Molecular mechanisms underlying cytokinin-induced ornamental flower morphology and its application in breeding: Using torenia as a model floricultural plant

For floricultural plants, flower morphology is one of the most important traits determining attractiveness and commercial value. Improvement of flower morphology toward more ornamental flowers is a major objective of breeding programs, but conventional breeding programs can be time-consuming, and the development of efficient breeding methods is desired. In recent studies, treatment with forchlorfenuron (CPPU), an inhibitor of the cytokinin degradation enzyme cytokinin oxidase/dehydrogenase (CKX), induces morphological changes in flowers of torenia (Torenia fournieri L.) depending on the floral stage of CPPU treatment. In this study, we aimed to elucidate the molecular mechanism responsible for CPPU-induced morphological changes in torenia flowers. Furthermore, we employed floral organ-specific promotion of cytokinin biosynthesis using transgenic technologies to produce torenia with ornamental flower morphologies. In the work described in Chapter 2, we investigated the temporal and spatial distributions of cytokinin signals in CPPU-treated flower buds as indicated by type-A response regulator (RR) and CKX gene expression. Quantitative real-time PCR analysis showed that the expression of both TfRR1 and TfCKX5 was induced from 1 day after CPPU treatment in sepals, petals, stamens, and pistils and maintained at a high level until 5 days after treatment when the earliest morphological changes due to CPPU treatment were observed. In situ hybridization analysis showed weak expression of both genes in stamens and pistils through all floral stages of untreated flower buds. However, when CPPU was applied at the sepal development stage, expression of both genes was strongly induced at the abaxial side of the stamen primordia, which are sites of initiation of the wide paracorolla. When CPPU was applied during the early stage of corolla development, high expression of these genes was observed in the stamen and in the basal and middle parts of the petal, which are the sites of initiation of the narrow paracorolla. When CPPU was applied during the middle corolla development stage, strong expression of these genes was detected in the middle to apical parts of the petal, which is the site of changes in the distribution pattern of vascular bundles and the resulting serrated margins. In the work described in Chapter 3, we investigated the morphological properties and the role of floral homeotic genes in the formation of two CPPU-induced types of paracorolla, wide and narrow paracorolla. The morphology of epidermal cells and distribution pattern of vascular bundles were the same in wide paracorolla as in petals; however, in the narrow paracorolla, the morphology of epidermal cells was either petal-like or stamen-like, and the distribution pattern of vascular bundles was stamen-like. In situ hybridization analysis of floral homeotic genes showed that a class A gene, T. fournieri SQUAMOSA (TfSQUA), and the class B genes, TfDEFICIENS (TfDEF) and TfGLOBOSA (TfGLO), were expressed in the broad region of the primordia of the wide paracorolla, as in petals. Class C genes, TfPLENA1 (TfPLE1) and TfFARINELLI (TfFAR), were only expressed at margins of the paracorolla primordia. However, in primordia of the narrow paracorolla, TfSQUA and one of the class C genes (TfPLE1) was expressed only at the margin of the primordia, whereas the class B genes were expressed in a broad region of the primordia, similar to the case of the primordia of the wide paracorolla. Thus, this expression pattern in the narrow paracorolla was intermediate between that of petals and stamens. Furthermore, these expression patterns were similar to those at the paracorolla initiation sites. In the work described in Chapter 4, we introduced Arabidopsis isopentenyltransferase 4 (AtIPT4) into torenia under the control of the APETALA1 (AP1) or APETALA3 (AP3) promoter to characterize the relationship between organ-specific promotion of cytokinin biosynthesis within flower buds and flower morphology. AP1::AtIPT4 plants had an increased number of petals, whereas AP3::AtIPT4 plants had expanded corolla, paracorolla, and serrated petal margins along with an increased number of petals. In AP3::AtIPT4 plants, marked receptacle enlargement was observed when flower buds were in the early corolla development stage in which the paracorolla primordia differentiate. As expected, AtIPT4 was expressed in the sepals and petals of AP1::AtIPT4 plants and in the petals and stamens of AP3::AtIPT4 plants. Cytokinin signals as revealed by TfRR1 and TfCKX5 expression were elevated in the floral organs in which the transgene was expressed. The results described above suggest that the paracorolla and serrated petal margins are induced by high localized levels of cytokinin signals at the site of those morphological changes (Chapter 2). The expression patterns of floral homeotic genes at the early stage of paracorolla development determine paracorolla morphology, and the expression pattern is determined by the site within flower buds where the paracorolla is formed (Chapter 3). Localized cytokinin signals in sepals and petals increase in the petal number, whereas those signals in petals and stamens are necessary to induce corolla expansion, paracorollas and serrated petal margins (Chapters 4 and 5). Furthermore, both receptacle enlargement and localization of elevated cytokinin signals to the paracorolla initiation site are necessary for stable induction of the paracorollas (Chapter 5). These findings may aid in the development of efficient breeding methods for improvement of flower morphology.