Planta Expression analysis in Pak-choi and Arabidopsis proteins all had a conserved MADS-box domain in the N terminus(Fig.la).Based on our previous transcriptome For cold treatment.1-month-old Pak-choi cultivar wuyue- sequencing data of five developmental stages in three Pak- man plants were transferred to a novel growth chamber, choi cultivars (NHCC001-suzhouging,002-aijiaohuang exposed to 4 C for 0,1,2,3,4 and 5 weeks and harvested and 004-wuyueman)(Song et al.2014),we only found at the same time point.Plants grown in the culture room two FLC homologous genes,CabbageG_a_f_g006153 without vernalization treatment were used as a control. and CabbageG_a_f_g011915.The expression of the two Three biological replications were performed in each sam- genes was higher in the seedling stage and almost unde- ple.For organ-specific expression analysis,the root,stem, tectable in the flowering stage in the three Pak-choi cul- leaf,style,stamen,petal and sepal tissues of the flowering tivars (Table S4).Phylogenetic analysis suggested that Pak-choi cultivar wuyueman were sampled.To investigate CabbageG_a_f_g006153 and CabbageG_a_f_g011915 the changes in downstream gene expression,the seeds of showed high homology to BrFLC2 and BrFLC3 and the transgenic and NC plants were grown on MS medium may have similar roles to those of BrFLC2 and BrFLC3, with 35 mg/L hygromycin and harvested after 15 days.Total respectively (Fig.1b).In addition,BrFLC2 was reported RNA was extracted,reverse-transcribed,and used for gPCR as the key FLC gene based on the previous report(Xiao as described in our previous report(Huang et al.2016).The et al.2013).Thus,we further investigated CabbageG_a_f Pak-choi and Arabidopsis actin genes were used as the inter- g006153 and designated it as BcFLC2.The full-length nal control.Primers for gPCR were designed using Primer 5 cDNA sequence of BcFLC2 is 591-bp long,encoding a and are listed in Table S1. putative protein of 196 amino acids with a molecular mass of 21.9 kDa and a pl of 8.84. Yeast one-hybrid assay For the yeast one-hybrid assay,the Matchmaker Gold Expression pattern of BcFLC2 in Pak-choi Yeast One-Hybrid System was used.The 1000-,1243-, 2000-and 1507-bp promoter sequences of BcTEMI, To investigate whether the BcFLC2 transcript was affected BcSOCI,BcMAF2 and BcSPL15 were inserted into the by vernalization,we performed gPCR to analyze its pAbAi reporter vector to form the bait vectors.The informa- expression pattern in the leaves of the Pak-choi cultivar tion for the promoters of BcTEMI,BcSOCI,BcMAF2 and wuyueman.The expression level of BcFLC2 declined dur- BcSPL15 is shown in Table S3.To detect whether BcFLC2 ing the process of vernalization,suggesting that BcFLC2 could bind to the CArG box in the BcMAF2 promoter,we was repressed by vernalization(Fig.2a).We found that mutated the CArG box in the BcMAF2 promoter using the its expression had a small peak at 2 weeks of treatment. Fast Mutagenesis System (Transgen Biotechnology,Bei- The results indicated that BcFLC2 responded to vernaliza- jing,China).The bait vectors were then integrated into the tion and may play a role in preventing premature flower- yeast genome (strain YIH Gold),separately.The recom- ing under short-term cold exposure.We further detected binant yeast cells were separately plated on SD medium the tissue-specific expression of BcFLC2.BcFLC2 was lacking uracil supplemented with different concentrations expressed in all detected tissues,including the root,stem, of Aureobasidin A(AbA)to select the minimal inhibitory leaf,style,stamen,petal and sepal tissues.BcFLC2 expres- concentration.The full-length ORF of BcFLC2 without the sion was higher in the roots,stems,leaves and stamens termination codon was constructed in the pGADT7 vector. than in other tissues(Fig.2b). The pGADT7-BcFLC2 plasmid was then transformed into bait strains.Transformants were screened by growing them on SD medium lacking leucine supplemented with 300 ng/ Subcellular localization of BcFLC2 protein mL AbA at 30 C for 3 days. The subcellular localization of a protein will help us to understand its possible functions.To examine the subcel- Results lular localization of BcFLC2,the 35S:BcFLC2-GFP and 35S:GFP constructs were transiently introduced into tobacco Molecular characterization of BcFLC2 leaves,separately.The GFP fluorescence of the cells trans- formed with 35S:GFP was detected in both,the nucleus and We identified three FLC homologous genes, the cytoplasm(Fig.3).The GFP fluorescence of the cells CabbageG_a_f_g052019,CabbageG_a_f_g006153 and transformed with 35S:BcFLC2-GFP was co-observed with CabbageG_a_f_g011915,in Pak-choi.Alignment of pro- DAPI in the nucleus,indicating that BcFLC2 is a nuclear tein sequences revealed that these three FLC homologous protein similar to other transcription factors. SpringerPlanta 1 3 Expression analysis