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\begin{document}

\title{A HY5-COL3-COL13 regulatory chain for controlling hypocotyl elongation
in Arabidopsis}



\author[1]{Bin Liu}%
\author[2]{Hong Long}%
\author[2]{Jing Yan}%
\author[2]{Lili Ye}%
\author[2]{Qin Zhang}%
\author[2]{Hongmei Chen}%
\author[2]{Sujuan Gao}%
\author[2]{Yaqin Wang}%
\author[2]{Xiaojing Wang}%
\author[2]{Shulan Sun}%
\affil[1]{Shanghai Jiao Tong University - Minhang Campus}%
\affil[2]{South China Normal University}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
CONSTANS-LIKE (COL) family members are commonly implicated in light
signal transduction during early photomorphogenesis. However, some of
their functions remain unclear. Here, we propose a role for COL13 in
hypocotyl elongation in Arabidopsis thaliana. We found that COL13 RNA
accumulates at high levels in hypocotyls and that a disruption in the
COL13 function via a T-DNA insertion or RNAi led to the formation of
longer hypocotyls of Arabidopsis seedlings under red light. On the
contrary, overexpression of COL13 resulted in the formation of shorter
hypocotyls. Using various genetic, genomic, and biochemical assays, we
proved that another COL protein, COL3, directly binds to the promoter of
COL13, and the promoter region of COL3 was targeted by the transcription
factor LONG HYPOCOTYL 5 (HY5), to form an HY5-COL3-COL13 regulatory
chain for regulating hypocotyl elongation under red light. Additionally,
further study demonstrated that COL13 interacts with COL3, and COL13
promotes the interaction between COL3 and CONSTITUTIVE PHOTOMORPHOGENIC1
(COP1), suggesting a possible COP1-dependent COL3-COL13 feedback
pathway. Our results provide new information regarding the gene network
in mediating hypocotyl elongation.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Light is one of the most important environmental cues influencing the
early stages of post-germination plant development (Kami, Lorrain,
Hornitschek, \& Fankhauser, 2010; Olle \& Vir\selectlanguage{polish}š\selectlanguage{english}ile, 2013; Wu, Cameron,
Ljung, \& Spalding, 2010). Light-grown seedlings exhibit a developmental
response termed photomorphogenesis, resulting in short hypocotyls and
expanded green cotyledons. In contrast, dark-grown seedlings are
characterized by long hypocotyls and unexpanded etiolated cotyledons;
this process is called skotomorphogenesis (Josse \& Halliday, 2008;
McNellis \& Deng, 1995; Smith, 2000).

As a central light signal repressor, the RING finger protein CONSTITUTIVE
PHOTOMORPHOGENIC1 (COP1) is involved in many light-regulated responses
and is responsible for the ubiquitination and degradation of several
positive transcription factors, including B-BOX CONTAINING PROTEINs
(BBXs) and ELONGATED HYPOCOTYL 5 (HY5) (Dornan et al., 2004; Duek,
Elmer, van Oosten, \& Fankhauser, 2004; Lau \& Deng, 2012; Osterlund,
Hardtke, Wei, \& Deng, 2000; Seo, Watanabe, Tokutomi, Nagatani, \& Chua,
2004; Seo et al., 2003).

BBXs family has 32 members (Kumagai et al., 2008), which are divided
into five groups based on whether their respective proteins contain one
or two BBX motifs and whether or not they possess a CCT domain (Khanna
et al., 2009). BBX family members, some of which have been characterized
and implicated in light signal transduction during early
photomorphogenesis (Cheng \& Wang, 2005; Graeff et al., 2016; Li et al.,
2014; Park et al., 2011; Preuss et al., 2012; Wang, Guthrie, Sarmast, \&
Dehesh, 2014; Xu, Jiang, Li, Holm, \& Deng, 2018; Xu et al., 2016; Yang
et al., 2014). The first BBX protein identified in
\emph{Arabidopsisthaliana} was CONSTANS (CO) (Putterill, Robson, Lee,
Simon, \& Coupland, 1995)\emph{.} In addition to CO, 16 other CO-Like
(COL) proteins have been identified, which contain one or two B-box
domains at the N-terminus and a CCT domain at the C terminus (Cheng \&
Wang, 2005). However, most of their functions remain unclear. COP1 can
interact with BBXs proteins. For example, COP1 interact with COL3, which
acts as a positive regulator under red light and localizes to nuclear
speckles. The \emph{col3} mutant partially suppresses the \emph{cop1}
mutation, suggesting that COL3 acts genetically downstream of COP1
(Datta, Hettiarachchi, Deng, \& Holm, 2006). The loss-of-function
\emph{col3}mutant has longer hypocotyls and flowers early and exhibits a
reduced number of lateral branches (Datta et al., 2006). COL3 also
directly interacts with BBX32, which is regulated by the circadian clock
to mediate flowering (Tripathi, Carvallo, Hamilton, Preuss, \& Kay,
2017). Interestingly, both COL3 and BBX32 belong to the BBXs family.

HY5 is a positive regulator under far-red, red, blue, and UV-B light
conditions (Ang et al., 1998; Delker et al., 2014; Hardtke et al.,
2000). It mediates about one-third of genes expression throughout
the\emph{Arabidopsis} genome, including \emph{BBX} s (Lee et al., 2007).
For example, HY5 binds to the promoters of \emph{BBX30} and \emph{BBX31}
, both of which negatively regulate light response (Heng et al., 2019).
Meanwhile, the expression of \emph{HY5} is regulated by BBXs. For
instance, BBX21, BBX22, and BBX23 directly binds to the HY5 promoter to
activate its transcription and promote photomorphogenesis (Datta et al.,
2008; Zhang et al., 2017; Job et al., 2018), whereas BBX24, BBX25 and
BBX28 repress HY5 action (Gangappa et al., 2013; Job et al., 2018; Lin
et al. , 2018). Therefore, BBXs and HY5 constitute a light signal
regulatory network that was essential for promoting photomorphogenesis.

