Regulatory Function Of Exogenous Melatonin On Flowering Time In Arabidopsis Thaliana

Melatonin (N-acetyl-5-methoxytryptamine) is a kind of hormone which exists extensively in various organisms, contains indole heterocycle and is similar in molecular structure with indole acetic acid (IAA). The N-acetyl and 5-methoxy functional groups make melatonin an amphiphilic molecule and determine its binding specificity with receptors. In animals, melatonin participates in regulation of circadian rhythms and photoperiodical reactions, it can enhance the body immunity and help reverse the aging process. In plants, melatonin can improve the resistances of individuals against different adverse circumstances and alleviate the oxidative damage induced by biotic and abiotic stresses.Previous research works indicated that more than 180 genes were involved in floral transition and floral organogenesis in Arabidopsis, and the regulation mechanisms of flowering times have been summed up as six major pathways, including photoperiod, age, vernalization, autonomous, ambient temperature and gibberellin pathways. These pathways can influence the process of floral transition by converging the signals generated in different environmental conditions to a small number of floral integrator genes. Up to now, the defined floral integrators include FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1). In other words, FT, LFY and SOC1 can merge the flowering signals come from different pathways and promote the transition of vegetative growth to reproductive growth. It is possible that melatonin plays a role in photoperiodical reactions and has inductive effects to morphogenesis of floral organs. Nevertheless, functional identification of melatonin in flowering regulation is only at the beginning stage.The results of the present work showed that exogenous treatment with melatonin solution of a certain concentration could overcome the barriers of photoperiod and accelerate flowering in Arabidopsis plants growing under facultative illumination conditions (12 h light/12 h dark). Quantitative RT-PCR (qRT-PCR) analyses of the RNA samples extracted from untreated and melatonin-treated plants at different time points indicated that application of exogenous melatonin could upregulate the expression of AP1、CCA1、CO、FT、FD、FLC、 FRI、LYF、LHY、PRR7、SOC1、SPL4、SVP、TOC1、TSF and TOC1, whereas the transcription of GI was inhibited and the expression level of PHYB was basically unchanged, in comparison with the controls. All these results suggested that melatonin could affect the transcription of the genes associated with floral transition and could regulate the flowering time obviously.CO could be induced approximately in 1 h after melatonin treatment. The expression level of it increased gradually and peaked at 48 h. Subsequently, the transcription of the downstream genes of CO, including FT, TSF, FD, SOC1, LFY and API, was enhanced obviously within a few hours. The expression levels of four upstream genes of CO functioned in controlling of circadian clock, including CCA1, LHY, TOC1 and PRR7, could be activated successively by melatonin application, and the upregulation of them occurred in a sequential order. Specifically, the expression of TOC1 peaked at 12 h after melatonin treatment, the transcription levels of CCA1 and LHY reached to the maximum value at 24 h after melatonin treatment, and PPR7 could be induced sufficiently in 48 h by melatonin. After achieving to the highest levels, the expression of CCA1, LHY, TOC1 and PRR7 all started down, indicating the downstream genes possessed an effect of feedback inhibition on the expression of the upstream genes. The increase of the transcription levels of the circadian clock genes could promote the expression of CO. PHYB encodes a phytochrome involved in sensing of the red light, no significant changes could be detected in transcription of this gene, suggesting the expression of it could not be induced by melatonin. GI can promote the expression of CO indirectly. However, the transcription of GI was repressed by melatonin after 0.5 h of treatment, indicating the positive effect of melatonin in activation of CO was not related with GI. These results suggested that application of exogenous melatonin could influence the photoperiod pathway of floral transition in Arabidopsis. Melatonin might promote flowering by acting on circadian clock genes located upstream of CO at first and upregulation of the circadian clock genes could activate CO in turn.In regulatory network formed by Arabidopsis flowering genes, FT, SOC1 and LFY encode the integrators of flowering signals. They can collect the regulatory signals come from different flowering pathways and their expression can be affected by upstream genes. The transcript abundances of FT, SOC1 and LFY increased significantly after treatment with exogenous melatonin and activation of these genes could promote flowering due to their influences on a number of downstream genes. SVP, FRI and FLC encode the main repressors of flowering in Arabidopsis, but their expression also could be enhanced by treatment with exogenous melatonin. It is possible that SVP, FRI and FLC have feedback effects on FT and SOC1, and they can make the Arabidopsis plants enter next developmental stage successfully once the floral transition is completed. In addition, melatonin treatment could upregulate the expression of SPL4, suggesting melatonin also participated in regulation of ageing pathway in flowering induction of Arabidopsis. API is associated with formation of inflorescences and development of floral organs, the upregulation effect of melatonin on API indicated that melatonin was probably involved in organogenesis of Arabidopsis flowers except its regulatory function to flowering time.