Introduction

Control of flowering time is critical for plant development, especially of horticultural crops. Regulation of flowering is a complex network, including both environment factors and internal regulatory signals. Photoperiod is a vital environmental factor in plant flowering regulation. Robson et al. (2001) identified numerous genes, which participate in regulation of flowering. The CCT family genes are concerned with photoperiod-induced flowering modulation and light-triggered signaling (Putterill et al. 1995; Wenkel et al. 2006). The CCT domain initially represented a motif at the C-terminus of CONSTANS (CO), CO-like and TIMING OF CAB1 (TOC1) in Arabidopsis. Previous studies have classified CCT genes into three families: the COL gene family, encoded one or two zinc-finger B-box domains and a CCT domain; the CCT motif family (CMF) with a only CCT domain; the pseudo-response regulator (PRR) gene family with a CCT domain and two conserved regions-pseudo receiver domain (Cockram et al. 2012). AtCO was the first CCT family gene which consisted of two B-box domains and a CCT domain cloned in Arabidopsis ( Robson et al. 2001). Surveys on photoperiod pathway showed that the transcription factor CO promoted flowering by increasing the transcripts of FLOWERING LOCUS T (FT) under long day (LD) condition (Putterill et al. 1995) . Genetic analyses had uncovered that CO/FT was the core component in photoperiod-mediated flowering control (Nakamichi 2015). CO gene integrated the circadian clock and light signals to control plant flowering (Samach et al. 2000; Suarez-Lopez et al. 2001). CO-like (COL) genes were downstream component of circadian clock measuring day length. They cooperated with FT and GIGANTEA (GI) , as central functional components in photoperiod pathway (Song et al. 2012). In Arabidopsis, 17 COL genes were identified (Robson et al. 2001; Khanna et al. 2009). It is reported that AtCO, AtCOL3, AtCOL5, and AtCOL9 take part in flowering time regulation in Arabidopsis (Putterill et al. 1995; Cheng and Wang 2005; Datta et al. 2006; Hassidim et al. 2009). AtCO gene accelerated flowering in response to long photoperiods in Arabidopsis, which repressed photomorphogenesis in darkness (Putterill et al. 1995). The overexpression of AtCOL5 could advance flowering time by raising the transcripts of FT (Hassidim et al. 2009). AtCOL9 repressed flowering by decreasing the transcripts of CO and FT. AtCOL9 overexpression transgenic lines showed late flowering phenotype in LD condition (Cheng and Wang 2005). The function of CO gene was conserved between dicots and monocots in photoperiodic floral induction pathway in Arabidopsis and rice (Wenkel et al. 2006). Heading date 1 (Hd1) was the homologue of AtCO, it was revealed that Hd1 could promote the rice heading in SD condition and inhibiting the rice heading in LD condition (Yano et al. 2000). Ghd7 was a CMF gene, involved in heading date and grains development in rice (Xue et al. 2008). Ghd7 delayed the rice heading by repressing the transcription of Early heading date1 (Ehd1) in the photoperiodic flowering pathway under LD conditions (Xue et al. 2008; Nakamichi 2015). Studies in rice showed that such genes were relatively common, they demarcation this group of genes to the CMF genes(Cockram et al. 2012). CMF genes had similarity function with COL in plant flowering regulation. OsCCT1 was a new CMF gene repressing the expression of Ehd1 and Hd3a to delay the flowering time (Zhang et al. 2015).

Onion (Allium cepa L.) is one of the main vegetables with economic production of bulb biennially. In 2018, onion production was 103.3 million tons harvested in 5.3 million hectares throughout the world (http://www.fao.org). The life cycle of onion is strictly regulated by light. There are multiple ecotype of onion dependent on the planting environment, as LD type, SD type and day-neutral. In previous study, an AcCOL was obtained, but it did not exhibit discernible circadian expression pattern (Taylor et al. 2010). AcCOL2 showed a circadian expression pattern in common with AtCO that possibly regulated the expression of AcFT1 (Rashid and Thomas 2020). In our previous study, AcCOL7 was cloned which involved in photoperiod pathway, as well as it likely played a significant role in promoting flowering (Sheng et al. 2018).

The CMF genes have not been identified in onion. In this study, a CMF gene was isolated from onion named AcCMF1. For the purpose of investigating the role of AcCMF1 in flowering regulation, AcCMF1 was transformed to Arabidopsis. AcCMF1 played similar roles in flowering regulation. Overexpression AcCMF1 could partly complement the function of co mutant in Arabidopsis. These results proposed that CMF gene was involvement in the flowering regulation of onion.

