**4. Differential activities of** *FT* **genes in the ALSV vector**

*FT* genes derived from various plant species are expressed by ALSV vectors in Arabidopsis, tobacco and soybean (**Figure 6A**) [11]. These *FT* genes are designated *AtFT* and *AtTSF* (derived from Arabidopsis), *VvFT* (grapevine), *GtFT1* and *GtFT2* (Japanese gentian), *InFT1* and *InFT2* (morning glory), *CsFT* (cucumber), *PmFT* (Japanese apricot), *GmFT2a* and *GmFT5a* (soybean), *LeFT* (tomato), *CiFT* (unshu mikan/satsuma mandarin), *PsFTa1* and *PsFTc* (pea), *MdFT1* and *MdFT2* (apple), *ComFT2* (squash) and *PtFT1* (aspen). Sizes of these FT proteins range from 172 to 184 amino acid residues, but their sizes have no correlation with their activities. A previous report described the degrees of inductions of Arabidopsis and tobacco flowering by these ALSV vectors [11]. **Figure 6B** summarizes these data, together with unpublished data on the induction of soybean flowering.

The average number of leaves at flowering was 30 in plants infected by the control wtALSV vector. The number of leaves was more than halved (less than 15) when plants were infected by the ALSV vectors expressing 10 *FT* genes, whereas, the number of leaves was more than 15 when plants were infected by the ALSV vectors expressing the remaining 9 *FT* genes. Among the 19 *FT* genes examined, *AtFT* and *VvFT* most strongly induced flowering, whereas *GtFT2* and *MdFT1* scarcely induced flowering. What determines the differences in the degree of flowering induction by these *FT* genes? **Figure 6C** shows the phylogenetic tree of the 19 FT proteins. The five FT proteins most strongly inducing flowering (indicated by circles) and the five FT proteins most weakly inducing flowering (indicated by triangles) are evenly distributed within the tree. This suggests that the activities of FT proteins were modified after species differentiation, rather than that their activities were evolutionarily fixed before species

**4. Differential activities of** *FT* **genes in the ALSV vector**

induction of soybean flowering.

78 Plant Engineering

*FT* genes derived from various plant species are expressed by ALSV vectors in Arabidopsis, tobacco and soybean (**Figure 6A**) [11]. These *FT* genes are designated *AtFT* and *AtTSF* (derived from Arabidopsis), *VvFT* (grapevine), *GtFT1* and *GtFT2* (Japanese gentian), *InFT1* and *InFT2* (morning glory), *CsFT* (cucumber), *PmFT* (Japanese apricot), *GmFT2a* and *GmFT5a* (soybean), *LeFT* (tomato), *CiFT* (unshu mikan/satsuma mandarin), *PsFTa1* and *PsFTc* (pea), *MdFT1* and *MdFT2* (apple), *ComFT2* (squash) and *PtFT1* (aspen). Sizes of these FT proteins range from 172 to 184 amino acid residues, but their sizes have no correlation with their activities. A previous report described the degrees of inductions of Arabidopsis and tobacco flowering by these ALSV vectors [11]. **Figure 6B** summarizes these data, together with unpublished data on the

**Figure 5.** Additional example of early-flowering plants. Petunia seedlings which exhibit early flowering via infection by the ALSV-XSB(AtFT) vector. Left plants are infected by wtALSV. The photograph was originally reported in Ref. [10].

The average number of leaves at flowering was 30 in plants infected by the control wtALSV vector. The number of leaves was more than halved (less than 15) when plants were infected by the ALSV vectors expressing 10 *FT* genes, whereas, the number of leaves was more than 15 when plants were infected by the ALSV vectors expressing the remaining 9 *FT* genes. Among the 19 *FT* genes examined, *AtFT* and *VvFT* most strongly induced flowering, whereas *GtFT2* and *MdFT1* scarcely induced flowering. What determines the differences in the degree of flowering induction by these *FT* genes? **Figure 6C** shows the phylogenetic tree of the 19 FT proteins. The five FT proteins most strongly inducing flowering (indicated by circles) and the five FT proteins most weakly inducing flowering (indicated by triangles) are evenly distributed within the tree. This suggests that the activities of FT proteins were modified after species differentiation, rather than that their activities were evolutionarily fixed before species

