Floral zygomorphy is thought of as an essential adaptation to specific pollinators. The CYCLOIDEA (CYC)‐like genes belonging to the plant‐specific TCP transcription factor family are the key regulators of floral zygomorphy. Their expression differentiations bring about diverse forms of floral zygomorphy. However, the regulatory mechanism underlying their expression differentiation remains unknown. In this study, we selected Chirita pumila D. Don, a zygomorphic member of Gesneriaceae, as a model to address this question. Mutant analysis shows that the CYC gene in C. pumila (CpCYC) plays an important role in controlling floral zygomorphy. Further functional investigation of CpCYC intron and 5′ regulatory regions indicates that an 899 bp promoter region is a key determinant of the dorsal‐specific expression of CpCYC. In this region, using electrophoretic mobility shift assay and transient expression system, we identified three kinds of cis‐regulatory elements with putative binding factors, that is, auxin responsive element, WRKY binding site, LEAFY binding site, and an element with an unknown binding factor. We undertook functional analyses of the two putative LEAFY binding sites, and found that LEAFY could directly activate and regulate CpCYC for its dorsal‐specific expression in patterning floral zygomorphy. This research would shed significant light on the regulatory and evolutionary pathways underlying the spatial‐specific expression of CYC‐like genes for the development of zygomorphic flowers.
The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.
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