Terpene scent compounds biosynthesis in rose – ROSASCENT

Roses are widely used as garden ornamental plants, as cut flowers and potted flowers. Essential oil is produced for the perfume industries. One character that is being taken increasingly into account is flower scent, for which there is a clear demand from consumers worldwide. However, roses bred for cut flowers often lack scent. We discovered that rose flowers use an alternative route to produce scent, by employing an enzyme, RhNUDX1, of a completely unexpected family. In this ANR program, we propose to gain an in-depth understanding of this new biosynthetic pathway. Using a combination of chemical, biochemical, molecular and genetic analyses, we want to discover all the proteins playing a part in this new pathway, from the biosynthesis of geranyl diphosphate to the geraniol derivatives. We want to characterize in detail the functions of these proteins in scent biosynthesis.
We will investigate:

1. The precise functions of RhNUDX1-1 and RhNUDX1-2
RhNUDX1-1 is involved in the production of geraniol, which is a monoterpene. We recently isolated another rose NUDX1 gene, RhNUDX1-2. We will functionally characterize this second gene, using enzymatic assays and correlation analyses of its expression and the presence of scent compounds in rose cultivars and species. We will look for correlations between NudX1 alleles and presence of terpenes in F1 progeny and old cultivated rose collection. A search for key amino acids, involved in this special function will be made for in RhNUDX1-1 and RhNUDX1-2, by comparison with the Arabidopsis protein, which is not involved in scent production. We will also sequence RhNUDX1-1 in a rose collection of the 19th century to look for a trace of signature of selection. Finally, sequencing of RhNUDX1-1 and RhNUDX1-2 in different rose species, and analysis of expression in such roses will allow us to precise if this special function has appeared recently in the Rosa genus during domestication or if it is present in all the Rosa species and maybe in other Rosaceae.

2. The regulation of RhNUDX1-1
One key feature of RhNUDX1-1 is its very high expression in cultivars emitting scent and its barely detectable expression in non-scented cultivars. The promoters responsible for this petal specific expression will be isolated and dissected. Transcription factors, putatively involved in this upregulation in petals will be searched for by comparing the 2 types of roses (with and without terpenes). QTL of expression (eQTL) will be also performed to identify trans factors controlling NUDX1 expression.

3. The partners of RhNUDX1-1
RhNUDX1-1 is responsible for the first step leading from geranyl diphosphate (GPP) to geranyl monophosphate. The enzyme responsible for the second step, from geranyl monophosphate to the volatile scented compound geraniol is unknown, although it can be hypothesized that it is a phosphatase. A search for phosphatases overexpressed in petals of cultivars producing terpenes will be made. We will also characterize the prenyltransferases responsible for the production of GPP. It will be particularly interesting to know the localization of such proteins, as geranyl diphosphate synthases are generally localized in plastids and RhNUDX1-1 is in the cytosol. We will also try to figure out which pathway, the plastidial “Methylerythritol phosphate pathway” or the cytosolic “Mevalonate pathway” is providing isopentenyl diphosphate and dimethylallyl diphosphate for the biosynthesis of GPP. Finally, transcriptome comparisons will also be used to isolate candidate reductases, involved in the reduction of geraniol into citronellol, another valuable monoterpene.

The proposed program may explain for the first time why some rose cultivars lack the typical rose scent. The sequences characterized during the program could be used as markers for the selection of fragrant roses. It could also open up all sorts of possibilities for production of terpenes of high value for cosmetic and fragrance industries.