Research ArticleEVOLUTIONARY BIOLOGY

An algal enzyme required for biosynthesis of the most abundant marine carotenoids

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Science Advances  04 Mar 2020:
Vol. 6, no. 10, eaaw9183
DOI: 10.1126/sciadv.aaw9183
  • Fig. 1 A vdl knockout mutant of N. oceanica no longer synthesizes the allenic vaucheriaxanthin acyl esters.

    (A) HPLC analyses (system IIb) of pigment extracts from wild type, the vdl mutant, and a vdl mutant complemented with its native VDL gene (vdl + VDL, clone #4) demonstrate that knockout of the VDL gene results in loss of vaucheriaxanthin acyl esters (peaks 3 and 4) and a concomitant increase in violaxanthin (2). Chromatograms were normalized to chlorophyll a (5); other peaks were identified as latoxanthin (1) and β-carotene (6). For detailed pigment stoichiometries in the wild type, the vdl mutant, and two VDL-complemented strains of the vdl mutant, see table S1. (B) Scheme showing the insertion site of the 1.8-kb zeocin resistance cassette (ZeoR) within the second exon of the VDL gene in the vdl mutant. Binding sites of the primers used for differentiation of wild-type (WT) and mutated VDL gene are indicated by black arrows. (C) Agarose gel showing 1.8-kb size difference of polymerase chain reaction (PCR) products of WT or the VDL-deficient mutant (vdl) using PCR primers specified in (B). (D) Agarose gel showing additional band of WT VDL fragment at 1.4 kb for PCR products from successfully complemented transformants of the vdl mutant; clones #2 and #4 were used for pigment analyses. Other experimental details are described in Materials and Methods.

  • Fig. 2 Transient expression of VDL1 from P. tricornutum in tobacco leaves results in accumulation of neoxanthin.

    HPLC analysis (system Ib) of pigment extracts from untreated leaves (control) and leaves after Agrobacterium-mediated transformation with VDL1 or VDL2 from P. tricornutum. Chromatograms were normalized to chlorophyll a (peak 8), and the parts showing neoxanthin (1), violaxanthin (2), 9′-cis-neoxanthin (3), deepoxyneoxanthin (4), and antheraxanthin (5) were magnified four times. Other peaks were identified as lutein (6), chlorophyll b (7), β-carotene (9), and 9-cis-β-carotene (10). For detailed pigment stoichiometries in leaf samples from the three treatments, see table S2.

  • Fig. 3 Violaxanthin-neoxanthin tautomerase activity of VDL proteins from further chromalveolate algae in tobacco leaves and in vitro.

    (A) HPLC analyses (system Ib) of pigment extracts from tobacco leaves transiently expressing VDL from algae with diadinoxanthin cycle (brown-colored chromatograms) or from algae that use the violaxanthin cycle for photoprotection (green-colored chromatograms). Depicted are details of chromatograms normalized to the chlorophyll a peak showing trans-neoxanthin (Nx), violaxanthin (Vx), and 9′-cis-neoxanthin (cNx). Blue label indicates that trans-neoxanthin is only found in leaves transformed with the algal VDL genes. Note the stronger accumulation of trans-neoxanthin when leaves are transformed with VDL from algae with diadinoxanthin cycle. Detailed pigment stoichiometries in leaf samples from all treatments are given in table S3. (B) HPLC analyses (system II) of samples from in vitro assays with recombinant algal VDL proteins using violaxanthin as substrate and incubation times of 3 hours [chromatogram colors and peak labels as in (A), reaction products labeled blue; values below species names indicate VDL concentrations in assay; activity of VDL from P. minimum was measured using bacterial lysate]. For each VDL, at least two different preparations were examined with similar results. VDL concentrations in the assays varied because of different expression efficiencies of VDL proteins in E. coli that could not be overcome by changing expression conditions. VDL proteins from algae with diadinoxanthin cycle showed substantial in vitro activities that were correlated with protein concentrations in the assays, while VDL proteins from algae with violaxanthin cycle showed minor or no activity independent of the protein concentrations used. Other experimental details are described in Materials and Methods.

  • Fig. 4 Functional comparison of VDE and VDL1 from P. tricornutum.

    (A) Proposed pathway of carotenoid biosynthesis in chromalveolate algae and reactions catalyzed by VDE (red arrows) and VDL (blue arrows). In addition, the two putative pathways to 9′-cis-neoxanthin—a neoxanthin isomer specific to land plants and green algae—are indicated by broken arrows (green-colored paths; enzymes unknown). (B to D) HPLC analyses (system II) of in vitro assays with recombinant VDE showing (B) enzymatic de-epoxidation of Vx to Ax and Zx and of Ddx to Dtx (reaction products labeled red); (C) pH dependence of VDE activity; and (D) dose-response curve of VDE inhibition by reductant DTT. (E to G) In vitro assays with recombinant VDL1 showing (E) enzymatic tautomerization of Vx to Nx and of Nx to Vx (reaction products labeled blue); (F) pH dependence of VDL1 activity; and (G) dose-response curve of VDL1 inhibition by DTT. The experimental details are described in Materials and Methods.

