Dissecting the cytokinin genetic pathway and the main gene regulatory networks in Cichorium endivia leaves: fundamental biology in leafy crops

Cytokinins (CKs) regulate important aspects of plant development including leaf shape. Being potent antagonists of senescence, CKs might be exploited to extend the shelf life properties of leafy vegetables. Endives (Cichorium endivia var. crispum) and escaroles (C. endivia var. latifolium) are leafy crops belonging to the Cichorieae tribe which leaves are widely consumed as fresh, minimally processed and cooked food. Similar to lettuce, their leaves display a wide range of morphology diversity, ranging from smooth (escaroles) to extremely curly leaves (endives). CK homeostasis and responses may also contribute to leaf morphology diversity among C. endivia cultivars. We assembled de novo the transcriptome of C. endivia and reconstructed the genetic pathway of cytokinins: genes of CK metabolism, transport, signaling and response were annotated, and their expression analyzed in edible leaves of two endive and two escarole cultivars by RNAseq. Metabolite analysis identified cis-zeatin as the main cytokinin type in mature leaves, which accumulated as reversibly inactivated zeatin O-glycosylated storage forms. Metabolite-transcript correlation identified candidate genes for cis- and trans-zeatin reversible O-glycosylation, and for the two-step interconversion of CK bases, nucleosides and nucleotides, which have remained elusive for a long time. Gene Co-expression Network (GCN) analyses identified an AHK4/CRE1 receptor-based module as a major CK regulatory network in edible leaves. K-means clustering and GCN analyses of transcription factors (TFs) and target pathways (CK, photosynthesis, senescence, leaf development) identified eight major modules of co-regulated genes, amongst which the two most abundant ones were anti-correlated and comprised genes promoting photosynthesis or genes involved in oxidative stress/senescence responses. Most CK-related genes placed within these two clusters. Developmental genes at the boundary between photosynthesis and leaf development were identified by a "guilt by association" approach. Finally, differential expression analysis between broad and curly leaves identified novel candidate TFs putatively involved in leaf shape diversity.