A previous study showed that COL3 played multiple roles in plant
development (e.g., flowering, hypocotyl elongation, and lateral root
formation) (Datta et al., 2006). Although COL3 is known to interact with
BBX32 to regulate flowering (Tripathi et al., 2017), there has been
little research on how COL3 regulates hypocotyl elongation and the
respective downstream pathways are uncharacterized. In the present
study, we proposed a role for COL13 and an HY5-COL3-COL13 regulatory
chain for controlling hypocotyl growth in \emph{A. thaliana} .

\textbf{Materials and methods}

\textbf{Plant materials and growth conditions}

The \emph{A. thaliana} mutant \emph{col13} (GK657F04-023194) in the
Columbia background (Col-0) was purchased from Germany (GABI-Kat, Max
Planck Institute for Plant Breeding Research, Carl-von-Linn\selectlanguage{ngerman}é-Weg 10,
50829 Köln, Germany) (Rosso et al., 2003), whereas \emph{col3} was
generously provided by Professor Magnus Holm (Datta et al., 2006).
Col-0, Ws, and their F1 hybrids were used as wild-type controls. Seed
sterilization and growth were performed as previously described (Liu et
al., 2016).

\textbf{Hypocotyl Experiments}

The light-response assays were performed as previously described with
some changes (Datta et al., 2006). Plates were treated at 4 °C for 72 h
and then moved to white light for 10 h to induce uniform germination.
After that, the plates were transferred to different light conditions
(dark, white, red, and blue light) and incubated at 22 °C for 3--6 d for
hypocotyl measurement. Red and blue lights were generated by light
emission diodes at 670 and 470 nm, respectively, (model E-30LED;
Percival Scienti\selectlanguage{english}fic). The light intensity was approximately 100
\selectlanguage{greek}μ\selectlanguage{english}mol/m\textsuperscript{2} s ppfd. The hypocotyl lengths of seedlings
were measured and counted using ImageJ software.

\textbf{Plasmid construction}

Constructs for overexpression and RNAi assays: To construct
the\emph{COL13} overexpression construct, the predicted
full-length\emph{COL13} cDNA was cloned and inserted into the
pCAMBIA1390 vector between the \emph{Sal} I and \emph{Eco} RI
restriction sites. To generate the \emph{COL13} -RNAi transgenic plants,
two fragments of the\emph{COL13} coding sequence were amplified by PCR
using primers containing \emph{Pst} I (5' end) and \emph{Mlu} I (3' end)
restriction sites, and \emph{Hin} dIII (5' end) and \emph{Bam} HI (3'
end) restriction sites. The two fragments were inserted into the pRNAi-0
vector in reverse orientation.

Constructs for GUS-staining assays: To construct the p\emph{COL13}
-GUS-2000 construct, a region comprising the 2000-bp promoter sequence
of \emph{COL13} was cloned and inserted into the pBI121 vector between
the \emph{Hin} dIII and \emph{Bam} HI sites. To construct the
p\emph{COL13} -GUS-2812 construct, a region comprising the 2812-bp
promoter sequence of \emph{COL13} was cloned and inserted into the
``1301 vector'' between the \emph{Sac} I and \emph{Sal} I sites. To
construct the p\emph{COL3} -GUS construct, a region comprising the
967-bp promoter sequence of \emph{COL3} was cloned and inserted into the
pBI101 vector between the \emph{Hind} III and \emph{Xba} I sites.

Constructs for yeast assays: To make the \emph{COL3} -pGBKT7,\emph{COP1}
-pGBKT7, \emph{COL13} -pGBKT7, \emph{COL3} -pGADT7,\emph{COP1} -pGADT7,
and \emph{COL13} -pGADT7 constructs, the\emph{COL3} , \emph{COP1,} and
\emph{COL13} fragments were subcloned into the pGBKT7 vector (Gal4 DNA
binding domain, Cat. No. 630489, Clontech) and pGADT7 (Gal4 activation
domain, Cat. No. 630442, Clontech), as appropriate. To construct the
COL3-pBbidge and COL3-COL13-pBbidge constructs, the \emph{COL3} and
\emph{COL13} fragments were subcloned into the
pBbidge\textsuperscript{TM} vector (Cat. No. 630404, Clontech), as
appropriate. To construct the GAD-HY5 or GAD-COL3 fusion protein in
yeast, the HY5 or COL3 coding sequence was subcloned into pJG4-5 with
the \emph{EcoRI} and \emph{XhoI} sites. To construct the COL3-LacZ
reporter gene, the 1119-bp COL3 promoter region was amplified from
genomic DNA and cloned into the pLacZi2u vector with the HindIII and
XhoI sites (Lin et al., 2007). Other LacZ reporter gene plasmids
containing various truncated COL13 promoters were similarly constructed
using the primers listed in Supplementary Table S1.

Constructs for GFP, CFP, and YFP assays: To construct the\emph{COL13}
-GFP construct, the full-length \emph{COL13} coding region was cloned
and inserted into the pBEGFP vector between the \emph{Xba} I and
\emph{Kpn} I restriction sites. To construct the COL3-CFP, COP1-CFP,
COL13-CFP, COL3-YFP, COP1-YFP, and COL13-YFP constructs, the full-length
coding regions of \emph{COL3} , \emph{COP1,} and \emph{COL13} were
cloned and inserted into the pBluescript II Phagemid vector (Y. Liu et
al., 2016), as appropriate.

Constructs for Co-IP assays: To construct the 35S:\emph{COL3-} HA
construct, the full-length \emph{COL3} cDNA was cloned and inserted into
the pCAMBIA1390-HA vector (Fang et al., 2019).

Constructs for dual-luciferase assays: Fragments of the COL3 or COL13
promoter were cloned into pGREEN0800-LUC to generate reporter vectors. A
modified pBluescript vector (pBS) was used as an effector (Han et al.,
2017).

The primers used are listed in Supplementary Table 1.

\textbf{Plant transformation}

Constructs in binary vectors were introduced into the\emph{Agrobacterium
tumefaciens} strain LBA4404 and transformed into\emph{Arabidopsis} wild
type (WT) or mutant plants by the floral-dip method (Clough \& Bent,
1998). Approximately 30 T1 transgenic plants for each transgene were
screened on MS medium supplemented with the appropriate antibiotics, and
phenotypic analyses were performed on T2 or later generations.

