Plant Biotechnology 40, 51–62 (2023)
DOI: 10.5511/plantbiotechnology.23.0122a

Original Paper

Molecular characterization of Satsuma mandarin(Citrus unshiu Marc.) VASCULAR PLANT ONE-ZINC FINGER2(CuVOZ2) interacting with CuFT1 and CuFT3
Nazmul Hasan1, Naoki Tokuhara2, Takayuki Noda3, Nobuhiro Kotoda1,2,3,*
1
The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan; 2 Graduate
School of Advanced Health Sciences, Saga University, Saga 840-8502, Japan; 3 Graduate School of Agriculture, Saga
University, Saga 840-8502, Japan* E-mail: koto@cc.saga-u.ac.jp, nobu_fruits@yahoo.co.jp Tel: +81-952-28-8744

Received December 1, 2022; accepted January 22, 2023 (Edited by R. Hayama)

Abstract Shortening the juvenility is a burning issue in breeding fruit trees such as Satsuma mandarin (Citrus unshiu
Marc.). Decreasing the breeding period requires a comprehensive understanding of the flowering process in woody plants.
Throughout the Arabidopsis flowering system, FLOWERING LOCUS T (FT) interacts with other transcription factors(TFs) and functions as a transmissible floral inducer. In a previous study, a VASCULAR PLANT ONE-ZINC FINGER1(VOZ1)-like TF from the Satsuma mandarin, CuVOZ1, showed protein–protein interaction with two citrus FTs in a yeast
two-hybrid (Y2H) system and precocious flowering in Arabidopsis. In this study, another VOZ, CuVOZ2, was isolated fromthe Satsuma mandarin ‘Aoshima’ and protein–protein interaction was confirmed between CuVOZ2 and CuFTs. No apicalmeristem (NAM) and zinc coordination motifs were identified within the N-terminal of CuVOZ2. Docking simulation
predicted that interactions between CuVOZ2 and CuFTs might occur in domain B of CuVOZ2, which contains a zinc finger
motif. According to docking predictions, the distances between the amino acid residues involved ranged from 1.09 to 4.37 Å,
indicating weak Van der Waals forces in the interaction. Cys216, Cys221, Cys235, and His239 in CuVOZ2 were suggested tobond with a Zn2+ in the Zn coordination motif. Ectopic expression of 35SΩ:CuVOZ2 in Arabidopsis affected the flowering
time, length of inflorescence and internode, and number of siliques, suggesting that CuVOZ2 might regulate both vegetativeand reproductive development, act as a trigger for early flowering, and be involved in the elongation of inflorescence possibly
in a slightly different way than CuVOZ1.
Key words:

citrus, docking simulation, FLOWERING LOCUS T, transcription factor, yeast two-hybrid.

Introduction
Citrus accounts for a significant portion of commerciallycultivated fruit crops around the globe. The selection ofideal commercial and cultural traits in citrus throughtraditional breeding methods is time-consuming. A long
juvenile period of five to ten years hinders conventional
breeding, heredity improvement, genetic studies, andultimately the production of citrus depending on thespecies (Albrigo et al. 2019; Krajewski and Rabe 1995).
It is still unknown what molecular mechanisms underlie
the shifts from the juvenile vegetative to the adultreproductive phase in woody plants like citrus, whichmight provide clues on how citrus breeding may be
accelerated.
In a typical flowering paradigm, FLOWERING LOCUS
T (FT), which integrates photoperiod and vernalization
pathways, functions as a transmissible floral inducer,
similar to florigen, during the transitions from thevegetative to the reproductive phase in Arabidopsis(Corbesier et al. 2007; Jaeger and Wigge 2007; Mathieu
et al. 2007; Notaguchi et al. 2008). In the companion cellsof leaves at dusk, FT is induced by photoperiodic signals
through CONSTANS (CO) (Andrés and Coupland2012; Koornneef et al. 1991; Liu et al. 2013; Mathieu etal. 2007; Samach et al. 2000; Wigge 2011). Induced FTthen translocates to the shoot apical meristem (SAM)
through the sieve tube system and forms a complex withtranscription factors (TFs), inducing expression of thefloral meristem identity genes and promoting flowering(Abe et al. 2005; An et al. 2004; Jaeger and Wigge 2007;
Lin et al. 2007; Liu et al. 2012; Putterill and VarkonyiGasic 2016; Tamaki et al. 2007; Wigge et al. 2005).
Another member of the PEBP-like gene family known
as TERMINAL FLOWER1 (TFL1) functions in the
repression of flowering (Bradley et al. 1997; Ohshima
et al. 1997). FT and TFL1 contain a potential ligandbinding pocket region within the fourth exon. Ahn etal. (2006) demonstrated that the fourth exon, especiallysegments B and C in that region, plays a critical role in

Published online March 17, 2023
Copyright © 2023 Japanese Society for Plant Biotechnology. This is an open access article distributed under the terms of
the Creative Commons Attribution 4.0 License (CC BY, https://creativecommons.org/licenses/by/4.0/).

