《园艺作物育种学》课程教学资源（学术研究）Isolation and functional characterization of a foral repressor, BcFLC2, from Pak‑choi（Brassica rapa ssp. chinensis）

Planta https:/doi.org/10.1007/s00425-018-2891-0 ORIGINAL ARTICLE CrossMark Isolation and functional characterization of a floral repressor,BcFLC2, from Pak-choi (Brassica rapa ssp.chinensis) Feiyi Huang'.Tongkun Liu1.Jin Wang'.Xilin Hou Received:8 November 2017/Accepted:5 April 2018 Springer-Verlag GmbH Germany,part of Springer Nature 2018 Abstract Main conclusion BcFLC2 functioned as a repressor of flowering by directly regulating BcTEMI,BcMAF2,BcSOCI and BcSPL15 in Pak-choi. FLOWERING LOCUS C(FLC)plays an important role in regulating flowering time.Here,we functionally described an FLC homologous gene,BcFLC2,that negatively regulated flowering in Pak-choi(Brassica rapa ssp.chinensis).The sequence comparison to Arabidopsis FLC showed that BcFLC2 also had a MADS-box domain at the N terminus.BcFLC2 was highly expressed in the leaves,roots,stems and stamens,and its expression was repressed by vernalization in Pak-choi.Interestingly, BcFLC2 expression exhibited a small peak at 2 weeks of vernalization treatment,suggesting that BcFLC2 may be involved in preventing premature flowering under short-term cold exposure in Pak-choi,which is different from the AtFLC expression pattern.Overexpression of BcFLC2 in Arabidopsis caused late flowering,while silencing of BcFLC2 in Pak-choi caused early flowering.BcFLC2 localized to the cell nucleus and functioned as a transcription factor.Yeast one-hybrid analysis revealed that BcFLC2 could bind to the promoters of Pak-choi Tempranillo I(BcTEMI),SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1(BcSOC1),SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15(BCSPL15)and MADS AFFECT- ING FLOWERING 2(BcMAF2).Taken together,the present results suggested that BcFLC2 played a key role in flowering regulation as a negative regulator by controlling BcTEMI,BcMAF2,BcSOCI and BcSPL15 expression. Keywords FLOWERING LOCUS C 2.Flowering time.Late flowering.MADS AFFECTING FLOWERING 2.Short- term cold exposure.TEMPRANILLO I.Vernalization Abbreviations PDS Phytoene desaturase AbA Aureobasidin A SPL15 SQUAMOSA PROMOTER BINDING PROTEIN- FLC FLOWERING LOCUS C LIKE 15 FT FLOWERING LOCUS T SOCI SUPPRESSOR OF OVEREXPRESSION OF MAF MADS AFFECTING FLOWERING CONSTANS I NC Negative control TEM1 TEMPRANILLO I Feiyi Huang and Tongkun Liu contributed equally to this work. Introduction Electronic supplementary material The online version of this Flowering is an essential process in the life cycle of higher article(https://doi.org/10.1007/s00425-018-2891-0)contains supplementary material,which is available to authorized users. plants so that plants can switch from the vegetative to repro- ductive phase,which is vital to agricultural production.It ☒Xilin Hou is a complex process determined by multiple environmen- hxl@njau.edu.cn tal and developmental signals,such as temperature,light State Key Laboratory of Crop Genetics and Germplasm and phytohormonal levels,which all ensure that flowering Enhancement/Key Laboratory of Biology and Germplasm occurs at the appropriate time (Boss et al.2004).In Arabi- Enhancement of Horticultural Crops in East China,Ministry dopsis,floral induction is mainly regulated by four pathways, of Agriculture,College of Horticulture.Nanjing Agricultural namely the photoperiod-,vernalization-,gibberellin-and University,Nanjing 210095,China Published online:14 May 2018 ②Springer