\textbf{Semi-quantitative reverse transcriptase (RT)-PCR and
quantitative reverse transcriptase (qRT)-PCR}

Semi-quantitative RT-PCR and qRT-PCR analyses were performed as
previously described (Zhang, Liu, et al., 2014). RNA was extracted from
5-d-old seedlings. Three biological and three technical repetitions were
performed for each combination of cDNA samples and primer pairs. The
primers used are listed in Supplementary Table 1.

\textbf{Dual-luciferase assay}

Protoplasts were isolated, and the dual-luciferase assay was performed
as previously described (Han et al., 2017). Transformed protoplasts were
incubated at room temperature (\textasciitilde{}22) for 20-22 h and
luciferase activities were measured using the dual-luciferase assay
system (Dual-Luciferase\selectlanguage{ngerman}\textsuperscript{®} Reporter Assay, Promega,
United States) according to the manufacturer's instructions. Firefly
luciferase activity was normalized to \emph{Renilla} luciferase
activity. Three biological replicates were performed for all
experiments.

\textbf{Electrophoretic mobility shift assay (EMSA)}

EMSA was performed as previously described with the LightShift
Chemiluminescent EMSA Kit (Thermo Scienti\selectlanguage{english}fic, United States) (Han et al.,
2017). The dual-luciferase assay mapped the COL3 binding site to a 1059
bp region of the \emph{COL13} promoter, located between 676 and 1675 bp
upstream of the transcription start site (ATG). This promoter region was
used as a 5\selectlanguage{english}' end biotin-labeled probe and the same fragment, but
unlabeled, was used as a competitor. To investigate the core-binding
motif of the 1059 bp region, a series of EMSAs involving deletions of
this region were performed. We divided the 1059 bp promoter sequence
into five overlapping regions: -1675 to -1391~bp (probe 1), -1421 to
-1184~bp (probe 2), -1201 to -1040~bp (probe 3), -1060 to -868~bp (probe
4), and -898 to -616~bp (probe 5). The sequence of probes is listed in
Supplementary Table 2.

\textbf{Yeast assays}

Yeast one-hybrid assays were performed as described by Lin et al.
(2007). The yeast two-hybrid and three-hybrid assays were performed
using a Clontech kit (PT3024-1 (PR973283)). For yeast two-hybrid assays,
the bait vectors (pGBKT7 plus candidate genes) and prey vectors (pGAGT7
plus candidate genes) were transformed into the gold and Y187 yeast
strains, respectively, and then each colony was picked for mating, and
the mating solution was sprayed on SD/-Trp/-Leu/X-\selectlanguage{greek}α-\selectlanguage{english}Gal/AbA (DDO/X/A)
agar plates, and positive results were confirmed by growing them on
SD/-Ade/-His/-Trp/-Leu/X-\selectlanguage{greek}α-\selectlanguage{english}Gal/AbA (QDO/X/A) agar plates. For the yeast
three-hybrid assay, COP1-pGADT7 and COL3-COL13-pBbidge constructs were
co-transformed into the gold yeast strain. Each colony was picked and
grown in SD/-Leu/-Met/-Trp and SD/-Leu/-Trp solutions, respectively.
Normalized Miller units were calculated as a ratio of \selectlanguage{greek}α-\selectlanguage{english}galactosidase
activity in yeast. For all yeast assays, we used empty vectors as
controls.

\textbf{Co-immunoprecipitation (Co-IP)}

Co-IP was performed as previously described with some changes (Fiil,
Qiu, Petersen, Petersen, \& Mundy, 2008). 35S:COL3-HA and 35S:COL13-GFP
constructs were transformed into EHA105 \emph{Agrobacterium} cells and
then used to generate 35S:COL3-HA and 35S:COL3-HA::COL13-GFP transgenic
plants. Proteins were extracted from 18-d-old seedlings. Anti-GFP used
in this assay was bought from Abcam (ab290), Anti-HA used in this assay
was bought from Sigma (H6908).

A 0.5 g sample of \emph{Arabidopsis} seedlings were ground in liquid
nitrogen, 1 ml (2 volumes) of lysis buffer was added (50 mM Tris pH 8.0,
150 mM NaCl, and 1 mM EDTA, containing 0.01 volume of 1\selectlanguage{ngerman}× Protease
Inhibitor Cocktail {[}Sigma{]}) or protease inhibitors (1 mM PMSF, 1
\selectlanguage{greek}μ\selectlanguage{english}g/ml aprotinin, 1 \selectlanguage{greek}μ\selectlanguage{english}g/ml leupeptin, 1 \selectlanguage{greek}μ\selectlanguage{english}g/ml pepstatin). It was spun at
16,000 \emph{g} for 20 min at 4 \selectlanguage{ngerman}°C, and the supernatant was transferred
to a new microcentrifuge tube. Protein concentration was determined
using the Bradford reagent (Bio-Rad) and bovine serum albumin was used
as a standard. For immunoprecipitation (IP) reactions, 1 mg of total
protein was incubated with 5 \selectlanguage{greek}μ\selectlanguage{english}l of polyantiserum (or pre-immune serum)
in a total volume of 1 ml of lysis buffer and incubated for 1 h to
overnight at 4 \selectlanguage{ngerman}°C, and the sample was mixed by inversion. During
incubation, a 40 \selectlanguage{greek}μ\selectlanguage{english}l wash per reaction of Protein-A-Agarose beads with 1
ml of cold lysis buffer or phosphate-buffered saline (PBS) was used by
gentle vortexing and spinning in a microcentrifuge at 12,000 \emph{g}for
30 s. The supernatant was carefully removed by aspiration. This process
was repeated twice. A 50 \selectlanguage{greek}μ\selectlanguage{english}l sample of fresh lysis buffer or PBS was
added. The beads were ready to be used. To precipitate the immune
complexes, 50 \selectlanguage{greek}μ\selectlanguage{english}l of Protein-A-Sepharose (Amersham Pharmacia Biotech)
slurry was added and incubated for 2 h to overnight at 4 \selectlanguage{ngerman}°C, mixing the
sample head-over-tail. The beads were washed in the microcentrifuge tube
by resuspension in 700 \selectlanguage{greek}μ\selectlanguage{english}l of lysis buffer. It was then centrifuged at
12,000 \emph{g} for 15--30 s at 4 \selectlanguage{ngerman}°C. The effluent was discarded. Steps
7 and 8 were repeated three more times. Next, 40 \selectlanguage{greek}μ\selectlanguage{english}l of 2\selectlanguage{ngerman}× SDS sample
buffer was added to the beads. The beads were gently mixed (no
vortexing) to avoid spreading the beads on the column walls. The sample
was heated at 95 °C for 5 min to ensure that the Protein-A-Sepharose
complex was within the heating well. The sample was then centrifuged at
12,000 \emph{g} for 30 s. A sample of 10 \selectlanguage{greek}μ\selectlanguage{english}l of the eluted
immunoprecipitate was loaded on an SDS-PAGE gel (Bio-Rad). For standard
western blotting analysis, 50 \selectlanguage{greek}μ\selectlanguage{english}g of total protein was loaded. Proteins
were electroblotted onto PVDF membranes (Amersham), blocked for at least
1 h at room temperature in PBS-Tween containing 5\% (wt/vol) non-fat
dried milk. Primary antibodies were added to PBS-Tween containing 5\%
(wt/vol) non-fat dried milk and incubated for 1 h. Blots were developed
using an ECL Kit from Amersham Pharmacia Biotech.

