Molecular Mechanism Of Flower Transition Induced By Short-day In Chrysanthemum Lavandulifolium

Plants flower at a specific time, which is the result of long-term adaptation to the environment. The quality of flower is an important ornamental and cultivated characteristic. Chrysanthemums are one of the traditional flowers in China, which are typical short-day plants and most of the traditional cultivars flower from autumn to winter. Circadian clock genes can sense the day-length signal and transfer the day-length signal into flower signal according to the researches on the molecular mechanism in long-day plants and short-day plants. Therefore, it is highly desirable to effectively manipulate the circadian clock genes and downstream flower genes involved in flower transition to breed the new chrysanthemum varieties that are suitable for industrial production. Due to the complex genetic background of chrysanthemums, the progresses of researches on the molecular mechanism of flower transition have been slow. In this study, we employed [Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino], which is a species, closely related to chrysanthemum, as the experimental material to study the molecular mechanism of flower transition induced by short-day. We isolated circadian clock, CO and FT homologous genes based on the transcriptome and digital gene expression database and then researched the expression patterns of these genes in different photoperiod conditions, combining the researches on functions of key genes in order to obtain the molecular mechanism of flower transition induced by short-day.The main results are described as follows:(1) C. lavandulifolium is a typical short-day plant and the critical photoperiod is13h light/11h dark according to the results of characteristics of photoperiod induction of flowering. Plants which have fourteen true leaves are at the end of juvenile phase. It could make flower successfully if they have supplied with22d short-day (12h light/12h dark) induction although they are placed in long-day (16h light/8h dark) condition thereafter. It could make flower when the length of dark period is12h (16h light/12h dark or8h light/12h dark) although the light period is lengthened or shortened. It only needs14.25-16.25cycles to form floral buds and38.45-40.25cycles to make flower. Therefore, C. lavandulifolium is an obligate short-day plant and the length of dark period and days of induction by short-day are key factors to influence its flowering. (2) We analyzed the stability of nine reference genes that are expressed relatively stable in our digital gene expression profiling in different tissues under various developmental stages and leaves with various photoperiodic treatments. The SAND gene is the most stable gene in different tissues under various developmental stages and MTP gene is the most stable gene in leaves with various photoperiodic treatments. These results lay foundation to research the expression patterns of genes in further study.(3) We have isolated eleven circadian clock genes in C. lavandulifolium, and with the exception of CIELF3, CIPRR1and CIPRR73, most of the circadian clock genes are expressed more highly in leaves than in other tested tissues. The diurnal rhythms of these circadian clock genes in long-day (16h light/8h dark) or short-day (12h light/12h dark) conditions are similar to those of their homologs in Arabidopsis. The diurnal rhythms of most circadian clock genes continue cycling under LL conditions though the peak levels are dampened and the oscillating periods are changed, indicating that these circadian clock genes are synchronized by light. The peak levels of CIGIs at2h after light on disappear and the peak levels of ClFKF1decrease in non-inductive night break condition. The peak levels of CIGIs do not change while the peak levels of ClFKF1disappeare in non-inductive8h light/8h dark condition, indicating that ClFKF1itself or the synchronous expression of ClFKF1and ClGIs might be essential to initiate the flowering of C. lavandulifolium. The ectopic expression of the ClGI-1gene in wild type Arabidopsis results in early flowering, with high expression levels of endogenous CO and FT being observed in transgenic Arabidopsis. It is indicated that ClGI-1might be activating the expression of CO and FT to inducing flower in C. lavandulifolium.(4) We have isolated eleven CO homologous genes in C. lavandulifolium and named them as ClCOL1-11. CICOL1-5and CICOL10-11are highly expressed in leaves and shoot apices according to the results of tissus-specific expression. The expression levels of CICOL4-5and CICOL7-8are highly increased under inductive short-day condition compared to long-day condition. Overexpression of ClCOL1, which has the closest relationship to CO in Arabidopsis, accelarates the flowering meanwhile the rosette branching number, the plant height and the duration of flowering time are significantly changed. The ectopic expression of the CICOL5gene in wild type Arabidopsis only results in early flowering and the flower time is earlier than that of the transgenic plants with overexpression of ClCOL1. These results indicate that CICOL5gene has close relation to flower transition and ClCOL1may act redundantly with CICOL5to regulate the flower transition in C. lavandulifolium.(5) We have isolated two FT homologous genes in C. lavandulifolium and named them as ClFT1and CIFT2. CIFT1is expressed highly in leaves than in shoot apices while CIFT2is expressed highly in shoot apices than in leaves. The expression levels of ClFT1are increasing while those of CIFT2are decreasing under the inductive short-day condition. Overexpression of CIFT1accelerates flowering while overexpression of ClFT2suppresses flowering, indicating that these two genes play reverse fuctions in flower trasition.In summary, our research showed that C. lavandulifolium is a typical obligate short-day plant and the length of dark period has close relation to flower. The circadian clock genes maintain their rhythm under the alternation of day and night condition and the peak expression levels of CIGIs genes at the begginning of light period could activate the expression of ClCOL4/5and CIFT1genes and ClFKF1itself or the synchronous expression of ClFKF1and ClGIs could repress the expression of CIFT2gene when the length of dark period beyond the critical night length. When the expression levels of ClFT1gene exceed the levels of CIFT2gene the flower transition could be completed. This research could not only lay foundation for understanding the molecular mechanism of plant daylength measure but also provide the new idea to modify flowering time by manipulating the circadian clock and downstream flower time genes.