Vol.:(0123456789) 1 3 Planta https://doi.org/10.1007/s00425-018-2891-0 ORIGINAL ARTICLE Isolation and functional characterization of a foral repressor, BcFLC2, from Pak‑choi (Brassica rapa ssp. chinensis) Feiyi Huang1  · Tongkun Liu1  · Jin Wang1  · Xilin Hou1 Received: 8 November 2017 / Accepted: 5 April 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Main conclusion BcFLC2 functioned as a repressor of fowering by directly regulating BcTEM1, BcMAF2, BcSOC1 and BcSPL15 in Pak-choi. FLOWERING LOCUS C (FLC) plays an important role in regulating fowering time. Here, we functionally described an FLC homologous gene, BcFLC2, that negatively regulated fowering in Pak-choi (Brassica rapa ssp. chinensis). The sequence comparison to Arabidopsis FLC showed that BcFLC2 also had a MADS-box domain at the N terminus. BcFLC2 was highly expressed in the leaves, roots, stems and stamens, and its expression was repressed by vernalization in Pak-choi. Interestingly, BcFLC2 expression exhibited a small peak at 2 weeks of vernalization treatment, suggesting that BcFLC2 may be involved in preventing premature fowering under short-term cold exposure in Pak-choi, which is diferent from the AtFLC expression pattern. Overexpression of BcFLC2 in Arabidopsis caused late fowering, while silencing of BcFLC2 in Pak-choi caused early fowering. BcFLC2 localized to the cell nucleus and functioned as a transcription factor. Yeast one-hybrid analysis revealed that BcFLC2 could bind to the promoters of Pak-choi Tempranillo 1 (BcTEM1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BcSOC1), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (BcSPL15) and MADS AFFECT￾ING FLOWERING 2 (BcMAF2). Taken together, the present results suggested that BcFLC2 played a key role in fowering regulation as a negative regulator by controlling BcTEM1, BcMAF2, BcSOC1 and BcSPL15 expression. Keywords FLOWERING LOCUS C 2 · Flowering time · Late fowering · MADS AFFECTING FLOWERING 2 · Short￾term cold exposure · TEMPRANILLO 1 · Vernalization Abbreviations AbA Aureobasidin A FLC FLOWERING LOCUS C FT FLOWERING LOCUS T MAF MADS AFFECTING FLOWERING NC Negative control PDS Phytoene desaturase SPL15 SQUAMOSA PROMOTER BINDING PROTEIN￾LIKE 15 SOC1 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 TEM1 TEMPRANILLO 1 Introduction Flowering is an essential process in the life cycle of higher plants so that plants can switch from the vegetative to repro￾ductive phase, which is vital to agricultural production. It is a complex process determined by multiple environmen￾tal and developmental signals, such as temperature, light and phytohormonal levels, which all ensure that fowering occurs at the appropriate time (Boss et al. 2004). In Arabi￾dopsis, foral induction is mainly regulated by four pathways, namely the photoperiod-, vernalization-, gibberellin- and Feiyi Huang and Tongkun Liu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00425-018-2891-0) contains supplementary material, which is available to authorized users. * Xilin Hou hxl@njau.edu.cn 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Planta autonomous-dependent pathways.The four pathways reg- (Halevy 1989).In Brassica species,two major QTLs related ulate two flowering pathway integrators,FLOWERING to vernalization-responsive flowering time are collinear with LOCUS T(FT)and SOCI,which act prior to the activa- Arabidopsis FRI and FLC,suggesting that the homologs of tion of floral meristem identity genes to determine the exact FLC and FRI may play vital roles in flowering time dur- flowering time (Fujiwara et al.2003).These two integrators ing the process of vernalization in other Brassica species are antagonistically regulated by two upstream regulators: (Osborn et al.1997).Although numerous studies have FLC acts as a negative regulator,and CO acts as a positive reported the roles of FLC in some Brassica species,such regulator of flowering (Lee et al.2000;Samach et al.2000). as Brassica napus (Tadege et al.2001)and Brassica olera- Exposure to cold winter conditions renders biennial and cea(Schranz et al.2002).the regulation mechanism of FLC winter-annual plants responsive to the inductive photoper- in Pak-choi (Brassica rapa ssp.chinensis)is still not well iod,resulting in flowering in the spring,which is known as understood.Pak-choi,belonging to the B.rapa family,is one vernalization (Reeves and Coupland 2000).The vernaliza- of the most important vegetable crops and is widely culti- tion requirement mainly results from FLC and FR/GIDA vated in Asia(Huang et al.2016).Wuyueman was used in (FR/)(Michaels and Amasino 1999:Johanson and Dean this work,which normally requires 5 weeks of vernalization 2000).FLC encodes a MADS-box transcription factor and treatment(4C)and which flowers later than other cultivars. functions as a major repressor of flowering.FLC represses Here,we performed a series of studies,including sequence, SOCI,SPL15 and FT by directly binding to the promoters expression,subcellular localization,ectopic expression, of SOC/and SPL/5 or the first intron of FT(Helliwell et al. silencing and downstream gene analysis of BcFLC2 in Pak- 2006;Searle et al.2006).Arabidopsis ecotypes with high- choi.The results showed that BcFLC2 was repressed by ver- abundance FLC are usually late flowering,whereas ecotypes nalization and highly expressed in the leaves,roots,stems with low-abundance FLC are early flowering (Lister et al. and stamens.BcFLC2 encoded a MADS-box transcription 2005).Overexpression of FLC shows a late-flowering phe- factor and regulated the expression of BcSOCI,BcSPL15, notype in Arabidopsis and Brassica rapa (Kim et al.2007). BCMAF2,and BcTEMI by directly binding to their promot- FLC expression is activated by FRI and suppressed by the ers.Overexpression of BcFLC2 in Arabidopsis caused a late- vernalization and autonomous pathways (Choi et al.2011). flowering phenotype,while silencing of BcFLC2 in Pak-choi Repression of FLC by vernalization is initially regulated caused an early flowering phenotype.Our results indicated by VERNALIZATION INSENSITIVE 3(VIN3),encod- that BcFLC2 functioned as a flowering repressor by directly ing a PHD-domain protein that interacts with the member inhibiting BcSOCI and BcSPL15 and activating BcTEMI of the polycomb repressive complex 2(PRC2)(De Lucia and BcMAF2 in Pak-choi. et al.2008).PRC2 complexes catalyze the methylation of H3K27 at the FLC locus to promote an inactive chromatin state (Sung and Amasino 2004).In addition to VIN3,VER- Materials and methods NALIZATION1 (VRN1)and VRN2 are required to maintain the silenced state of FLC(Bastow et al.2004).Recent stud- Plant materials ies revealed that long non-coding RNAs are also involved in the epigenetic repression of FLC.COOLAIR enhances Arabidopsis wild type(WT)(obtained from Prof.Isabelle the down-regulation of FLC expression(Swiezewski et al. Jupin,University Paris 7,France)and BcFLC2-overex- 2009).and COLDAIR helps to recruit PRC2 to the FLC pressing seedlings were Col-0 ecotype background.Pak- locus(Heo and Sung 2011).This epigenetic repression can choi (Brassica rapa ssp.chinensis)cultivars wuyueman and be maintained through cell division after vernalization treat- 49caixin,kept at Nanjing Agricultural University,and all ment,which can only be "forgotten"in the next generation Arabidopsis plants were grown in plastic trays with a growth (Finnegan and Dennis 2007).MADS-box transcription fac- medium(vermiculite:loam,1:2,v:v)in a culture room under tors typically act as multimers with other MADS-domain long-day conditions (16 h light at 22 C/8 h dark at 18 C). proteins(de Folter et al.2005).SVP,another MADS-domain To extract mesophyll protoplasts,WT Arabidopsis seedlings protein,interacts directly with FLC.Research has shown that were grown under short-day conditions with an 8/16 h light/ the FLC-SVP complex directly binds to the FT and SOCI dark cycle for 1 month. to repress their expression (Li et al.2008).The loss of SVP partially suppresses FLC-mediated flowering inhibition and Cloning and sequence analysis does not influence flowering as greatly as the loss of FLC, and thus,there may be some redundancy in SVP function RNA extraction and cDNA synthesis were performed (Lee et al.2007). according to our previous report(Huang et al.2016). In addition to Arabidopsis,other members of Brassi- BcFLCI,BcFLC2 and BcFLC3(CabbageG_a_f_g052019. caceae can also regulate flowering through vernalization CabbageG a f g006153 and CabbageG a f g0l1915) Springer