\textbf{Histochemical GUS staining, GFP, and fluorescence resonance
energy transfer (FRET) experiments}

Histochemical GUS staining, GFP microscopy, and FRET were performed as
previously described with some modifications (Datta et al., 2006; Hou,
Wu, \& Gan, 2013; Zhang, Zhang, et al., 2014). For the GUS-staining
assay, the young seedlings (4 d after germination) were fixed and
incubated in GUS-staining solution for 24 h at 37 \selectlanguage{ngerman}°C. The stained
samples were then cleaned with 75\% ethanol and observed under a
dissecting microscope.

For the GFP assay, the fusion \emph{COL13} -GFP constructs were
transformed into protoplasts for transient expression as previously
described (Wu et al., 2009). Ten stable transgenic plants with
\emph{COL13} -GFP were obtained using the floral-dip method. Photographs
of GFP were taken using a confocal microscope Olympus.

For the FRET assay, images were acquired using an Olympus confocal
microscope, and protoplasts were visualized 16 h after transformation.
The CFP was excited by a laser diode 405 laser and YFP, by an argon-ion
laser. The target regions were bleached with 100 iterations using an
argon-ion laser at 100\% power.

\textbf{Statistical analysis}

Experimental data were analyzed using an ANOVA, and the statistical
significance of any differences between treatments was tested using
Duncan's test or t-tests. All analyses were conducted using SPSS for
Windows.

\textbf{Results}

\emph{COL13} \textbf{RNA accumulates at high levels in hypocotyls}

By searching the gene expression information in the Arabidopsis
Information Resource (TAIR) database (Klepikova, Kasianov, Gerasimov,
Logacheva, \& Penin, 2016), we found that \emph{COL13}(\emph{AT2G47890}
) is highly expressed in the hypocotyl. qRT-PCR analysis confirmed
that\emph{COL13} was expressed in most plant organs, with higher
expression in the hypocotyl and stem (Fig. 1a). To determine the spatial
patterns of \emph{COL13} expression in more detail, transgenic lines
expressing GUS driven by the 2812 bp \emph{COL13} promoter fragment were
generated. As shown in Fig. 1b, GUS expression was predominantly in the
hypocotyl.

\textbf{COL13 regulates hypocotyl elongation under red-light conditions}

To characterize the role of \emph{COL13} in plants, we obtained the
corresponding \emph{Arabidopsis} T-DNA insertion mutant
(GK-657F04-023194, termed \emph{col13} in the following; Fig. S1a) from
GABI-Kat, Max Planck Institute for Plant Breeding Research (Rosso et
al., 2003). The mutation was verified by PCR (Fig. S1b), which amplified
the\emph{Sulphonamide}(\emph{sul} ) gene by using the primers listed in
Supplementary Table 1. To con\selectlanguage{english}firm that the phenotype of the \emph{col13}
mutant was indeed caused by disruption of the \emph{COL13} gene, we
generated \emph{COL13}overexpression (OX) (Fig. 2a) and \emph{COL13}
RNAi transgenic lines (Fig. 2b) for comparison.

To examine whether \emph{COL13} was involved in light responses, the WT,
\emph{COL13} RNAi, and\emph{col13} seedlings were germinated and grown
under different light wavelengths (white, red, blue), as well as under
dark conditions. As shown in Fig. S1c, under white or red light, the
\emph{COL13} RNAi and\emph{col13} seedlings had longer hypocotyls than
that of the WT, whereas in blue light or dark conditions, the hypocotyl
length of all seedlings was not significantly different. Therefore, our
research focused on red light. For further study, \emph{COL13} OX,
\emph{COL13}RNAi, \emph{col13,} and WT seedlings were germinated and
grown under red light. We found that the \emph{COL13} OX seedlings had
shorter hypocotyls than the WT seedlings under red light (Fig. 2c, d).
In contrast, the \emph{COL13} RNAi and \emph{col13} seedlings had longer
hypocotyls than the WT seedlings under the same conditions (Fig. 2c, e).
These \selectlanguage{english}findings suggested that COL13 acts as a positive regulator of
red-light-mediated inhibition of hypocotyl elongation.

\textbf{Genetic interaction and physiological characterization of
hypocotyl elongation}

Given that the \emph{phyB} , \emph{hy5, col3,} and \emph{cop1} mutations
can affect hypocotyl elongation under red-light conditions (Datta et
al., 2006; Lee et al., 2007; Peter H Quail, 2002; von Arnim \& Deng,
1994), we investigated the expression of \emph{COL13} in the absence
of\emph{PHYB} , \emph{COL3} , \emph{HY5,} and \emph{COP1} .
Semi-quantitative RT-PCR and qRT-PCR analysis revealed that the
expression of \emph{COL13}in \emph{phyB} , \emph{col3,} or \emph{hy5}
knockout plants was signi\selectlanguage{english}ficantly reduced compared with that of the WT,
whereas the expression of \emph{COL13} in the \emph{cop1} mutants was
increased (Fig. 3a, b). As the expression of \emph{COL13} decreased the
most in the \emph{col3} mutant, we generated transgenic lines expressing
GUS under the control of the \emph{COL13} promoter with the
\emph{col3}mutant background. Interestingly, although the \emph{COL13}
promoter was active in the hypocotyls and cotyledons in the WT
seedlings, GUS expression was not detected in the hypocotyl in the
\emph{col3} mutant background (Fig. 3c).

