NFT1 involved in flowering initiation induced by heating 21,22,mxm to that of the FT-like genes in the FT-tFL family genes, with a identity of 70% and 77% to Arabidopsis FT and rice Hd3a, re- spectively, as well as 52% identity to TFL in Arabidopsis. Sequence comparison between NFT1 and the FT-TFL family proteins showed that NFT1 carries the functionally important K2 Storage under natural conditions FT signatures Tyr85(Y)(Tyr79 in NFT1)and Gln 140(Q)(GIn 134 in NFT1)(Fig. 4A)(Hanzawa et al. 2005). In addition, the gene Storage under natural conditions structure of NFT1 was similar to that of FT(Fig. 4B), and NFTI exhibited a region identical to most other FT genes within seg- 30C for 20 d ment B of the fourth exon(encoding an external loop of PEBP (Fig. 4A; Supplementary Fig. S1), which is important to FT vs. TFLI function in Arabidopsis(Ahn et al. 2006). Phylogeny re- constructions with other published FT-TFL genes clearly showed that NFT1 falls into the FT-like subfamily, rather thar the TFL- and MFT-like subfamily(Supplementary Fig. $2) Expression pattern of NFT1 in Chinese narcissus The spatial expression pattern of NFT1 was detected through in situ hybridization analysis. Mature leaf blades, shoot apice from 3-year-old growing plants and fower buds were used Treatments for in situ hybridization. The transcripts of Arabidopsis FT, Fig. 2 High temperature treatment can rescue the low flowering per- rice Hd3a and RFTi were mainly detected in the leaf phloem centage of 3-year-old bulbs caused by a short duration of storage (Tamaki et al. 2007, Komiya et al. 2009). Similarly, hybridization under natural conditions.(A)llustration of the different storage treat- with NFT1 RNA antisense probe revealed signals over vascular ments of narcissus bulbs, with temperature regimes shown below the bundles within transverse leaf sections(Fig 5A, B). The signal 号月已 corresponding date.(B)Effect of different planting dates on the fow- ering percentage of 3-year-old bulbs. Different lower case letters indi. was detected primarily in the phloem, xylem parenchyma and cate significant differences between different treatments(Pearson's muscle cells at high magnification(Fig 5B). NFT1 signal was too x, P<0.05, n=30-50). Detailed temperature regimes in(B low to be detected in the shoot apices of the 3-year-old bulbs shown in(A). during endo-dormancy(ie. from April to early July under nat- ural conditions)(data not shown). However, signal was de- tected in the apices from late July(Fig. 5C)and in early Sufficient high temperature treatment can Rower buds. Gene-specific quantitative real-time PCR improve the flowering rate of 2-year-old bulbs (qRT-PCR)analyses were performed on mRNA samples from Fewer than 25% of the 2-year-old bulbs flowered when stored Rowers, leaves, shoot apices and bulbs during active growth. natural temperature( Fig. 3B). Almost no plants flowered when Strong expression levels of NFT1 were detected in the leaves the 2-year-old bulbs were initially subjected to 30 C for 20d, and shoot apices, whereas low levels were detected in opening with storage at 22-25%C until planting. The maximum flower- flowers and bulbs. The bulbs have several layers of scales, and ing percentage of the bulbs treated at 30 C for 40 d, with stor- the expression levels in scales at different locations did not age at 22-25.C, was 38.6%; whereas the flowering percentage of show any significant difference(Fig. 5D) those subjected to treatment at 30oC for 80d was 86.1%.The esults for the 2-year-old bulbs stored at natural temperature Ectopic expression of NFT1 in A thaliana and those treated at 30C for 40 d, with storage at 22-25oC, advanced flowering were not signifcantly different(Fig. 3B). Obviously, longer Ectopic expression through the 35S promoter provided evi- treatment at high temperature improves the Owering rate of dence that NFT1 encodes a protein that acts as floral regulator 2-year-old bulbs in Arabidopsis. A total of nine lines with the 35S: NFTI con- struct in Col plants [ wild type(WT))and five lines with trans- Isolation of NFTl, a homolog of FT, from Chinese formation of the 355: NFTI construct directly into ft-3 mutant narcissus plants plants were selected for flowering time analysis. No develop Using degenerate primers and the RACE (rapid amplification of mental abnormality, except for flowering time, was caused in DNA ends) method, a full-length cDNA of the NFTI clone, these lines. The flowering time of most lines was significantly comprising the complete coding regions, was isolated. Analysis earlier than that of WT or ft-3 mutant plants under inductive of the sequences showed that NFT1 CDNA was 816 bp long and long-day conditions(Fig. 6A, B, E, F). The lines of the 35S: NFT1 encodes a protein with a predicted length of 174 amino acids. construct in Col flowered 7-14 d earlier and made 5-7 leave The amino acid sequence encoded by NFTI was highly similar fewer than those of Col controls( Fig. 6B). Although the lines of Plant Cell Physiol. 