Planta 1 3 autonomous-dependent pathways. The four pathways reg￾ulate two flowering pathway integrators, FLOWERING LOCUS T (FT) and SOC1, which act prior to the activa￾tion of foral meristem identity genes to determine the exact fowering time (Fujiwara et al. 2003). These two integrators are antagonistically regulated by two upstream regulators: FLC acts as a negative regulator, and CO acts as a positive regulator of fowering (Lee et al. 2000; Samach et al. 2000). Exposure to cold winter conditions renders biennial and winter-annual plants responsive to the inductive photoper￾iod, resulting in fowering in the spring, which is known as vernalization (Reeves and Coupland 2000). The vernaliza￾tion requirement mainly results from FLC and FRIGIDA (FRI) (Michaels and Amasino 1999; Johanson and Dean 2000). FLC encodes a MADS-box transcription factor and functions as a major repressor of fowering. FLC represses SOC1, SPL15 and FT by directly binding to the promoters of SOC1 and SPL15 or the frst intron of FT (Helliwell et al. 2006; Searle et al. 2006). Arabidopsis ecotypes with high￾abundance FLC are usually late fowering, whereas ecotypes with low-abundance FLC are early fowering (Lister et al. 2005). Overexpression of FLC shows a late-fowering phe￾notype in Arabidopsis and Brassica rapa (Kim et al. 2007). FLC expression is activated by FRI and suppressed by the vernalization and autonomous pathways (Choi et al. 2011). Repression of FLC by vernalization is initially regulated by VERNALIZATION INSENSITIVE 3 (VIN3), encod￾ing a PHD-domain protein that interacts with the member of the polycomb repressive complex 2 (PRC2) (De Lucia et al. 2008). PRC2 complexes catalyze the methylation of H3K27 at the FLC locus to promote an inactive chromatin state (Sung and Amasino 2004). In addition to VIN3, VER￾NALIZATION1 (VRN1) and VRN2 are required to maintain the silenced state of FLC (Bastow et al. 2004). Recent stud￾ies revealed that long non-coding RNAs are also involved in the epigenetic repression of FLC. COOLAIR enhances the down-regulation of FLC expression (Swiezewski et al. 2009), and COLDAIR helps to recruit PRC2 to the FLC locus (Heo and Sung 2011). This epigenetic repression can be maintained through cell division after vernalization treat￾ment, which can only be “forgotten” in the next generation (Finnegan and Dennis 2007). MADS-box transcription fac￾tors typically act as multimers with other MADS-domain proteins (de Folter et al. 2005). SVP, another MADS-domain protein, interacts directly with FLC. Research has shown that the FLC–SVP complex directly binds to the FT and SOC1 to repress their expression (Li et al. 2008). The loss of SVP partially suppresses FLC-mediated fowering inhibition and does not infuence fowering as greatly as the loss of FLC, and thus, there may be some redundancy in SVP function (Lee et al. 2007). In addition to Arabidopsis, other members of Brassi￾caceae can also regulate fowering through vernalization (Halevy 1989). In Brassica species, two major QTLs related to vernalization-responsive fowering time are collinear with Arabidopsis FRI and FLC, suggesting that the homologs of FLC and FRI may play vital roles in fowering time dur￾ing the process of vernalization in other Brassica species (Osborn et al. 1997). Although numerous studies have reported the roles of FLC in some Brassica species, such as Brassica napus (Tadege et al. 2001) and Brassica olera￾cea (Schranz et al. 2002), the regulation mechanism of FLC in Pak-choi (Brassica rapa ssp. chinensis) is still not well understood. Pak-choi, belonging to the B. rapa family, is one of the most important vegetable crops and is widely culti￾vated in Asia (Huang et al. 2016). Wuyueman was used in this work, which normally requires 5 weeks of vernalization treatment (4 °C) and which fowers later than other cultivars. Here, we performed a series of studies, including sequence, expression, subcellular localization, ectopic expression, silencing and downstream gene analysis of BcFLC2 in Pak￾choi. The results showed that BcFLC2 was repressed by ver￾nalization and highly expressed in the leaves, roots, stems and stamens. BcFLC2 encoded a MADS-box transcription factor and regulated the expression of BcSOC1, BcSPL15, BcMAF2, and BcTEM1 by directly binding to their promot￾ers. Overexpression of BcFLC2 in Arabidopsis caused a late￾fowering phenotype, while silencing of BcFLC2 in Pak-choi caused an early fowering phenotype. Our results indicated that BcFLC2 functioned as a fowering repressor by directly inhibiting BcSOC1 and BcSPL15 and activating BcTEM1 and BcMAF2 in Pak-choi. Materials and methods Plant materials Arabidopsis wild type (WT) (obtained from Prof. Isabelle Jupin, University Paris 7, France) and BcFLC2-overex￾pressing seedlings were Col-0 ecotype background. Pak￾choi (Brassica rapa ssp. chinensis) cultivars wuyueman and 49caixin, kept at Nanjing Agricultural University, and all Arabidopsis plants were grown in plastic trays with a growth medium (vermiculite:loam, 1:2, v:v) in a culture room under long-day conditions (16 h light at 22 °C/8 h dark at 18 °C). To extract mesophyll protoplasts, WT Arabidopsis seedlings were grown under short-day conditions with an 8/16 h light/ dark cycle for 1 month. Cloning and sequence analysis RNA extraction and cDNA synthesis were performed according to our previous report (Huang et al. 2016). BcFLC1, BcFLC2 and BcFLC3 (CabbageG_a_f_g052019, CabbageG_a_f_g006153 and CabbageG_a_f_g011915)

