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Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species.

Chung HH, Schwinn KE, Ngo HM, Lewis DH, Massey B, Calcott KE, Crowhurst R, Joyce DC, Gould KS, Davies KM, Harrison DK - Front Plant Sci (2015)

Bottom Line: In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II).The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports.A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay.

View Article: PubMed Central - PubMed

Affiliation: Centre for Native Floriculture, School of Agriculture and Food Sciences, The University of Queensland, Gatton QLD, Australia.

ABSTRACT
Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

No MeSH data available.


Related in: MedlinePlus

Presence of betalain pigments in A. majus petals transiently transformed with 35S:PmDOD and fed DOPA. HPLC analysis was conducted on a tissue extract and compared to a known beetroot control sample. Pigment retention times and absorption maximums were consistent with the major pigments being the same as those in beetroot – betanin (peak 2) and vulgaxanthin I (peak 1). The chromatograms shown are only for the solvent gradient optimized for betaxanthin separation and detection at 470 nm, conditions that are also adequate for separation and detection of the beetroot betacyanins. Chromatograms of A. majus control tissue (35S:PmDOD-bombarded tissue fed water, 35S:GFP bombarded tissue fed DOPA) show no pigment peaks.
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Figure 5: Presence of betalain pigments in A. majus petals transiently transformed with 35S:PmDOD and fed DOPA. HPLC analysis was conducted on a tissue extract and compared to a known beetroot control sample. Pigment retention times and absorption maximums were consistent with the major pigments being the same as those in beetroot – betanin (peak 2) and vulgaxanthin I (peak 1). The chromatograms shown are only for the solvent gradient optimized for betaxanthin separation and detection at 470 nm, conditions that are also adequate for separation and detection of the beetroot betacyanins. Chromatograms of A. majus control tissue (35S:PmDOD-bombarded tissue fed water, 35S:GFP bombarded tissue fed DOPA) show no pigment peaks.

Mentions: The potential for DOPA 4,5-extradiol cleavage activity by DOD and DOD-like was assayed using a biolistics-based transient gene expression assay (Harris et al., 2012). This method uses the formation of betalain pigments as an indicator of 4,5-DOPA cleavage and betalamic acid (BA) formation. Petals of the white-flowered nivea (chalcone synthase) mutant of the anthocyanin-producing species A. majus, which lack anthocyanins and other flavonoids, were bombarded with the P. mirabilis cDNA constructs driven by the 35S promoter and fed DOPA. A construct with a Green Fluorescent Protein (GFP) reporter gene (35S:GFP) was co-precipitated with the other constructs onto the gold particles in some experiments as an indicator of transformation frequency. Pigment production was observed only in tissue bombarded with 35S:DOD and not in tissue bombarded with 35S:DOD-like or in control tissue (bombarded tissue fed water; Figure 4). The pigmented zones showed green autofluorescence under blue light in the absence of GFP, a characteristic typical of betaxanthins (Gandía-Herrero et al., 2005). The results for 35S:DOD were typical of what was observed in (Harris et al., 2012) when using a construct for a Portulaca DOD gene confirmed to be betalain-related. The pigments produced in the bombarded tissue were identified as the betacyanin betanin and the glutamine-based betaxanthin vulgaxanthin I based on HPLC comparison to the pigments in a beetroot standard sample (Figure 5). The pigment accumulated in multicellular zones, likely due to DOD protein or BA/pigment moving from the single transformed cells into adjacent untransformed cells. No pigment was observed in tissue bombarded with 35S:DOD-like despite GFP fluorescence showing the transformation process was successful (blue light panels, Figure 4). The DOD-like cDNA used in these assays was from a different accession of P. mirabilis and has two amino acid changes (ORF E192Q and T265S, both outside the proposed catalytic domain) compared to the original DOD-like sequence, which may represent allelic variation. It was confirmed that these amino acid changes were not influencing the assay by repeating the experiment with a construct using the original DOD-like sequence, with the same results obtained (Supplementary Figure S1).


Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species.

Chung HH, Schwinn KE, Ngo HM, Lewis DH, Massey B, Calcott KE, Crowhurst R, Joyce DC, Gould KS, Davies KM, Harrison DK - Front Plant Sci (2015)

Presence of betalain pigments in A. majus petals transiently transformed with 35S:PmDOD and fed DOPA. HPLC analysis was conducted on a tissue extract and compared to a known beetroot control sample. Pigment retention times and absorption maximums were consistent with the major pigments being the same as those in beetroot – betanin (peak 2) and vulgaxanthin I (peak 1). The chromatograms shown are only for the solvent gradient optimized for betaxanthin separation and detection at 470 nm, conditions that are also adequate for separation and detection of the beetroot betacyanins. Chromatograms of A. majus control tissue (35S:PmDOD-bombarded tissue fed water, 35S:GFP bombarded tissue fed DOPA) show no pigment peaks.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493658&req=5

Figure 5: Presence of betalain pigments in A. majus petals transiently transformed with 35S:PmDOD and fed DOPA. HPLC analysis was conducted on a tissue extract and compared to a known beetroot control sample. Pigment retention times and absorption maximums were consistent with the major pigments being the same as those in beetroot – betanin (peak 2) and vulgaxanthin I (peak 1). The chromatograms shown are only for the solvent gradient optimized for betaxanthin separation and detection at 470 nm, conditions that are also adequate for separation and detection of the beetroot betacyanins. Chromatograms of A. majus control tissue (35S:PmDOD-bombarded tissue fed water, 35S:GFP bombarded tissue fed DOPA) show no pigment peaks.
Mentions: The potential for DOPA 4,5-extradiol cleavage activity by DOD and DOD-like was assayed using a biolistics-based transient gene expression assay (Harris et al., 2012). This method uses the formation of betalain pigments as an indicator of 4,5-DOPA cleavage and betalamic acid (BA) formation. Petals of the white-flowered nivea (chalcone synthase) mutant of the anthocyanin-producing species A. majus, which lack anthocyanins and other flavonoids, were bombarded with the P. mirabilis cDNA constructs driven by the 35S promoter and fed DOPA. A construct with a Green Fluorescent Protein (GFP) reporter gene (35S:GFP) was co-precipitated with the other constructs onto the gold particles in some experiments as an indicator of transformation frequency. Pigment production was observed only in tissue bombarded with 35S:DOD and not in tissue bombarded with 35S:DOD-like or in control tissue (bombarded tissue fed water; Figure 4). The pigmented zones showed green autofluorescence under blue light in the absence of GFP, a characteristic typical of betaxanthins (Gandía-Herrero et al., 2005). The results for 35S:DOD were typical of what was observed in (Harris et al., 2012) when using a construct for a Portulaca DOD gene confirmed to be betalain-related. The pigments produced in the bombarded tissue were identified as the betacyanin betanin and the glutamine-based betaxanthin vulgaxanthin I based on HPLC comparison to the pigments in a beetroot standard sample (Figure 5). The pigment accumulated in multicellular zones, likely due to DOD protein or BA/pigment moving from the single transformed cells into adjacent untransformed cells. No pigment was observed in tissue bombarded with 35S:DOD-like despite GFP fluorescence showing the transformation process was successful (blue light panels, Figure 4). The DOD-like cDNA used in these assays was from a different accession of P. mirabilis and has two amino acid changes (ORF E192Q and T265S, both outside the proposed catalytic domain) compared to the original DOD-like sequence, which may represent allelic variation. It was confirmed that these amino acid changes were not influencing the assay by repeating the experiment with a construct using the original DOD-like sequence, with the same results obtained (Supplementary Figure S1).

Bottom Line: In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II).The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports.A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay.

View Article: PubMed Central - PubMed

Affiliation: Centre for Native Floriculture, School of Agriculture and Food Sciences, The University of Queensland, Gatton QLD, Australia.

ABSTRACT
Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

No MeSH data available.


Related in: MedlinePlus