Materials and Methods

Plant materials

A LD type higher-generation inbred onion SA2 was used in this experiment. It was provided by the Onion and Garlic Research Group of Northeast Agricultural University. Arabidopsis thaliana wild-type (WT) accessions used were Col-0 and Ler. The Arabidopsis mutant col-5 (SALK_096361C, Col-0), gi (CS181, Ler-0) and ga3 (SALK-103671C, Col-0) were obtained from TAIR（http: //www.arabidopsis.org/. Plants were grown on soil in a plant incubator under a 16 h light and 8 h dark period at 22/18^oC. Tissue samples were collected from ten-leaf-stage onion on both vegetative and reproductive growth for relative expression of AcCMF1. Flowering time was measured by counting the total number of rosette leaves (RLs) and recording the days at bolting. Eight-week-old seedlings were used to measure plant height. Each independent line with three biological replicates was used to measrue number of RLs, flowering days and plant height. There were twenty plants per replication.

Cloning, sequence alignment and phylogenetic analysis of AcCMF1

Sequence of AcCMF1 was obtained from transcriptome database in our previous study (Yuan et al. 2018) . Trizol reagent (Invitrogen, USA) was used to extract total RNA from onion leaves, and cDNA was synthesized using M-MuLV reverse transcriptase (Thermo Scientific, USA). The primers are listed in Table S1.

Simple Modular Architecture Research Tool (SMART) was used to explore the conserved domains of AcCMF1 (http://smart.embl-heidelberg.de/). The CCT family proteins amino acid sequence of Arabidopsis and rice were obtained from NCBI. MEGA5 software was used to construct the multiple sequence alignments of AcCMF1 and related CCT family proteins. The phylogenetic tree was constructed through MEGA5 software using the Neighbor-Joining (NJ) method (Saitou and Nei 1987; Tamura et al. 2011).

The multiple sequence alignments were drawn using the BoxShade web site (http://www.ch.embnet.org/software/BOX_ form.html).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from onion using Trizol (Invitrogen, USA). cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO, Shanghai, China). qRT-PCR was carried out using KOD SYBR® qPCR Mix (TOYOBO, Shanghai, China). Acaction was the reference gene. Comparative threshold method (2^-△△Ct) was used to measure relative transcripts levels of genes (Livak and Schmittgen 2001). The primers in this study were listed in Table S1.

Subcellular localization in Arabidopsis mesophyll protoplast

The full length coding sequence (CDS) of AcCMF1 was transient expressed in Arabidopsis for subcellular localization (Yoo et al. 2007). The primers are shown in Table S1. The pG-eGFP vector (with GFP protein driven by CaMV35S promoter) to generate CaMV35S: eGFP-AcCMF1. The empty vector was used as control. Fluorescence microscope was used to observe the eGFP-AcCMF1 subcellular localization.

Ectopic expression of AcCMF1 in Arabidopsis

The CDS regions of AcCMF1 was inserted to the pCXSN1250-3301 vector, in which the target genes were controlled by CaMV35S promoter. The recombinant vector was transformed to Agrobacterium strain GV3101 and then used to infect Arabidopsis (Col-0, Ler, col-5, ga3 and gi) via Agrobacterium-mediated the floral dip method (Clough and Bent 2010). The transgenic lines were selected on MS medium with Glufosinate ammonium (PPT). PCR was used to select positive transgenic lines. Homozygous transgenic Arabidopsis seeds (T₃) were used for further study.

Statistical analysis

The values were obtained from three independent experiments and presented as the mean ± standard errors. Univariate ANOVA analysis was used to represent the significant differences of the data (P < 0.05).

Results

Cloning and phylogenetic analysis of onion AcCMF1

In this study, a novel CCT family gene was obtained based on the transcriptome database from our previously study (Yuan et al. 2018). The gene was identified to contain a CCT domain. It was annotated as CMF gene and named AcCMF1. According to phylogenetic analysis, CCT family protein from Arabidopsis and rice could be classified into four groups (Fig. 1). The members in group I contained two B-box motifs and a CCT domain. The group II members contained a B-box motif and a CCT domain. The group III members included a B-box domain, a diverse B-box domain and a CCT domain. AcCMF1 belonged to group IV without B-box domain had a closer evolutionary relationship with OsGHd7 (Fig. 1). The full length of AcCMF1 cDNA was 891 bp, encoding 296 amino acids. The sequence alignment of AcCMF1 compared with other members of CCT family was performed (Fig. 2). AcCMF1 showed 15.16 and 17.63% identity with OsGhd7 and ZmGhd7, which were CMF proteins from rice and maize. AcCMF1 had all the conserved amino acids of CCT domain (RX₅RYX₂KX₂RX₇YX₂RKX₂AX₃PRX₂GRF) (Fig. 2).