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. ②Springer

Planta 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

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. Springer

Planta 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

Planta a MADS box domain AtFLC VGRKKLEI EKARCL SVLCCASVALLVVS OOKA 80 CabbageG_a_f_g052019 SKRRN ARCL SVLCCASVALLVV SCKL DNL 80 CabbageG a_f g006153 NGRKKL EI ENKSSRCVT KA周SASA DOLKA 80 CabbageG a_f_g011915 KARCL SVLCDASVALLVVSSSCKLYS 80 AtFLC EN 160 CabbageG a f g052019 160 CabbageG_a_f_g006153 159 CabbageG_a_f_g011915 LKEKEK 160 AtFLC AS 196 CabbageG a f g052019 CI TKME JNDVSHEe iMFNEMARSYAPLLAVNCVSLLPLAANAACTPFHI ECCVYCCTCRFCF 240 CabbageG a f g006153 AS 196 CabbageG a f g011915 EVTLPLLN 197 AtFLC 196 CabbageG a_f g052019 ETI ATKYI TCAKLKI VCKDSVTLKSEVVCEAVTGRRGRYRVSVKGDRCDCCCLAVL VNSPI SNCCFPDPGRNTATVI LTR 320 CabbageG_a_f_g006153 196 CabbageG a_f_g011915 197 AtFLC 196 CabbageG_a_f_g052019 SNGAASTRHFANANCFFRDEPLRGCATLRKCYLAECDYRA 360 CabbageG a f g006153 196 CabbageG_a_f_g011915 197 AtFLC 80 BrFLC1 100 CabbageG_a_f_g052019 BrFLC2 100 CabbageG a f g006153 BrFLC3 100 CabbageG a f g011915 Fig.1 Multiple sequence alignment and phylogenctic analy- FLCs and FLC homologues from Arabidopsis and Brassica rapa. sis.a Alignment of AtFLC with three Pak-choi FLC homo- The tree was constructed using the maximum likelihood method logues (CabbageG a f g052019.CabbageG a f g006153 and in MEGA6.The gene accession numbers were as follows:AtFLC CabbageG a f gol1915).Perfectly matched residues.highly con- (AT5G10140).BrFLCI (Bra009055).BrFLC2 (Bra028599)and served residues and less conserved residues are marked in dark blue BrFLC3(Bra006051) pink and sky blue,respectively.b Phylogenetic tree of three Pak-choi Overexpression of BcFLC2 caused late flowering (Fig.4c).These results suggested that BcFLC2 may function and affected expression levels of flowering-related as a floral repressor. genes in Arabidopsis To identify the targets regulated by BcFLC2,the transcripts of some flowering-related genes were analyzed in the NC and Based on the above findings,we predicted that BcFLC2 may transgenic Arabidopsis lines.Of the genes examined,AtSOCI be a repressor of flowering.To test this hypothesis,we first and AtSPL15 were down-regulated.while AtTEM/.AtMAF2 overexpressed BcFLC2 in Arabidopsis to investigate its func- and AtFLC were up-regulated in the transgenic lines compared tion.Two homozygous T3 transgenic Arabidopsis lines (#1 to the NC plants(Fig.4d).These results indicated that BcFLC2 and #3)confirmed by PCR(Fig.S1)were selected for fur- may delay flowering by regulating the expression of AtSOCl, ther analyses.Compared to the NC,BcFLC2-overexpressing AtSPL15.AtTEMI and AtMAF2. lines presented obvious late flowering(Fig.4a).In addition. the rosette leaves number at the time of bolting for #1 and #3 Silencing of BcFLC2 in Pak-choi resulted in early plants was higher than that for the NC plants,with an average flowering of 46 and 51 leaves,respectively (Fig.4b).The opening time of the first flower of the #1 and #3 plants was later by approxi- To further clarify the function of BcFLC2 in Pak-choi flow- mately 27 and 31 days compared to the NC plants,respectively ering regulation,we used the TYMV-VIGS approach to Springer

