Overexpression Of The Cucumber LEAFY Homolog CFL And Hormone Treatments Alter Flower Development In Gloxinia (Sinningia Speciosa)

The mechanism of flowering is increasingly becoming the focus of botanic developemental biology research. During flower development in Arabidopsis, LEAFY(LFY) has been found to play a pivotal role, interacting with and coordinating other flowering-related genes. LFY can directly target APETALA1 (AP1), AGAMOUS (AG), and APETALA3 (AP3). All these LFY-targeted genes belong to the MADS-box gene family of transcription factors, which play distinct roles in flower development. Cucumber-FLO-LFY (CFL), a LFY-like gene from Cucumis sativus L., was introduced into Sinningia speciosa to establish the function of CFL and investigate its potential to alter flowering time of gloxinia. The results obtained are as following:1. Using recombination techniques, the gene fragment CFL, derived from pBluscriptSKII, was isolated and placed into pBI121, pCAMBIA13011 and pER8 vectors respectively. Different protocols were used in order to evaluate their effects on transformation efficiency. CFL gene was transformed into leaves of Sinningia speciosa mediated by ultrasonic 10seconds with Agrobacterium tumefaciens EHA105. Southern blot analysis reveal that the exogenous gene has been integrated into the plant genome.2. Here, we show in gloxinia plants that transgenic 35S::CFL can result in bushy growth habits as well as fewer branches, and promote early flowering (approximately 30 days earlier than that of wild type). Our results imply that CFL act as a functional homolog of LFY in gloxinia despite being derived from a species which is distant from gloxinia in the phylogenetic tree. Cucumber belongs to the order of Cucurbitales, but gloxinia is in the order of Lamiales. Our results also provide evidence for a broad functional conservation of LFY-like genes and the regulatory MADS-box genes downstream of LFY in dicotyledonous plants.3. Floral buds were regenerated directly from the sepals of 35S::CFL gloxinia, but not the wild type, indicating that the overexpression of CFL is sufficient for the formation of floral buds for the sepals. GA₃ supplement can also lead to the regeneration of floral buds from the sepals of the wild type, indicating that GA3 treatment and CFL overexpression has a similar effect on the regeneration of floral organs from sepals. ABA and 5%[w/v] sucrose induced senescence of flower buds, while it inhibited the expansion of flowers and the regeneration of floral buds from sepals.4. To reveal CFL functions in gloxinia, we isolated partial cDNA fragments of three CFL-targeted genes from gloxinia, using primers designed based on AP1, AP3, AG of Arabidopsis, respectively. These gloxinia cDNAs referered as to SsAP1-1, SsAP3-1 and SsAG-1, respectively shared a high similarity with their homologs in tobacco and Arabidopsis. Semi-quantitative RT-PCR showed that these three genes were expressed in the reproductive organ of wild-type gloxinia with different spatial and temporal patterns.5. Our results show that constitutive CFL overexpression significantly promote early flowering without gibberellin (GA₃) supplement, suggesting that CFL can serve functionally as a LFY homolog in gloxinia. Moreover, GA3 and abscisic acid (ABA) treatments could modulate the expression of MADS-box genes in opposite directions. GA3 resembles the overexpression of CFL in the expression of MADS-box genes and the regeneration of floral buds, but ABA inhibits the expression of MADS-box genes and flower development. These results suggest that CFL and downstream MADS-box genes involved in flower development are regulated by GA3 and ABA. Our results alos showed that IAA had a little effect on the expression CFL and three MADS-box genes and the regeneration of floral buds from the sepals of wild-type, but the inhibition of formation of red color in the flower buds. These results suggest that IAA is unlikely to be directly involved in the regulation of LFY homologs and their downstream genes in flower initiation.