54(2): 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 273Sufficient high temperature treatment can improve the flowering rate of 2-year-old bulbs Fewer than 25% of the 2-year-old bulbs flowered when stored at natural temperature (Fig. 3B). Almost no plants flowered when the 2-year-old bulbs were initially subjected to 30C for 20 d, with storage at 22–25C until planting. The maximum flower￾ing percentage of the bulbs treated at 30C for 40 d, with stor￾age at 22–25C, was 38.6%; whereas the flowering percentage of those subjected to treatment at 30C for 80 d was 86.1%. The results for the 2-year-old bulbs stored at natural temperature and those treated at 30C for 40 d, with storage at 22–25C, were not significantly different (Fig. 3B). Obviously, longer treatment at high temperature improves the flowering rate of 2-year-old bulbs. Isolation of NFT1, a homolog of FT, from Chinese narcissus plants Using degenerate primers and the RACE (rapid amplification of cDNA ends) method, a full-length cDNA of the NFT1 clone, comprising the complete coding regions, was isolated. Analysis of the sequences showed that NFT1 cDNA was 816 bp long and encodes a protein with a predicted length of 174 amino acids. The amino acid sequence encoded by NFT1 was highly similar to that of the FT-like genes in the FT-TFL family genes, with an identity of 70% and 77% to Arabidopsis FT and rice Hd3a, re￾spectively, as well as 52% identity to TFL in Arabidopsis. Sequence comparison between NFT1 and the FT-TFL family proteins showed that NFT1 carries the functionally important FT signatures Tyr85(Y) (Tyr79 in NFT1) and Gln140(Q) (Gln134 in NFT1) (Fig. 4A) (Hanzawa et al. 2005). In addition, the gene structure of NFT1 was similar to that of FT (Fig. 4B), and NFT1 exhibited a region identical to most other FT genes within seg￾ment B of the fourth exon (encoding an external loop of PEBP) (Fig. 4A; Supplementary Fig. S1), which is important to FT vs. TFL1 function in Arabidopsis (Ahn et al. 2006). Phylogeny re￾constructions with other published FT-TFL genes clearly showed that NFT1 falls into the FT-like subfamily, rather than the TFL- and MFT-like subfamily (Supplementary Fig. S2). Expression pattern of NFT1 in Chinese narcissus The spatial expression pattern of NFT1 was detected through in situ hybridization analysis. Mature leaf blades, shoot apices from 3-year-old growing plants and flower buds were used for in situ hybridization. The transcripts of Arabidopsis FT, rice Hd3a and RFT1 were mainly detected in the leaf phloem (Tamaki et al. 2007, Komiya et al. 2009). Similarly, hybridization with NFT1 RNA antisense probe revealed signals over vascular bundles within transverse leaf sections (Fig. 5A, B). The signal was detected primarily in the phloem, xylem parenchyma and muscle cells at high magnification (Fig. 5B). NFT1 signal was too low to be detected in the shoot apices of the 3-year-old bulbs during endo-dormancy (i.e. from April to early July under nat￾ural conditions) (data not shown). However, signal was de￾tected in the apices from late July (Fig. 5C) and in early flower buds. Gene-specific quantitative real-time PCR (qRT-PCR) analyses were performed on mRNA samples from flowers, leaves, shoot apices and bulbs during active growth. Strong expression levels of NFT1 were detected in the leaves and shoot apices, whereas low levels were detected in opening flowers and bulbs. The bulbs have several layers of scales, and the expression levels in scales at different locations did not show any significant difference (Fig. 5D). Ectopic expression of NFT1 in A. thaliana advanced flowering Ectopic expression through the 35S promoter provided evi￾dence that NFT1 encodes a protein that acts as floral regulator in Arabidopsis. A total of nine lines with the 35S::NFT1 con￾struct in Col plants [wild type (WT)] and five lines with trans￾formation of the 35S::NFT1 construct directly into ft-3 mutant plants were selected for flowering time analysis. No develop￾mental abnormality, except for flowering time, was caused in these lines. The flowering time of most lines was significantly earlier than that of WT or ft-3 mutant plants under inductive long-day conditions (Fig. 6A, B, E, F). The lines of the 35S::NFT1 construct in Col flowered 7–14 d earlier and made 5–7 leaves fewer than those of Col controls (Fig. 6B). Although the lines of Fig. 2 High temperature treatment can rescue the low flowering per￾centage of 3-year-old bulbs caused by a short duration of storage under natural conditions. (A) Illustration of the different storage treat￾ments of narcissus bulbs, with temperature regimes shown below the corresponding date. (B) Effect of different planting dates on the flow￾ering percentage of 3-year-old bulbs. Different lower case letters indi￾cate significant differences between different treatments (Pearson’s 2 , P < 0.05, n = 30–50). Detailed temperature regimes in (B) are shown in (A). Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 273 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from