**Figure 6.** Induction of early flowering with various *FT* genes. (A) ALSV vectors used in this analysis. 'XyFT' represents *FT* genes derived from various plant species (such as *AtFT* and *MdFT1*). Respective *FT* genes are inserted at the XSB site in independent vectors. (B) Flowering times of ALSV-infected plants (Arabidopsis, tobacco or soybean) counted by the number of true leaves at flowering. Arabidopsis was grown at 25°C under a short-day condition (8 h:16 h light/dark photoperiod), and tobacco and soybean were grown at 25°C under a long-day condition (16 h:8 h light/dark photoperiod). 'Average' represents the average values of the numbers of leaves at flowering in Arabidopsis, tobacco and soybean. 'wtALSV' represents the wild-type ALSV vector without any insertion. MdFT2, GtFT2, MdFT1 and wtALSV did not induce soybean flowering, then the numbers of leaves are conveniently set as 25. (C) Phylogenetic tree of the FT proteins expressed by the ALSV vectors in this analysis. In (B) and (C), the five FT proteins most strongly inducing flowering are indicated by filled circles, and the five FT proteins most weakly inducing flowering are indicated by filled triangles.

**Figure 7.** Activities of chimeric *FT* genes in Arabidopsis. (A) ALSV vectors used in this analysis. CnFT, chimeric *FT* genes. (B) Amino acid sequences of AtFT and MdFT1, and the three fragments (f1, f2 and f3) defined in this analysis. (C) Combinations of FT fragments in chimeric FT proteins. The fragments derived from AtFT are grey, and the fragments derived from MdFT1 are white. (D) Arabidopsis plants infected by ALSV vectors. NI, non-inoculated plant. Juvenile plants with three true leaves were inoculated by particle bombardment of viral RNA. Viral infection was confirmed by RT-PCR (reverse transcription-polymerase chain reaction) analysis. Photographs were taken 30 days after inoculation.

differentiation. Even the *MdFT1* gene, the weakest *FT* gene in this analysis, more or less shortened flowering time when expressed in transgenic Arabidopsis and apple [35], confirming its activity as florigen. The activities of *FT* genes examined in our analysis are inconsistent with grafting experiments using transgenic Arabidopsis plants ectopically expressing *AtFT* or *AtTSF* [36]. In this paper, they found that AtTSF is scarcely mobile from rootstock to scion, then fails to induce early flowering. In contrast, AtTSF was highly active when expressed by the ALSV vector in **Figure 6**. The activity of FT proteins as the regulator of gene expressions may matter when expressed by ALSV vectors, rather than their mobility along the phloem.

Chimeric *FT* genes between *AtFT* (strongest FT) and *MdFT1* (weakest FT) were constructed and investigated in order to determine which part of the *FT* gene regulates its activity. This analysis was performed by Yamagishi N, and is presented for the first time here. The ALSV vectors in this analysis express chimeric *FT* genes at the middle of the polyprotein in RNA2 (**Figure 7A**). **Figure 7B** shows the amino acid sequences of AtFT and MdFT1. FT proteins were divided into three fragments (f1, f2 and f3); f1 corresponds to the first exon, f2 corresponds to the second and the third exons and f3 corresponds to the fourth exon of the FT genes. Chimeric FT proteins are designated C1 FT, C<sup>2</sup> FT, C<sup>3</sup> FT, C<sup>4</sup> FT, C<sup>5</sup> FT and C6 FT (**Figure 7C**). All ALSV vectors including those expressing AtFT and MdFT1 were inoculated into *N. benthamiana*, but the vectors expressing C1 FT and C3 FT did not infect *N. benthamiana*. When virus-infected Arabidopsis plants were grown under a long-day condition, C<sup>2</sup> FT, C<sup>6</sup> FT, C<sup>4</sup> FT and AtFT strongly induced flowering, while C<sup>5</sup> FT and MdFT1 only weakly induced flowering (**Figure 7D**). This shows that the C-terminus (f3) of the FT protein determines its strength as a florigen. Although we typically use the ALSV vector expressing *AtFT* to shorten generation time in horticultural plants, chimeric *FT* genes such as *C2 FT* many more strongly induce flowering in some plant species.
