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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Enhancement of Carotenoid Biosynthesis in Transplastomic Tomatoes by Induced Lycopene-to-Provitamin A Conversion^1,[OA]

Max-Planck-Institut für Molekulare Pflanzenphysiologie, D–14476 Potsdam-Golm, Germany

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Carotenoids are essential pigments of the photosynthetic apparatus and an indispensable component of the human diet. In addition to being potent antioxidants, they also provide the vitamin A precursor β-carotene. In tomato (Solanum lycopersicum) fruits, carotenoids accumulate in specialized plastids, the chromoplasts. How the carotenoid biosynthetic pathway is regulated and what limits total carotenoid accumulation in fruit chromoplasts is not well understood. Here, we have introduced the lycopene β-cyclase genes from the eubacterium Erwinia herbicola and the higher plant daffodil (Narcissus pseudonarcissus) into the tomato plastid genome. While expression of the bacterial enzyme did not strongly alter carotenoid composition, expression of the plant enzyme efficiently converted lycopene, the major storage carotenoid of the tomato fruit, into provitamin A (β-carotene). In green leaves of the transplastomic tomato plants, more lycopene was channeled into the β-branch of carotenoid biosynthesis, resulting in increased accumulation of xanthophyll cycle pigments and correspondingly reduced accumulation of the -branch xanthophyll lutein. In fruits, most of the lycopene was converted into β-carotene with provitamin A levels reaching 1 mg per g dry weight. Unexpectedly, transplastomic tomatoes also showed a >50% increase in total carotenoid accumulation, indicating that lycopene β-cyclase expression enhanced the flux through the pathway in chromoplasts. Our results provide new insights into the regulation of carotenoid biosynthesis and demonstrate the potential of plastids genome engineering for the nutritional enhancement of food crops.

While the enzymology of the carotenoid biosynthetic pathways in plants and eubacteria is now reasonably well understood (Armstrong, 1997; Cunningham and Gantt, 1998; Hirschberg, 2001), understanding of the regulation of carotenoid biosynthesis is still rather poor (Bramley, 2002). Mainly using the tomato (Solanum lycopersicum) fruit as model system, the study of pigmentation mutants (Ronen et al., 2000; Isaacson et al., 2002; Galpaz et al., 2006) and transgenic approaches (Giuliano et al., 2000, 2008; Römer and Fraser, 2005; Fraser et al., 2007) have provided first insights into regulatory mechanisms operating in carotenogenesis. For example, constitutive expression of the phytoene desaturase (crtI) gene from the bacterium Erwinia uredovora resulted in elevated β-carotene accumulation in tomatoes, but also led to an unexpected reduction in total carotenoid levels (Römer et al., 2000). The reduction in total carotenoids is believed to be an effect of feedback regulation from β-carotene or one of its downstream metabolites (Bramley, 2002). However, fruit-specific overexpression of the native lycopene β-cyclase resulted in increased β-carotene accumulation, without a concomitant decrease in total carotenoids (Rosati et al., 2000). Why some genetic disturbances of carotenoid biosynthesis negatively affect total carotenoid accumulation and others do not (or even result in an increase; Dharmapuri et al., 2002; Fraser et al., 2002), remains to be established.

