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 22/18oC.
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.
References
Ben-Naim O, R Eshed, A Parnis, P
Teper-Bamnolker, A Shalit, G Coupland, A Samach, E Lifschitz (2006). The CCAAT binding
factor can mediate interactions between CONSTANS-like proteins and DNA. Plant J 46:462–476
Chaurasia AK, HB Patil, A Azeez, VR Subramaniam, B Krishna, AP Sane, PV Sane (2016). Molecular characterization of CONSTANS-Like (COL) genes
in banana (Musa acuminata L. AAA
Group, cv. Grand Nain). Physiol
Mol Biol Plants
22:1–15
Cheng XF, ZY Wang (2005). Overexpression of
COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and
FT in Arabidopsis thaliana. Plant J
43:758–768
Clough SJ, AF Bent (2010). Floral dip: A simplified method for
Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J 16:735–743
Cockram J, T Thiel, B Steuernagel, N Stein,
S Taudien, PC Bailey, DM O'Sullivan (2012).
Genome
dynamics explain the evolution of flowering time CCT domain gene families in
the Poaceae. PLoS One
7; Article e45307
Datta S, G Hettiarachchi, XW
Deng, M Holm (2006). Arabidopsis CONSTANS-LIKE3 is a positive regulator of red
light signaling and root growth. Plant Cell 18:70–84
Fowler S, K Lee, H Onouchi, A
Samach, K Richardson, B Morris, G Coupland, J Putterill (2014). GIGANTEA: A circadian clock-controlled gene that regulates
photoperiodic flowering in Arabidopsis and encodes a protein with several
possible membrane-spanning domains. EMBO J 18:4679–4688
Gangappa SN, JF Botto (2014). The BBX family of
plant transcription factors. Trends
Plant Sci 19:460–470
Griffiths S, RP Dunford, G Coupland, DA Laurie (2003). The evolution of
CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant Physiol 131:1855–1867
Hassidim M, Y Harir, E Yakir, I Kron, RM Green (2009). Over-expression of CONSTANS-LIKE 5 can induce flowering
in short-day grown Arabidopsis. Planta 230:481–491
Hwang DY, S Park, S Lee, SS Lee, T Imaizumi, YH Song (2019).
GIGANTEA regulates the timing stabilization of CONSTANS by altering the
interaction between FKF1 and ZEITLUPE. Mol
Cells 42:693–701
Imaizumi T, TF Schultz, FG Harmon, LA Ho, SA Kay (2005). FKF1F-BOX protein mediates cyclic degradation of a
repressor of CONSTANS in Arabidopsis. Science 309:293–297
Jung C, AE Muller (2009). Flowering time control and applications in plant
breeding. Trends Plant Sci
14:563–573
Ke YT, KF Lin, CH Gu, CH Yeh
(2020). Molecular characterization and expression profile of PaCOL1, a
CONSTANS-like gene in Phalaenopsis orchid.
Plants-Basel 9; Article 68
Khanna R, B Kronmiller, DR Maszle, G Coupland, M Holm, T Mizuno, SH Wu (2009). The Arabidopsis B-Box zinc finger family. Plant Cell 21:3416–3420
Li YP, ML Xu (2017). CCT family genes in cereal crops: A current
overview. Crop J
5:449–458
Li YF, YQ Zhao, M Zhang, GX Jia, M Zaccai (2018). Functional and
evolutionary characterization of the CONSTANS-like family in Lilium x formolongi. Plant Cell Physiol
59:1874–1888
Livak KJ , TD Schmittgen (2001). Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Meng X, MG Muszynski, ON Danilevskaya (2011). The FT-Like ZCN8 gene functions as a floral activator
and is involved in photoperiod sensitivity in maize. Plant Cell 23:942–960
Michaels SD (2009). Flowering time regulation produces much fruit. Curr Opin Plant Biol 12:75–80
Miller TA, EH Muslin, JE Dorweiler (2008). A maize CONSTANS-like gene, conz1, exhibits distinct
diurnal expression patterns in varied photoperiods. Planta 227:1377–1388
Mizoguchi T, L Wright, S Fujiwara, F Cremer, K Lee, H Onouchi, A
Mouradov, S Fowler, H Kamada, J Putterill, G Coupland (2005). Distinct roles of GIGANTEA in promoting flowering and
regulating circadian rhythms in Arabidopsis. Plant Cell 17:2255–2270
Nakamichi N (2015). Adaptation to the local environment by modifications of
the photoperiod response in crops. Plant
Cell Physiol 56:594–604
Nemoto Y, M Kisaka, T Fuse, M Yano, Y Ogihara (2003). Characterization and functional analysis of three wheat
genes with homology to the CONSTANS flowering time gene in transgenic rice. Plant J 36:82–93
Nemoto Y, Y Nonoue, M Yano, T
Izawa (2016). Hd1, a CONSTANS orthlog
in rice, functions as an Ehd1
repressor through interaction with monocot-specific CCT-domain protein Ghd7.