Planta 1 3 Overexpression of BcFLC2 caused late fowering and afected expression levels of fowering‑related genes in Arabidopsis Based on the above fndings, we predicted that BcFLC2 may be a repressor of fowering. To test this hypothesis, we frst overexpressed BcFLC2 in Arabidopsis to investigate its func￾tion. Two homozygous T3 transgenic Arabidopsis lines (#1 and #3) confrmed by PCR (Fig. S1) were selected for fur￾ther analyses. Compared to the NC, BcFLC2-overexpressing lines presented obvious late fowering (Fig. 4a). In addition, the rosette leaves number at the time of bolting for #1 and #3 plants was higher than that for the NC plants, with an average of 46 and 51 leaves, respectively (Fig. 4b). The opening time of the frst fower of the #1 and #3 plants was later by approxi￾mately 27 and 31 days compared to the NC plants, respectively (Fig. 4c). These results suggested that BcFLC2 may function as a foral repressor. To identify the targets regulated by BcFLC2, the transcripts of some fowering-related genes were analyzed in the NC and transgenic Arabidopsis lines. Of the genes examined, AtSOC1 and AtSPL15 were down-regulated, while AtTEM1, AtMAF2 and AtFLC were up-regulated in the transgenic lines compared to the NC plants (Fig. 4d). These results indicated that BcFLC2 may delay fowering by regulating the expression of AtSOC1, AtSPL15, AtTEM1 and AtMAF2. Silencing of BcFLC2 in Pak‑choi resulted in early fowering To further clarify the function of BcFLC2 in Pak-choi fow￾ering regulation, we used the TYMV-VIGS approach to Fig. 1 Multiple sequence alignment and phylogenetic analy￾sis. a Alignment of AtFLC with three Pak-choi FLC homo￾logues (CabbageG_a_f_g052019, CabbageG_a_f_g006153 and CabbageG_a_f_g011915). Perfectly matched residues, highly con￾served residues and less conserved residues are marked in dark blue, pink and sky blue, respectively. b Phylogenetic tree of three Pak-choi FLCs and FLC homologues from Arabidopsis and Brassica rapa. The tree was constructed using the maximum likelihood method in MEGA6. The gene accession numbers were as follows: AtFLC (AT5G10140), BrFLC1 (Bra009055), BrFLC2 (Bra028599) and BrFLC3 (Bra006051)

Planta 0.6 the NC plants(Table S5).The transcript levels of the pre- BcFLC2 dicted downstream genes were also detected by qPCR.Com- pared to the NC plants,the transcript levels of BcSOC/and BcSPL15 were higher and the transcript levels of BcTEMI 0.4 and BcMAF2 were lower in the BcFLC2-silenced plants. In addition,the expression of BcFLCI and BcFLC3 in the BcFLC2-silenced plants was detected,which exhibited no 0.2 almost change.These results suggested that the early flower- ing phenotype might be specifically caused by the reduction of BcFLC2(Fig.5d). 0.0 BcFLC2 directly bound to the promoters of BcMAF2, 2 BcTEM1,BcSOC1 and BcSPL15 Treatment time (week) 6 FLC can bind to the CArG box(CC(A/T)GG)in the pro- BcFLC2 moters of its targets (Deng et al.2011).We found that the CArG box was present in the promoters of BcSOC/, BcSPL15,BcTEMI and BcMAF2 (Table S3).The tran- scripts of AtSOCl,AtSPL15,AtTEMI and AtMAF2 were significantly altered in BcFLC2-overexpressing Arabi- dopsis seedlings.In addition,the transcripts of BcSOCI, BcSPL15,BcTEMI and BcMAF2 were also significantly altered in BcFLC2-silenced Pak-choi seedlings.To deter- mine whether BcFLC2 could directly bind to the promot- ers of BcSOC1,BcTEMI,BcSPL15 and BcMAF2,the yeast one-hybrid assay was performed.Based on these results, Root Stem Leaf Style Stamen Petal Sepal the promoter fragments of BcSOCI,BcSPL15,BcTEMI and BcMAF2 that contained the CArG box were selected.As Fig.2 gPCR analysis of BcFLC2 transcript level in vernalization treatment(a)and different tissues(b)of Pak-choi.Data shown are the shown in Figs.6 and 7a,the yeast cells.containing BcSOC/. means+SE of three biological replications BcSPL15,BcTEMI or BcMAF2 promoter fragments,trans- formed with pGADT7-BcFLC2 could grow on SD/-Leu/ AbA*.The results indicated that BcFLC2 might directly bind to the promoters of BcSOCI,BcSPL/5,BcTEMI and generate BcFLC2-silenced Pak-choi plants.Three weeks BCMAF2.As we know,FLC cannot bind to the CO pro- after the '49caixin'plants underwent particle gun bombard- moter,we used the BcCO promoter as the negative control ment,the photobleaching or mosaic leaf phenotype that is promoter.The yeast cells,containing the BcCO promoter typical of PDS deficiency or TYMV was visible on the upper fragment,transformed with pGADT7-BcFLC2,could not leaves of pTY-BcPDS,pTY-BcFLC2 or NC plants,suggest- grow on SD/-Leu/AbA*(Fig.7a),suggesting that BcFLC2 ing that TYMV-mediated gene silencing was effective in does not bind indiscriminately. Pak-choi.The silencing efficiency was examined using Since the relationships among SOCI,SPL15,TEMI and qPCR by analyzing the abundance of BcPDS or BcFLC2 FLC have been studied(Deng et al.2011),we further ana- in the BcPDS-silenced or BcFLC2-silenced plants.Overall. lyzed the relationship between BcMAF2 and BcFLC2.To the silencing efficiency of BcPDS or BcFLC2 was approxi- investigate whether BcFLC2 could bind to the CArG box in mately 50%(Fig.5b,c).As expected,the BcFLC2-silenced the BcMAF2 promoter,we mutated the CArG box(Fig.7b). plants exhibited an early flowering phenotype compared to When the CArG box was mutated,there was no binding the NC plants (Fig.5a).The bolting time in pTY-BcFLC2 (Fig.7a).Together,our results indicated that BcFLC2 could Pak-choi was approximately 15-17 days earlier than that in Springer

