Mechanisms of bullseye boundary formation in Hibiscus trionum petal patterning

Abstract

Flower corollas often display striking patterns combining pigmentation, cell shape and cuticular ornamentation. These patterns contribute to plant-pollinator interactions but how spatial control of cell fate specification is achieved across the petal epidermis remains obscure. Hibiscus trionum is an emerging model system producing flowers with a striking bullseye pattern on the adaxial petal epidermis. Cells in the proximal region are flat, elongated with cuticular striations and accumulate purple anthocyanin pigments, while cells in the distal region are white, conical and smooth. However, the exact mechanisms specifying cell identity along the proximo-distal petal axis are unknown. In particular, how a sharp boundary emerges to separate the two contrasting regions that makes the bullseye remains to be understood. My PhD adds to the current understanding of petal patterning mechanisms by investigating the genetic and cellular mechanisms of H. trionum bullseye boundary formation. Homeodomain-leucine zipper (HD-ZIP) transcription factors comprise four subgroups fulfilling diverse roles in plant lateral organ development. Several HD-ZIP genes were found to be differentially expressed along the proximo-distal axis of the H. trionum petal during early development. None of these candidate genes are from subgroup III which specifies lateral organ adaxial-abaxial polarity, but are instead from subgroups I, II and IV. Combining detailed microscopy analyses with gene over- expression and genome editing using CRISPR/Cas9, I found that mis-expressing some of these candidate genes can perturb cell fate acquisition and bullseye boundary specification along the proximo-distal axis of the H. trionum petal. This reveals a possible role for HD-ZIP homologs in orchestrating proximodistal cell differentiation in H. trionum petals. Next, I investigated epidermis cell behaviour during bullseye pattern boundary formation. Before visible pattern features emerge, the largest cells are found at the one-third position from the petal base, possibly marking the future bullseye boundary. To examine whether progenitor boundary cells with peak cell area are the earliest cells to stop dividing and start differentiating, I developed a DAPI (4′,6- diamidino-2-phenylindole) staining protocol. As an increase in cell ploidy due to endoreduplication often accompanies cell differentiation, this will allow me to test for a correlation between cell size and nuclei size across the petal epidermis. Besides using cell geometry to track boundary establishment, I also identified genetic markers of the boundary and isolated their promoter regions to develop boundary reporter lines. To start dissecting the signaling processes that participate in bullseye boundary formation, I performed mechanical disruption experiments. I found that punctures in the proximal region of young petals disrupt correct boundary formation, whereas punctures in the distal compartment do not, and only impair growth instead. My preliminary results indicate that localised changes in cell geometry and cell polarity due to petal punctures depend on the distance and position along the proximo-distal axis relative to the puncture. Hence, mechanical disruption experiments can help us understand how epidermis cell behaviour depends on yet unknown intercellular positional signals along the H. trionum petal proximodistal axis.