Functional and complementation analysis of hop genes in heterologous systems

Several hop genes involved in the phenylpropanoid pathway responsible for the production of prenylated chalcones (e.g. xanthohumol) with significant bioactivity and anticancerogenic properties have been identified. These include genes encoding for the crucial enzymes of this pathway, like chalcone synthases (CHS_H1) or o-methyltransferase (OMT) and putative genes encoding for transcription factors (TFs) regulating their expression. The regulators act in ternary MBW complexes (s-HlMYB3 or HlMyb2/HlbHLH2/HlWDR1) as well as binary (s-HlMYB3/HlbHLH2; HlMYB2/HlbHLH2; HlbHLH2/HlWDR1) interactions. To investigate the function of these TFs in vivo, we cloned the appropriate genes into Agrobacterium tumefaciens vectors under control of constitutive promoter 35S to reach their stable overexpression in plants. Because the hop transformation is labor intensive and time consuming, we performed such analysis firstly on heterologous systems Arabidopsis thaliana, Nicotiana sp. and Petunia hybrid. Overexpression of the hop TF genes showed that they are able to affect plant morphology (i.e. enhanced branching for 35S:HlMyb3 in Arabidopsis, pleiotropic effects for 35S:HlWDR1 regardless the plant recipient and increased level of anthocyanins in tobacco flowers overexpressing HlbHLH2). To get more knowledge about the hop TFs function, we used known Arabidopsis mutants of genes involved in similar pathways as presumed for hop genes to perform complementation study. The results showed that hop TFs genes are able to regulate the flavonoid and/or anthocyanin biosynthesis pathway. For instance, HlWDR1 gene complemented ttg1 mutant lacking trichomes and anthocyanins in the seed coats. HlbHLH2 gene complemented tt8 mutant, member of TTG1/TT8/GL2 complex, analogical to hop MBW complex. gl1 mutant was complemented by HlMyb1 gene, which suggests involvement in trichome formation regulated in analogous manner as flavonoid pathway in Arabidopsis.

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We acknowledge the use of research infrastructure that has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 316304.