Spatiotemporal dynamics of CYCLOIDEA expression during early floral development in monkeyflower

The establishment of bilateral symmetry in flowers depends on the precise regulation of CYCLOIDEA (CYC) gene expression along the dorsal-ventral axis. Although auxin and BLADE-ON-PETIOLE (BOP) have been implicated as regulators of CYC, when, where, and how they affect CYC expression remain unclear. Here, we combined transgenic manipulation and fluorescent confocal imaging in the monkeyflower species Mimulus parishii to capture the spatiotemporal dynamics of CYC (MpCYC2a and MpCYC2b) expressions in the floral meristems (FMs) of both wild-type (WT) and bop mutant (mpbop) plants. Our imaging data revealed three distinct phases of CYC expression in relation to auxin response pattern and FM size: initiation, maintenance, and specification. During the initiation phase, no auxin response maxima were detectable, but MpCYC expressions were already dorsally localized in the FM and showed no difference between the WT and mpbop. After the initiation phase, auxin response maxima were highly dynamic during FM expansion, when MpCYC expressions remained dorsally restricted in WT but expanded broadly in mpbop FMs. These findings suggest that early asymmetry establishment in the FM is guided by positional cues independent of auxin or BOP, which are instead essential for refining and maintaining dorsal-specific CYC expression during later FM enlargement. Our work illustrates how combining advanced imaging with emerging model systems can unveil new, "hidden" information on even extensively studied developmental genes and lays the groundwork for further elucidating the mechanisms underlying the repeated evolution of bilateral floral symmetry.