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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.


Sequence alignment of catalytic domain and adjacent regions of plant LigB homologs. The sequences are shown divided into Classes I and II. The catalytic domain is boxed and the asterisks mark the acidic residues that may be important for DOD activity/DOPA recognition. For the Class II genes that have DOD in their name (aside from this study) the gene names used are those in the GenBank annotations, which have generally been based only on sequence homology to DOD. 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. Accession numbers are given in Supplementary Figure S4.
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Figure 7: Sequence alignment of catalytic domain and adjacent regions of plant LigB homologs. The sequences are shown divided into Classes I and II. The catalytic domain is boxed and the asterisks mark the acidic residues that may be important for DOD activity/DOPA recognition. For the Class II genes that have DOD in their name (aside from this study) the gene names used are those in the GenBank annotations, which have generally been based only on sequence homology to DOD. 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. Accession numbers are given in Supplementary Figure S4.

Mentions: Given the differences in gene expression and protein function of the DOD and DOD-like cDNAs, the LigB gene family was examined in more detail for P. mirabilis and other Caryophyllales species. Two transcriptome databases for P. mirabilis, formed using 454 Next Generation Sequencing for RNA from either the violet (22,665 transcript contigs) or yellow (21,189 contigs) regions of the petal, were interrogated for sequences with similarity to LigB sequences. In addition to the previously identified DOD and DOD-like sequences, a third candidate LigB sequence was identified (named PmLigB) that contained the H-N-L-R motif characteristic of Class I LigB proteins (Christinet et al., 2004), Figure 7, and Supplementary Figure S3). This had 59.6 and 67.4% deduced amino acid identity to PmDOD and PmDOD-like, respectively. Given the sequence divergence this suggests that there are at least three functionally distinct LigB genes in P. mirabilis.


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)

Sequence alignment of catalytic domain and adjacent regions of plant LigB homologs. The sequences are shown divided into Classes I and II. The catalytic domain is boxed and the asterisks mark the acidic residues that may be important for DOD activity/DOPA recognition. For the Class II genes that have DOD in their name (aside from this study) the gene names used are those in the GenBank annotations, which have generally been based only on sequence homology to DOD. 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. Accession numbers are given in Supplementary Figure S4.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Sequence alignment of catalytic domain and adjacent regions of plant LigB homologs. The sequences are shown divided into Classes I and II. The catalytic domain is boxed and the asterisks mark the acidic residues that may be important for DOD activity/DOPA recognition. For the Class II genes that have DOD in their name (aside from this study) the gene names used are those in the GenBank annotations, which have generally been based only on sequence homology to DOD. 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. Accession numbers are given in Supplementary Figure S4.
Mentions: Given the differences in gene expression and protein function of the DOD and DOD-like cDNAs, the LigB gene family was examined in more detail for P. mirabilis and other Caryophyllales species. Two transcriptome databases for P. mirabilis, formed using 454 Next Generation Sequencing for RNA from either the violet (22,665 transcript contigs) or yellow (21,189 contigs) regions of the petal, were interrogated for sequences with similarity to LigB sequences. In addition to the previously identified DOD and DOD-like sequences, a third candidate LigB sequence was identified (named PmLigB) that contained the H-N-L-R motif characteristic of Class I LigB proteins (Christinet et al., 2004), Figure 7, and Supplementary Figure S3). This had 59.6 and 67.4% deduced amino acid identity to PmDOD and PmDOD-like, respectively. Given the sequence divergence this suggests that there are at least three functionally distinct LigB genes in P. mirabilis.

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.