Here we have used tomato plastid transformation to address the regulation of carotenoid biosynthesis exerted at the level of lycopene to β-carotene conversion by the enzyme lycopene β-cyclase (Fig. 1A ). We show that plastid expression of a plant lycopene β-cyclase does not only trigger efficient conversion of lycopene to β-carotene, but unexpectedly also results in a >50% increase in total carotenoid accumulation. This contrasts moderately increased β-carotene levels and reduced total carotenoid accumulation upon expression of a bacterial lycopene β-cyclase (Wurbs et al., 2007) and suggests lycopene β-cyclase activity as an important regulatory point in plant and microbial carotenoid biosynthesis.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Engineering of the carotenoid biosynthetic pathway by plastid transformation. A, Carotenoid biosynthetic pathway in higher plants. The pathway splits into an -branch and a β-branch immediately downstream of lycopene, the major storage carotenoid of tomato fruits. The enzyme expressed from the tomato plastid genome in this study, lycopene β-cyclase, leads into the β-branch. B, Physical maps of the targeting region in the plastid genome (ptDNA) and the plastid transformation vectors pEcrtY and pNLyc constructed in this study. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic region between the trnfM and trnfG genes (Ruf et al., 2001). The selectable marker gene aadA is driven by a chimeric rRNA operon promoter (Prrn; Svab and Maliga, 1993), fused to the 3'-UTR from the psbA gene (TpsbA), and flanked by two loxP sites to allow marker removal by Cre-mediated site-specific recombination (Zhou et al., 2008). The transgene expression cassette consists of the ribosomal RNA operon promoter fused to the 5' leader from the gene 10 of phage T7 (Prrn-G10L; Kuroda and Maliga, 2001) and the 3'-UTR of the rps16 gene (Trps16). Restriction sites used for cloning or RFLP analysis are indicated, and the psaB-derived hybridization probe is denoted by a horizontal bar. Sites lost due to ligation to heterologous ends are in parentheses. C, Southern-blot analysis of tomato transplastomic lines carrying the lycopene β-cyclase gene from daffodil (S.l.-pNLyc) or from E. herbicola (S.l.-pEcrtY). Total cellular DNA was digested with BglII and hybridized to a radioactively labeled probe detecting the psaB region of the plastid genome, which flanks the transgene insertion site (section B). Fragment sizes are given in kb. wt, Wild type. D, Alignment (produced with ClustalW2) of the amino acid sequences of the lycopin β-cyclases from daffodil (Np) and E. herbicola (Eh). Asterisk (*) denotes residues identical in both sequences (marked in bold), colon (:) indicates conserved substitutions, and a dot indicates semiconserved substitutions. The N-terminal extension of the Np sequence is likely to harbor the transit peptide for protein import into plastids. The amino acids that changed due to correction of the Lyc sequence from daffodil (published sequence: GenBank accession no. X98796.1; corrected sequence: accession no. GQ327929) are underlined. The corrections improve the sequence similarity in the N-terminal domains of the Np and Eh sequences.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

A recent transcriptomics and translatomics analysis of plastid gene expression during tomato fruit ripening revealed that most genes in the plastid genome are drastically down-regulated during fruit development (Kahlau and Bock, 2008). However, this study also identified a small number of plastid expression elements (promoters and translation initiation signals) that remain active in chromoplasts (Kahlau and Bock, 2008). As a previous attempt to express the lycopene β-cyclase gene (crtY) from the carotenoid-producing eubacterium Erwinia herbicola has resulted only in a moderate increase in β-carotene accumulation (to 280 ng/mg dry weight), we wanted to test if transgene expression levels can be improved by using a promoter that retains higher activity in chromoplasts than the previously used atpI promoter (Wurbs et al., 2007). As the ribosomal RNAs represent the most abundant plastid RNA species in chromoplasts (Kahlau and Bock, 2008), we selected the rRNA operon promoter and combined it with the strongest known ribosome-binding site, the Shine-Dalgarno sequence from gene 10 of bacteriophage T7 (Kuroda and Maliga, 2001; Oey et al., 2009; Fig. 1B). In addition to the lycopene β-cyclase gene from E. herbicola, we also cloned the Lyc lycopene β-cyclase gene from the higher plant daffodil (Narcissus pseudonarcissus; Al-Babili et al., 1996; kindly provided by Drs. Peter Beyer and Salim Al-Babili, University of Freiburg, Germany) into the same expression cassette (Fig. 1B). When we resequenced both genes after having finished vector construction, we noted three single-nucleotide deviations from the published Lyc sequence (GenBank accession no. X98796.1). All three deviations were also present in the original Lyc clone and presumably represent errors from band compressions in sequencing gels: a C was missing after position 442 (three Cs instead of two Cs in the published sequence), a G was missing after position 447 (three Gs instead of two Gs in the published sequence), and a C was missing after position 480 (two Cs instead of one C in the published sequence). These insertions change the published protein sequence over a stretch of 12 amino acids (Fig. 1D; as there are altogether three nucleotides missing, the published sequence falls back into the correct reading frame at nucleotide position 481). The corrected Lyc sequence was deposited in the GenBank database (accession no. GQ327929).