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Molecular characterization of Satsuma mandarin CuVOZ2determining the function of TFL1 or FT in Arabidopsis.
Our previous study demonstrated a protein–protein
interaction of MdVOZ1a, apple VASCULAR PLANT
ONE-ZINC FINGER (VOZ) 1, a member of the NAC
family subgroup VIII-2 TF, with MdFT1 and MdFT2,
two apple FT proteins, in the Y2H system, suggesting
the possible involvement of MdVOZ1a in leaf and fruit
development through the binding with MdFTs (Mimida
et al. 2011).
VOZ is a multifunctional gene regulating various
biological processes, such as flower induction anddevelopment, pathogen defense, and abiotic stressresponses (Li et al. 2020). VOZ transcription factors arepresent exclusively in higher plants, including vascularplants (Mitsuda et al. 2004). In Arabidopsis, VOZ1 andVOZ2 have a zinc-finger motif and transcriptional
activator activities (Jensen et al. 2010; Mitsuda et al.
2004). AtVOZ1 is especially expressed in the phloem,
whereas AtVOZ2 is abundantly expressed in Arabidopsis
roots. Despite the differences in the lengths of theamino acid sequences, molecular weights, and sequencesimilarities between AtVOZ1 and AtVOZ2, they are
redundantly involved in flowering (Celesnik et al. 2013;
Kumar et al. 2018; Yasui and Kohchi 2014; Yasui et al.
2012). In addition, it is reported in Arabidopsis that theloss-of-function mutation in VOZ genes confers cold anddrought tolerance but causes growth inhibition, higher
temperature and salt sensitivities, and susceptibility
to pathogens such as fungi and bacteria (Koguchi etal. 2017; Nakai et al. 2013a; Prasad et al. 2018; Song etal. 2018). In rice, OsVOZ1 and OsVOZ2 act as negativeand positive regulators, respectively, to resist the fungal
pathogen Magnaporthe oryzae (Wang et al. 2021). VOZs
individually suppress FLOWERING LOCUS C (FLC), a
floral repressor, and activate FT to promote flowering.
Inducing FT expression and photoperiodic floweringin Arabidopsis also results from the modification of thefunction of CO protein by VOZs (Koguchi et al. 2017;
Kumar et al. 2018; Nakai et al. 2013a, 2013b; Song et al.
2018; Yasui and Kohchi 2014; Yasui et al. 2012). Previous
reports suggest that VOZ transcription factors play
crucial roles in regulating plant growth and development
and have important application values in the genetic
improvement of stress resistance in crops.
However, in woody plants, the mechanism ofprotein–protein interaction between VOZs and FT
and further regulation of flowering/fruiting have yet
to be investigated. In addition to the protein–protein
interaction between MdVOZ1a and MdFTs (Mimida
et al. 2011), we recently found the interaction between
citrus CuVOZ1 and CuFTs (CuFT1 and CuFT3) in the
Y2H system (Hasan et al. 2023). Therefore, in this study,
the protein–protein interactions between the other VOZ(CuVOZ2) and CuFTs from the Satsuma mandarin
were elucidated with some methods of Y2H, deletion
Copyright © 2023 Japanese Society for Plant Biotechnology

mutation, and docking simulation. The characterizationof CuVOZ2 was also performed using transgenic
Arabidopsis.

Materials and methods
Plant materials
The adult vegetative and reproductive tissues of the Satsuma
mandarin (Citrus unshiu Marc.) ‘Aoshima’ were collected
in 2018 from the Saga Prefectural Fruit Tree Experiment
Station in Ogi, Japan as described by Hasan et al. (2023).
The juvenile vegetative tissues were collected in August 2019from 20-month-old seedlings grown at Saga University. Seedsof ‘Aoshima’ were collected in January 2020. Transgenic
Arabidopsis plants were grown under long-day (LD) conditionsin an incubator (Biotron; Nippon Medical and Chemical
Instruments Co., Ltd., Tokyo, Japan). All collected tissues/plants
were immediately frozen with liquid nitrogen and preserved ina freezer at −70°C until RNA extraction.