To understand the functional relationship and genetic interaction
between \emph{COL13} and \emph{COL3} and their role in the regulation of
hypocotyl growth, we generated a \emph{col13} \emph{col3} double mutant
and examined hypocotyl length under red-light conditions. Given
that\emph{col13} was in Col-0 and \emph{col3} was in the WS background,
crossing lines from different backgrounds would likely affect hypocotyl
length. To reduce the effect of the background, we used the F1 hybrid of
Col-0\emph{x} WS as the WT. We found that, although hypocotyl length in
the double-mutant \emph{col13 col3} was longer than in the WT seedlings,
it was not significantly different from hypocotyl length in the single
mutants, \emph{col13} or \emph{col3.} (Fig. 3d). To confirm this result,
we created the RNAi lines of \emph{COL13} in the \emph{col3} mutant
background (Fig. 3e), and we obtained the same result as in Fig. 3d.
Additionally, we also generated a \emph{COL13} -OX line in
the\emph{col3} mutant background and showed that the hypocotyl length in
this strain was similar to that of the WS and significantly shorter than
that of the \emph{col3} mutant (Fig. 3e)\emph{.} In other
words,\emph{COL13} overexpression rescued the phenotype exhibited by
the\emph{col3} mutant. Taken together, our results suggest
that\emph{COL13} might be downstream of \emph{COL3} in the
red-light-mediated signaling pathway.

\textbf{HY5-COL3-COL13 regulatory chain}

Based on the genetic data, the \emph{col3 hy5} double mutant behaved
like the \emph{hy5} mutation (Datta et al., 2006), and \emph{COL13}might
be downstream of \emph{COL3} in regulating hypocotyl elongation. In
addition, it has been well established that HY5 binds to ``CACG'' and
``GACGTG'' in the promoters of light-responsive genes (Lee et al., 2007;
Nawkar et al., 2017). COL3 is a light-responsive gene, and we examined
the promoter sequence of the COL3 gene and found that it contains two
``CACG'' and one ``GACGTG''. Thus, we hypothesized that there would be
an HY5-COL3-COL13 regulatory chain for controlling hypocotyl
growth\emph{.} To test this hypothesis, the HY5 and \emph{COL3} coding
sequences, as well as a deletion series of the \emph{COL13} promoter,
were cloned into the dual-luciferase system (Fig. 4a). As shown in
Figure 4b, these dual-luciferase experiments and yeast-one hybrid assays
con\selectlanguage{english}firmed the ability of HY5 to bind to the \emph{COL3} promoter and COL3
to bind to the \emph{COL13} promoter. Additionally, these experiments
also mapped the COL3 target regions (1059 bp) to between -1675 bp and
-616 bp of the \emph{COL13} promoter (Fig. 4b). To investigate the
core-binding motif of the 1059 bp region, a series of EMSAs involving
deletions of this region were performed. We divided the 1059 bp promoter
sequence into five overlapping regions (Fig. 4c): -1675 to -1391~bp
(probe 1), -1421 to -1184~bp (probe 2), -1201 to -1040~bp (probe 3),
-1060 to -868~bp (probe 4), and -898 to -616~bp (probe 5), and showed
that probe 2 (-1421 to -1184~bp) was essential for binding of COL3 to
the \emph{COL13} promoter (Fig. 4d). The \emph{in vivo} interaction of
COL3 with probe 2 was further con\selectlanguage{english}firmed by EMSA competition experiments
that were conducted by adding excess amounts of the competitor (5-, 10-,
and 25-fold higher amounts) (Fig. 4e). Next, we analyzed the sequence of
probe 2, and found that in the 238 bp fragment, there were three light
responsive elements (ATCT-motif, G-Box and TCT-motif) and one core
promoter element for transcription start (TATA-box) (Fig. 4f). Then we
wondered if these promoter elements were required for binding with COL3.
To address this question, yeast one-hybrid assays were performed and the
results showed that a 49-bp region of \emph{COL13} promoter containing
G-Box and TCT-motif were the binding requirement (Fig, 4g).

\textbf{COL13 is located in the nucleus}

Transformation of \emph{Arabidopsis} protoplasts with a construct
expressing COL13-CFP indicated that COL13 is located in the nucleus
(Fig. 5a), and a similar result was obtained when the root apical cells
of stable COL13-GFP transgenic plants were examined (Fig. 5b).

\textbf{COL13 interacts with COL3} \textbf{, but not COP1}

According to previous reports, both COL3 and COL13 are CONSTANS
(CO)-like proteins, which are related to CO (Robson et al., 2001), and
as shown for COL13 above, COL3 also positively regulates
red-light-mediated inhibition of hypocotyl elongation
in\emph{Arabidopsis} (Datta et al., 2006). We also demonstrated that
COL13 shares the same subcellular localization as COL3 (Fig. 5a, b).
Given that COL3 can interact with BBX32 and that COL13 also belongs to
the BBX zinc finger TF family, we hypothesized that COL3 might interact
with COL13. This idea was supported by a two-hybrid assay revealing that
COL3 interacts with COL13 protein in yeast (Fig. 6a). Next, we examined
the interaction in transgenic plants expressing both COL3 and COL13 and
showed that COL13 was co-immunoprecipitated with COL3 from seedling
tissues (Fig. 6b). The phenotypes of 35S:COL3-HA and 35S:COL13-GFP
transgenic plants were the same as 35S:COL3 and 35S:COL13 transgenic
plants, respectively, which produced shorter hypocotyl than WT grown in
the presence of red light (Fig. S2). The interaction between COL13 and
COL3 was also demonstrated in plant cells in a FRET assay (Fig. 6c-f).
As shown in Fig. 6c, both cyan \selectlanguage{english}fluorescent protein (CFP)-fused COL3 and
yellow \selectlanguage{english}fluorescent protein (YFP)-fused COL13 were observed in the nucleus
after excitation with a 405 nm or 514 nm laser, respectively. After
bleaching an area of interest with the 514 nm laser, YFP-COL13
fluorescence was reduced dramatically, whereas there was a clear
increase in CFP-COL3 emission in the same area (Fig. 6d), indicating
that FRET had occurred. The relative intensities of emissions from
CFP-COL3 and YFP-COL13 in the area of interest, before and after
bleaching, are shown in Fig. 6e, f.

