaporated, and saponified with methanol (MeOH) including 5% potassium
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Fig. 4 Proposed carotenoid biosynthetic pathway in M. polymorpha thalli. The names of carotenoids detected in M. polymorpha are shown with red letters.
Lycopene cyclase genes in liverwort
Cloning of the lycopene b- and e-cyclase genes from M. polymorpha To find sequences homologous to the known LCYb and LCYe genes, homology searches of the genome, cDNA and EST sequences of liverwort (T. Kohchi et al. personal communication) were performed. Based on the homologous sequences obtained, the primers were designed and the coding regions of each gene were amplified by PCR of liverwort cDNAs. The following primers were used: MpLCYbF, 50 -CATATGAGCTCGAC GAGGTATG-30 ; MpLCYbR: 50 -GGATCCTTATTGCCGCTTCAT CAATG-30 ; MpLCYeF, 50 -CATATGGGAACCATAGATCGG-30 ; and MpLCYeR, 50 -CTCGAGTCATCTCATCTCCTTCGC-30 (the underlined sequences were added for cloning). Then PCR products were cloned into the plasmid vector and sequenced.
Sequence analysis Homology search was performed by BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi). Amino acid alignments and a phylogenetic tree were constructed using CLUSTAL W (http://www.clus tal.org/). Transit peptides of the gene products were predicted by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/).
Expression of liverwort LCYb and LCYe genes in Escherichia coli The coding regions of the liverwort LCYb (MpLCYb) and LCYe (MpLCYe) were cloned into pET21a (Merck Millipore) independently. These plasmids were designated pET21-MpLCYb and pET21-MpLCYe. For complementary expression of the MpLCYb and MpLCYe genes, they were cloned simultaneously into pETDuet (Merck Millipore) and called pETDuet-MpLCYb/ MpLCYe. Each plasmid was introduced into the lycopeneproducing E. coli [BL21 (DE3)], which carried plasmid pACCRT-EIB for the expression of the crtE, crtB and crtI genes from the bacterium Pantoea ananatis (Misawa et al. 1995). Each transformed E. coli was grown in 2YT medium at 37 C until an optical density of 0.8–1.0 was achieved, induced with 0.05 mM of isopropyl-b-D-thiogalactopyranoside (IPTG) and further cultured at 21 C for 2 d.
Extraction and analysis of carotenoids from E. coli cells Extraction of carotenoids from recombinant E. coli was performed by the method described by Fraser et al. (2000). Escherichia coli cultures were centrifuged and cell pellets were extracted with MeOH using a mixer for 5 min. Tris–HCl (50 mM, pH 7.5) (containing 1 M NaCl) was added and mixed. Chloroform was then added to the mixture and incubated for 5 min. After centrifugation, the chloroform phase was collected and dried by centrifugal evaporation. Dried residues were re-suspended with ethyl acetate, and applied to HPLC with a Waters Alliance 2695–2996 (PDA) system. HPLC was carried out according to the method described (Yokoyama and Miki 1995) using TSKgel ODS-80Ts (4.6 150 mm, 5 mm; Tosoh). This was developed at a flow rate of 1.0 ml min1 at 25 C with solvent A (water : MeOH, 5 : 95, v/v) for 5 min, followed by a linear gradient from solvent A to solvent B (tetrahydrofuran : MeOH, 3 : 7, v/v) for 5 min, and solvent B alone for 8 min. Individual carotenoids were identified by comparing retention times and absorption spectra with those of the authentic standards. a-Carotene, d-carotene and e-carotene were purified from the recombinant E. coli cells according to a method similar to that of analysis of carotenoids in liverwort, and chemical structures were confirmed by UV-VIS, high resolution electrospray ionization MS (HR-ESI-MS), CD and 1H-NMR (500 MHz) (Maoka et al. 2007, Maoka et al. 2008).
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
hydroxide at room temperature. This was re-extracted with hexane : Et2O (1 : 1). Each carotenoid extract was then subjected to HPLC on silica gel (Cosmosil 5SL-II, 4.6 250 mm; Nacalai tesque Inc.), developed with acetone : hexane (2 : 8) at a flow rate of 1.0 ml min1, and monitored at 450 nm. Carotenes eluted from silica gel HPLC were further subjected to reversephase HPLC to separate b-carotene and a-carotene. The reverse-phase HPLC was carried out using a YMC-Pack ODS-A column (4.6 250 mm ID; YMC Co.), developed with chloroform : methanol (15 : 85) at a flow rate of 1.0 ml min1 and monitored at 450 nm. Carotenoid compositions were estimated by peak area in HPLC. Individual carotenoids were fractionated during HPLC, as needed, and analyzed by UV-VIS, fast atom bombardment mass spectrometry and 1H-nuclear magnetic resonance (NMR; 500 MHz, CDCl3).
Spectroscopic data a-Carotene [(60 R)-b,e-carotene]: UV-VIS (Et2O) 423 (shoulder), 445, 473 nm; 1H-NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4368; CD (Et2O) nm (e) 240 (+5.5), 260 (+4.5), 280 (0), 350 (+0.5). d-Carotene [(6R)-e,c-carotene]: UV-VIS (Et2O) 430, 454, 487 nm; 1H-NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4371; CD (Et2O) nm (e) 255 (0), 270 (+6.5), 290 (0), 303 (–15). e-Carotene [(6R)-e,e-carotene]: UV-VIS (Et2O) 417, 440, 468 nm; 1H NMR (CDCl3) shown in Supplementary Table S1; HR-ESI-MS calculated for C40H56 (M+) 536.4382, found 536.4387; CD (Et2O) nm (e) 235 (0), 267 (+9), 285 (0), 300 (–1.8), 330 (–0.5).