Isolation of CuVOZ2 from the Satsuma mandarin‘Aoshima’Total RNA was extracted from tissues using thecetyltrimethylammonium bromide (CTAB) method (Kotoda
et al. 2000). A reverse transcription (RT) reaction was
performed using ReverTra Ace® qPCR RT Master Mix FSQ301 (TOYOBO Co., Ltd., Osaka, Japan) with the extracted
RNA from new leaves. The cDNA was amplified from the RT
product by polymerase chain reaction (PCR) using KOD Plus
Neo (TOYOBO). After attachment of the adenine nucleotide,
the amplified PCR products were cloned into pGEM®-T Easy
vectors (Promega, Madison, WI, USA). Several clones weresequenced and confirmed using an ABI PRISM 3130xl Genetic
Analyzer (Life Technologies, Carlsbad, CA, USA). The isolated
genes were designated as CuVOZ2 (acc. no. LC729265). The
oligonucleotide primer sets used in gene cloning are listed in
Supplementary Table S1.

Phylogenetic analysis and comparison of aminoacid sequences
Putative amino acid sequences were analyzed using theClustalX2 multiple-sequence alignment program ver. 2.0.5(Jeanmougin et al. 1998) and the BioEdit program ver. 7.2.5(Hall 1999) with known genes from other plant species toclarify the phylogenetic relationship of the VOZ2 gene in citrus.
A phylogenetic tree was produced by the neighbor-joining (NJ) method for the deduced amino acid sequences of VOZ genes
from apple [Malus×domestica (AB531023, MDP0000729316,
MDP0000879912, and MDP0000296843)], Arabidopsis(AT1G28520 and AT2G42400), Clementine [Citrus clementina(Ciclev10015064m and Ciclev10015076m)], maidenhair fern
[Adiantum capillus-veneris (KAI5077272)], Lombardy poplar[Populus nigra (Potri.004G050900 and Potri.019G092800)],
rice [Oryza sativa (LOC_Os01g54930 and LOC_Os05g43950)],
Satsuma mandarin (LC729264 and LC729265), and sweet		orange [Citrus sinensis (XP006467710 and XP006469061)].
The deduced proteins of the TFL1/FT family from plant species
used in amino acid comparison are MdFT1 (AB161112) andMdFT2 (AB458504) in apple; AtFT (AT1G65480) and AtTSF(AT4G20370) in Arabidopsis; VvFT (DQ871590) in grapevine(Vitis vinifera); PnFT1 (AB106111), PnFT1a (AB161109),
and PnFT3c (AB110009) in Lombardy poplar; CuFT1(LC729261) and CuFT3 (LC729263) in Satsuma mandarin;and LeSFT (AY186735) in tomato (Solanum lycopersicum). The
phylogenetic tree for VOZ2 was displayed using the N-J plot
with bootstrap values for 1,000 trials in each branch.

Yeast two-hybrid (Y2H) assay
The attB-flanked PCR products for the entire coding regionsof CuVOZ2 or CuFTs (CuFT1 and CuFT3) or the truncatedregion of CuVOZ2 were introduced into a pDONR221 vector(Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) inframe as a donor vector. Each entry clone was introduced into a
pDEST22 (ampicillinR) to construct the expression vector with
the GAL4 activation domain (GAL4 AD) (prey). The constructs
were designated as CuVOZ2 (AD), CuFT1 (AD) and CuFT3(AD). The truncations were constructed by gradual deletion of
the C-terminal region or deletion of the N-terminal region. The
truncated version of CuVOZ2 was designated as CuVOZ2N1(AD) (1–100aa), CuVOZ2DelN1 (AD) (101–485aa),
CuVOZ2N2 (AD) (1–200aa), CuVOZ2N3 (AD) (1–300aa)
and CuVOZ2N4 (AD) (1–400aa). Similarly, the constructs
produced with pDEST32 (gentamicinR) for GAL4 DBD (bait)
were designated as CuVOZ2 (BD), CuFT1 (BD) and CuFT3(BD). Yeast (Saccharomyces cerevisiae) strain MaV203 (MATα,leu2-3,112, trp1-901, his3∆200, ade2-101, gal4∆, gal80∆,
SPAL10::URA3, GAL1::lacZ, HIS3UAS GAL1::HIS3@LYS2, can1R,
cyh2R) (Vidal 1997) was transformed with several combinationsof AD and BD plasmids according to the lithium method (Gietz
and Woods 2002). Single AD yeast transformants were screened
on a selective agar medium with a minimal synthetic defined(SD) base with a –L dropout (DO) supplement (Clontech
Laboratories Inc., Mountain View, CA, USA) containing 2%
glucose. Single BD transformants were screened on SD –W
media. Yeast co-transformed with AD and BD constructs was
selected on SD –L/–W media. Co-transformed yeasts wereincubated on SD –L/–W/–H media to confirm induction of
the HIS3 gene and the protein–protein interaction. To prevent
self-activation of the HIS3 reporter gene, 25 mM of 3-amino1,2,4-triazole (3-AT), a HIS3 inhibitor, was added to the media.
The yeasts with the same combination were grown on SD –L/–W/–U media to confirm the activity of the URA3 reporter
gene. For selection against positive interactions, 0.2%
5-fluoroorotic acid (5FOA) was used. All the yeasts were grown
at 30°C for 48 h. The protein–protein interactions between
CuVOZ2 and CuFTs were also confirmed by the colony filter
lift assay.

