ANTHOCYANIN BIOSYNTHESIS IN DISTINCT GENOTYPES OF CHINA ASTER (CALLISTEPHUS CHINENSIS)

Flower colour in China aster is determined by accumulation of chalcones (yellow), flavones and flavonols (cream) and anthocyanins (red to blue). Several genes (A, Ch, F, G, M and R) are involved in biosynthesis of these pigments (Fig. 1) [1]. Enzymatic studies revealed that the genes Ch, F, R and M control the activities of the enzymes chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5-glucosyltransferase (A5GT), respectively [2, 3, 4, 5]. The genes A and G could not be correlated with specific enzymes but most probably concern late steps in the anthocyanidin pathway. Mutants concerning chalcone synthase (CHS), flavanone 3-hydroxylase (FHT), flavone synthase II (FNSII) and flavonol synthase (FLS) have not yet been described.

A flower-specific cDNA library was constructed using the wildtype line 06. Using heterologous probes, full-length clones of CHS, FHT, DFR and anthocyanidin synthase (ANS) as well as an almost full-length clone of CHI were isolated and characterized (Tab. 1). The ANS cDNA clone was found to contain a 208 bp intron.

The cDNA clones were used for further characterization of the wildtype line 06 and of the genetically stable anthocyaninless mutant lines 07, 08 and 10h. Southern blot analyses revealed that the genes encoding CHI, FHT, DFR, and ANS are single copy genes. In confirmation of the enzymatic studies, northern blot analyses revealed that the yellow coloured mutant lines 07 and 10h are CHI-deficient, and line 10h is additionally DFR-deficient (Fig. 2). Lines 07 and 08 with recessive alleles of gene A were found to be ANS-deficient (Fig. 3), confirming our hypothesis that a late step of the pathway is controlled by this gene. The gene G seems to exert a regulatory effect, at least on DFR. Thus, in line 08 with dominant F-alleles but recessive alleles of gene G, DFR gene transcription was found to be extensively reduced (Fig. 4). Tab. 2 summarises the genotypes and phenotypes (flower colours) of the examined lines.

Further investigations will be concerned with the enzymatic and molecular characterization of an unstable mutation leading to deep blue or red stripes on a lavender or orange background. Instability might be due to the presence of transposable elements.