1999 MPIZ Report

Research area of the group of Bernd Weisshaar at MPIZ Koeln

Functional analysis of transcription factors mediating the formation of flavonols in Arabidopsis thaliana

Plants are exposed to sun light which contains significantamounts of potentially harmful high-energy photons. A potentialprotective mechanism against this deleterious radiation is theaccumulation of pigments, and it has long been proposed that flavonoidsare such pigments.

Flavonoids, and more specifically flavonol glycosides, absorbUV-B light. They accumulate after an inductive light treatmentin their glycosylated form in the vacuoles of epidermal cells(Weisshaar and Jenkins, 1998). Four 'structural' genes are required forthe formation of the C-15 basic flavonol aglycon from centralbiosynthesis intermediates. 4-coumaroyl-CoA is the main substrateof the first enzyme specific for flavonoid biosynthesis, chalconesynthase (CHS). Chalcone-flavanone isomerase (CFI) and flavanone3-hydroxylase (F3H) are required for synthesis of 3-hydroxy-flavanone,which serves as a substrate for either dihydroflavonol reductase(DFR) or flavonol synthase (FLS). DFR reduces the heterocyclicC-ring of dihydroflavonols and leads to the formation of pigmentsabsorbing in the visible range of the spectrum, such as anthocyanins,phlobaphenes, or condensed tannins. In contrast, FLS oxidisesthe C-ring and results in the formation of colourless flavonolswhich predominantly absorb UV-light. UV and blue light stimulatethe transcription of the CHS, CFI, F3H, andFLS genes, but they are also activated at certain developmentalstages and in specific tissues. The various signals are integratedand transmitted to the nucleus, and the set of genes requiredfor accumulation of the respective flavonol(s) must become activein a coordinated way. Our studies concentrated on the coordinatedactivation of a number of genes, but include also experimentsto analyse the biochemisty and physiology of flavonoid biosynthesis.

Initial experiments to study light-dependent CHS gene activationwere performed with parsley (Petroselinum crispum). A light-regulatoryunit 1 (LRU1PcCHS) was defined which is sufficient to mediatelight-induced CHS gene expression and consists of at leasttwo distinct cis-acting elements, ACEPcCHS and MREPcCHS(ACE: ACGT-containing element; MRE: MYB recognition element).A number of factors of the bZIP and MYB type were isolated which,respectively, recognise these elements (Feldbr├╝ggeet al., 1997; Kircher et al., 1998). From the biochemicaldata collected with parsley plants and cultured parsley cellsit became clear that genetic tools will be needed to further unravelthe mechanisms controling flavonol accumulation in plants. Therefore,work with Arabidopsis thaliana was initiated.

In parallel to using genetic and functional genomics approachesto find the transcription factor(s) co-regulating flavonol biosynthesisgenes, we also analysed transparent testa (tt) mutantsof A. thaliana. While A. thaliana wildtype plantsproduce dark brown seeds, tt mutant seeds show a yellowto pale brown color. This is due to the absence of condensed tannins(flavonoid-derived brown pigments) from the seed coat. The TTloci include structural genes required for flavonoid biosynthesis.However, for some of the so far unkown TT genes (TT1,TT2, TT8) there are indications for a regulatoryfunction.

(text from the MPIZ research report for 1999 - still valid)