Light-induced anthocyanin pigmentation in transgenic Lc petunia : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

Introduction of Leaf colour (Lc). a bHLH transcription factor from maize, under the control of the CaMV35S promoter into petunia (cv. Mitchell) plants resulted in enhanced anthocyanin pigmentation in vegetative tissues. Anthocyanin biosynthesis was observed to be dependent on the level of light the plants were grown under: plants grown in a plastic greenhouse remained green, while plants exposed to high-light were dark purple. The nature of this response to light and the associated molecular mechanisms were the focus of this investigation. Molecular analysis of gene expression in Mitchell petunia showed that light induced the expression of the early flavonoid structural genes, as well as flavonol synthase (FLS) required for flavonol production. Light induced both the early and late structural genes required for anthocyanin biosynthesis in the transgenic Lc Mitchell petunia plants, but reduced the expression of FLS. Light-induced flavonoid gene expression was examined under three light treatments: shade (50 - 350 µmol m -2 sec -1 ); ambient-greenhouse (300 - 750 µmol m -2 sec -1 ) and high-light (750 µmol m -2 sec -1 ). The level of flavonoid gene expression was dependent upon light intensity. High-light was required to maximally activate anthocyanin pigmentation in Lc petunia. Expression of the Lc transgene remained unchanged irrespective of light intensity, indicating that the light-induced changes in anthocyanin synthesis were not due to variable expression of the transgene. Anthocyanin regulation occurs primarily at the transcriptional level, and two classes of transcription factors. Myb and bHLH. are generally involved. Transient expression studies using several exogenous Myb transcription were carried out using shade-grown (non-induced) Lc petunia material. The induction of coloured cells in the treated tissue supports the idea that the bHLH transgene (LC) is interacting with an endogenous Myb under high-light conditions, resulting in the activation of the flavonoid biosynthetic pathway and accumulation of anthocyanin pigments. A partial sequence of a candidate endogenous Myb transcription factor from petunia was cloned. It was light-induced and shares structural features with other anthocyanin-regulating Myb transcription factors, particularly An2 from petunia. This Myb in combination with LC may be responsible for the light-induced anthocyanin pigmentation observed in Lc petunia