Cloning And Functional Analysis Of Flower Development Genes In Platanus Acerifolia

London plane(Platanus acerifolia Willd) is a hybrid of American sycamore(P. occidentalis) and Oriental plane (P. orientalis). It is an important city landscape tree because its vegetative characteristics (e.g. innate disease-resistance and ability to grow rapidly in urban soils) improve the urban environment. A negative trait of Platanus, however, is the release of a large quantity of pollen during the flowering period, and this has been linked to symptoms of pollinosis and seasonal asthma in the human population (Subiza et al.,1994; Varela et al.,1997). Furthermore, the release of seed hairs later in the year produces other environmental pollutants. Thus, these aerial contaminants associated with the reproductive cycle of Platanus significantly detract from the value of the trees. The ability to control flowering in this species would, therefore, have wide-spread application.The aim of this study is to identify the key genes directing flower development in Platanus, by which to expand our knowledge of the molecular mechanisms underlying floral development in London Plane, and the woody species in general. The long-term aim of such work is to facilitate breeding and genetic-manipulation programs for the control of flower development in Platanus urban trees. The major results as following:1. Two types of FT-orthologous Genes were isolated from London Plane tree, namely PaFT and PaTSF. We revealed that PaFT, is atypical of most known FT orthologs in that it undergoes complex post-transcriptional regulation by alternative splicing (AS). The expression patterns of the different PaFT AS forms were related to organ-type and developmental stage. Ectopic expression of the archetypal splice form of PaFT-A in tobacco and Arabidopsis developed multiple altered phenotypes, notably early flowering and attenuation of apical dominance. The evidence in this study indicates that PaFT may have a broad role in London Plane tree development that is fine-tuned to the particular developmental requirements by complicated AS regulation. However, PaTSF appears not to be related to AS regulation. The expression pattern of PaTSF was not overlap with PaFT, thus implied that the two genes may have different regulatory roles in London Plane tree. Ectopic expression of PaTSF in tobacco also caused early flowering, but the phenotype of attenuation of apical dominance was not obviously.2. Five PISTILLATA (PI) homoeologous genes (PaPI1-to-5) and two AGAMOUS (AG) homoeologous genes (PaAG1 and PaAG2) were isolated from London Plane tree. Genetic alterations have been found among the five PaPI genes. PaPI1 and PaPI2 show a similar genomic structure to other known PI homoeologs, but PaPI3/4/5 lack intron sequences. In addition, PaPI5 lacks the third, fourth and fifth exons which encode the K-domain. PaPI2 appears micro-regulated by alternative splicing, displaying three splice forms (PaPI2a, PaPI2b and PaPI2c). Expression analyses and functional characterization supported the conclusion that PaPI transcripts encoding full-length polypeptides functions as a B-class MADS box homolog in London Plane tree. By contrast the PaPI5 form, which lacks the K-domain, had no apparent effect on flower development. In vitro studies also demonstrated that the K-domain is required to form PaPI/PaAP3 heterodimers. Phenotypic characterization of PaAG was also conducted through the heterologous expression system. Unlike other AG-homoeologous genes, ectopic expression of PaAG in tobacco developed a novel phenotype, i.e. a conversion of both sepals and petals into carpels. The results suggest that PaAG may have a more specific role in regulating the fourth whorl organ development.3. We constructed a complex vector, i.e. pMOG22-PaFT1A-BpFULL1::BARNASE, with the aim to apply it in molecular breeding of London Plane tree. The transformed pMOG22-PaFT1A-BpFULL1::BARNASE tobacco lines failed to form early flower buds compare with the 35S:PaFT1A lines, thereby indicating functionality of this construct. The results show that the pMOG22-PaFT1A-BpFULL1::BARNASE construct has potential biotechnological applications in tree species.4. A 3.5kb promoter region of PaAG gene was obtained using the TAIL-PCR (Thermal asymmetric interlaced PCR) technique. Sequence analysis and promoter prediction all supported the conclusion that this promoter sequence has transcriptional activity.