NFT1 involved in flowering initiation induced by heating Shanghai are 20.8, 25.0, 29. 2, 28.5 and 251@C in May, June, July, NFT-A28(5-CTACACCCTGGTGATGGTRGAYCCNGA-3. A August and September, respectively. The highest and lowest partial cDNA clone was isolated from Chinese narcissus emperatures are shown in Fig. 1B. BLAST searches in public databases, with the conceptual Three-year-old bulbs were stored under natural conditions amino acid sequence of the clone as query sequences, and planted on different dates, specifically, July 25, August 15 showed that the clone shared high sequence identity with and September 1(Nos CK1, CK2 and CK3 in Fig. 2A). The FT-like genes from various plants, and thus was named NFTI percentages of the flowering plants were recorded to analyze(Narcissus FT-1). The 3 and 5 regions corresponding to the e effects of different planting dates on flowering. The per- DNAs of NFTI were isolated using the RACE method(Roche centages of the flowering plants treated at 30C for 20 d stored Diagnostics) cDNA and dna clones with a complete open under natural conditions and planted on July 25(No. 1 in reading frame were isolated via PCR, with primers located in Fig 1A)were compared with those stored under natural con- the 5 and 3 regions ditions. The experiment was repeated three times: in 2006, 2007 d2008 Plant transformation and transgenic plant analysis Two-year-old bulbs were initially subjected to high tempera- An EcoRI fragment containing the full-length cDNA for the ture(30 C)for 20, 40 and 80 d, and then stored between 22 and NFT1 gene was introduced into the binary T-DNA vector 25 C until planting to determine the effects of different high pMon530(Monsanto), driven by the 35S promoter. Genet temperature durations on fowering. The results for the transformation lines were obtained, as previously reported(Li 2-year-old bulbs stored at natural temperature and with treat- et al. 2009). The phenotypic effects of NFT1 in transgenic Col ment at 30 C for 40d, with storage from 22 to 25C(Nos 1-4 plants were analyzed in nine independent transgenic lines. Five in Fig 3A), were also compared to test the effect of different independent lines were created by directly transforming the temperatures on Lowering. The experiment was conducted in 35S: NFT1 construct into ft-3 mutant plants, and were selected triplicate with independent materials and was repeated twice, for flowering time analy in2010and2011 Temperature-controlled incubators were used for different Sequence alignments 88号月 temperature treatments. The samples in each treatment con- Predicted amino acid sequences were used for phylogenetic sisted of at least 40 bulbs, unless otherwise stated. The detailed analysis. Protein sequences were aligned using Clustal w were planted in a controlled greenhouse with a short photo- (http://www.clustalorg/),thenmanuallyadjusted period (10 h/14 h light/dark) at 18-20oC. The growing condi- RT-PCR tions and planting order were previously described (Li et al 2012) Total RNA from different tissues was extracted from frozen tissue using TRIZOL reagent(Invitrogen) and purifed on Scanning electron microscopy RNeasy columns(QIAGEN). The first CDNA strand was gener ated according to the instructions for the Superscript RT SMs of 3-year-old bulbs were collected weekly after their har- (Toyobo ). PCR amplification was performed with gene-specific vest. Apices were dissected under an anatomical lens as rapidly primers,NFT (5-TAGCCCAAGTGACCCAAACT-3 )and as possible, and fixed in formaldehyde-acetic acid(cor NFT R2(5-GGACTCCACCCCCAAATTAT-3) ACTIN ex of 63% ethanol, 5% formaldehyde and 6% acetic acid) at 4"c sion levels were monitored to serve as a quantifying control, overnight. Afterwards, the samples were dehydrated in an etha- and the experiments were repeated at least three times ol series, critical-point dried in liquid COz, sputter-coated with qRT-PCR was performed with 3-6 independent biological gold-palladium,analyzed, and photographed with a Philips XL replicates and three technical replicates for each sampleData 30 FEG SEM. Five to 10 samples of each treatment were de- were analyzed via the tected at each time point, and the number of samples at dif- Schmittgen and Livak( 2008). Statistical significance between ferent development stages was analyzed any pair of relative expression means was determined by a t-test. The experiment was repeated with samples from another Cloning of NFT1 cDNAs year with a similar result. Partial cDNAs were isolated from younger inflorescences of Chinese narcissus using degenerate primers according to the In situ hybridization consensus-degenerate hybrid oligonucleotide primer method Samples were fixed in 4%(v/v) paraformaldehyde and 0.5%(v/v) (Rose et al. 1998, Rose et al. 2003, Staheli et al. 2011). The glutaraldehyde for 24 h at 4oC. Then samples were dehydrated first PCR was conducted using a pair of degenerated primers, through an ethanol series, embedded in Histoplast and sectioned NFT-A64(5-GCCGGGGGCGTANACNGTYTG-3)and NFT- at 7 um using a rotary microtome. a probe for NFTI was ampli- A20(5-GCACTGGCTGGTGACNGATATHCC-3) Nested PCr fied with primers 5-gaattcATGAGTAGGGATCCTTTGGTT-3 as performed using another pair of degenerate primers, and 5-gaattcTCATCATTAGGGGTACATCCTC-3. Digoxigenin- NFT-A65 (5-CCAGCCGGGGGCRTANACNGTYT-3) and labeled sense and antisense rna probes were synthesize Plant Cell Physiol. 