Poster: Secondary Metabolism

Abs # 293: A new dioxygenase involved in plant betalain biosynthesis

Presenter:	Zryd, Jean-Pierre H, jzryd@ie-pc.unil.ch
Authors	Zryd, Jean-Pierre H (A)   Christinet, Laurent  (A)   Zaiko, Maia  (A)
Affiliations:	(A): University of Lausanne - Institute of Ecology
Web Site:	http://www.unil.ch/lpc

Betalains are violet and yellow chromo-alkaloid pigments specific of plants from the order Caryophyllales and also present in fungi (Amanita and Hygrocybe). We already identified the dodA gene coding for a specific DOPA-dioxygenase from Amanita muscaria (AmDOD), but numerous attempts to identify a homolog in plants failed. In order to isolate specific plant genes, subtractive cDNA libraries were built with total RNA from immature petals of white, yellow and respectively violet inbred lines of Portulaca grandiflora (Pg). Specific clones were first detected by northern blot analysis using RNA from petals of the white and colored genotypes. Positive candidates were submitted to further transcription analysis in colored, green stems and leaves in order to assess their specific expression. A full-length cDNA, possessing a low level of homology with a LigB domain (pfam02900) from a bacterial dioxygenase (Sphingomonas), was identified in silico as an extradiol dioxygenase, due to the strict conservation of the catalytic and iron-binding sites amino acids. Transient over-expression of the Pgdod gene after biolistic transformation of Pg white petals from homozygous recessive plants produced violet or yellow spots related to the local accumulation of betalains. HPLC analysis of these spots confirmed the complementation of the recessive allele by the Pgdod gene. Southern blot analysis showed that this gene is a single copy-gene in Pg. PgDOD protein has no sequence similarity with the AmDOD protein, which also complement Pg white petals. We will discuss the evolution of this new gene family in angiosperms.