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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

Differential activity of BvDODA and BvDODA1 in a transient expression assay. White petal tissue of A. majus was bombarded with constructs for the genes (driven by the 35S promoter) followed by supplementation with DOPA or water. Representative petal samples are shown. Pigmentation was only visible in BvDODA1 bombarded tissue that was also fed DOPA. The pigmented regions also had fluorescence in blue light characteristic of betaxanthins. The only phenotype observed in BvDODA-bombarded tissue was a small number of cells in the DOPA-fed samples with weak green fluorescence in blue light. Control tissue bombarded with both BvDODA and GFP constructs and fed DOPA lacked pigment formation while GFP fluorescence indicated transformation had occurred. Scale bar = 1 mm. Hashed squares show regions depicted at higher magnification in panels with 200 μm scale bars.
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Figure 9: Differential activity of BvDODA and BvDODA1 in a transient expression assay. White petal tissue of A. majus was bombarded with constructs for the genes (driven by the 35S promoter) followed by supplementation with DOPA or water. Representative petal samples are shown. Pigmentation was only visible in BvDODA1 bombarded tissue that was also fed DOPA. The pigmented regions also had fluorescence in blue light characteristic of betaxanthins. The only phenotype observed in BvDODA-bombarded tissue was a small number of cells in the DOPA-fed samples with weak green fluorescence in blue light. Control tissue bombarded with both BvDODA and GFP constructs and fed DOPA lacked pigment formation while GFP fluorescence indicated transformation had occurred. Scale bar = 1 mm. Hashed squares show regions depicted at higher magnification in panels with 200 μm scale bars.

Mentions: Given the differential biological activities and phylogenetic separation of PmDOD and PmDOD-like, the biological activity of Amaranthaceae genes with sequence similarity to DOD or DOD-like was examined. The characterized sequences from B. vulgaris (BvDODA and BvDODA1) were used along with a Class II homolog isolated from green leaf tissue of Ptilotus (PhybDOD-like), another Amaranthaceae species. Based on the amino acid sequences, BvDODA is closer to PhybDOD-like (80.2% identity) than it is to BvDODA1 (70.1% identity). Outside of the Amaranthaceae (Table 2), BvDODA is also more similar to PmDOD-like than PmDOD (or BvDODA1). The differences in % identities when examining BvDODA1 and PmDOD are less conclusive (Table 2), but in general they suggest that BvDODA, PhybDOD-like, and PmDOD-like may have a divergent enzymatic activity to that of BvDODA1 and PmDOD. Previous analysis has suggested BvDODA1 is the key LigB gene for betalain biosynthesis in B. vulgaris (Hatlestad et al., 2012), although BvDODA is also expressed in colored tissues (Hatlestad et al., 2012) and recombinant protein has some DOPA 4,5-cleavage activity (Sasaki et al., 2009; Gandía-Herrero and García-Carmona, 2012). The transient gene expression assay was used with constructs driven by the 35S promoter. Pigment production upon supplementation with DOPA was observed only in 35S:BvDODA1-bombarded tissue and not when 35S:BvDODA or 35S:PhybDOD-like was used (Figure 9). However, as seen with PmDOD-like (Figure 4), tissue bombarded with these other constructs had a low frequency of single cells with pale green autofluorescence under blue-light, but no visible pigmentation under white light (Figure 9). The transient expression assay was repeated for PhybDOD-like using Allium cepa (onion) epidermal cells, with PmDOD as a positive control. After DOPA feeding, yellow multicellular zones developed in PmDOD bombarded tissue, while sporadic brown cells and a single pale yellow cell developed in tissue bombarded with PhybDOD-like (Figure 6B). This demonstrated that the differential activity was not contingent on the host tissue used in the assay. These results provide further evidence of Class II LigB proteins in betalain producing taxa that are functionally different to DOD. These DOD-like enzymes, however, may conditionally exhibit limited DOPA cleavage activity that leads to BA.


