Karakterisering en moleculaire studie van verkleuringen bij witloof

Witloof chicory (Cichorium intybus var. foliosum) is a leafy vegetable crop of Belgian heritage that is renowned for its typical bitter taste and its crunchy leaves. To produce chicons of consistently high quality, chicory growers and breeders are continuously searching for ways to optimise the cultivation process. This covers the selection of healthy, well-performing cultivars on the one hand and the entire process of chicory production on the other hand. The latter includes root cultivation in the field, root storage, forcing, harvest and finally postharvest storage of the mature chicons. For this project, we focussed our attention on one of the key areas that determine chicon quality and shelf life i.e. forcing and chicon storage. A major concern during this final phase of witloof production is the development of physiological colour disorders. These disorders come in many shapes and sizes and include red, brown and black discolourations of the leaves, as well as red and brown discolourations of the pith (the developing flowering stem in the centre of the chicon). Depending on the type and intensity of the disorder, the presence of colour disorders can cause a substantial reduction of product quality, both from a nutritional and an economical point of view. Moreover, colour disorders may affect up to 50% of chicons when the conditions are unfavourable, thus resulting in significant food waste and economic losses for growers. To address this issue, it is essential to understand what takes place on a macroscopic, microscopic and molecular level during the development of colour disorders. So far, research on discolourations in witloof chicory has characterised a wide range of discolouration types and identified a number of physiological and biochemical processes that may contribute to their development. Importantly, polyphenol oxidases (PPOs) are thought to play a major role by oxidising phenolic compounds after cellular damage or wounding. However, little efforts have been made to explore the role of genetics in the development of discolouration symptoms, even though its importance has long been recognised. We built on the existing research by analysing chicory discolourations on different scales of complexity, from the visible to the molecular and back to the visible. Discolourations in chicory are surprisingly complex and their frequency and intensity are subject to considerable variability. By contrasting two cultivars with a high and a low sensitivity to colour disorders, we established that genetic makeup is a dominant factor in discolouration variability. In addition, temporal variability of postharvest disorders appears to be predominantly determined by the time of forcing within one season i.e. the duration of root storage. In contrast, preharvest disorders show more year-to-year variability, and are therefore more dependent on environmental conditions during root cultivation. On a microscopic level, a combination of light microscopy and µ-CT tomography shed new light on the microstructure of important colour disorders and indicate that postharvest storage is associated with porosity changes in leaf and pith tissue. These changes may lead to cellular disruption, which could in turn provide a starting point for the development of postharvest discolourations such as internal red. Moreover, microscopic analysis corroborates previous hypotheses based on observational evidence, such as the rupture of laticifers in the development of external red. In addition, the results reveal structural and possibly biochemical similarities between different discolouration types, such as apple pith and layered core browning. In a last step towards higher complexity, we used untargeted metabolic profiling and a transcriptome analysis in a sensitive cultivar to explore the molecular side of internal red discolouration from an unbiased perspective. The results suggest that discolouration coincides with extensive environmental adaptation. Furthermore, they indicate involvement of light exposure, osmotic stress and intricate regulation of the phenylpropanoid metabolism. Finally, we identified members of the PPO gene family in chicory, which were subsequently targeted for selective gene editing with a CRISPR/Cas9. In the future we will build on this work by identifying regenerants with reduced PPO gene expression and by comparing their discolouration sensitivity to that of wild-type plants. As such, we aim to provide functional evidence for the role of PPO in witloof discolourations. With this work, we contribute to the understanding of discolourations in witloof chicory and provide new insights and perspectives for further research to work towards a future with fewer food waste and superior witloof quality.