Successful protoplast transfection in Cichorium and the search for bitterness related genes

Cichorium intybus var. sativum (chicory) and var. foliosum (witloof) are economically important crops with a high nutritional value. Many specialized metabolites have been isolated, such as polyphenols and terpenoids, with beneficial effects on human health. Cichorium plants are rich in sesquiterpene lactones (SL) which are responsible for a bitter taste, limiting the use of these crops for industrial purposes. Editing specific genes from the SL pathway in these Cichorium species would lead to changes in the SL metabolite pathway and thus result in altered bitterness.

In this study CRISPR/Cas9 was successfully used on witloof, using protoplast transfection with plasmid DNA of a CRISPR/Cas9 vector targeting the first exon in the phytoene desaturase gene (CiPDS). CiPDS mutation frequencies of up to 20% were observed in callus-stage and albino and dwarf phenotypes were obtained. Hence, our developed protocol for successful protoplast transfection and regeneration in Cichorium can be used to alter the SL pathway genes.

Three genes have already been identified to control the production of SLs: germacrene A synthase (GAS), germacrene A oxidase (GAO) and costunolide synthase (COS). To discover new genes related to the SL pathway, a set of samples was generated from three different species: chicory, witloof and lettuce. Two week old seedlings were elicitated with the phyotohormone jasmonate (MeJA), a known activator of SL biosynthesis, and harvested at three different time points (2h, 6h and 24h). Using GAS, GAO and COS as target genes, RT-qPCR experiments showed the highest upregulation in seedlings harvested after 6 hours of MeJA treatment. This optimal time point in the three different species was selected for RNA-sequencing. Co-expression analysis with the GAS, GAO and COS genes will be used to identify candidate genes of the SL pathway.

To confirm the functional activity of newly identified candidate enzymes, a functional yeast expression assay will be carried out. The candidate sequences will be cloned into a yeast expression vector and tested by co-transformation with GAS, GAO and COS genes. Yeast extracts will be analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and can confirm enzymatic activity of the encoding genes. Identifying these new genes related to the SL pathway and using the successful CRISPR/Cas9 transfection and regeneration protocol opens opportunities to change the bitterness of the Cichorium species.