The localisation, intracellular transport, and biosynthetic regulation of betalain plant pigments
This thesis investigates the localisation, transport and biosynthetic control of betalain plant pigments to compare with the extensively researched anthocyanins. Anthocyanins and betalains appear similar, yet no plant naturally contains both pigment types. Due to this mutual exclusivity, betalain pigments are thought to functionally replace anthocyanins in many Caryophyllales. However, minimal research has been conducted to support this replacement hypothesis, resulting in limited knowledge of betalain pigment distribution and biosynthesis. The following series of experiments have added to this body of knowledge. Localisation of betalains was compared with that reported for anthocyanins. Histological analyses of 12 different betalain-producing species revealed similar pigment localisation to that of anthocyanic species. Similarities in pigment localisation suggest that these pigment types may have similar functional roles. The histological analyses also found that betacyanins and betaxanthins had differential localisation in several taxa. Organ- or tissue-specific distribution of betalain compounds suggests differing biological functions for betaxanthins and betacyanins. Hypotheses on betalain transport were tested using transgenic Arabidopsis thaliana lines capable of producing anthocyanins (PAP1-5), betalains (DOD-6), or both (DOD-6 x PAP1-5). Betaxanthins appeared to use vesicular transport, as betaxanthins were detected in small circular bodies within the cytoplasm. Furthermore, this observation suggests that betaxanthin formation occurred outside of the vacuole. DOD-6 was also crossed with Arabidopsis mutants, tt12 and tt19, which are deficient in proteins required for flavonoid vacuolar transport. Betaxanthin accumulation was reduced in both lines. In addition, DOD-6 was treated with transport inhibitors that affect anthocyanin accumulation. These experiments demonstrated that betaxanthins can utilise known flavonoid transport mechanisms, at least in this artificial pigment system. Regulation of betalain biosynthesis was analysed using Swiss chard (Beta vulgaris subsp. cicla cv. ‘Bright Lights’). Betalain production was induced through physical wounding of the lamina in red and white Swiss chard lines. Betalain pigments were produced around the wounding sites in the red line but not in the white line. Transcript level changes of betalain and flavonoid biosynthetic genes in these tissues were measured using real-time quantitative polymerase chain reaction analyses. Betalain biosynthetic genes were not up-regulated in the red line even though red pigments visibly accumulated. Rather, these genes were already expressed in the red line prior to wounding. Biosynthetic control of betalains may either be earlier in the pathway or at the post-transcriptional level. In contrast, all three flavonoid biosynthetic genes were up-regulated in response to wounding, indicating that expression of flavonoid and betalain biosynthetic genes are not co-regulated in Swiss chard. The final set of experiments examined the function of the two Beta vulgaris DOD genes (DODA and DODA1). Both genes were transiently expressed in nivea Antirrhinum majus dorsal petals and vacuum infiltrated with the betalain precursor L-DOPA. Expression of DODA1 but not DODA appeared to produce betalains. DODA-like genes have been found in anthocyanin-producing species, suggesting that this gene may not be involved in betalain biosynthesis. The overall findings from this research indicate that betalain pigment evolution of may have involved the utilization of anthocyanin transport machinery, but the regulatory control of the two pathways appears different.