\textbf{COL13 promotes interaction between COL3 and COP1}

Interestingly, although COL13 and COL3 have similar structures,
containing two N-terminal tandemly repeated B-box domains and a CCT
domain in the C-terminal, only COL3 can interact with COP1, and COL13
does not bind to COP1 (Fig. 6a). These results were also demonstrated by
the FRET assay (Fig. S3). To investigate whether COL13 influences the
interaction between COP1 and COL3, we performed a yeast three-hybrid
assay. In this yeast system, the COL3-COL13-pBridge construct allowed
expression of \emph{COL3-BD} /bait and \emph{COL13} in yeast, and
\emph{COL13} was expressed only in the absence of methionine (Met). As
shown in Fig. 7a, the growth of yeast carrying indicated constructs on
selective medium (+Met or -Met) along with an \selectlanguage{greek}α-\selectlanguage{english}galactosidase assay that
showed that COP1 and COL3 had a stronger binding activity with the
expression of COL13. Based on a previous report, COP1 interacted with
COL3 and inhibited the production of COL3 (Datta et al., 2006). By
combining our results above, we propose a possible COP1-dependent
COL3-COL13 feedback pathway (Fig. 7b), which is involved in the
regulation of hypocotyl elongation.

\textbf{Discussion}

Light regulates photomorphogenesis in plants. A large number of genes
that are involved in such photomorphogenesis processes have been
identified as light receptors (Datta et al., 2006; Kircher et al., 2002;
Peter H. Quail, 2002), signal transduction factors (Gangappa et al.,
2013; Osterlund et al., 2000) or degradation proteins (Crocco, Holm,
Yanovsky, \& Botto, 2010; Crocco et al., 2015; Delker et al., 2014). One
of the immediate questions is how these genes act in a network to
mediate various light-related phenotypes. It has been shown that
multiple pathways are interlinked to form a gene network of
photomorphogenesis (Lau \& Deng, 2012; Lee, Park, Ha, Baldwin, \& Park,
2017). Among these pathways, it is worth mentioning the ones formed by a
subset of family genes termed the COL genes (Cheng \& Wang, 2005). These
family of genes plays multiple roles in plant development (Datta et al.,
2006; Graeff et al., 2016; Muntha et al., 2018; Tripathi et al., 2017;
Wang et al., 2014). As an effort toward COL networking, we investigated
the relationship between\emph{COL3} and \emph{COL13} and provided
evidence that these two COLs and HY5 were connected together to form an
HY5-COL3-COL13 regulatory chain that controls hypocotyl elongation in
\emph{Arabidopsis}(Fig. 7b). Hypocotyl elongation is a genetically
well-controlled process that responds to light. In \emph{Arabidopsis} ,
several key genes are required for hypocotyl growth. Among these, COP1
is a negative regulator (McNellis, von Arnim, \& Deng, 1994), whereas
HY5 and COL3 are considered to be positive (Datta et al., 2006; Hardtke
et al., 2000). A previous study showed that COL3 plays a role in
flowering and hypocotyl elongation (Datta et al., 2006), and COL3 is
known to interact with B-BOX32 to regulate flowering (Tripathi et al.,
2017). However, there has been no research on how COL3 regulates
hypocotyl elongation. In this study, we demonstrated that \emph{COL13} ,
whose RNA accumulated to a high level in the hypocotyl (Fig. 1), was one
more positive regulator in the regulation of hypocotyl elongation under
red-light conditions. For example, overexpression of \emph{COL13} or
knockdown of its transcript resulted in a shorter or longer hypocotyl,
respectively, (Fig. 2).

As a positive regulator under far-red, red, blue, and UV-B light
conditions (Ang et al., 1998; Delker et al., 2014; Hardtke et al.,
2000), HY5 mediates about one-third of genes expression throughout
the\emph{Arabidopsis} genome (Lee et al., 2007). This study revealed
that HY5 bound to the promoter of \emph{COL3} and upregulate its
transcription (Fig. 4b), indicating that \emph{COL3} also acted as a
downstream target of HY5. Interestingly, COL3 bind to the
\emph{COL13}promoter and positively regulate its expression (Fig.4b-g),
suggesting that COL3 are positive regulators of COL13. Thus, these
findings suggest that HY5, COL3, and COL13 constitute a hypocotyl
regulatory pathway.

BBX family members are commonly involved in photomorphogenesis and that
they can interact with other BBX proteins to regulate plant growth
(Tripathi et al., 2017; Wang et al., 2014). For example, COL3 belong to
the BBX family and COL3 interact with BBX32 to regulate flowering
(Tripathi et al., 2017). In this study, we demonstrated that COL13 could
interact with COL3 (Fig. 6). Furthermore, we found that the expression
of COL13 promoted the interaction between COP1 and COL3 (Fig. 7a). To
our knowledge, COP1 is responsible for the degradation of several
positive TFs, such as COL3, in the dark (Datta et al., 2006; Dornan et
al., 2004; Duek et al., 2004; Lau \& Deng, 2012; Osterlund et al., 2000;
Seo et al., 2004; Seo et al., 2003). Increasing the binding activity of
COP1 and COL3 would lead to the degradation of COL3. As a result, there
would be less COL3 to activate the expression of COL13 (Fig. 7b).
Therefore, we proposed a possible COP1-dependent COL3-COL13 feedback
pathway as a hypothesis to optimize the HY5-COL3-COL13 regulatory
pathway (Fig. 7b). This feedback pathway could enrich the regulation
network in hypocotyl elongation.

