Planta were isolated from the leaf cDNA of the Pak-choi cultivar Generation of BcFLC2-overexpressing Arabidopsis wuyueman with three pairs of primers-BcFLC1-S and lines BcFLC1-A.BcFLC2-S and BcFLC2-A.and BcFLC3- S and BcFLC3-A-based on homology cloning.The Arabidopsis(Col-0)was transformed with Agrobacterium primers were designed based on the BcFLC homologue tumefaciens (strain GV3101)harboring 35S:BcFLC2- genes Bra009055(BrFLCI),Bra028599(BrFLC2)and GFP or 35S:GFP(negative control,NC)using the floral Bra006051(BrFLC3).Then,the PCR products were dip method(Clough and Bent 1998).The seeds of the To cloned into the pMD18-T vector before sequencing.The transgenic Arabidopsis were sowed on 1/2 MS medium amino acid sequences of BcFLCs and the other FLCs containing 35 mg/L hygromycin for selection.Four from Arabidopsis and Brassica rapa were used for phy- transgenic Arabidopsis lines were obtained (#1,#2,#3 logenetic analysis.The protein sequences were obtained and #4).To confirm the presence of BcFLC2 in the four from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). transgenic Arabidopsis lines,we isolated cDNA from the Phylogenetic analysis and multiple sequence alignment seedlings of the NC and BcFLC2-overexpressing lines. were performed according to our previous report (Huang The 35S:BcFLC2-GFP plasmid was used as the positive etal.2016). control(PC).Then,PCR was performed using a pair of The open reading frame (ORF)sequences of BcSOCl, specific primers(O1 and 02).However,seeds were only BcSPL15,BcTEMI and BcMAF2 were also obtained obtained from two positive lines (#1 and #3),and thus, according to the above methods.The genomic DNA of two T3 homozygous transgenic lines were used for sub- the Pak-choi cultivar wuyueman was isolated with the sequent experiments.The days from sowing to opening Plant Genomic DNA Kit(Tiangen,Beijing,China).The of the first flower were counted.The number of rosette genomic sequences of BcSOCI,BcSPL15,BcTEMI leaves was counted at the time of bolting.Each experiment and BcMAF2 were cloned using four pairs of primers- was calculated from 30 plants.Values are expressed as the BcSOCl-S and BcSOC1-A.BcSPL15-S and BcSPL15- means+standard deviation.The differences between the A,BcTEMI-S and BcTEM1-A.and BcMAF2-S and lines were separated using the least significant difference BcMAF2-A-from genomic DNA.Based on the genomic (LSD)test at P<0.01. sequences,the predicted promoter regions were ampli- fied using Self-Formed Adaptor(SEFA)PCR using a KX Genome Walking Kit (Zoman Biotechnology,Bei- Virus-induced gene silencing(VIGS)in Pak-choi jing,China).The predicted promoter region of BcCO was for silencing BcFLC2 amplified using the same method.The primers used in the study are listed in Table S1.The CArG boxes in the pro- A specific 40-bp fragment of the BcFLC2 coding region moters of BcSOCI.BCSPL15.BCTEMI and BcMAF2 were and its antisense sequence were synthesized and inserted analyzed using Softberry (http://www.softberry.com/). into the pTY-S(pTY)vector of the turnip yellow mosaic virus-induced gene silencing (TYMV-VIGS)system to form a BcFLC2-silencing construct by the company (Gen- Subcellular localization Script,Nanjing,China)(Pflieger et al.2008).pTY-BcPDS was constructed to examine the efficiency of the silencing The full-length ORF of BcFLC2 without the termination protocol in the Pak-choi seedlings.The empty pTY plasmid codon was obtained by PCR using the primers Ol and was used as the NC.The sequences of oligonucleotides O2.The target fragment and linear pCambia 1302 vector used for VIGS are listed in Table S2.The 2-week-old Pak- (35S:GFP)with Xbal and BamHI were purified using a choi cultivar 49caixin plants,which usually bolt at 8 weeks double enzyme digestion reaction.The recombinant fusion and do not require vernalization,were used for VIGS.The vector 1302-BcFLC2(35S:BcFLC2-GFP)was generated pTY,pTY-BcPDS and pTY-BcFLC2 plasmids(5 ug)coated by ligation using a DNA ligation kit (Takara,Beijing, on gold particles were bombarded into 4-5 Pak-choi plants China).35S:GFP was used as a control.The 35S:BcFLC2- using particle gun bombardment(Bio-Rad,PDS1000/He) GFP and 35S:GFP plasmids were separately transformed based on the previous protocol with some modification into Agrobacterium tumefaciens (strain GV3101)by (Hamada et al.2017).Three biological replicates were electroporation for transformation of the tobacco leaves performed.Three weeks later,leaves showing virus symp- (Zhang et al.2012).DAPI(nucleus specific dye)was used toms were sampled for detection.Two BcFLC2-silencing to stain the nuclei.After incubation for 48 h at 25 C,GFP Pak-choi plants,pTY-BcFLC2-3 and pTY-BcFLC2-4,were in tobacco leaves was detected using confocal microscopy confirmed by qPCR and used for the following experi- (Leica,TCS SP2,Wetzlar,Germany). ments.The days from sowing to the time of bolting were counted. ②SpringerPlanta 1 3 were isolated from the leaf cDNA of the