Planta 1 3 generate BcFLC2-silenced Pak-choi plants. Three weeks after the ‘49caixin’ plants underwent particle gun bombard￾ment, the photobleaching or mosaic leaf phenotype that is typical of PDS defciency or TYMV was visible on the upper leaves of pTY-BcPDS, pTY-BcFLC2 or NC plants, suggest￾ing that TYMV-mediated gene silencing was efective in Pak-choi. The silencing efficiency was examined using qPCR by analyzing the abundance of BcPDS or BcFLC2 in the BcPDS-silenced or BcFLC2-silenced plants. Overall, the silencing efciency of BcPDS or BcFLC2 was approxi￾mately 50% (Fig. 5b, c). As expected, the BcFLC2-silenced plants exhibited an early fowering phenotype compared to the NC plants (Fig. 5a). The bolting time in pTY-BcFLC2 Pak-choi was approximately 15–17 days earlier than that in the NC plants (Table S5). The transcript levels of the pre￾dicted downstream genes were also detected by qPCR. Com￾pared to the NC plants, the transcript levels of BcSOC1 and BcSPL15 were higher and the transcript levels of BcTEM1 and BcMAF2 were lower in the BcFLC2-silenced plants. In addition, the expression of BcFLC1 and BcFLC3 in the BcFLC2-silenced plants was detected, which exhibited no almost change. These results suggested that the early fower￾ing phenotype might be specifcally caused by the reduction of BcFLC2 (Fig. 5d). BcFLC2 directly bound to the promoters of BcMAF2, BcTEM1, BcSOC1 and BcSPL15 FLC can bind to the CArG box (CC(A/T)6GG) in the pro￾moters of its targets (Deng et al. 2011). We found that the CArG box was present in the promoters of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 (Table S3). The tran￾scripts of AtSOC1, AtSPL15, AtTEM1 and AtMAF2 were significantly altered in BcFLC2-overexpressing Arabi￾dopsis seedlings. In addition, the transcripts of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 were also signifcantly altered in BcFLC2-silenced Pak-choi seedlings. To deter￾mine whether BcFLC2 could directly bind to the promot￾ers of BcSOC1, BcTEM1, BcSPL15 and BcMAF2, the yeast one-hybrid assay was performed. Based on these results, the promoter fragments of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 that contained the CArG box were selected. As shown in Figs. 6 and 7a, the yeast cells, containing BcSOC1, BcSPL15, BcTEM1 or BcMAF2 promoter fragments, trans￾formed with pGADT7-BcFLC2 could grow on SD/-Leu/ AbA*. The results indicated that BcFLC2 might directly bind to the promoters of BcSOC1, BcSPL15, BcTEM1 and BcMAF2. As we know, FLC cannot bind to the CO pro￾moter, we used the BcCO promoter as the negative control promoter. The yeast cells, containing the BcCO promoter fragment, transformed with pGADT7-BcFLC2, could not grow on SD/-Leu/AbA* (Fig. 7a), suggesting that BcFLC2 does not bind indiscriminately. Since the relationships among SOC1, SPL15, TEM1 and FLC have been studied (Deng et al. 2011), we further ana￾lyzed the relationship between BcMAF2 and BcFLC2. To investigate whether BcFLC2 could bind to the CArG box in the BcMAF2 promoter, we mutated the CArG box (Fig. 7b). When the CArG box was mutated, there was no binding (Fig. 7a). Together, our results indicated that BcFLC2 could Fig. 2 qPCR analysis of BcFLC2 transcript level in vernalization treatment (a) and diferent tissues (b) of Pak-choi. Data shown are the means±SE of three biological replications

Planta a 35S:GFP CaMV35S HygR NOS CaMV35S GFP NOS LB RB 35S:BcFLC2-GFP CaMV35S HygR NOS CaMV35S BcFLC2 GFP HNOS LB RB b DAPI Fluorescence Bright field Merge 35S:GFP 35S:BcFLC2-GFP Fig.3 Subcellular localization of BcFLC2 protein.a 35S:GFP and 35S:BcFLC2-GFP constructs.b Transient expression of 35S:GFP and 35S:BcFLC2-GFP fusion protein in tobacco leaves.Scale bars=50 um not bind to the BcMAF2 promoter when its CArG box was by AtFLC.AtFLC was primarily expressed in the shoot api- mutated. cal meristem (SAM)and leaves to control flowering time in Arabidopsis(Searle et al.2006).Interestingly,although BcFLC2 was also negatively regulated by vernalization, Discussion its transcript slightly increased at 2 weeks of vernalization treatment,which was different from the expression pattern The elucidation of the underlying mechanism of flowering of AtFLC,suggesting that BcFLC2 may prevent premature regulation is important for Pak-choi.In the present study, flowering under short-term cold in Pak-choi. a new FLC homologous gene,BcFLC2,was isolated and The ectopic and constitutive expression of BcFLC2 in functionally characterized in Pak-choi.Arabidopsis only has Arabidopsis exhibited a higher expression level of AtTEMI a single FLC gene(Deng et al.2011),but Pak-choi has three and lower expression levels of AtSOC/and AtSPL15,caus- FLC genes,of which BcFLC2 functioned as a key player ing significantly late flowering (Fig.4d).The silencing of according to our previous transcriptome data and reports BcFLC2 in Pak-choi led to the up-regulation of BcSOCI and (Xiao et al.2013;Song et al.2014).BcFLC2 was a nuclear BcSPLI5 and down-regulation of BcTEMI,resulting in early protein and functioned as a transcription factor(Fig.3).To flowering(Fig.5c).These findings suggested that BcTEMI, investigate the regulatory roles of BcFLC2 in Pak-choi,we BcSOC/and BcSPL/5 acted downstream of BcFLC2.In analyzed its expression pattern with vernalization treatment Arabidopsis,TEMI,SOCI and SPL15 are controlled by and in different tissues.We observed that BcFLC2 expres- FLC via direct binding to their promoters (Deng et al.2011; sion was relatively higher in the roots,stems,leaves and Tao et al.2012).In Arabidopsis,direct up-regulation of stamens(Fig.2b),similar to the expression pattern displayed TEM/by both FLC and SVP contributes to the eventual Springer