The plastid expression cassettes containing the crtY and Lyc transgenes were inserted into plasmid pKP9 (Zhou et al., 2008), generating the chloroplast transformation vectors pEcrtY and pNLyc, respectively (Fig. 1B). The two constructs were introduced into the plastid genome of the commercial tomato cv IPA-6 by biolistic chloroplast transformation (Svab and Maliga, 1993; Ruf et al., 2001). Eleven transplastomic lines were obtained with pEcrtY (from 400 selection plates with bombarded leaf pieces) and 22 transplastomic lines were obtained with pNLyc (from 350 selection plates). Three independently generated lines per construct were characterized in detail. The lines will be subsequently referred to as S.l.-pNLyc (for Solanum lycopersicum harboring the Narcissus Lyc gene) and S.l.-pEcrtY (for Solanum lycopersicum harboring the Erwinia crtY gene).

Analysis of Transplastomic Tomato Plants Harboring Lycopene β-Cyclase Transgenes

Putative chloroplast transformants were purified to homoplasmy by passing them through additional regeneration cycles under antibiotic selection. Transformation of the chloroplast genome, correct integration of the transgenes via homologous recombination, and homoplasmy of the transplastomic lines (i.e. absence of residual copies of the wild-type chloroplast genome) were assessed by RFLP analysis (Fig. 1C) and homoplasmy was additionally confirmed by seed assays (Fig. 2A ). Lack of segregation of the antibiotic resistance in the T1 generation demonstrated homoplasmy (Fig. 2A) and confirmed maternal transgene inheritance, as expected for a plastid-encoded trait in tomato. Homoplasmic T1 plants from S.l.-pNLyc and S.l.-pEcrtY transplastomic lines were indistinguishable from the wild-type control (Fig. 2B), indicating that transgene expression is phenotypically neutral.

View larger version (100K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Homoplasmy and phenotypes of transplastomic tomato lines. A, Examples of seed tests to confirm homoplasmy. Seeds from the wild-type (S.l.-wt) and transplastomic plants generated with constructs pEcrtY and pNLyc were germinated on medium with spectinomycin (100 mg/L). Antibiotic resistance and lack of segregation in the T1 generation confirms the homoplasmic state of the transplastomic lines. B, Wild-type-like phenotype of homoplasmic tomato lines expressing lycopene β-cyclase transgenes from their plastid genomes.

View larger version (43K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Analysis of lycopene β-cyclase mRNA accumulation in leaves (A) and ripe fruits (B) of transplastomic tomato plants. Total cellular RNA was hybridized to radiolabeled probes corresponding to the coding regions of crtY or Lyc. With each probe, two major transcript species are detected. While the bottom band represents mature monocistronic lycopene β-cyclase mRNA (1.3 kb for crtY and 1.7 kb for Lyc), the top band most probably represents a stable read-through transcript, as has been observed before with pKP9-derived vectors (Zhou et al., 2008; Oey et al., 2009). Sizes of marker bands are indicated in kb. Note that crtY and Lyc do not cross-hybridize due to insufficient sequence similarity (Fig. 1D). wt, Wild type.

View larger version (74K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Herbicide resistance assays to test for lycopene β-cyclase expression. The herbicide CPTA was used as specific inhibitor of the lycopin β-cyclase activity. Aseptically grown tomato plants were watered with 2.5 mL CPTA solution to obtain the final concentrations indicated (50, 100, 150, 250, or 500 µM). Phenotypic comparison with a water-treated control (0 µM) was done after 7 d. wt, Wild type.

The dark-red carotenoid lycopene represents the main storage carotenoid in tomato fruits. Presence of elevated levels of lycopene β-cyclase activity in fruits should result in enhanced conversion of lycopene into the orange carotenoid β-carotene (provitamin A; Fig. 1A). Inspection of ripe fruits from our transplastomic tomato plants revealed that the S.l.-pNLyc tomatoes were bright orange instead of dark red (Fig. 5 ). In contrast, S.l.-pEcrtY transplastomic tomatoes were virtually indistinguishable from wild-type tomatoes (Fig. 5). Taken together with the results from the CPTA assays, this suggested that the daffodil cyclase was expressed to high levels and was highly active in tomato plastids.

View larger version (39K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Phenotypes of tomato fruits from transplastomic tomato plants expressing lycopene β-cyclase transgenes. Fruits from a wild-type plant (S.l.-wt), an S.l.-pEcrtY line, and an S.l.-pNLyc line were harvested at different ripening stages and photographed from the side (top row) and from the bottom (bottom row). The orange color of ripe S.l.-pNLyc fruits indicates efficient conversion of red lycopene into orange β-carotene (provitamin A).