Docking simulation
The three-dimensional (3D) structure of the proteins and theN. Hasan et al.

protein–protein interaction were simulated based on the cDNA
sequences of CuVOZ2, CuFT1, and CuFT3 to better understandthe results obtained in the Y2H system. A deep-learning neuralnetwork was applied to predict possible docking regions tounderstand how CuVOZ2 and CuFTs interact. The protein
modeling and interaction were predicted in AlphaFold2_
advanced.ipynb (Jumper et al. 2021), a Web-based iPython
Notebook service for interactive coding supported by Google.
The artificial intelligence predicted and constructed models
of protein–protein complex structures using several protein
sequence databases and multiple-sequence alignment. The
metal ion-binding site was predicted with the help of the Metal
Ion-Binding site prediction and modeling server (MIB2) (Luet al. 2012). Visualizations of protein models and predictionsof docking regions were created using ChimeraX ver. 1.5(Pettersen et al. 2021).

Vector construction and Arabidopsis transformation
The oligonucleotide primers of VOZ2_XbaI_F and VOZ2_
KpnI_R (Supplementary Table S1) were used to amplify thecoding region of CuVOZ2 to construct a binary vector forplant transformation. The amplified product was digested withXbaI and KpnI and cloned into the corresponding restriction
enzyme site of the 35SΩ/pSMAK193E plant-transformation
vector (Kotoda et al. 2010). The floral-dip method (Cloughand Bent 1998) was used to transform wild-type Arabidopsis
with Agrobacterium tumefaciens EHA101. Kanamycin-resistanttransgenic seedlings were transplanted from the plate tomoistened soil (vermiculite : perlite=​1 : 1) at the two-leaf stage
and incubated in a growth chamber (Biotron; Nippon Medical
and Chemical Instruments Co., Ltd.) at 22°C under long-day(LD) conditions (16 h photoperiod; cool white fluorescent light,
72.36 µmol m−2 s−1). Transgenic plants with an empty vector ofpSMAK251 (indicated as PSM251) were used as the control.
Morphological and expression analyses were performed on
the third-generation (T3) transgenic plants. The number ofdays to bolting and flowering was tracked. Rosette and cauline
leaves were counted at flowering time. The length of the firstinflorescence was measured at flowering and 40 days after
incubation (DAI). The branches, siliques, nodes, and internodes
were also counted or measured 40 DAI. An entire transgenic orcontrol plant was collected for qRT-PCR analysis.

Expression analysis in the Satsuma mandarin‘Aoshima’ and transgenic Arabidopsis
Expression analysis of CuVOZ2 was carried out via qRT-PCR.
One microgram of RNA was used to synthesize the firststrand cDNA in 20 µl of a reaction mixture using a ReverTra
Ace® qPCR RT Master Mix (TOYOBO). One microliter ofthe first-strand cDNA was used as a template in SYBR® Green
RealTime PCR Master Mix (TOYOBO) for a total volume of
12.5 µl. The qRT-PCR was performed using a LightCycler® 96
System (Roche Diagnostics, Mannheim, Germany) as follows:
95°C for 1 min, 40 cycles of 95°C for 15 s and 60°C for 1 min for
CuVOZ2, CuActin, and AtTUB4. Three technical replicates were
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Molecular characterization of Satsuma mandarin CuVOZ2maintained for each analysis. CuActin was used as a reference
gene to normalize the transcript levels of CuVOZ2 (Kotoda etal. 2016). AtTUB4 was used as a reference gene to normalize
the transcript levels of CuVOZ2 in transgenic Arabidopsis. The
primer sets used in the experiment are listed in Supplementary
Table S1.