Planta 1 3 not bind to the BcMAF2 promoter when its CArG box was mutated. Discussion The elucidation of the underlying mechanism of fowering regulation is important for Pak-choi. In the present study, a new FLC homologous gene, BcFLC2, was isolated and functionally characterized in Pak-choi. Arabidopsis only has a single FLC gene (Deng et al. 2011), but Pak-choi has three FLC genes, of which BcFLC2 functioned as a key player according to our previous transcriptome data and reports (Xiao et al. 2013; Song et al. 2014). BcFLC2 was a nuclear protein and functioned as a transcription factor (Fig. 3). To investigate the regulatory roles of BcFLC2 in Pak-choi, we analyzed its expression pattern with vernalization treatment and in diferent tissues. We observed that BcFLC2 expres￾sion was relatively higher in the roots, stems, leaves and stamens (Fig. 2b), similar to the expression pattern displayed by AtFLC. AtFLC was primarily expressed in the shoot api￾cal meristem (SAM) and leaves to control fowering time in Arabidopsis (Searle et al. 2006). Interestingly, although BcFLC2 was also negatively regulated by vernalization, its transcript slightly increased at 2 weeks of vernalization treatment, which was diferent from the expression pattern of AtFLC, suggesting that BcFLC2 may prevent premature fowering under short-term cold in Pak-choi. The ectopic and constitutive expression of BcFLC2 in Arabidopsis exhibited a higher expression level of AtTEM1 and lower expression levels of AtSOC1 and AtSPL15, caus￾ing signifcantly late fowering (Fig. 4d). The silencing of BcFLC2 in Pak-choi led to the up-regulation of BcSOC1 and BcSPL15 and down-regulation of BcTEM1, resulting in early fowering (Fig. 5c). These fndings suggested that BcTEM1, BcSOC1 and BcSPL15 acted downstream of BcFLC2. In Arabidopsis, TEM1, SOC1 and SPL15 are controlled by FLC via direct binding to their promoters (Deng et al. 2011; Tao et al. 2012). In Arabidopsis, direct up-regulation of TEM1 by both FLC and SVP contributes to the eventual Fig. 3 Subcellular localization of BcFLC2 protein. a 35S:GFP and 35S:BcFLC2-GFP constructs. b Transient expression of 35S:GFP and 35S:BcFLC2-GFP fusion protein in tobacco leaves. Scale bars=50 μm

Planta a 0 Rosette leaves at bolting 60 40 20 0 NC 教料 #3 Opening of first flower 100 80 60 40 NC #1 #3 20 NC #1 #3 2.5 1.5 AtFLC AtTEM1 AtSPL15 2.0 1.0 3 5 2 0.5 0.5 0.0 00 NC #1 #3 NC 1 #3 NC 1.5 2.5 AtSOC1 AtMAF2 2.0 1.0 "S 1.0 0.5 0.5 0.0 0.0- NC #1 #3 NC #1 #3 Fig.4 Overexpression of BcFLC2 in Arabidopsis.a The NC and sent the standard deviation of the mean number of 30 plants for each 35S:BcFLC2-GFP #and #3 plants grown in a chamber (16 h line.**indicates significant differences from control (P<0.01).d light/8 h dark photoperiod at 22 C/18 C).Scale bars=1.5 cm. Expression analysis of predicted downstream genes in the NC and Rosette leaf number at bolting (b)and opening of first flower (c)in 35S:BcFLC2-GFP #I and #3 plants the NC and 35S:BcFLC2-GFP #and #3 plants.Error bars repre- ②Springer

Planta 1 3 Fig. 4 Overexpression of BcFLC2 in Arabidopsis. a The NC and 35S:BcFLC2-GFP #1 and #3 plants grown in a  chamber (16  h light/8  h dark photoperiod at 22  °C/18  °C). Scale bars=1.5  cm. Rosette leaf number at bolting (b) and opening of frst fower (c) in the NC and 35S:BcFLC2-GFP #1 and #3 plants. Error bars repre￾sent the standard deviation of the mean number of 30 plants for each line. ** indicates signifcant diferences from control (P<0.01). d Expression analysis of predicted downstream genes in the NC and 35S:BcFLC2-GFP #1 and #3 plants

Planta b 1.51 BcPDS 1.0 0.5 0.0 NC pTY-BcPDS 1.5 BcFLC2 1.0 0.5 NC pTY-BcFLC2-3 pTY-BcFLC2-4 pTY-BCPDS 0.0 NC d 1.5 15 BcSOC1 BcTEM1 BcSPL15 1.0 10 0.5 5 0.0 NC NC NC 1.5 1.5 1.5 BcMAF2 BcFLC1 BcFLC3 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 NC NC 3 NC 3 Fig.5 a Early flowering phenotype of the BcFLC2-silencing Pak- pTY-BcPDS plants.c Expression analysis of BcFLC2 in the NC and choi plants.Pak-choi plants bombarded with pTY (NC).pTY-BcFLC2 pTY-BcFLC2 plants.d QPCR analysis of predicted downstream (pTY-BcFLC2-3 and pTY-BcFLC2-4)and pTY-BcPDS plasmids. genes in the NC and pTY-BcFLC2 plants Scale bars=2.5 cm.b Expression analysis of BcPDS in the NC and suppression of both,FT and SOCI (Tao et al.2012).TEMI pathways(Moon et al.2003).SPL/5 promotes flowering and functions as a direct FT repressor and acts upstream of FT is involved in the transition from the vegetative to the repro- (Ikeda and Ohme-Takagi 2009).Overexpression of TEM/ ductive phase (Hyun et al.2016).Here,a yeast one-hybrid in Arabidopsis results in a late-flowering phenotype,while analysis showed that BcFLC2 could also bind to the promot- the tem/-I mutation exhibits early flowering(Castillejo and ers of BcTEMI,BcSOCI and BcSPL15(Fig.6).BcFLC2, Pelaz 2008).SOCI is a positive regulator of flowering and BcSOCI and BcSPLI5 were predominantly expressed in the has been proven as the common target in multiple flowering leaves,while BcFLC2 and BcTEMI were predominantly Springer