in Pak‑choi and Arabidopsis For cold treatment, 1-month-old Pak-choi cultivar wuyue￾man plants were transferred to a novel growth chamber, exposed to 4 °C for 0, 1, 2, 3, 4 and 5 weeks and harvested at the same time point. Plants grown in the culture room without vernalization treatment were used as a control. Three biological replications were performed in each sam￾ple. For organ-specifc expression analysis, the root, stem, leaf, style, stamen, petal and sepal tissues of the fowering Pak-choi cultivar wuyueman were sampled. To investigate the changes in downstream gene expression, the seeds of the transgenic and NC plants were grown on MS medium with 35 mg/L hygromycin and harvested after 15 days. Total RNA was extracted, reverse-transcribed, and used for qPCR as described in our previous report (Huang et al. 2016). The Pak-choi and Arabidopsis actin genes were used as the inter￾nal control. Primers for qPCR were designed using Primer 5 and are listed in Table S1. Yeast one‑hybrid assay For the yeast one-hybrid assay, the Matchmaker® Gold Yeast One-Hybrid System was used. The 1000-, 1243-, 2000- and 1507-bp promoter sequences of BcTEM1, BcSOC1, BcMAF2 and BcSPL15 were inserted into the pAbAi reporter vector to form the bait vectors. The informa￾tion for the promoters of BcTEM1, BcSOC1, BcMAF2 and BcSPL15 is shown in Table S3. To detect whether BcFLC2 could bind to the CArG box in the BcMAF2 promoter, we mutated the CArG box in the BcMAF2 promoter using the Fast Mutagenesis System (Transgen Biotechnology, Bei￾jing, China). The bait vectors were then integrated into the yeast genome (strain Y1H Gold), separately. The recom￾binant yeast cells were separately plated on SD medium lacking uracil supplemented with diferent concentrations of Aureobasidin A (AbA) to select the minimal inhibitory concentration. The full-length ORF of BcFLC2 without the termination codon was constructed in the pGADT7 vector. The pGADT7-BcFLC2 plasmid was then transformed into bait strains. Transformants were screened by growing them on SD medium lacking leucine supplemented with 300 ng/ mL AbA at 30 °C for 3 days. Results Molecular characterization of BcFLC2 We identified three FLC homologous genes, CabbageG_a_f_g052019, CabbageG_a_f_g006153 and CabbageG_a_f_g011915, in Pak-choi. Alignment of pro￾tein sequences revealed that these three FLC homologous proteins all had a conserved MADS-box domain in the N terminus (Fig. 1a). Based on our previous transcriptome sequencing data of fve developmental stages in three Pak￾choi cultivars (NHCC001-suzhouqing, 002-aijiaohuang and 004-wuyueman) (Song et al. 2014), we only found two FLC homologous genes, CabbageG_a_f_g006153 and CabbageG_a_f_g011915. The expression of the two genes was higher in the seedling stage and almost unde￾tectable in the fowering stage in the three Pak-choi cul￾tivars (Table S4). Phylogenetic analysis suggested that CabbageG_a_f_g006153 and CabbageG_a_f_g011915 showed high homology to BrFLC2 and BrFLC3 and may have similar roles to those of BrFLC2 and BrFLC3, respectively (Fig. 1b). In addition, BrFLC2 was reported as the key FLC gene based on the previous report (Xiao et al. 2013). Thus, we further investigated CabbageG_a_f_ g006153 and designated it as BcFLC2. The full-length cDNA sequence of BcFLC2 is 591-bp long, encoding a putative protein of 196 amino acids with a molecular mass of 21.9 kDa and a pI of 8.84. Expression pattern of BcFLC2 in Pak‑choi To investigate whether the BcFLC2 transcript was afected by vernalization, we performed qPCR to analyze its expression pattern in the leaves of the Pak-choi cultivar wuyueman. The expression level of BcFLC2 declined dur￾ing the process of vernalization, suggesting that BcFLC2 was repressed by vernalization (Fig. 2a). We found that its expression had a small peak at 2 weeks of treatment. The results indicated that BcFLC2 responded to vernaliza￾tion and may play a role in preventing premature fower￾ing under short-term cold exposure. We further detected the tissue-specifc expression of BcFLC2. BcFLC2 was expressed in all detected tissues, including the root, stem, leaf, style, stamen, petal and sepal tissues. BcFLC2 expres￾sion was higher in the roots, stems, leaves and stamens than in other tissues (Fig. 2b). Subcellular localization of BcFLC2 protein The subcellular localization of a protein will help us to understand its possible functions. To examine the subcel￾lular localization of BcFLC2, the 35S:BcFLC2-GFP and 35S:GFP constructs were transiently introduced into tobacco leaves, separately. The GFP fuorescence of the cells trans￾formed with 35S:GFP was detected in both, the nucleus and the cytoplasm (Fig. 3). The GFP fuorescence of the cells transformed with 35S:BcFLC2-GFP was co-observed with DAPI in the nucleus, indicating that BcFLC2 is a nuclear protein similar to other transcription factors