Subcellular localization of AcCMF1

The fusion expression vector used to investigate the intracellular localization of AcCMF1 was constructed as pGII-eGFP-AcCMF1. The empty vector pGII-eGFP was also transformed to Arabidopsis as control. Arabidopsis protoplasts were extracted and used for observation. We detected strong GFP fluorescence in the nucleus when eGFP-AcCMF1 plasmid was transformed to Arabidopsis, while GFP fluorescence was observed in whole Arabidopsis protoplast when empty vector pGII-eGFP plasmid transformed (Fig. 3). These results confirmed that AcCMF1 was nuclear-localized protein. CO worked as the transcription factor to promote flowering under LD (Putterill et al. 1995). AcCMF1 suggested to be as characteristic transcription factor.

Characterization of AcCMF1 expression

To characterize the organ specific expression of AcCMF1, qRT-PCR was performed in various onion organs at reproductive phase under LD condition (Fig. 4). Although AcCMF1 expressed throughout the growth cycle of the plant, the transcript level was the highest in the young leaves before bolting, followed by a high expression level in the young flower stems (Fig. 4). A high expression of the gene in young leaves before bolting also indicated that AcCMF1 was an important component receiving optical signal in photoperiod pathway and played an important role in plant flowering regulation.

Results from qRT-PCR showed that AcCMF1 has double peaks of transcription under both LD and SD conditions. AcCMF1 was mainly expressed under dark condition. Under LD condition, the expression of AcCMF1 was peak at 6:00 am and 8:00 pm. The transcripts of AcCMF1 reached peak at 10:00 am and 8:00 pm under SD condition (Fig. 5).

Role of AcCMF1 in plant flowering regulation

Fig. 1: The phylogenetic relationship and conserved domain analysis of CCT homologs. Neighbor-joining tree of CCT family genes, AcCMF1, AcCOL, AcCOL7, AtCOLs and OsCOLs. Bootstrap values from 1000 replicates were used to assess the robustness of the tree. AcCMF1 from onion was indicated in red boxes

Fig. 2: Conserved protein domains alignment of AcCMF1 with other CMF proteins. The identical and similar residues were shown in black and gray, respectively. The CCT domain was highlighted in red line

AcCMF1 was transformed to Arabidopsis to investigate its function on flowering regulation. The wild-type (WT) Arabidopsis was bolting with 16 rosette leaves (RLs) on average, at about 32-day-stage on average and the plant height was 37 cm on average. AcCMF1-OE-WT lines showed more rosettes, but there was no significantly different on flowering days and plant height between the wild type and AcCMF1 transgenic Arabidopsis (Supplementary Fig. S1). Compared with the wild-type A. thaliana, co mutant plants showed dwarf phenotype and their flowering time of was delayed (Fig. 6A). A. thaliana co mutant was bolting with 18 RLs and at 38-day-stage on average (Fig. 6BC). AcCMF1 was overexpressed in Arabidopsis co mutant under the control of CaMV35S promoter to further verify the function of AcCMF1. Compared to co mutant, the flowering time of AcCMF1-OE-co lines were advanced. The transgenic plants flowered at about 32-day-stage (Fig. 6BC). Plant height of AcCMF1-OE-co lines was rescued, which was 37 cm on average (Fig. 6D). The transgenic plants displayed advanced bolting time compared to co mutant (Fig. 6). AcCMF1 not only could promote the plant flowering, but also participated in plants development regulation.

Fig. 3: Subcellular localization of AcCMF1. The left verticals are green fluorescence images, middle verticals are bright-filed images, and right verticals are merged images of bright field and green fluorescence. Scale bars in this figure are 10 μm

Fig. 4: The expression patterns of AcCMF1. The tissue expression patterns of AcCMF1. Relative expression levels were determined by qRT-PCR. CV, cauloin in vegetative phase; BV, bulb in vegetative phase; LBB, leaf before bolting; LAB, leaf after bolting; TFS, tender floral stem; MS, mature floral stem; INF, inflorescence; CR, cauloid in reproductive phase; BR, bulb in reproductive phase

The gi is upstream gene of CO in the photoperiod pathway of plant flowering regulation. The gi mutants showed longer vegetative growth time, thicker stem, longer flowering time and less lateral branches than the wild-type plants. In order to explore the relationship between CO genes and other flowering regulation pathways, AcCMF1 was overexpressed in gi. The gi mutant was bolting at 35-day-stage on average. There was no significant difference in flowering time and plant morphology between AcCMF1-OE-gi plants and gi mutant (Supplementary Fig. S2). Arabidopsis thaliana ga3 mutant is a GA synthesis blocked mutant. The plant growth of ga3 was weaker than the wild type. But the flowering time was similar to the wild one. AcCMF1 overexpressed in ga3 mutant did not affect the flowering time of ga3 mutant (Supplementary Fig. S3).