Statistical analysis
Data on the number of days to bolting and flowering; thenumbers of leaves, nodes, and siliques; and the length of thefirst inflorescence and internodes were analyzed using one-way
ANOVA, and the multiple comparison was done by Tukey’s
honestly significant difference (HSD) test. Statistical analyses
were performed at the significance level of p<0.05 using
RStudio v.1.2.5089 (RStudio Team 2020).

Figure 1. Phylogenetic analysis of CuVOZ2 and comparison of the deduced protein sequences of VOZ2 or FT in several plant species. (A)Schematic representation of the genomic organization of CuVOZ2. The numbers that appear below the exon region are based on the cDNA sequenceof CuVOZ2. (B) Relationship of the predicted VOZ-family proteins between Satsuma mandarin and other plant species. Phylogenetic tree of VOZfamily proteins of apple, Arabidopsis, citrus, maidenhair fern, poplar, rice, and tomato species. The amino acid sequences of Arabidopsis VOZ-family
proteins were obtained from TAIR, and others were obtained from NCBI, phytozome and Genome Database for Rosaceae (GDR). Numbers along thebranches are bootstrap values (1,000 replicates). The unit for the scale bars indicates branch lengths (0.05 substitutions/site). (C) The deduced protein
sequence of CuVOZ2 with those of the VOZ2 family proteins from apple, Arabidopsis, and citrus species. The genes used for comparison are MdVOZ2(MDP0000296843, apple), AtVOZ2 (AT2G42400, Arabidopsis), CcVOZ2 (Ciclev10015076m, Clementine), CuVOZ2 (LC729265, Satsuma mandarin),
and CsVOZ2 (XP006469061, sweet orange). Amino acids in black and gray are identical and similar, respectively. The dashes indicate the lack ofamino acids. The triangles show the intron positions. The green and purple regions show the predicted motif regions. Two domains, Domain A andDomain B were represented by black lines, defined by Mitsuda et al. (2004). Asterisks represent conserved residues possibly forming a functional zinccoordinating motif (Mitsuda et al. 2004). Amino acids in blue are in the predicted docking regions when CuVOZ2 interacts with CuFTs. Amino acids
with a dot on the top are the amino acid residues predicted to bind with AtFT. The amino acids in red are the amino acid residues predicted to havea hydrogen bond. (D) The deduced protein sequences of CuFT1 and CuFT3 with those of the TFL1/FT family from apple, Arabidopsis, grapevine,and Lombardy poplar species. The genes used for analysis are as follows: MdFT1 (AB161112, apple), MdFT2 (AB458504, apple), AtFT (AT1G65480,
Arabidopsis), AtTSF (AT4G20370, Arabidopsis), VvFT (DQ871590, grapevine), PnFT1 (AB106111, Lombardy poplar), PnFT1a (AB161109, Lombardy
poplar), PnFT3c (AB110009, Lombardy poplar), CuFT1 (LC729261, Satsuma mandarin), CuFT3 (LC729263, Satsuma mandarin), and LeSFT(AY186735, tomato). Putative amino acid sequences were aligned using the ClustalX2 multiple-sequence alignment program ver.2.0.5 (Jeanmougin etal. 1998). Red and black lines on the fourth exon of FT family proteins indicate segment B and C regions, respectively, as defined by Ahn et al. (2006).

Copyright © 2023 Japanese Society for Plant Biotechnology		

N. Hasan et al.

Results
Isolation of CuVOZ2 from the Satsuma mandarin‘Aoshima’

The cDNA of CuVOZ2 was isolated from the Satsuma
mandarin ‘Aoshima’ and the function of the gene wasinvestigated. The CuVOZ2 gene consisted of four exonsof 225, 156, 514, and 557 base pairs (bp) encoding aputative protein of 483 amino acids, which was also
confirmed by next-generation sequencing (NGS)of the ‘Aoshima’ genome (Figure 1A). The length ofthe coding sequence was 1,452 bp. To investigate theevolutionary link among the VOZ genes, a phylogenetic
analysis was carried out using the putative amino acidsequences corresponding to VOZ genes from apple,
Arabidopsis, citrus (Clementine, Satsuma mandarin, andsweet orange), maidenhair fern, poplar, and rice. The
phylogenetic tree revealed that CuVOZ2 was closely
clustered with the VOZ2 of Clementine and sweet orange
and with the same clade as AtVOZ2, MdVOZ2, and
PtVOZ2 (Figure 1B).
The sequence of CuVOZ2 was 100% identical to thatof CcVOZ2, 99.17% identical to that of CsVOZ2 and53.86% identical to that of previously isolated CuVOZ1(LC729264) at the amino acid level. The motif searchtool using the Pfam database showed that the aminoacid sequence of CuVOZ2 contains a C-terminal beta
pleated sheet identified as a no apical meristem (NAM)(289–388aa) motif (Finn et al. 2016) (Figure 1C, green
regions). One motif, named the phosphoethanolaminebinding protein (PBP), was identified in the latter region
in the sequence of TFL1/FT family proteins including
CuFT1 (LC729261) and CuFT3 (LC729263) (Figure 1D).
Expression pattern of CuVOZ2The tissue-specific expression pattern of CuVOZ2 was
examined using qRT-PCR. CuVOZ2 was expressed invarious tissues, as shown in Figure 2. The expression
was relatively high in shoot apices in both juvenile andadult phases and stems in the juvenile phase. However,
the expression was lower in the reproductive tissues,
such as juice sacs and peels in November. The expression
was moderate in other tissues at the vegetative andreproductive stages.
Interactions of CuVOZ2 with CuFT1 or CuFT3 in
the Y2H system