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. HPLC analysis of pigment accumulation in fruits and leaves from wild-type plants (S.l.-wt) and transplastomic S.l.-pEcrtY and S.l.-pNLyc plants. A, Comparison of lycopene and β-carotene contents (in ng/mg dry weight [DW]) in ripe fruits as determined by HPLC. Values represent means from 12 measurements: three fruits harvested from three to four independent plant lines per construct. B, Comparison of carotenoid contents in leaves. Values represent means from nine measurements, which included leaves harvested from at least four independent transplastomic lines per construct. The SD is shown as error bar. Asterisks indicate significant differences compared to the wild type (P value < 0.05).

We next wanted to determine whether or not lycopene β-cyclase overexpression alters carotenoid biosynthesis also in leaves. When leaf tissue from the transplastomic plants expressing the Erwinia cyclase gene was analyzed, carotenoid composition was found to be unaltered (Fig. 6B). In contrast, leaves from S.l.-pNLyc plants displayed pronounced changes in pigment composition. While chlorophyll contents were identical to the wild type (data not shown), the carotenoid spectra showed significantly elevated levels of the xanthophyll cycle carotenoids violaxanthin, antheraxanthin, and zeaxanthin and a significant reduction in lutein accumulation (Fig. 6B). These changes are proportional in that they do not entail a significant alteration of total leaf carotenoid contents. This suggests that in S.l.-pNLyc plants, the flux through the β-branch of the carotenoid biosynthetic pathway (Fig. 1A) is enhanced and the flux through the -branch is proportionately reduced.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In this work, we have expressed two lycopene β-cyclase genes from the plastid genome of tomato. While the gene from the eubacterium Erwinia did not result in a significant change in carotenoid accumulation, expression of the lycopene β-cyclase from the higher plant daffodil did not only trigger efficient lycopene-to-provitamin A conversion, but also led to a massive increase in total fruit carotenoid content.

The lack of efficient expression of the Erwinia gene was surprising, because an earlier attempt to convert lycopene to β-carotene by expression of this gene from the plastid genome had resulted in at least a moderate increase in β-carotene accumulation (to 286 µg/g dry weight), although total carotenoid levels declined by >10% (Wurbs et al., 2007). The lycopene β-cyclases from Erwinia and Narcissus share significant sequence similarity at the protein level (Fig. 1D), but the similarity at the DNA level is very low. The low CPTA tolerance of the S.l.-pEcrtY plants (Fig. 4) clearly points to a problem with protein accumulation. The promoter and 5'-untranslated region (UTR) used here should have triggered stronger expression than the previously used atpI promoter (Kahlau and Bock, 2008; Oey et al., 2009) and it is not entirely clear why this was not the case. One possible reason could be that accessibility of the Shine-Dalgarno sequence to the plastid ribosomes is impaired in the Prrn-G10L sequence context, for example, by aberrant secondary structure formation between the 5'-UTR and the crtY coding region. Masking of the Shine-Dalgarno sequence by RNA secondary structure formation has been demonstrated to effectively prevent translation in both bacteria (Hall et al., 1982) and plastids (Hirose and Sugiura, 1997). As aberrant RNA folding is notoriously difficult to predict and nearly impossible to analyze in vivo, its possible involvement in the lack of crtY expression from the Prrn-G10L cassette was not investigated further.

Interestingly, plastid expression of the daffodil lycopene β-cyclase led to a strong elevation of total fruit carotenoid content. This was surprising, because previous expression of the lycopene β-cyclase from Erwinia had resulted in a lower total carotenoid content (Wurbs et al., 2007). A plausible explanation could be that the daffodil enzyme is less susceptible to negative feedback regulation by β-carotene than the bacterial enzyme. This would also be compatible with the high β-carotene (and total carotenoid) accumulation that the daffodil enzyme triggers in flower chromoplasts. The daffodil lycopene β-cyclase, therefore, may provide a valuable tool for enhancing the carotenoid metabolic pathway in fruits.