Fig. 5: The diurnal rhythm expression pattern of AcCMF1 in onion leaves under different photoperiod

Fig. 6: Overexpression of AcCMF1 in Arabidopsis co mutant. (A) Phenotype (B) Flowering time (C) Number of rosette leaves (D) Plant height of co mutant, wild type, AcCMF1-OE lines under LD condition. Error bars indicate the standard errors. Asterisks indicate the significant differences (P < 0.05)

Discussion

Plant flowering is an important developmental process in plant life cycle precisely controlled by various environmental signals especially in commercial crops (Nemoto et al. 2003; Miller et al. 2008; Jung and Muller 2009; Michaels 2009). CCT family genes exist broadly in monocot and dicoty plants. Most members of CCT gene family take effect in plant flowering control. Plant CCT genes were distributed into three categories: COL family, CMF family and PRR family (Cockram et al. 2012). COL family and CMF genes were classified into four types. Type I included two normal B-box motifs, such as AtCO and AtCOL1 to AtCOL5; AtCOL6 to AtCOL8 and AtCOL16 belonged to type II had a B-box motif and a CCT domain; type III had a B-box motif and a second diverse B-box motif, such as AtCOL9-AtCOL15; and CMF genes belonged to type IV with only a CCT domain but no B-box domain (Griffiths et al. 2003; Cockram et al. 2012; Gangappa and Botto 2014; Wu et al. 2017). It has been reported that most type I COL homologs played a positive role in regulation of flowering (Zhang et al. 2015; Chaurasia et al. 2016). Nevertheless, CCT family genes display multiple functions in flowering regulation. For instance, AtCOL9 played a negative role in Arabidopsis flowering control, repressing CO expression (Cheng and Wang 2005). The CMF genes encoded proteins contain a single CCT domain and are critical for domestication and adaptation in cereal crops (Li and Xu 2017). OsGhd7 was a CMF gene delayed heading under LD conditions but not SD conditions in rice (Xue et al. 2008). In this study, a novel CCT family gene, AcCMF1, was isolated from onion. AcCMF1 contained a single CCT domain without other structures and taken part in onion flowering regulation (Fig. 1, 2).

CO localized in nucleus, which could bind the promoter of FT to trigger its expression to promote flowering (Wenkel et al. 2006; Tiwari et al. 2010; Nemoto et al. 2016). Recent studies suggested that full length of Phalaenopsis orchid PaCOL1 protein localized in nucleus. PaCOL1 was still localized in nucleus without B-box domain, but it was localized in cytoplasm and nucleus without CCT motif (Ke et al. 2020). In our study, AcCMF1 was localized in nucleus (Fig. 3). This implied that AcCMF might take part in flowering regulation as a transcription factor. Leaf is the most important tissue for plant to intercept light. AtCO was the first certified CCT family gene which control flowering in Arabidopsis as a phloem-specific transcription factor (Robson et al. 2001). All Populus PtCOL genes were preferentially expressed in leaves (Li et al. 2020). In bamboo, PvCO1 showed abundant transcripts in immature and mature leaves, as well as, PvCO2 only expressed in bamboo leaves (Xiao et al. 2018). AcCMF1 showed high expression in leaf before bolting (Fig. 4). CO was degraded by the ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) at the posttranscriptional level in dark. In Arabidopsis, the expression of CO showed circadian rhythms pattern (Suarez-Lopez et al. 2001; Shim et al. 2017). Previous study revealed that the transcripts of CO was peaking at dawn and dusk in LD condition, which was crucial for the stabilization of CO (Imaizumi et al. 2005; Turck et al. 2008). Hassidim et al. (2009) showed AtCOL5 overexpression complemented the late flowering phenotype of co mutant. Hd1 was a homolog of CO gene in rice. OsHd1 had a comparable circadian expression pattern with AtCO, while OsHd1 acted as a flowering repressor in SD condition (Takeshi et al. 2002). A putative CO homolog was cloned and designated AcCOL in onion, but AcCOL did not display a observable circadian expression pattern (Taylor A et al. 2010). AcCOL2 displayed well diurnal expression pattern in accordance with photoperiod detecting (Rashid and Thomas 2020). In onion, the transcript level of AcCMF1 showed double peaks 24 h period (Fig. 5). AcCMF1 might regulat onion flowering by capturing and transforming light signal. The expression of CO was controlled by day and night cycles (Meng et al. 2011). AcCMF1 showed the similar expression pattern with LfCOL6 in Lilium× formolongi, which played positive role in triggering flowering induction under LD (Li et al. 2018). This expression pattern was not completely consistent speculating that CCT family genes in onion had different function in flowering regulation and the circadian clock was modulated by different CCT genes.