Auxotrophy assays for HIS3 and URA3 reporter genes,
sensitivity tests for 5FOA, and X-gal filter lift assays
for the LacZ reporter gene were performed using theyeast transformants harboring a pair of bait and prey
plasmids [CuFT1 (AD)/CuVOZ2 (BD), CuFT1 (BD)/CuVOZ2 (AD), CuFT3 (AD)/CuVOZ2 (BD), andCuFT3 (BD)/CuVOZ2 (AD)] to investigate the protein–protein interactions in the Y2H system. All the yeast

Figure 2. Expression pattern of CuVOZ2 in various tissues in theSatsuma mandarin ‘Aoshima’ by quantitative real-time RT-PCR. The
samples (left to right) are as follows: the seeds (Sd), juvenile roots (JR),juvenile stems (JSm), leaves from the juvenile plant (JL), and juvenile
shoot apices (JSA) of one-year-old seedlings in the juvenile phase; thenew leaves in April (NL), old leaves in August (OL), stems with a nodein August (Sm), and shoot apices in April (SA) in the adult phase; andthe flower buds in April (FB), young fruits in May (YF), juice sacs inAugust and November (JS), and peels in August and November (PL)in the adult reproductive phase. Expression levels were normalizedby citrus actin. Values are the means±SD of the results from threetechnical replicates. A column without an error bar indicates that thestandard deviation fell within the symbol.

constructs that grew on SD –L/–W/–H+​25 mM 3-AT
showed the induction of the HIS3 reporter gene in theHis auxotrophy assays (Figure 3A). Growth inductionof the yeasts containing CuFT1 (AD)/CuVOZ2 (BD)and CuFT3 (AD)/CuVOZ2 (BD) constructs on an SD–L/–W/–U media plate in the Ura auxotrophy assay
and growth inhibition of all constructs on an SD –L/–
W+​5FOA media plate in the 5FOA sensitivity assay
confirmed the activity of the URA3 reporter gene.
However, the yeasts containing CuVOZ2 (AD)/CuFT1(BD) and CuVOZ2 (AD)/CuFT3 (BD) constructs did
not show growth induction on the SD –L/–W/–U media
plate. The indigo coloration in the X-gal filter assay
confirmed the activity of the LacZ reporter gene. The
growth inhibition of all the BD constructs tested on SD–W or SD –L/–H media plates, and the colony filter lift
assay showed that any single constructs were not selfactivated (Figure 3B).
Identification of binding regions using the Y2HsystemA series of truncated CuVOZ2 proteins were fusedwith the GAL4 activation domain (GAL4 AD) to
identify the region of interaction between CuVOZ2 andCuFT1 (Figure 3C) and between CuVOZ2 and CuFT3(Figure 3D). In Y2H assays, constructs of the CuVOZ2
truncation series (AD)/CuFTs (BD) showed inhibited
growth in the SD –L/–W/–H+​25 mM 3-AT media andless blue coloration in the X-gal assay except for the
CuVOZ2N4 (AD)/CuFT (BD) constructs. Both the
CuVOZ2N4 (AD)/CuFT1 (BD) and CuVOZ2N4 (AD)/CuFT3 (BD) constructs showed normal growth in the
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Molecular characterization of Satsuma mandarin CuVOZ2Figure 3. Protein–protein interactions between CuVOZ2 with CuFTs detected in a yeast two-hybrid system. (A) Yeast competent cell MaV203 wastransformed with a pair of bait [CuVOZ2(BD) or CuFTs(BD)] and prey [CuVOZ2(AD) or CuFTs(AD)] plasmids, using the lithium method. The
transformants were grown at 30°C for 48 h. The selection test for the HIS3 reporter gene of yeast transformants was performed on a selective agar
medium with a Minimal SD Base and –L–W–H DO supplement containing 25 mM 3-amino-1,2,4-triazole (3-AT) and 2% glucose. The selection test
for the URA3 reporter gene was performed on an SD –L/–W/–Ura DO supplement containing 0.2% 5-fluoroorotic acid (5FOA). A subsequent X-gal(5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) filter lift assay for β-galactosidase activity was performed using yeast transformant cells to test
the LacZ reporter gene. (B) No effect of self-activation was detected in the constructed vector. The interaction between the truncated CuVOZ2 andfull-length CuFT1 (C) and CuFT3 (D) in yeast is shown. The left panels of (C) and (D) show the growth of the transformed yeast cells on SD/–L–W,
SD/–L–W–H, SD/–L–W–H+10 mM 3-AT, and SD/–L–W–H+25 mM 3-AT and a filter assay for the β-galactosidase activity of the transformants
grown on SD/–L–W–H. The right panels of (C) and (D) illustrate the full-length or truncated (N1, DelN1, N2–4) version of CuVOZ2. An RalGDSWT(AD)/Krev1(BD) construct was used as a strong positive interaction control, an RalGDS-m1(AD)/Krev1(BD) construct as a weak positive
interaction control, and an RalGDS-m2(AD)/Krev1(BD) construct as a negative interaction control.

