Betalains are a unique group of tyrosine-derived, vacuolar pigments found in the plant order Caryophyllales. Betalains exhibit yellow-orange (betaxanthins) and red-purple (betacyanins) colours. Betalamic acid, the chromophore of betaxanthins and betacyanins, is synthesized by DOPA 4,5-dioxygenase encoded by the DOD gene. Various amino acids or amines react with betalamic acid to form the betaxanthins. Cyclo-DOPA, formed through DOPA oxidation, reacts with betalamic acid to form betacyanins. The aim of this study was to better understand betalain synthesis in flowers and leaf tissues using the native Australian plant, Ptilotus. The project consisted of the following five experimental components: (1) cloning partial cDNAs of DOD homologues from flowers of different coloured Ptilotus cultivars; (2) correlating betalain content, DOD gene expression and DOPA oxidase activity from different flower development stages to better understand betalain synthesis in Ptilotus flowers; (3) characterising the regulation of betalain synthesis in vegetative tissues in response to the abiotic stressors; (4) correlating DOD gene expression and DOPA oxidase activity with stress induction of betalains in Ptilotus leaves; and, (5) cloning the full-length Ptilotus DOD sequence and conducting transient expression assays to test the functionality of the cloned Ptilotus DOD gene.
Betalain pigmentation, DOD gene expression and DOPA oxidase activity were characterised at five serial flower development stages for Ptilotus cvs. Passion, Purity, Little Princess and Joey. Whilst there was a partial correlation between betacyanin content and DOD expression in pink flowered cultivars, there was no correlation between betaxanthin content and DOD gene expression in the cream flowered cultivar, Purity. There was no correlation between DOPA oxidase activity and betalain content for any cultivar studied. In vitro DOPA feeding of excised florets rapidly produced betaxanthin pigments suggesting that the betalain synthesis in Ptilotus flowers may be controlled at the first step in the pathway [i.e. the putative tyrosine hydroxylase (TH)] and that there is endogenous DOD activity in flowers of all the cultivars tested. There was no correlation between DOPA oxidase activity and betalain content. It is possible that the assay for DOPA oxidase may not be specific to the enzyme involved in betalain synthesis. This possibility is consistent with a recent discovery of a cytochrome P450 enzyme involved in the oxidation of DOPA in the betalain pathway of Beta vulgaris (Hatlestad et al., 2012).
Ptilotus cv. Little Princess plants were subjected to either cool or warm temperature regimes and to UVC stress. Betacyanin content increased in response to cool (22/12 °C) temperature stress without the UVC irradiation. With the addition of UVC (2.7, 4.0 and 5.3 kJ m-2), betacyanin content increased by ~ 2 fold. A similar phenomenon has been observed from anthocyanin pigment research in response to cool or UV radiation. Extrapolating, it is plausible that betalains may have a physiological role in protecting the plants against harmful active oxygen species, such as hydrogen peroxide (H2O2) or singlet oxygen (O2). DOD gene expression and DOPA oxidase activity showed no correlation with betalain content. Alternatively, it is possible that another DOD homologue is involved in betalain synthesis, similar to that recently found in B. vulgaris (Hatlestad et al., 2012). The time-course analysis of DOD expression showed a maximum expression at 8 h post cool-stress treatment, this being ca. 7.5-fold higher compared to the control treatment. Similarly, there was a ca. 7.5-fold increase in DOD expression 2 h after commencing the cool temperature regime and with UVC irradiation (5.3 kJ m-2).
Protein sequence of DOD from green leaf tissue of Ptilotus cv. Little Princess revealed amino acid polymorphisms at the fourth and fifth positions of the conserved H-P-(S/A)-(N/D)-x-T-P catalytic motif when compared to the DOD homologues of other betalain containing plants. Ptilotus DOD had amino acids Asn and Gly substituting Asp or Glu, which are normally observed at the fourth and fifth positions of the functional DOD’s. To test the functionality of the Ptilotus DOD, transient expression was performed on white onion tissues with DOPA substrate feeding. The biolistic experiment revealed multi-cell brown coloured foci and a single pale yellow coloured cell, in contrast to the expected yellow multi-cell foci. It is possible that the brown cells may be a byproduct of betaxanthin degradation whereas the pale yellow colour might represent formation of an unknown intermediate compound instead of betalamic acid. These observations suggest that while the Ptilotus DOD has DOPA cleavage activity, but it may not necessarily be involved in betalain synthesis. Further examination of the brown and pale yellow phenotype through HPLC is suggested.
In short, this thesis makes an important contribution to new knowledge on the regulation of betalain synthesis in flowers and stressed vegetative tissues using Ptilotus as a novel plant model. The findings demonstrate that DOD and DOPA oxidase may not be the key players of the betalain biosynthesis pathway and suggest involvement of a putative TH gene in the first step of the pathway.