In order to verify the effect of AcCMF1 family genes on flowering, AcCMF1 overexpression vector was constructed and transformed to A. thaliana. The Arabidopsis co mutant performed late flowering phenotype. Nevertheless, overexpressed AcCMF1 in Arabidopsis co mutant could supplement the late flowering phenotype of co mutant under LD condition (Fig. 6). OsGhd7 was an LD-specific repressor played a crucial role in increasing rice yields and controlling heading dates containing only a CCT domain (Xue et al. 2008). The in vivo investigation indicated Ghd7 and Hd1 were interaction to bind the promoter region of Ehd1 to repress its expression in photoperiod induced flowering pathway (Nemoto et al. 2016). Ghd7 could inhibit the expression of the flowering time pathway genes in conjunction with Hd1 in Poaceae (Nemoto et al. 2016). Expression of floral repressor SbGhd7, the orthologs of rice Ghd7, inhibited SbCO transcriptional activity and delayed the flower in sorghum under LD conditions (Yang et al. 2014). AcCMF1 restored the late phenotype of co mutant and promote the flowering of onion under LD condition. It is suggestted that AcCMF1 played positive role in onion flowering regulation and involved in different pathways compared to cereal crops. Further study is essential to gain more knowledge of regulatory mechanism of AcCMF1 in onion. GIGANTEA (GI) protein modulates the stability of FKF1, which is related to the stabilization of CO in the afternoon of long days (Park et al. 1999; Mizoguchi et al. 2005; Fowler et al. 2014; Hwang et al. 2019). It had been reported overexpression of CO could restore the late floral phenotype of gi mutants under long and short sunshine conditions in Arabidopsis (Ben-Naim et al. 2006; Sawa et al. 2007). We transformed AcCMF1 into gi mutant of Arabidopsis. Overexpressed AcCMF1 in gi mutants had little effect on flowering (Supplementary Fig. S2). There were no significant difference between gi mutant and AcCMF1-OE lines on flowering time. It was speculated that gi did not regulated the accumulation of AcCMF1 and there were other members of onion CCT family involved in the regulation of flowering via gi. To verify the interaction between AcCMF1 and other flowering regulatory genes, AcCMF1 was overexpressed in ga3 mutant in Arabidopsis. There were no significant changes between the transgenic plants and ga3 mutants (Supplementary Fig. S3). It indicated that AcCMF1 did not take part in the gibberellin pathway of flowering regulation. Previous study mentioned that CCT domain was the essential structure of CO to bind the particular cis-elements of FT promoter directly (Tiwari et al. 2010). AcCMF1 might be involved in onion flowering regulation by adjusting the transcripts of CO and FT. The mechanism of AcCMF1 reaction with other CCT or flowering related genes control the onion flowering was unclear and should be explored in further study.

Conclusion

AcCMF1 belonged to onion CCT family. The function of AcCMF1 in plant flowering regulation was revealed by using AcCMF1 Arabidopsis transgenic lines. AcCMF1 was expressed highest in young leaves before bolting. AcCMF1 advanced the flowering time of co mutant in Arabidopsis, which also played a positive role in plant flowering.

Acknowledgments

The authors gratefully acknowledge the financial support for this study provided by the grants from the Open Project of Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, China (neauhc201805); Northeast Agricultural University SIPT Project. We are grateful to The Key Laboratory of Vegetable Biology of Heilongjiang Province and the Key Laboratory of Northern region Horticultural Crop Genetic Improvement and Facility Cultivation of Heilongjiang Province.

Author Contributions

Yong Wang designed the experiments, participated in generation of transgenics; Shouyi Ren participated in the cloning experiments and gene expression analysis; Cuicui Zhang participated in qRT-PCR; Yuqi Zhang and Yang Xu participated in sequence alignment and phylogenetic analysis; Jiru Wang participated in subcellular localization; Xiaochen Cong participated in collecting phenotypic data; Lei Qin helped conceiving the study, participated in its coordination and manuscript writing and editing.

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