Identification and characterization of CYC2-like genes related to floral symmetric development in Tagetes erecta (Asteraceae).

Marigold (Tagetes erecta) is an annual herbaceous flower belonging to Asteraceae, whose capitulum is composed of bilateral symmetry ray florets on the outer periphery and radial symmetry disk florets on the inside. The flower symmetry evolution from radial symmetry to bilateral symmetry has changed the morphology, inflorescence architecture and function of florets among several lineages in Asteraceae. Several studies have identified that CYC2 genes in TCP transcription factor family are the key genes regulating the flower morphogenesis, such as corolla symmetry and stamen development. Here, seven TeCYC2 genes were cloned and phylogenetically grouped into the CYC2 branch of TCP transcription family. TeCYC2c and TeCYC2d were found to be expressed specifically in ray florets, TeCYC2b was strongly expressed in both ray and disk florets, TeCYC2g was significantly higher expressed in ray florets than in disk florets, while TeCYC2a, TeCYC2e1 and TeCYC2e2 were significantly expressed in disk florets, according to an examination of the expression profile. Among the ectopic expression lines of seven TeCYC2 genes in Arabidopsis thaliana, the flower symmetry of all transgenic lines was changed from radial symmetry to bilateral symmetry, and only the reproductive growth of TeCYC2c lines was affected. In TeCYC2c transgenic Arabidopsis, the pollen sac was difficult to crack, and the filaments were shorter than the pistils, resulting in a significant decrease in the seed setting rate. All TeCYC2 proteins were localized in the nucleus. Eight pairs of interactions between TeCYC2 proteins were validated by Y2H and BiFC assays, indicating the possibility of TeCYC2 proteins forming homodimers or heterodimers to improve functional specificity. Our findings verified the main regulatory role of TeCYC2c on the development of corollas and stamen in marigold, and analyzed the interaction network of the formation mechanism of floral symmetry in two florets, which provided more insights into the expansion of CYC2 genes in the evolution of Asteraceae inflorescence and contributed to elucidate the complex regulatory network, as well as the molecular breeding concerning flower form diversity in marigold.

Functional conservation and divergence of SEPALLATA-like genes in the development of two-type florets in marigold.

Marigold (Tagetes erecta), as one member of Asteraceae family, bears a typical capitulum with two morphologically distinct florets. The SEPALLATA genes are involved in regulating the floral meristem determinacy, organ identity, fruit maturation, seed formation, and plant architecture. Here, five SEP-like genes were cloned and identified from marigold. Sequence alignment and phylogenetic analysis demonstrated that TeSEP3-1, TeSEP3-2, and TeSEP3-3 proteins were grouped into SEP3 clade, and TeSEP1 and TeSEP4 proteins were clustered into SEP1/2/4 clade. Quantitative real-time PCR analysis revealed that TeSEP1 and TeSEP3-3 were broadly expressed in floral organs, and that TeSEP3-2 and TeSEP4 were mainly expressed in pappus and corollas, while TeSEP3-1 was mainly expressed in two inner whorls. Ectopic expression of TeSEP1, TeSEP3-2, TeSEP3-3, and TeSEP4 in arabidopsis and tobacco resulted in early flowering. However, overexpression of TeSEP3-1 in arabidopsis and tobacco caused no visible phenotypic changes. Notably, overexpression of TeSEP4 in tobacco decreased the number of petals and stamens. Overexpression of TeSEP1 in tobacco led to longer sepals and simpler inflorescence architecture. The comprehensive pairwise interaction analysis suggested that TeSEP proteins had a broad interaction with class A, C, D, E proteins to form dimers. The yeast three-hybrid analysis suggested that in ternary complexes, class B proteins interacted with TeSEP3 by forming heterodimer TePI-TeAP3-2. The regulatory network analysis of MADS-box genes in marigold further indicated that TeSEP proteins played a "glue" role in regulating floral organ development, implying functional conservation and divergence of MADS box genes in regulating two-type floret developments. This study provides an insight into the formation mechanism of floral organs of two-type florets, thus broadening our knowledge of the genetic basis of flower evolution.

Identification, characterization and functional analysis of AGAMOUS subfamily genes associated with floral organs and seed development in Marigold (Tagetes erecta).

BACKGROUND: AGAMOUS (AG) subfamily genes regulate the floral organs initiation and development, fruit and seed development. At present, there has been insufficient study of the function of AG subfamily genes in Asteraceae. Marigold (Tagetes erecta) belongs to Asteraceae family whose unique inflorescence structure makes it an important research target for understanding floral organ development in plants. RESULTS: Four AG subfamily genes of marigold were isolated and phylogenetically grouped into class C (TeAG1 and TeAG2) and class D (TeAGL11-1 and TeAGL11-2) genes. Expression profile analysis demonstrated that these four genes were highly expressed in reproductive organs of marigold. Subcellular localization analysis suggested that all these four proteins were located in the nucleus. Protein-protein interactions analysis indicated that class C proteins had a wider interaction manner than class D proteins. Function analysis of ectopic expression in Arabidopsis thaliana revealed that TeAG1 displayed a C function specifying the stamen identity and carpel identity, and that TeAGL11-1 exhibited a D function regulating seed development and petal development. In addition, overexpression of both TeAG1 and TeAGL11-1 leaded to curling rosette leaf and early flowering in Arabidopsis thaliana. CONCLUSIONS: This study provides an insight into molecular mechanism of AG subfamily genes in Asteraceae species and technical support for improvement of several floral traits.