Expression of the daffodil lycopene β-cyclase also resulted in altered carotenoid composition in leaves. Accumulation of the xanthophyll cycle pigments zeaxanthin, antheraxanthin, and violaxanthin was increased and accumulation of the -branch xanthophyll lutein was correspondingly reduced (Fig. 1A). In leaves, conversion of lycopene to β-carotene and -carotene is usually complete in that no lycopene is detectable. Obviously, there is substrate competition between lycopene β-cyclase and lycopene -cyclase and presence of elevated amounts of lycopene β-cyclase is, therefore, sufficient to channel more lycopene into the β-branch of carotenoid biosynthesis. This alteration in flux through the pathway did not result in any phenotypic effect, which is not unexpected, because lutein, the pigment species reduced in S.l.-pNLyc plants, was shown previously to be dispensable for photosynthesis in higher plants (Pogson et al., 1996).

Both elevating the provitamin A content and increasing the total carotenoid content of tomatoes represent important goals of breeding and genetic engineering efforts (Ye et al., 2000; Giuliano et al., 2008; Mayer et al., 2008). Having obtained, by plastid genome engineering in a commercial tomato variety, provitamin A levels approaching 1 mg/g dry weight and, at the same time, a >50% increase in total fruit carotenoid content, therefore, is also an encouraging step forward in metabolic engineering of the carotenoid pathway in tomato. Given that approximately 10 mg per day of β-carotene provides the adult recommended dietary allowance for vitamin A (http://www.nlm.nih.gov/medlineplus/druginfo/natural/patient-vitamina.html), S.l.-pNLyc tomatoes provide a very rich source of provitamin A. Expression of the daffodil lycopene β-cyclase represents only the second (and the to date most successful) example of metabolic engineering by plastid transformation in a food crop (Wurbs et al., 2007). This is because tobacco (Nicotiana tabacum) is still the only plant in which plastid transformation is routine. Generation of transplastomic plants in the few other species that can be transformed is much more difficult, laborious, and time consuming (Ruf et al., 2001; Dufourmantel et al., 2005). However, as the transplastomic technology offers unique advantages, including high-level foreign protein accumulation (of up to >70% of the plant's total soluble protein; Oey et al., 2009), absence of epigenetic effects, convenient transgene stacking in operons, and greatly increased transgene containment due to the maternal inheritance of plastid genes (Ruf et al., 2007), there is clearly a persuasive case for the further development of plastid transformation technology and its application in agriculture and biotechnology. Our data presented here demonstrate that if appropriate combinations of transgenes and expression elements are chosen, efficient metabolic engineering via plastid transformation is also possible in non-green tissues.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Aseptically grown tomato (Solanum lycopersicum ‘IPA-6’) plants were obtained from surface-sterilized seeds germinated and grown on agar-solidified Murashige and Skoog medium (Murashige and Skoog, 1962) with 20 g/L Suc. Homoplasmic transplastomic lines were rooted and propagated on the same media. Rooted homoplasmic plants were transferred to soil and grown to maturity under standard greenhouse conditions (250 µmol quanta m^–2 s^–1). Fruits were harvested at different stages of ripening.

Cloning Procedures

The coding region of the crtY gene from Erwinia herbicola was excised as NcoI/XbaI fragment from a previously described plasmid (Wurbs et al., 2007), cloned into a Prrn-G10L-driven expression cassette (Kuroda and Maliga, 2001; Oey et al., 2009), and then integrated as SacI/HindIII fragment into the previously described plastid transformation vector pKP9 (Zhou et al., 2008), generating vector pEcrtY (Fig. 1C). A cloned lycopene β-cyclase gene (Lyc) from daffodil (Narcissus pseudonarcissus; kindly provided by Drs. Peter Beyer and Salim Al-Babili, University of Freiburg, Germany) was amplified by PCR with primers PNlyc5' (5'-TTTTCCATGGATACTCTATTGAGAACCCA-3') and PNlyc3' (5'-TTTTACTAGTCCCTATCTTGAACTAAGTTA-3'). With the primer sequences, a 5' NcoI restriction site and a 3' SpeI site were introduced (underlined). The entire coding region of the Lyc gene (including the putative transit peptide sequence) was cloned into the same Prrn-G10L expression cassette (as NcoI/SpeI fragment into the NcoI/XbaI-digested vector) and subsequently integrated into pKP9 as SacI/ApaI fragment, generating transformation vector pNLyc (Fig. 1C).