Plant J 86:221–233
Park DH, DE Somers, YS Kim, YH Choy, HK Lim, MS Soh, HJ Kim, SA
Kay, HG Nam (1999). Control of circadian rhythms and photoperiodic
flowering by the Arabidopsis GIGANTEA gene. Science
285:1579–1582
Putterill J, F Robson, K Lee, R Simon, G Coupland (1995). The Constans gene of Arabidopsis promotes flowering and
encodes a protein showing similarities to zinc-finger transcription factors. Cell 80:847–857
Rashid MHA, B Thomas (2020). Diurnal expression of Arabidopsis gene homologs during
daylength-regulated bulb formation in onion (Allium cepa L.). Sci Hortic 261; Article 108946
Robson F, MMR Costa, SR Hepworth, I Vizir, M Pineiro, PH Reeves,
J Putterill, G Coupland (2001). Functional importance of conserved domains in the
flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and
transgenic plants. Plant J 28:619–631
Saitou N, M Nei (1987). The neighbor-joining method: A new method for
reconstructing phylogenetic trees. Mol Biol
Evol 4:406–425
Samach A, H Onouchi, SE Gold,
GS Ditta, Z Schwarz-Sommer, MF Yanofsky, G Coupland (2000). Distinct roles of CONSTANS target genes in reproductive
development of Arabidopsis. Science
288:1613–1616
Sawa M, DA Nusinow, SA Kay, T Imaizumi (2007). FKF1 and GIGANTEA complex formation is required for
day-length measurement in Arabidopsis. Science
318:261–265
Sheng J, C Yang, X Wu, Q Yuan, D Chen, D Zhang, Y Wang (2018). Molecular cloning and functional identification of
photoperiod pathway transcription factor gene AcCOL7 in Allium cepa. Acta Hortic Sin 45:493–502
Shim JS, A Kubota, T Imaizumi (2017). Circadian clock and photoperiodic flowering in Arabidopsis:
CONSTANS is a hub for signal integration. Plant Physiol 173:5–15
Song YH, RW Smith, BJ To, AJ
Millar, T Imaizumi (2012). FKF1 conveys timing information for CONSTANS stabilization in
photoperiodic flowering. Science 336:1045–1049
Suarez-Lopez P, K Wheatley, F Robson, H Onouchi, F Valverde, G Coupland (2001). CONSTANS mediates between the circadian clock and the
control of flowering in Arabidopsis. Nature
410:1116–1120
Takeshi I, O Tetsuo, S Nobuko, T Takatoshi, Y Masahiro, S Ko (2002). Phytochrome mediates
the external light signal to repress FT orthologs in photoperiodic flowering of
rice. Genes Dev 16:2006–2020
Tamura K, D Peterson, N Peterson, G Stecher, M Nei, S Kumar (2011). MEGA5: Molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
Taylor A, AJ Massiah, B Thomas (2010). Conservation of Arabidopsis
thaliana photoperiodic flowering time genes in onion (Allium cepa L.). Plant
Cell Physiol 51:1638–1647
Tiwari SB, Y Shen, HC Chang, YL Hou, A Harris, SF Ma, M
McPartland, GJ Hymus, L Adam, C Marion, A Belachew, PP Repetti, TL Reuber, OJ Ratcliffe (2010). The flowering time regulator CONSTANS is recruited to
the FLOWERING LOCUS T promoter via a unique cis-element.
New Phytol 187:57–66
Turck F, F Fornara, G Coupland (2008). Regulation and identity of florigen: FLOWERING LOCUS T
moves center stage. Annu Rev
Plant Biol 59:573–594
Wenkel S, F Turck, K Singer, L Gissot, JL Gourrierec, A Samach, G Coupland (2006). CONSTANS and the CCAAT box binding complex share a
functionally important domain and interact to regulate flowering of
Arabidopsis. Plant Cell 18:2971–2984
Wu W, XM Zheng, D Chen, Y Zhang, W Ma, H Zhang, L Sun, Z
Yang, C Zhao, X Zhan, X Shen, P Yu, Y Fu, S Zhu, L Cao, S Cheng (2017). OsCOL16, encoding a CONSTANS-like protein, represses flowering
by up regulating Ghd7 expression in
rice. Plant Sci 260:60–69
Xue WY, YZ Xing, XY Weng, Y Zhao, WJ Tang, L Wang, HJ Zhou, SB Yu, CG Xu, XH Li, QF Zhang (2008). Natural variation in
Ghd7 is an important regulator of
heading date and yield potential in rice. Nat Genet
40:761–767
Yang S, RL Murphy, DT Morishige, PE Klein, WL Rooney, JE Mullet (2014). Sorghum phytochrome B
inhibits flowering in long days by activating expression of SbPRR37 and SbGHD7,
repressors of SbEHD1, SbCN8 and SbCN12. PLoS One 9; Article e105352
Yano M, Y Katayose, M
Ashikari, U Yamanouchi, L Monna, T Fuse, T Baba, K Yamamoto, Y Umehara, Y Nagamura (2000). Hd1, a major photoperiod
sensitivity quantitative trait locus in rice, is closely related to the
Arabidopsis flowering time gene CONSTANS, Plant Cell 12:2473–2483
Yoo SD, YH Cho, J Sheen (2007). Arabidopsis mesophyll protoplasts: A versatile cell
system for transient gene expression analysis. Nat Protoc 2:1565–1572
Yuan QL, C Song, LY Gao, HH Zhang, CC Yang, J Sheng, J Ren, D Chen, Y. Wang (2018). Transcriptome de
novo assembly and analysis of differentially expressed genes related to
cytoplasmic male sterility in onion. Plant Physiol Biochem
125:35–44
Zhang R, J Ding, CX Liu, CP Cai, BL Zhou, TZ Zhang, WZ Guo (2015). Molecular evolution and phylogenetic analysis of eight
COL superfamily genes in group I related to photoperiodic regulation of
flowering time in wild and domesticated cotton (Gossypium) species. PLoS One 10; Article e118669