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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

Maximum likelihood analysis of LigB genes. Analysis used the nucleotide sequences of the putative ORFs. The Amaranthaceae species names are in red, species of other betalain producing Caryophyllales families in blue, an anthocyanin taxa within the Caryophyllales (Dianthus) in green, and the non-Caryophyllales species in black. Sequences for McDOD1 and McDOD2 are not full-length but span the catalytic domain. Bootstrap analysis used 1000 datasets, with results shown for nodes that have at least 70% support. The branch-lengths indicate the average number of substitutions per site, with the scale bar given at the bottom of the figure. See Supplementary Figure S4 for the accession numbers. The Class I grouping is indicated. For the Class II sequences, the published DOD of Portulaca grandiflora is indicated along with the activities of sequences assayed in this study.
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Figure 8: Maximum likelihood analysis of LigB genes. Analysis used the nucleotide sequences of the putative ORFs. The Amaranthaceae species names are in red, species of other betalain producing Caryophyllales families in blue, an anthocyanin taxa within the Caryophyllales (Dianthus) in green, and the non-Caryophyllales species in black. Sequences for McDOD1 and McDOD2 are not full-length but span the catalytic domain. Bootstrap analysis used 1000 datasets, with results shown for nodes that have at least 70% support. The branch-lengths indicate the average number of substitutions per site, with the scale bar given at the bottom of the figure. See Supplementary Figure S4 for the accession numbers. The Class I grouping is indicated. For the Class II sequences, the published DOD of Portulaca grandiflora is indicated along with the activities of sequences assayed in this study.

Mentions: The relationships within the LigB family were examined by forming phylogenetic trees on the DNA sequences (Figure 8). The resultant tree shows a clear separation of Class I LigB sequences away from the additional LigB sequences found in betalain-producing species, with close to 100% bootstrap support. The additional LigB sequences form distinct clades consisting of those from the Amaranthaceae and those from the other betalain families examined, again with bootstrap support close to 100%. Within the non-Amaranthaceae clade, PmDOD and PmDOD-like are in separate groups with high bootstrap support, supporting the transient assay results that they have different biological activities. Within the Amaranthaceae species, the Beta vulgaris DODA1 separates from BvDODA, with BvDODA being close to PhybDOD-like (of Ptilotus), suggesting they too may have distinct activities. Trees formed on the amino acid sequences, with or without outgroup rooting, gave a very similar outcome to that from the DNA sequence (Supplementary Figure S4).


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)

Maximum likelihood analysis of LigB genes. Analysis used the nucleotide sequences of the putative ORFs. The Amaranthaceae species names are in red, species of other betalain producing Caryophyllales families in blue, an anthocyanin taxa within the Caryophyllales (Dianthus) in green, and the non-Caryophyllales species in black. Sequences for McDOD1 and McDOD2 are not full-length but span the catalytic domain. Bootstrap analysis used 1000 datasets, with results shown for nodes that have at least 70% support. The branch-lengths indicate the average number of substitutions per site, with the scale bar given at the bottom of the figure. See Supplementary Figure S4 for the accession numbers. The Class I grouping is indicated. For the Class II sequences, the published DOD of Portulaca grandiflora is indicated along with the activities of sequences assayed in this study.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Maximum likelihood analysis of LigB genes. Analysis used the nucleotide sequences of the putative ORFs. The Amaranthaceae species names are in red, species of other betalain producing Caryophyllales families in blue, an anthocyanin taxa within the Caryophyllales (Dianthus) in green, and the non-Caryophyllales species in black. Sequences for McDOD1 and McDOD2 are not full-length but span the catalytic domain. Bootstrap analysis used 1000 datasets, with results shown for nodes that have at least 70% support. The branch-lengths indicate the average number of substitutions per site, with the scale bar given at the bottom of the figure. See Supplementary Figure S4 for the accession numbers. The Class I grouping is indicated. For the Class II sequences, the published DOD of Portulaca grandiflora is indicated along with the activities of sequences assayed in this study.
Mentions: The relationships within the LigB family were examined by forming phylogenetic trees on the DNA sequences (Figure 8). The resultant tree shows a clear separation of Class I LigB sequences away from the additional LigB sequences found in betalain-producing species, with close to 100% bootstrap support. The additional LigB sequences form distinct clades consisting of those from the Amaranthaceae and those from the other betalain families examined, again with bootstrap support close to 100%. Within the non-Amaranthaceae clade, PmDOD and PmDOD-like are in separate groups with high bootstrap support, supporting the transient assay results that they have different biological activities. Within the Amaranthaceae species, the Beta vulgaris DODA1 separates from BvDODA, with BvDODA being close to PhybDOD-like (of Ptilotus), suggesting they too may have distinct activities. Trees formed on the amino acid sequences, with or without outgroup rooting, gave a very similar outcome to that from the DNA sequence (Supplementary Figure S4).

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