  • Fig. 5 Phylogeny and summary of enzymatic activities in tobacco or in vitro of VDL proteins from selected dinophyte, haptophyte, and heterokont algae.

    Investigated VDL proteins are underlined, and their violaxanthin-neoxanthin tautomerase activities in tobacco or in vitro were indicated as strong (++), medium (+), weak (o), or not detectable (−) based on the results shown in Fig. 3. Also indicated are the type of xanthophyll cycle (Xan cycle) and the major allenic light-harvesting carotenoid (LH car) present in the respective algal species [for abbreviations of carotenoids, see Fig. 4]. Midpoint-rooted maximum likelihood tree of VDE family proteins from selected chromalveolate algae shows bootstrap supports (100 replicates) above 50% for major nodes. “VDL1” or “1” behind species name indicates additional presence of a VDL2 in that species. For VDE, VDL2, and VDR, values in brackets indicate the number of sequences included in the phylogenetic analysis. The full tree is shown in fig. S2.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaw9183/DC1

    Fig. S1. Tentative identification of latoxanthin from N. oceanica.

    Fig. S2. Midpoint-rooted maximum likelihood tree of VDE family proteins from selected species of chromalveolate algae and Viridiplantae (land plants and green algae).

    Fig. S3. In vitro assays with PtVDL2 using violaxanthin or diadinoxanthin as substrate.

    Fig. S4. Investigation of other carotenoids than violaxanthin as potential substrates of PtVDL1.

    Fig. S5. Kinetics of tautomerization of violaxanthin to neoxanthin and of antheraxanthin to deepoxyneoxanthin by PtVDL1.

    Fig. S6. In vitro activity of PtVDL1 or PtVDE with and without addition of ascorbate.

    Fig. S7. Pigment composition of chromalveolate algae for which VDL proteins were functionally characterized.

    Table S1. Pigment stoichiometries in N. oceanica wild type, the vdl mutant, and two strains of the vdl mutant complemented with the native VDL gene (vdl + VDL).

    Table S2. Pigment stoichiometries in leaves from N. benthamiana transiently expressing PtVDL1 fused either to transit peptide tpNtVDE for luminal targeting or to tpAtZEP for stromal targeting and in leaves expressing PtVDL2 fused with tpNtVDE for luminal targeting.

    Table S3. Pigment stoichiometries in leaves from N. benthamiana transiently expressing either VDL from algae with diadinoxanthin cycle or VDL from algae with violaxanthin cycle.

    Data file S1. Results of targeting prediction for VDL and VDE proteins.

    Data file S2. Algal sources and database accessions of VDE family protein sequences analyzed in this work.

    Data file S3. PCR templates and primers used for generation of expression constructs used in this work.

    Data file S4. Strain-specific single nucleotide polymorphisms in the genes amplified in this work.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Tentative identification of latoxanthin from N. oceanica.
    • Fig. S2. Midpoint-rooted maximum likelihood tree of VDE family proteins from selected species of chromalveolate algae and Viridiplantae (land plants and green algae).
    • Fig. S3. In vitro assays with PtVDL2 using violaxanthin or diadinoxanthin as substrate.
    • Fig. S4. Investigation of other carotenoids than violaxanthin as potential substrates of PtVDL1.
    • Fig. S5. Kinetics of tautomerization of violaxanthin to neoxanthin and of antheraxanthin to deepoxyneoxanthin by PtVDL1.
    • Fig. S6. In vitro activity of PtVDL1 or PtVDE with and without addition of ascorbate.
    • Fig. S7. Pigment composition of chromalveolate algae for which VDL proteins were functionally characterized.
    • Table S1. Pigment stoichiometries in N. oceanica wild type, the vdl mutant, and two strains of the vdl mutant complemented with the native VDL gene (vdl + VDL).
    • Table S2. Pigment stoichiometries in leaves from N. benthamiana transiently expressing PtVDL1 fused either to transit peptide tpNtVDE for luminal targeting or to tpAtZEP for stromal targeting and in leaves expressing PtVDL2 fused with tpNtVDE for luminal targeting.
    • Table S3. Pigment stoichiometries in leaves from N. benthamiana transiently expressing either VDL from algae with diadinoxanthin cycle or VDL from algae with violaxanthin cycle.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Results of targeting prediction for VDL and VDE proteins.
    • Data file S2 (Microsoft Excel format). Algal sources and database accessions of VDE family protein sequences analyzed in this work.
    • Data file S3 (Microsoft Excel format). PCR templates and primers used for generation of expression constructs used in this work.
    • Data file S4 (Microsoft Excel format). Strain-specific single nucleotide polymorphisms in the genes amplified in this work.

    Files in this Data Supplement:

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