Regulatory pathways of CYC‐like genes in patterning floral zygomorphy exemplified in Chirita pumila

Abstract

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.

An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae)

“…As described before [ 29 ], wild type zygomorphic flowers produced three different types of petals (two dorsal, two lateral and one ventral petals) and only two fertile stamens in the ventral region. By contrast, transgenic flowers were actinomorphic with five petals identical in both size and shape, all stamens fertile and the corolla tube ventralized ( Figure 7 a–h), similar to the naturally occurred peloria flowers [ 47 ]. In addition, the yellow spot characteristic of the ventral corolla tube of wild type flowers has extended to all over the corolla tube in transgenic flowers ( Figure 7 a,c,e,g).…”

“…pumila . The GUS signals in positive transgenic lines were mainly observed in developing dorsal petals, dorsal, and lateral staminodes ( Figure 7 q,r) [ 47 ], demonstrating the role of CpCYC in controlling the development of these floral organs. This GUS staining patterns are consistent with the expression domains of CpCYC tested by qRT-PCR.…”

“…pumila , may regulate the shape of dorsal petals and repress the development of dorsal and lateral stamens in C . pumila , but this needs further functional tests [ 47 ]. In this study, we carried out functional analyses by silencing CpCYC using RNAi technology in C .…”

“…Our results show that transgenic plants with dorsal floral organs escaping from CpCYC expression produce inheritable fully actinomorphic flowers with five equal petals and five fertile stamens, similar to the natural mutant C . pumila flowers [ 47 ] and the cyc dich double mutants in Antirrhinum [ 11 ]. Our results provide a direct functional evidence for the role of CpCYC in controlling the development of dorsal petals and dorsal/lateral staminodes in C .…”

“…While extensive expression data in various species suggest important roles of CYClike genes in zygomorphic floral development, functional evidence is very limited. Our previous study on gene expression and phenotypic analyses on the basis of natural peloria indicates that CpCYC, a CYC-like gene in C. pumila, may regulate the shape of dorsal petals and repress the development of dorsal and lateral stamens in C. pumila, but this needs further functional tests [47]. In this study, we carried out functional analyses by silencing CpCYC using RNAi technology in C. pumila.…”