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)

Differential activity of BvDODA and BvDODA1 in a transient expression assay. White petal tissue of A. majus was bombarded with constructs for the genes (driven by the 35S promoter) followed by supplementation with DOPA or water. Representative petal samples are shown. Pigmentation was only visible in BvDODA1 bombarded tissue that was also fed DOPA. The pigmented regions also had fluorescence in blue light characteristic of betaxanthins. The only phenotype observed in BvDODA-bombarded tissue was a small number of cells in the DOPA-fed samples with weak green fluorescence in blue light. Control tissue bombarded with both BvDODA and GFP constructs and fed DOPA lacked pigment formation while GFP fluorescence indicated transformation had occurred. Scale bar = 1 mm. Hashed squares show regions depicted at higher magnification in panels with 200 μm scale bars.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Differential activity of BvDODA and BvDODA1 in a transient expression assay. White petal tissue of A. majus was bombarded with constructs for the genes (driven by the 35S promoter) followed by supplementation with DOPA or water. Representative petal samples are shown. Pigmentation was only visible in BvDODA1 bombarded tissue that was also fed DOPA. The pigmented regions also had fluorescence in blue light characteristic of betaxanthins. The only phenotype observed in BvDODA-bombarded tissue was a small number of cells in the DOPA-fed samples with weak green fluorescence in blue light. Control tissue bombarded with both BvDODA and GFP constructs and fed DOPA lacked pigment formation while GFP fluorescence indicated transformation had occurred. Scale bar = 1 mm. Hashed squares show regions depicted at higher magnification in panels with 200 μm scale bars.
Mentions: Given the differential biological activities and phylogenetic separation of PmDOD and PmDOD-like, the biological activity of Amaranthaceae genes with sequence similarity to DOD or DOD-like was examined. The characterized sequences from B. vulgaris (BvDODA and BvDODA1) were used along with a Class II homolog isolated from green leaf tissue of Ptilotus (PhybDOD-like), another Amaranthaceae species. Based on the amino acid sequences, BvDODA is closer to PhybDOD-like (80.2% identity) than it is to BvDODA1 (70.1% identity). Outside of the Amaranthaceae (Table 2), BvDODA is also more similar to PmDOD-like than PmDOD (or BvDODA1). The differences in % identities when examining BvDODA1 and PmDOD are less conclusive (Table 2), but in general they suggest that BvDODA, PhybDOD-like, and PmDOD-like may have a divergent enzymatic activity to that of BvDODA1 and PmDOD. Previous analysis has suggested BvDODA1 is the key LigB gene for betalain biosynthesis in B. vulgaris (Hatlestad et al., 2012), although BvDODA is also expressed in colored tissues (Hatlestad et al., 2012) and recombinant protein has some DOPA 4,5-cleavage activity (Sasaki et al., 2009; Gandía-Herrero and García-Carmona, 2012). The transient gene expression assay was used with constructs driven by the 35S promoter. Pigment production upon supplementation with DOPA was observed only in 35S:BvDODA1-bombarded tissue and not when 35S:BvDODA or 35S:PhybDOD-like was used (Figure 9). However, as seen with PmDOD-like (Figure 4), tissue bombarded with these other constructs had a low frequency of single cells with pale green autofluorescence under blue-light, but no visible pigmentation under white light (Figure 9). The transient expression assay was repeated for PhybDOD-like using Allium cepa (onion) epidermal cells, with PmDOD as a positive control. After DOPA feeding, yellow multicellular zones developed in PmDOD bombarded tissue, while sporadic brown cells and a single pale yellow cell developed in tissue bombarded with PhybDOD-like (Figure 6B). This demonstrated that the differential activity was not contingent on the host tissue used in the assay. These results provide further evidence of Class II LigB proteins in betalain producing taxa that are functionally different to DOD. These DOD-like enzymes, however, may conditionally exhibit limited DOPA cleavage activity that leads to BA.

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