\textbf{Acknowledgements}

We are grateful to Prof. Magnus Holm (Gothenburg University, Sweden) for
providing the \emph{col3} mutant seeds. This work was supported by the
National Natural Science Foundation of China (31572161 and 31672188),
the Youth Foundation of the National Natural Science Foundation of China
(30900107), and China International Postdoctoral Program.

\textbf{Author contributions}

S.S. and W.X. proposed the project. S.S., W.X., and L.B. designed the
experiments. L.B., L.H., Y.J., Y.L., Z.Q., C.H., and G.S. performed the
experiments. L.B. and S.S. analyzed the data. L.B. wrote the manuscript,
with the participation of S.S. and W.Y.

\textbf{Conflict of Interest Statement}

The authors declare no conflict of interest.

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Experimental Botany, 65} (12), 3201-3213.

\textbf{Figure legends}

\textbf{Fig. 1 \emph{COL13} RNA accumulates at high levels in the
hypocotyl.} (a) Quantitative real-time PCR analysis of
\emph{COL13}transcript abundance in different tissues. R=Root; S=Stem;
L=Leaf; SAM=Shoot apical meristem; H=Hypocotyl; F=Flower. (b) Activity
of the COL13 promoter revealed by \selectlanguage{greek}β-\selectlanguage{english}glucuronidase (GUS) staining
in\emph{Arabidopsis} seedlings. Bar = 100 mm.

\textbf{Fig. 2 COL13 regulates hypocotyl elongation under red-light
conditions.} (a) Relative expression of \emph{COL13} in Col-0 and
overexpression (OX) lines. (b) Relative expression of \emph{COL13} in
Col-0, T-DNA mutant (\emph{col13} ) and RNAi lines (R1-1 etc.).
Asterisks indicate that the expression of \emph{COL13} in OX9, OX11,
OX20,\emph{col13} , and \emph{COL13} RNAi lines are significantly
different with Col-0 under red light (P \textless{} 0.05). (c)-(e)
Phenotypic analysis seedlings of the indicated genotypes were grown in
the presence of red light. Images of representative seedlings are shown
in (c), white bar=0.5cm. The hypocotyl lengths of the indicated
genotypes were measured at the 5\textsuperscript{th} or
3\textsuperscript{rd} day, and are shown in (d) and (e), respectively.
Error bars indicate SD (n \textgreater{}15). Asterisks indicate that
hypocotyl lengths in OX9, OX11, OX20, \emph{col13} , and \emph{COL13}
RNAi lines are significantly different with Col-0 under red light (P
\textless{} 0.05).

\textbf{Fig. 3 Genetic interaction and physiological characterization of
hypocotyl elongation. (a)} Semi-quantitative reverse transcriptase
(RT)-PCR analyses of \emph{COL13} expression in \emph{phyB} ,\emph{col3}
, \emph{hy5} and \emph{cop1} mutants. (\textbf{b} ) qRT-PCR analyses of
\emph{COL13} expression in \emph{phyB} , \emph{col3} ,\emph{hy5} and
\emph{cop1} mutants. (\textbf{c} ) Activity of the\emph{COL13} promoter
revealed by \selectlanguage{greek}β-\selectlanguage{english}glucuronidase (GUS) staining in WT and \emph{col3} mutant
backgrounds. (\textbf{d} ) Hypocotyl length in WT, single- and
double-mutant plants. Here we use the F\textsubscript{1}hybrid of
Col-0\selectlanguage{ngerman}×WS as WT. (\textbf{e} ) Hypocotyl length in WT and\emph{col3}
plants compared to transgenic plants with \emph{COL13} RNAi or
\emph{COL13} overexpression (OX) in the \emph{col3} background. Here we
use WS as WT. Error bars indicate SD (n\selectlanguage{english} \textgreater{}15). Lower-case
letters indicate significantly different data groups (hypocotyl length)
of the indicated seedlings grown in red light.

\textbf{Fig. 4 Analysis of the binding of HY5 to the COL3 promoter and
COL3 to COL13 promoter truncations.} (a) Diagram of constructs used in
this study. For luciferase system, the AD-HY5 or AD-COL3 fusion proteins
driven by the 35S promoter produces a potential effector protein, while
the AD protein alone represents a negative control for basal activity
of\emph{COL3} promoter or each \emph{COL13} promoter truncation.
The\emph{LUC} gene driven by the series of \emph{COL3} promoter
or\emph{COL13} promoter truncations tests the ability of the AD-HY5 or
AD-COL3 fusion protein to bind to each promoter truncation. For
yeast-one hybrid system, the GAD-HY5 or GAD-COL3 fusion proteins driven
by the \emph{GAL1} (P\textsubscript{GAL1}) promoter serves as effectors.
The GAD protein served as negative control to see if there exits the
self activity of \emph{COL3} or \emph{COL13} promoters. The
\emph{LacZ}gene driven by \emph{COL3} or \emph{COL13} promoter
truncations served as the reporter to test the binding activity of the
GAD-HY5 or GAD-COL3 fusion protein to individual promoter truncations.
(b) For luciferase assay, the fusion protein AD-HY5 can up-regulate
\emph{LUC} expression from the \emph{COL3} promoter, but not from
\emph{COL13} promoter; and the fusion protein AD-COL3, but not AD alone,
can up-regulate \emph{LUC}expression from some of the \emph{COL13}
promoter truncations. For Yeast-one hybrid assay, the fusion protein
GAD-HY5 can strongly bind to the promoter of \emph{COL3} , but not
\emph{COL13} to direct \emph{LacZ}expression in yeast cells that turns
5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside to blue compound; and
the fusion protein AD-COL3, but not AD alone, can bind to some of
the\emph{COL13} promoter truncations. (c) The 1059-bp promoter (-1675 bp
to -616 bp ) of \emph{COL13} was devided into five overlapping regions:
-1675 to -1391 bp (probe 1), -1421 to -1184 bp (probe 2), -1201 to -1040
bp (probe 3), -1060 to -868 bp (probe 4), and -898 to -616 bp (probe 5).
(d) Electrophoretic mobility shift assay (EMSA) analysis showing the
binding of COL3 on the -1421 to -1184 bp promoter of \emph{COL13} (probe
2) in vitro. The \textbf{+} and \textbf{--} represent the positive and
negative control, respectively. (\textbf{e} ) Electrophoretic mobility
shift assay (EMSA) analysis showing the binding of COL3 on the -1421 to
-1184 bp promoter of \emph{COL13} in vitro. The black arrow indicates
binding of COL3 to the biotin-labeled \emph{COL13} promoter.
The\textbf{+} and \textbf{--} represent the presence and absence of
corresponding components, respectively. Competition experiments were
carried out by adding 5-, 10- and 25-fold excessive competitor. (f) In
probe 2, there were three light responsive elements (ATCT-motif, G-Box
and TCT-motif) and one core promoter element for transcription start
(TATA-box). (g) The fusion protein GAD-COL3, but not GAD alone, can
strongly bind to a promoter truncation of \emph{COL13} to direct
\emph{LacZ} expression in yeast cells. The 238-bp promoter region (-1421
bp to -1184 bp ) of\emph{COL13} was devided into four fragments: -1421
to -1356 bp, -1355 to -1307 bp, -1306 to -1242 bp, and -1241 to -1184
bp. The colour rectangles represented indicated promoter elements in
(f).