Transformation of Tomato Chloroplasts

Plastid transformation was carried out using a biolistic protocol (Svab and Maliga, 1993; Ruf et al., 2001). Young leaves from tomato plants grown under aseptic conditions were bombarded with plasmid DNA-coated 0.6-µm gold particles using a biolistic gun with a Hepta adaptor (PDS1000He; Bio-Rad). Spectinomycin-resistant tomato lines were selected on a modified Murashige and Skoog medium (Wurbs et al., 2007) containing spectinomycin (500 mg/L). For each construct, several independent transplastomic tomato lines were subjected to three additional rounds of regeneration on spectinomycin-containing medium to obtain homoplasmic tissue. Homoplasmy was confirmed by inheritance assays (Bock, 2001) in which seeds were germinated on Murashige and Skoog medium containing spectinomycin (100 mg/L).

Isolation of Nucleic Acids and Gel-Blot Analyses

Total plant DNAs were isolated from fresh leaf tissue samples by a cetyltrimethylammoniumbromide-based method (Doyle and Doyle, 1990). Total cellular RNA was extracted using the peqGOLD TriFast reagent (Peqlab GmbH). For Southern-blot analysis, samples of 5 µg total DNA were digested with the restriction enzyme BglII, separated by gel electrophoresis on 1% agarose gels, and transferred onto Hybond nylon membranes (GE Healthcare) by capillary blotting. Total cellular RNA samples were electrophoresed in formaldehyde-containing 1% agarose gels and blotted onto Hybond nylon membranes. Hybridizations were performed at 65°C in Church buffer (Church and Gilbert, 1984). Hybridization probes were purified by agarose gel electrophoresis following extraction of the DNA fragments of interest from excised gel slices using the GFX PCR (DNA and gel band purification) kit (GE Healthcare). A 550 bp PCR product generated by amplification of a portion of the psaB coding region using primers P7247 and P7244 (Wurbs et al., 2007) was used as an RFLP probe to verify plastid transformation and assess homoplasmy. Transgene-specific probes for northern-blot analyses were generated by excising the Erwinia and Narcissus lycopene β-cyclase coding regions from plasmid clones with NcoI and XbaI.

Herbicide Tolerance Assays

Synthesis of the chemical CPTA, as a specific lycopene cyclase inhibitor, was described previously (Schuetz and Baldwin, 1958; Wurbs et al., 2007). Tolerance of transplastomic tomato plants to CPTA was assessed by adding 2.5 mL CPTA solution to plants grown under aseptical conditions in boxes on 2x Murashige and Skoog medium (to obtain final herbicide concentrations of 50, 100, 150, 250, or 500 µM) followed by phenotypic comparison with a water-treated control after 7 d.

HPLC Analyses of Pigments

Carotenoids and chlorophylls were isolated from leaf tissue (that was frozen, ground, and lyophilized) by extraction with 80% acetone followed by two additional extractions with 100% acetone and combination of the three extracts. Tomato fruit tissue harvested at the onset of fruit softening was frozen, ground, lyophilized, and extracted as described above. Separation, identification, and quantification of carotenoids were performed by HPLC using an Agilent 1100 Series HPLC system with a diode array detection unit (Agilent). For all separations, a YMC ODS-A 250 x 4.6 mm column + precolumn was used. Separation was performed as described previously (Wurbs et al., 2007). All pigment species were identified and quantified by comparison with known amounts of pure standards.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number GQ327929.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Peter Beyer and Salim Al-Babili (University of Freiburg, Germany) for providing a daffodil lycopene cyclase clone. We thank Dr. Stephanie Ruf and Steffi Seeger (Max-Planck-Institut für Molekulare Pflanzenphysiologie) for help with plant transformation, Helga Kulka (Max-Planck-Institut für Molekulare Pflanzenphysiologie Green Team) for plant care and cultivation, Josef Bergstein (Max-Planck-Institut für Molekulare Pflanzenphysiologie) for photography, and Yossi Hirschberg (The Hebrew University of Jerusalem) for helpful discussion and critically reading the manuscript.

Received April 28, 2009; accepted July 6, 2009; published July 8, 2009.

	FOOTNOTES

 
¹ This work was supported by the Max Planck Society and by the Deutsche Forschungsgemeinschaft (grant no. BO 1482/11–1 to R.B.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ralph Bock (rbock{at}mpimp-golm.mpg.de).

^[OA] Open access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.140533

^* Corresponding author; e-mail rbock{at}mpimp-golm.mpg.de

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Peter V. Minorsky
Plant Physiol. 2009 151: 1-2. [Full Text]  

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