SD –L/–W/–H+​25 mM 3-AT media, and the indigo
coloration in the X-gal assay was similar to that of the
full-length CuVOZ2 (AD)/CuFT (BD) constructs.
These observations indicated that CuFTs showed little
interaction with C-terminal truncations of CuVOZ2[(N1, 1–100aa), (N2, 1–200aa), and (N3, 1–300aa)] andan N-terminal truncation of CuVOZ2 (DelN1, 101–
485aa) on the SD –L/–W/–H+​25 mM 3-AT media plateand X-gal assay but showed an apparent interaction with
the CuVOZ2N4 region, which consisted of 1–400aa in
the N-terminal region (Figure 3C, D).
Docking simulation

The predicted amino acid residues associated with
the interaction between CuVOZ2 and CuFT1 in the
CuVOZ2–CuFT1 complex were as follows: Pro198,
Asn199, Leu201, Ile203, His205, Arg223, Ser236, Phe238,
Arg255, and Pro256 in CuVOZ2 (Figures 1C, 4A, B)
and Arg62, Ser100, Phe101, Gln103, Glu104, Asn107,
Arg130, Gln131, Tyr134, Gly137 and Trp138 in CuFT1(Figures 1D, 4A, B). The predicted amino acid residues
associated with the interaction between CuVOZ2 andCopyright © 2023 Japanese Society for Plant Biotechnology

CuFT3 in the CuVOZ2–CuFT3 complex were as follows:
Ser200, Leu201, Pro202, Ile203, His205, and Phe235
in CuVOZ2 (Figures 1C, 4C, D) and Pro75, Asp79,
Ser80, Leu81, Pro113, Thr114, Met115, and Gln140 in
CuFT3 (Figures 1D, 4C, D). The common amino acidresidues associated with docking in both complexes
were Leu201, Ile203, His205, and Phe238 in CuVOZ2(Figure 1C). No common amino acid residues involvedin docking were found for CuFTs. All the predicted
amino acid residues of CuVOZ2 involved in docking in
the CuVOZ2–CuFT complexes were in the third exon.
In the CuVOZ2–CuFT3 complex, one hydrogen bondwas observed between the Ser200 of CuVOZ2 and thePro113 of CuFT3 (Figures 1C, D, 4E). The possibility of
the interaction between CuVOZ2 and AtFT in transgenic
Arabidopsis was also predicted. Ile203, Ile204, His205,
Pro206, Ala210, Phe211, Asp220, Thr222, Arg223,
Leu226, Ser236, Phe238, Arg256, and Pro257 for
CuVOZ2 (Figures 1C, 4F, G) and Val11, Arg13, Asp17,
Asp42, Pro77, Ser78, Ile117, Arg126, Leu128, Thr132,
Tyr134, Arg145, Glu149, Ile150, Asn152, Asn163, Cys170
and Gly171 for AtFT (Figures 1D, 4F, G) were involved

N. Hasan et al.

		