\textbf{Fig. 5 Subcellular localization of COL13} (a) COL13-CFP
localizes to the nucleus in protoplasts. (d) COL13-GFP localizes to the
nucleus in root tip cells.

\textbf{Fig. 6 COL13 interacts with COL3.} (a) Yeast two-hybrid assay
between COL13 and COL3. DDO=double dropout; QDO=quadruple dropout;
pGADT7=prey plasmid; pGBKT7=bait plasmid. (b) Co-immunoprecipitation
(Co-IP) in \emph{Arabidopsis} Immunoprecipitations (IPs) were performed
on proteins extracted from 10 d-old \emph{Arabidopsis} seedlings grown
under long-day illumination (16L: 8D) at 22 degC. Leaf tissues were
harvested 1 h after the light cycle commenced. IP was performed using an
anti-HA antibody and COL13 was co-immunoprecipitated with an anti-GFP
antibody. A 5\% input was used. Western blots were performed on 10\%
(wt/vol) precast gels (Bio-Rad). (c) COL3-CFP and COL13-YFP colocalize
to the nucleus in protoplasts in the light and dark. Bar=5um. (d-f) FRET
between CFP-COL3 and YFP-COL13 analyzed by acceptor bleaching in the
nucleus. Bar=5um. The top panels in (d) show a representative pre-bleach
nucleus co-expressing YFP-COL13 and CFP-COL3 excited with either a 514
or a 405 nm laser in light and dark, resulting in emission from YFP
(yellow) or CFP (blue), respectively. The bottom panels in (d) show the
same nucleus post-bleaching after excitation with a 514 or a 405 nm
laser. The relative intensities of both YFP and CFP were measured before
and after bleaching, as indicated in (e) and (f), respectively.

\textbf{Fig. 7 COL13 promotes the interaction between COL3 and COP1.}

(a) Yeast three-hybrid analysis of COP1-COL3 interaction in the presence
of COL13. Normalized Miller units were calculated as a ratio of
\selectlanguage{greek}α-\selectlanguage{english}galactosidase activity in yeast. Additionally, normalized Miller units
are reported separately for yeast grown on media with or without 1 mM
methionine (Met), corresponding to induction (-Met) or repression (+Met)
of \emph{Met25} promoter-driven COL13 expression, respectively. Means
and standard errors of the means for three biological repetitions are
shown. Lower-case letters indicate significant differences in
\selectlanguage{greek}α-\selectlanguage{english}galactosidase. (b) A model representing the HY5-COL3-COL13 regulatory
chain and COP1-dependent COL3-COL13 feedback pathway in the regulation
of hypocotyl elongation.

\textbf{Supporting Information}

\textbf{Table S1 List of primers and their uses.}

\textbf{Table S2 Probes used in EMSA assay.}

\textbf{Fig. S1 COL13 regulates hypocotyl elongation under red-light
conditions.} (a) Scheme of the \emph{Arabidopsis} COL13 gene (AT2G47890)
showing the T-DNA insertion position. (b) Identification of the
sulfonamide resistance gene (SUL) in the col13 mutant by PCR. (c) The
hypocotyl lengths of the wild type (WT), COL13 RNAi, and col13 seedlings
under different light conditions. Error bars indicate the standard
deviations (n \textgreater{}15). Lower-case letters indicate significant
differences (P \textless{} 0.05).

\textbf{Fig. S2 Hypocotyl lengths of the indicated genotypes were
measured at the 5\textsuperscript{th} day.} Error bars indicate SD
(n \textgreater{}15). Asterisks indicate that hypocotyl lengths in
35S:COL-HA and 35S:COL13-GFP are significantly different with Col-0
under red light (P \textless{} 0.05).

\textbf{Fig. S3 COP1 can interact with COL3, but not COL13.} (a)
COL3-CFP and COP1-YFP co-localized to the nucleus in protoplasts under
both light and dark conditions. (b) FRET between CFP-COL3 and YFP-COP1
analyzed by acceptor bleaching in the nucleus. The top panels in b show
a representative pre-bleach nucleus co-expressing YFP-COP1 and CFP-COL3
excited with either a 514 or a 405 nm laser in light and dark, resulting
in emission from YFP (yellow) or CFP (blue), respectively. The bottom
panels in (b) show the same nucleus after bleaching following excitation
with a 514 or a 405 nm laser. The relative intensities of both YFP and
CFP were measured once before and twice after bleaching, as indicated in
(c) and (d). (e) COL13-CFP and COP1-YFP co-localized to the nucleus in
protoplasts in light and dark. (f) FRET between CFP-COL13 and YFP-COP1
analyzed by acceptor bleaching in the nucleus. The top panels in f show
a representative pre-bleach nucleus co-expressing YFP-COP1 and CFP-COL13
excited with either a 514 or a 405 nm laser in light and dark, resulting
in emission from YFP (yellow) or CFP (blue), respectively. The bottom
panels in f show the same nucleus after bleaching following excitation
with a 514- or a 405-nm laser. The relative intensities of both YFP and
CFP were measured once before and twice after bleaching, as indicated in
(g) and (h).\selectlanguage{english}
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