Molecular analysis of anthocyanin biosynthetic pathway genes in Cymbidium orchids : a thesis presentation in partial fulfilment of the requirements for the degree of Master of Science in Plant Molecular Biology at Massey University, Palmerston North, New Zealand

Abstract

Anthocyanin biosynthesis was examined in cymbidium orchid (Cymbidium Swartz). Cymbidium orchids lack true red and purple/blue flowers, because they accumulate cyanidin and peonidin (pink) anthocyanins, but not pelargonidin (red) or delphinidin (blue). Transient gene expression studies showed that the use of a heterologous flavonoid biosynthetic gene, dihydroflavonol reductase (DFR) from Anthurium, enhanced production of pelargonidin in cymbidium floral tissues. Similarly, delphinidin was produced when a pansy flavonoid 3', 5'-hydroxylase (F3'5'H) was introduced. The maize Lc/C1 transcription factors in combination with DFR or F3'5'H was necessary in order to observe upregulation of the anthocyanin pathway and changes in anthocyanin pigment accumulation. The presence of the new anthocyanins was confirmed by TLC and HPLC. cDNA clones of the flavonoid biosynthetic genes chalcone synthase (CHS), DFR and flavonoid 3' hydroxylase (F3'H) were isolated using PCR. The full length cymbidium CHS (1173 bp) was ~85% identical at DNA level with CHS from Oncidium 'Gower Ramsey', a Phalaenopsis hybrid cultivar, as well as bibenzyl synthase from Phalaenopsis sp. The 792 bp partial-length F3'H sequence was ~66% identical with F3'H from Pelargonidiumxhortorum, Verbena hybrida and Sorghum bicolor, while the DFR sequence was highly homologous with the published cymbidium DFR. The deduced protein sequences contained domains or conserved residues typical of CHS and F3'H. Southern analysis showed both cymbidium CHS and F3'H are represented by small gene families, with CHS consisting of at least three members and F3'H up to three genes. By contrast, DFR is likely to be presented as a single gene. Using different coloured cymbidium cultivars, it was shown that DFR expression correlated with cyanidin production in the flower. The CHS clone was most highly expressed in leaf tissues and in late developmental stages in floral tissues of Vanguard Mas Beauty (a green cultivar). This expression pattern did not correlate with pigment production, and hence this gene is unlikely to be involved in anthocyanin production in flowers. F3'H expression was not detected in leaf and floral tissues at any developmental stages examined. DFR and CHS promoters were isolated by genome walking, in an attempt to identify organ specific promoters suitable for use in cymbidium. A 1544 bp DFR promoter and a 1561 bp CHS promoter were cloned upstream of a GFP reporter gene and transient gene expression studies showed that CHS promoter had the ability to drive GFP production in white and pink petal tissues and in leaf tissues. However, the DFR promoter activated GFP expression only in the white petal tissues. These transient gene expression studies also demonstrated that maize Lc/C1 transcription factors greatly enhanced the activities of both CHS and DFR promoters. The success of this transient expression system indicates that MYB and bHLH transcription factor are likely to be involved in anthocyanin production in cymbidium. Together, the results of this study confirm that a suite of molecular strategies to modify flower colour in cymbidium are feasible, as well as providing essential information on flavonoid and anthocyanin genes that expand our knowledge and understanding of this complex flowering plant.