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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

Cell phenotypes in tissue bombarded with DOD-like constructs and fed DOPA. Tissue bombarded with constructs for PmDOD-like and PhybDOD-like. The GFP construct was not included. (A)A. majus petal tissue used. Single green fluorescent cells at low frequency were observed under blue light. A few of these cells were possibly pale pink under white light in the PmDOD-like bombarded sample. Hatched squares show regions depicted at higher magnification in the panels on the right side. Scale bar is the same size in all. (B) White onion scale tissue used. No pigments observed when bombarded with gold particles only (i); conspicuous multicellular yellow zones observed when bombarded with the PmDOD construct (positive control; ii); brown cells (iii) and a rare pale yellow cell (iv) was observed when bombarded with the PhybDOD-like construct. Scale bar = 500 μm (A), 25 μm (B).
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Figure 6: Cell phenotypes in tissue bombarded with DOD-like constructs and fed DOPA. Tissue bombarded with constructs for PmDOD-like and PhybDOD-like. The GFP construct was not included. (A)A. majus petal tissue used. Single green fluorescent cells at low frequency were observed under blue light. A few of these cells were possibly pale pink under white light in the PmDOD-like bombarded sample. Hatched squares show regions depicted at higher magnification in the panels on the right side. Scale bar is the same size in all. (B) White onion scale tissue used. No pigments observed when bombarded with gold particles only (i); conspicuous multicellular yellow zones observed when bombarded with the PmDOD construct (positive control; ii); brown cells (iii) and a rare pale yellow cell (iv) was observed when bombarded with the PhybDOD-like construct. Scale bar = 500 μm (A), 25 μm (B).

Mentions: When the transient assay was repeated for DOD-like without including the GFP internal control, a phenotype was revealed under blue-light. Single cells with weak green autofluorescence were observed at low frequency (Figure 6A). These cells were generally not visibly pigmented under white light, except for a few that possibly had a very pale pink color, suggesting extremely limited betalain production. No cells with these phenotypes were observed in any control tissue; viz. bombarded tissue fed water or tissue bombarded with empty vector or the GFP reporter construct and fed DOPA (data not shown). In an attempt to enhance any weak phenotypes, the GFP-free experiment was repeated using the higher concentration of 10 mM DOPA (rather than 1 mM). Close examination of the sporadic blue light fluorescent foci revealed that some foci had more white-light visible pigmentation than had been observed in the previous experiment (Supplementary Figure S1), albeit it was still very weak. Compared to DOD, which gave abundant and multicellular betalain-pigmented foci that were readily apparent even under much lower magnification (Figure 4, Supplementary Figure S2), both DOD-like constructs gave very limited activity. The low frequency of the cells precluded biochemical analysis of the phenotype from DOD-like, but these results suggest that DOD-like has, at best, only a very limited capacity for a DOPA cleavage that would allow BA formation.


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)

Cell phenotypes in tissue bombarded with DOD-like constructs and fed DOPA. Tissue bombarded with constructs for PmDOD-like and PhybDOD-like. The GFP construct was not included. (A)A. majus petal tissue used. Single green fluorescent cells at low frequency were observed under blue light. A few of these cells were possibly pale pink under white light in the PmDOD-like bombarded sample. Hatched squares show regions depicted at higher magnification in the panels on the right side. Scale bar is the same size in all. (B) White onion scale tissue used. No pigments observed when bombarded with gold particles only (i); conspicuous multicellular yellow zones observed when bombarded with the PmDOD construct (positive control; ii); brown cells (iii) and a rare pale yellow cell (iv) was observed when bombarded with the PhybDOD-like construct. Scale bar = 500 μm (A), 25 μm (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Cell phenotypes in tissue bombarded with DOD-like constructs and fed DOPA. Tissue bombarded with constructs for PmDOD-like and PhybDOD-like. The GFP construct was not included. (A)A. majus petal tissue used. Single green fluorescent cells at low frequency were observed under blue light. A few of these cells were possibly pale pink under white light in the PmDOD-like bombarded sample. Hatched squares show regions depicted at higher magnification in the panels on the right side. Scale bar is the same size in all. (B) White onion scale tissue used. No pigments observed when bombarded with gold particles only (i); conspicuous multicellular yellow zones observed when bombarded with the PmDOD construct (positive control; ii); brown cells (iii) and a rare pale yellow cell (iv) was observed when bombarded with the PhybDOD-like construct. Scale bar = 500 μm (A), 25 μm (B).
Mentions: When the transient assay was repeated for DOD-like without including the GFP internal control, a phenotype was revealed under blue-light. Single cells with weak green autofluorescence were observed at low frequency (Figure 6A). These cells were generally not visibly pigmented under white light, except for a few that possibly had a very pale pink color, suggesting extremely limited betalain production. No cells with these phenotypes were observed in any control tissue; viz. bombarded tissue fed water or tissue bombarded with empty vector or the GFP reporter construct and fed DOPA (data not shown). In an attempt to enhance any weak phenotypes, the GFP-free experiment was repeated using the higher concentration of 10 mM DOPA (rather than 1 mM). Close examination of the sporadic blue light fluorescent foci revealed that some foci had more white-light visible pigmentation than had been observed in the previous experiment (Supplementary Figure S1), albeit it was still very weak. Compared to DOD, which gave abundant and multicellular betalain-pigmented foci that were readily apparent even under much lower magnification (Figure 4, Supplementary Figure S2), both DOD-like constructs gave very limited activity. The low frequency of the cells precluded biochemical analysis of the phenotype from DOD-like, but these results suggest that DOD-like has, at best, only a very limited capacity for a DOPA cleavage that would allow BA formation.

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