The long juvenile period of perennial woody plants is a major constraint in breeding programs. FLOWERING LOCUS T (FT) protein is an important mobile florigen signal that induces plant flowering. However, whether FT can be transported in woody plants to shorten the juvenile period is unknown, and its transport mechanism remains unclear. In this study, trifoliate orange FT (ToFT) and Arabidopsis FT (AtFT, which has been confirmed to be transportable in Arabidopsis) as a control were transformed into tomato and trifoliate orange, and early flowering was induced in the transgenic plants. Long-distance and two-way (upward and downward) transmission of ToFT and AtFT proteins was confirmed in both tomato and trifoliate orange using grafting and western blot analysis. However, rootstocks of transgenic trifoliate orange could not induce flowering in grafted wild-type juvenile scions because of the low accumulation of total FT protein in the grafted scions. It was further confirmed that endogenous ToFT protein was reduced in trifoliate orange, and the accumulation of the transported ToFT and AtFT proteins was lower than that in grafted juvenile tomato scions. Furthermore, the trifoliate orange FT-INTERACTING PROTEIN1 homolog (ToFTIP1) was isolated by yeast two-hybrid analysis. The FTIP1 homolog may regulate FT transport by interacting with FT in tomato and trifoliate orange. Our findings suggest that FT transport may be conserved between the tomato model and woody plants. However, in woody plants, the transported FT protein did not accumulate in significant amounts in the grafted wild-type juvenile scions and induce the scions to flower.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDβ). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDβ in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDβ and CiFDα. Interestingly, CiFDα and CiFDβ were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDβ can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDβ can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought-and low-temperature-induced citrus flowering through different regulatory patterns.