Complex color patterns on petals are widespread in flowering plants, yet the mechanisms underlying their formation remain largely unclear.

Here, by conducting detailed morphological, anatomical, biochemical, optical, transcriptomic, and functional studies, we investigated the cellular bases, chromogenic substances, reflectance spectra, developmental processes, and underlying mechanisms of complex color pattern formation on Nigella orientalis petals.

We found that the complexity of the N. orientalis petals in color pattern is reflected at multiple levels, with the amount and arrangement of different pigmented cells being the key. We also found that biosynthesis of the chromogenic substances of different colors is sequential, so that one color/pattern is superimposed on another. Expression and functional studies further revealed that a pair of R2R3-MYB genes function cooperatively to specify the formation of the eyebrow-like horizontal stripe and the Mohawk haircut-like splatters. Specifically, while NiorMYB113-1 functions to draw a large splatter region, NiorMYB113-2 functions to suppress the production of anthocyanins from the region where a gap will form, thereby forming the highly specialized pattern.

Our results provide a detailed portrait for the spatiotemporal dynamics of the coloration of N. orientalis petals and help better understand the mechanisms underlying complex color pattern formation in plants.