Planta 1 3 suppression of both, FT and SOC1 (Tao et al. 2012). TEM1 functions as a direct FT repressor and acts upstream of FT (Ikeda and Ohme-Takagi 2009). Overexpression of TEM1 in Arabidopsis results in a late-fowering phenotype, while the tem1-1 mutation exhibits early fowering (Castillejo and Pelaz 2008). SOC1 is a positive regulator of fowering and has been proven as the common target in multiple fowering pathways (Moon et al. 2003). SPL15 promotes fowering and is involved in the transition from the vegetative to the repro￾ductive phase (Hyun et al. 2016). Here, a yeast one-hybrid analysis showed that BcFLC2 could also bind to the promot￾ers of BcTEM1, BcSOC1 and BcSPL15 (Fig. 6). BcFLC2, BcSOC1 and BcSPL15 were predominantly expressed in the leaves, while BcFLC2 and BcTEM1 were predominantly Fig. 5 a Early fowering phenotype of the BcFLC2-silencing Pak￾choi plants. Pak-choi plants bombarded with pTY (NC), pTY-BcFLC2 (pTY-BcFLC2-3 and pTY-BcFLC2-4) and pTY-BcPDS plasmids. Scale bars=2.5 cm. b Expression analysis of BcPDS in the NC and pTY-BcPDS plants. c Expression analysis of BcFLC2 in the NC and pTY-BcFLC2 plants. d QPCR analysis of predicted downstream genes in the NC and pTY-BcFLC2 plants

Planta Fig.6 Binding activities of SD/-Leu SD/-Leu/AbA* BeFLC2 protein with the promoters of BcTEMI.BcSOCI and BcSPL/5 detected by yeast one-hybrid assays.Yeast cells proBcTEM1-pAbAi+pGADT7 were grown on an SD/-Leu medium plate supplemented with or without 300 ng/mL AbA proBcTEM1-pAbAi+pGADT7-BcFLC2 proBcSPL15-pAbAi+pGADT7 proBcSPL15-pAbAi+pGADT7-BcFLC2 proBcSOC1-pAbAi+pGADT7 proBcSOC1-pAbAi+pGADT7-BcFLC2 expressed in the stamens of Pak-choi(Figs.2,S2).Thus, promoter.In addition,BcFLC2 and BcMAF2 were all pre- we suggested that BcFLC2 might directly repress BcSOCI dominantly expressed in the leaves(Figs.2,S2).Thus,we and BcSPL15 and activate BcTEM/expression in Pak-choi. hypothesized that BcFLC2 might directly activate BcMAF2 MAF2 encodes a floral repressor and can prevent pre- expression in Pak-choi.Although FLC predominantly acts mature vernalization under short periods of cold exposure. as a repressor,it can also positively regulate genes.For The maf2 mutants flower earlier than the wild type after example,FLC activates the expression of SMZ and TOE3 to short periods of cold exposure but retain a normal vernaliza- repress flowering (Deng et al.2011). tion response (Ratcliffe et al.2003).The expression level of In conclusion,the present work provides evidence that AtMAF2 was up-regulated in the BcFLC2-overexpressing BcFLC2 is a transcription factor and plays a crucial role in Arabidopsis plants,while the expression level of BcMAF2 repressing flowering in Pak-choi (Fig.8).BcFLC2 could was down-regulated in BcFLC2-silenced Pak-choi (Figs.4d, directly bind to the promoters of BcSOCI,BcSPL15, 5c),which suggested that BcFLC2 might positively regulate BcTEMI and BcMAF2 to regulate their expression.This the expression of BcMAF2.In addition,the yeast one-hybrid study provides an important clue to the BcFLC2-mediated assay showed that BcFLC2 directly bound to the BcMAF2 regulatory mechanism of fowering time in Pak-choi. Springer

Planta 1 3 expressed in the stamens of Pak-choi (Figs. 2, S2). Thus, we suggested that BcFLC2 might directly repress BcSOC1 and BcSPL15 and activate BcTEM1 expression in Pak-choi. MAF2 encodes a foral repressor and can prevent pre￾mature vernalization under short periods of cold exposure. The maf2 mutants fower earlier than the wild type after short periods of cold exposure but retain a normal vernaliza￾tion response (Ratclife et al. 2003). The expression level of AtMAF2 was up-regulated in the BcFLC2-overexpressing Arabidopsis plants, while the expression level of BcMAF2 was down-regulated in BcFLC2-silenced Pak-choi (Figs. 4d, 5c), which suggested that BcFLC2 might positively regulate the expression of BcMAF2. In addition, the yeast one-hybrid assay showed that BcFLC2 directly bound to the BcMAF2 promoter. In addition, BcFLC2 and BcMAF2 were all pre￾dominantly expressed in the leaves (Figs. 2, S2). Thus, we hypothesized that BcFLC2 might directly activate BcMAF2 expression in Pak-choi. Although FLC predominantly acts as a repressor, it can also positively regulate genes. For example, FLC activates the expression of SMZ and TOE3 to repress fowering (Deng et al. 2011). In conclusion, the present work provides evidence that BcFLC2 is a transcription factor and plays a crucial role in repressing fowering in Pak-choi (Fig. 8). BcFLC2 could directly bind to the promoters of BcSOC1, BcSPL15, BcTEM1 and BcMAF2 to regulate their expression. This study provides an important clue to the BcFLC2-mediated regulatory mechanism of fowering time in Pak-choi. Fig. 6 Binding activities of BcFLC2 protein with the promoters of BcTEM1, BcSOC1 and BcSPL15 detected by yeast one-hybrid assays. Yeast cells were grown on an SD/-Leu medium plate supplemented with or without 300 ng/mL AbA