54(2) 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 279Shanghai are 20.8, 25.0, 29.2, 28.5 and 25.1C in May, June, July, August and September, respectively. The highest and lowest temperatures are shown in Fig. 1B. Three-year-old bulbs were stored under natural conditions and planted on different dates, specifically, July 25, August 15 and September 1 (Nos. CK1, CK2 and CK3 in Fig. 2A). The percentages of the flowering plants were recorded to analyze the effects of different planting dates on flowering. The per￾centages of the flowering plants treated at 30C for 20 d stored under natural conditions and planted on July 25 (No. 1 in Fig. 1A) were compared with those stored under natural con￾ditions. The experiment was repeated three times: in 2006, 2007 and 2008. Two-year-old bulbs were initially subjected to high tempera￾ture (30C) for 20, 40 and 80 d, and then stored between 22 and 25C until planting to determine the effects of different high temperature durations on flowering. The results for the 2-year-old bulbs stored at natural temperature and with treat￾ment at 30C for 40 d, with storage from 22 to 25C (Nos. 1–4 in Fig. 3A), were also compared to test the effect of different temperatures on flowering. The experiment was conducted in triplicate with independent materials and was repeated twice, in 2010 and 2011. Temperature-controlled incubators were used for different temperature treatments. The samples in each treatment con￾sisted of at least 40 bulbs, unless otherwise stated. The detailed methods are illustrated in Figs. 1–3. After treatment, all bulbs were planted in a controlled greenhouse with a short photo￾period (10 h/14 h light/dark) at 18–20C. The growing condi￾tions and planting order were previously described (Li et al. 2012). Scanning electron microscopy SMs of 3-year-old bulbs were collected weekly after their har￾vest. Apices were dissected under an anatomical lens as rapidly as possible, and fixed in formaldehyde–acetic acid (composed of 63% ethanol, 5% formaldehyde and 6% acetic acid) at 4C overnight. Afterwards, the samples were dehydrated in an etha￾nol series, critical-point dried in liquid CO2, sputter-coated with gold–palladium, analyzed, and photographed with a Philips XL 30 FEG SEM. Five to 10 samples of each treatment were de￾tected at each time point, and the number of samples at dif￾ferent development stages was analyzed. Cloning of NFT1 cDNAs Partial cDNAs were isolated from younger inflorescences of Chinese narcissus using degenerate primers according to the consensus-degenerate hybrid oligonucleotide primer method (Rose et al. 1998, Rose et al. 2003, Staheli et al. 2011). The first PCR was conducted using a pair of degenerated primers, NFT-A64 (50 -GCCGGGGGCGTANACNGTYTG-30 ) and NFT￾A20 (50 -GCACTGGCTGGTGACNGATATHCC-30 ). Nested PCR was performed using another pair of degenerate primers, NFT-A65 (50 -CCAGCCGGGGGCRTANACNGTYT-30 ) and NFT-A28 (50 -CTACACCCTGGTGATGGTRGAYCCNGA-30 ). A partial cDNA clone was isolated from Chinese narcissus. BLAST searches in public databases, with the conceptual amino acid sequence of the clone as query sequences, showed that the clone shared high sequence identity with FT-like genes from various plants, and thus was named NFT1 (Narcissus FT-1). The 30 and 50 regions corresponding to the cDNAs of NFT1 were isolated using the RACE method (Roche Diagnostics). cDNA and DNA clones with a complete open reading frame were isolated via PCR, with primers located in the 50 and 30 regions. Plant transformation and transgenic plant analysis An EcoRI fragment containing the full-length cDNA for the NFT1 gene was introduced into the binary T-DNA vector pMon530 (Monsanto), driven by the 35S promoter. Genetic transformation lines were obtained, as previously reported (Li et al. 2009). The phenotypic effects of NFT1 in transgenic Col plants were analyzed in nine independent transgenic lines. Five independent lines were created by directly transforming the 35S::NFT1 construct into ft-3 mutant plants, and were selected for flowering time analysis. Sequence alignments Predicted amino acid sequences were used for phylogenetic analysis. Protein sequences were aligned using Clustal W (http://www.clustal.org/), then manually adjusted. RT-PCR Total RNA from different tissues was extracted from frozen tissue using TRIZOL reagent (Invitrogen) and purified on RNeasy columns (QIAGEN). The first cDNA strand was gener￾ated according to the instructions for the Superscript RT (Toyobo). PCR amplification was performed with gene-specific primers, NFT F2 (50 -TAGCCCAAGTGACCCAAACT-30 ) and NFT R2 (50 -GGACTCCACCCCCAAATTAT-30 ). ACTIN expres￾sion levels were monitored to serve as a quantifying control, and the experiments were repeated at least three times. qRT-PCR was performed with 3–6 independent biological replicates and three technical replicates for each sample. Data were analyzed via the 2–Ct method, as described by Schmittgen and Livak (2008). Statistical significance between any pair of relative expression means was determined by a t-test. The experiment was repeated with samples from another year with a similar result. In situ hybridization Samples were fixed in 4% (v/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde for 24 h at 4C. Then samples were dehydrated through an ethanol series, embedded in Histoplast and sectioned at 7 mm using a rotary microtome. A probe for NFT1 was ampli- fied with primers 50 -gaattcATGAGTAGGGATCCTTTGGTT-30 and 50 -gaattcTCATCATTAGGGGTACATCCTC-30 . Digoxigenin￾labeled sense and antisense RNA probes were synthesized Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 279 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from