Expression analysis of the MpLCyb and MpLCYe genes Total RNA of liverwort thalli was extracted using an RNeasy Plant mini kit (Qiagen) and treated with DNase I. A 1 mg aliquot of total RNA was reverse-transcribed with oligo(dT) primer using ReverTra Ace (TOYOBO). PCR was performed using the following primers: MpLCYbF3, 50 -CGAGGTTGACTGCCATCC30 ; MpLCYbR1, 50 -GGATCCTTATTGCCGCTTCATCAATG-30 ; MpLCYeF3, 50 -GTTGAGTGCGAGAACTTC-30 ; MpLCYeR1,
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
199
M. Takemura et al.
50 -CTCGAGTCATCTCATCTCCTTCGC-30 ; MpACT1F, 50 -TTCA ACGTTCCTGCCATGTA-30 ; and MpACT1R, 50 -GATCTCCCTT GTCATACGGT-30 MpACT (Marchantia polymorpha ACTIN) was used as an internal control.
Supplementary data Supplementary data are available at PCP online.
Acknowledgments
Disclosures The authors have no conflicts of interest to declare.
References Alque´zar, B., Zacarı´as, L. and Rodrigo, M.J. (2009) Molecular and functional characterization of a novel chromoplast-specific lycopene b-cyclase from Citrus and its relation to lycopene accumulation. J. Exp. Bot. 60: 1783–1797. Bai, L., Kim, E., DellaPenna, D. and Brutnell, T.P. (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J. 59: 588–599. Chaundhary, N., Nijhawan, A., Khurana, J.P. and Khurana, P. (2010) Carotenoid biosynthesis genes in rice: structural analysis, genomewide expression profiling and phylogenetic analysis. Mol. Genet. Genomics 283: 13–33. Cunningham, F.X. Jr. and Gantt, E. (1998) Gene and enzymes of carotenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 557–583. Cunningham, F.X. Jr. and Gantt, E. (2001) One ring or two? Determination of ring number in carotenoids by lycopene e-cyclases. Proc. Natl Acad. Sci. USA 98: 2905–2910. Cunningham, F.X. Jr., Pogson, B., Sun, Z., McDonald, K.A., DellaPenna, D. and Gantt, E. (1996) Functional analysis of the b and e lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8: 1613–1626. Demmig-Adams, B., Gilmore, A.M. and Adams, W.W. III (1996) In vivo functions of carotenoids in higher plants. FASEB J. 10: 403–412.
200
Plant Cell Physiol. 55(1): 194–200 (2014) doi:10.1093/pcp/pct170 ! The Author 2013.
Downloaded from http://pcp.oxfordjournals.org/ at North Dakota State University on October 28, 2014
We thank Dr. Takayuki Kohchi, Dr. Katsuyuki T. Yamato and Dr. Kimitsune Ishizaki for their assistance in searching liverwort sequences.
Fraser, P.D. and Bramley, P.M. (2004) The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43: 228–265. Fraser, P.D., Elisabete, M., Pinto, S., Holloway, D.E. and Bramley, P.M. (2000) Application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J. 24: 551–558. Maoka, T., Akimoto, N., Ishiguro, K., Yoshinaga, M. and Yoshimoto, M. (2007) Carotenoids with a 5,6-dihydro-5,6-dihydroxy-b-end group, from yellow sweet potato ‘Benimasari’, Ipomoea batatas LAM. Phytochemistry 68: 1740–1745. Maoka, T., Akimoto, N., Kuroda, Y., Hashimoto, K. and Fujiwara, Y. (2008) Pittosporumxanthins, cycloaddition products of carotenoids with a-tocopherol from seeds of Pittosporum tobia. J. Nat. Prod. 71: 622–627. Mendes, A.F.S., Chen, C., Gmitter, F.G. Jr., Moore, G.A. and Costa, M.G.C. (2011) Expression and phylogenetic analysis of two new lycopene b-cyclases from Citrus paradisi. Physiol. Plant 141: 1–10. Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T. et al. (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177: 6575–6584. Okada, S., Fujisawa, M., Sone, T., Nakayama, S., Nishiyama, R., Takenaka, M. et al. (2000) Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha. Plant J. 24: 421–428. Phillip, D. and Young, A.J. (1995) Occurrence of the carotenoid lactucaxanthin in higher plant LHC II. Photosynth. Res. 43: 273–282. Ronen, G., Carmel-Goren, L., Zamir, D. and Hirschberg, J. (2000) An alternative pathway to b-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc. Natl Acad. Sci. USA 97: 11102–11107. Ronen, G., Cohen, M., Zamir, D. and Hirschberg, J. (1999) Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant. Delta Plant J. 17: 341–351. ´ . and Rodrı´guez-Concepcio´n, M. (2012) Carotenoid bioRuiz-Sola, M.A synthesis in Arabidopsis: a colorful pathway. The Arabidopsis Book e0158. doi: 10.1199/tab.0158. Takenaka, M., Yamaoka, S., Hanajiri, T., Shimizu-Ueda, Y., Yamato, K.T., Fukuzawa, H. et al. (2000) Direct transformation and plant regeneration of the haploid liverwort Marchantia plymorpha L. Transgenic Res. 9: 179–185. Yokoyama, A. and Miki, W. (1995) Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin-producing bacterium Agrobacterium aurantiacum. FEMS Microbiol. Lett. 128: 139–144.
