Expression changes of miRNA-regulated genes associated with the formation of the leafy head in cabbage (Brassica oleracea var. capitata).

Cabbage (B. oleracea var. capitata) is an economically important vegetable crop grown for its leafy head. Cabbage plants’ vegetative development goes from seedling to rosette, folding and heading stages. Leaves that form the rosette are large and mostly flat, acting as the major photosynthetic organs. In the following developmental stages, the plants produce leaves that curve inward, overlapping the shoot apex to produce the leafy head. These leaves are shielded from the light and act as sink organs. We investigated the role of miRNA-regulated genes in the transition from the rosette to the heading stage. We first phenotyped leaf formation and -expansion for two contrasting (round and pointed) heading cabbage morphotypes to define the rosette and heading developmental stages. This allowed us to collect tissue from young expanding leaves that would develop into, respectively, rosette and heading leaves for miRNA and gene expression analyses. Young leaf tissue of two time points of a non-heading collard green (B. oleracea var. acephala) morphotype was used as a control to distinguish the transcripts (miRNAs/genes) that regulate plant age from those defining the transition from rosette to heading leaves in cabbages. For both round and pointed cabbages, we compared the miRNA and mRNA abundances between rosette and heading leaves to identify differentially expressed miRNAs (DEMs) and –genes (DEGs). After correcting for miRNAs and genes related to plant age, we identified 33 DEMs and 1,998 DEGs with roles in the transition from rosette to heading stage. We predicted the target genes of these 33 DEMs and focused on the subset that were DEGs between rosette and heading stage leaves to construct miRNA-target gene interaction networks. Our main finding is a role for miR396b-5p targeting two Arabidopsis thaliana orthologues of GROWTH REGULATING FACTORs 3 (GRF3) and -4 (GRF4) in pointed cabbage head formation.