Pak-choi cultivar wuyueman with three pairs of primers—BcFLC1-S and BcFLC1-A, BcFLC2-S and BcFLC2-A, and BcFLC3- S and BcFLC3-A—based on homology cloning. The primers were designed based on the BcFLC homologue genes Bra009055 (BrFLC1), Bra028599 (BrFLC2) and Bra006051 (BrFLC3). Then, the PCR products were cloned into the pMD18-T vector before sequencing. The amino acid sequences of BcFLCs and the other FLCs from Arabidopsis and Brassica rapa were used for phy￾logenetic analysis. The protein sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Phylogenetic analysis and multiple sequence alignment were performed according to our previous report (Huang et al. 2016). The open reading frame (ORF) sequences of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 were also obtained according to the above methods. The genomic DNA of the Pak-choi cultivar wuyueman was isolated with the Plant Genomic DNA Kit (Tiangen, Beijing, China). The genomic sequences of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 were cloned using four pairs of primers— BcSOC1-S and BcSOC1-A, BcSPL15-S and BcSPL15- A, BcTEM1-S and BcTEM1-A, and BcMAF2-S and BcMAF2-A—from genomic DNA. Based on the genomic sequences, the predicted promoter regions were ampli￾fied using Self-Formed Adaptor (SEFA) PCR using a KX Genome Walking Kit (Zoman Biotechnology, Bei￾jing, China). The predicted promoter region of BcCO was amplifed using the same method. The primers used in the study are listed in Table S1. The CArG boxes in the pro￾moters of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 were analyzed using Softberry (http://www.softberry.com/). Subcellular localization The full-length ORF of BcFLC2 without the termination codon was obtained by PCR using the primers O1 and O2. The target fragment and linear pCambia 1302 vector (35S:GFP) with XbaI and BamHI were purifed using a double enzyme digestion reaction. The recombinant fusion vector 1302-BcFLC2 (35S:BcFLC2-GFP) was generated by ligation using a DNA ligation kit (Takara, Beijing, China). 35S:GFP was used as a control. The 35S:BcFLC2- GFP and 35S:GFP plasmids were separately transformed into Agrobacterium tumefaciens (strain GV3101) by electroporation for transformation of the tobacco leaves (Zhang et al. 2012). DAPI (nucleus specifc dye) was used to stain the nuclei. After incubation for 48 h at 25 °C, GFP in tobacco leaves was detected using confocal microscopy (Leica, TCS SP2, Wetzlar, Germany). Generation of BcFLC2‑overexpressing Arabidopsis lines Arabidopsis (Col-0) was transformed with Agrobacterium tumefaciens (strain GV3101) harboring 35S:BcFLC2- GFP or 35S:GFP (negative control, NC) using the foral dip method (Clough and Bent 1998). The seeds of the T0 transgenic Arabidopsis were sowed on 1/2 MS medium containing 35  mg/L hygromycin for selection. Four transgenic Arabidopsis lines were obtained (#1, #2, #3 and #4). To confrm the presence of BcFLC2 in the four transgenic Arabidopsis lines, we isolated cDNA from the seedlings of the NC and BcFLC2-overexpressing lines. The 35S:BcFLC2-GFP plasmid was used as the positive control (PC). Then, PCR was performed using a pair of specifc primers (O1 and O2). However, seeds were only obtained from two positive lines (#1 and #3), and thus, two T3 homozygous transgenic lines were used for sub￾sequent experiments. The days from sowing to opening of the frst fower were counted. The number of rosette leaves was counted at the time of bolting. Each experiment was calculated from 30 plants. Values are expressed as the means±standard deviation. The diferences between the lines were separated using the least signifcant diference (LSD) test at P<0.01. Virus‑induced gene silencing (VIGS) in Pak‑choi for silencing BcFLC2 A specifc 40-bp fragment of the BcFLC2 coding region and its antisense sequence were synthesized and inserted into the pTY-S (pTY) vector of the turnip yellow mosaic virus-induced gene silencing (TYMV-VIGS) system to form a BcFLC2-silencing construct by the company (Gen￾Script, Nanjing, China) (Pfieger et al. 2008). pTY-BcPDS was constructed to examine the efciency of the silencing protocol in the Pak-choi seedlings. The empty pTY plasmid was used as the NC. The sequences of oligonucleotides used for VIGS are listed in Table S2. The 2-week-old Pak￾choi cultivar 49caixin plants, which usually bolt at 8 weeks and do not require vernalization, were used for VIGS. The pTY, pTY-BcPDS and pTY-BcFLC2 plasmids (5 μg) coated on gold particles were bombarded into 4–5 Pak-choi plants using particle gun bombardment (Bio-Rad, PDS1000/He) based on the previous protocol with some modifcation (Hamada et al. 2017). Three biological replicates were performed. Three weeks later, leaves showing virus symp￾toms were sampled for detection. Two BcFLC2-silencing Pak-choi plants, pTY-BcFLC2-3 and pTY-BcFLC2-4, were confrmed by qPCR and used for the following experi￾ments. The days from sowing to the time of bolting were counted