Figure 4. Predicted protein–protein interaction between CuVOZ2 and CuFTs or AtFT. (A) Blue and green areas indicate CuVOZ2 and CuFT1,
respectively. (B) The amino acid residues involved in docking in CuVOZ2–CuFT1 complex. (C) Blue and red areas indicate CuVOZ2 and CuFT3,
respectively. (D) The amino acid residues involved in docking in CuVOZ2–CuFT3 complex. (E) A predicted hydrogen bond between CuVOZ2 andCuFT3 is shown. (F) Blue and pink areas indicate CuVOZ2 and AtFT, respectively. Met1, Val254, and Tyr483 for CuVOZ2 and Met1, Gly102, andArg173 for FTs are displayed to confirm the relative position among the complexes. (G) The amino acid residues involved in docking in CuVOZ2–AtFT complex. (H) The predicted Zn2+ binding site in the CuVOZ2 protein is shown. (I) Comparative representation of CuVOZ1 and CuVOZ2
proteins. Purple and blue ribbons indicate CuVOZ1 and CuVOZ2, respectively. The orange colored region represents the extra α-helix or β-pleatedsheet region on CuVOZ2. The yellow regions in the middle of the two proteins indicate possible docking regions in (A), (C), and (F). The predictionof protein–protein interaction was calculated with AlphaFold.ipnyb (Jumper et al. 2021) and visualized with ChimeraX ver. 1.5 (Pettersen et al. 2021).
The Zn2+ binding site was predicted using the Metal Ion-Binding site prediction and modeling server (MIB2) (Lu et al. 2012). Some amino acids
might be hidden in the background.

in the interaction in the protein complexes. The Zn2+
ion binding site for CuVOZ2 was predicted in the third
exon, consisting of Cys216, Cys221, Cys235, and His239(Figure 4H). Ten α-helices and five β-pleated sheets were
found in the CuVOZ1 protein, whereas CuVOZ2 had 11
α-helices and six β-pleated sheets (Figure 4I).
Analysis of CuVOZ2 in transgenic Arabidopsis

The constitutive expression construct of 35SΩ:CuVOZ2(Figure 5A) was introduced into wild-type Arabidopsis
to elucidate the possible role of CuVOZ2 in transgenic
Arabidopsis using the heterologous system. About 30
independent transgenic lines were obtained. The T 3
transgenic plants were grown in an incubator under
LD conditions, and five independent lines (n=14) were
phenotyped together with the controls (Figure 5B).
In addition to the phenotypic analyses, the transgene
expression in five independent lines was confirmed via
qRT-PCR. All transgenic lines with 35SΩ:CuVOZ2
expressed the transgene. Among the transgenic plants,
lines 2 and 9 showed higher expression as compared tolines 1, 4, and 10 (Figure 5C). Several parameters weretaken into consideration to analyze the characteristicsof transgenic Arabidopsis. 35SΩ:CuVOZ2 induced early
flowering in all transgenic lines (Figure 5B, D). The

inflorescence was significantly elongated in lines 1, 9, and
10 at flowering and in lines 2 and 9 40 DAI (Figure 5E).
The number of branches in line 1 was higher, whereasthat of line 10 was lower (Figure 5F). The number ofsiliques in lines 1, 2, 4, and 9 was significantly higher(Figure 5G). Significant difference was not observedin the number of nodes for transgenic lines 1, 2, and
4 (Figure 5H), whereas the length of internodes intransgenic lines 2 and 10 was significantly higher (Figure5I). The number of rosette or cauline leaves did notshow any significant differences at flowering time except
in one line as compared with the control (Figure 5J,
K). The lines with a higher expression of the transgene
showed a phenotype of significantly early flowering, long
inflorescence, more siliques, and long internodes.

Discussion
Although Jensen et al. (2010) classified VOZ as a memberof NAC family subgroup VIII-2, Gao et al. (2018) classified
VOZ genes as an independent gene family. In the VOZfamily, to date, 635 members have been identified in 166
plant species and listed in PlantTFDB, a plant TF database(Jin et al. 2017; Tian et al. 2020). The exon/intron structural
diversity plays a crucial role in the evolution of plant gene
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Molecular characterization of Satsuma mandarin CuVOZ2Figure 5. Characterization of transgenic Arabidopsis ectopically expressing CuVOZ2. (A) Schematic representation of a constructed transformation
vector. 35S, promoter region of cauliflower mosaic virus 35S; Ω, Ω sequence responsible for enhancing translation (Gallie and Walbot 1992); andTrbcS, 3′ region of Arabidopsis rbcS-2B gene. (B) Typical phenotypes of transgenic Arabidopsis with CuVOZ2 at flowering time in a growth chamber
under LD conditions. (C) Expression analysis for transgenes in whole plants of transgenic Arabidopsis. The photo was taken on the 22 days after
incubation (DAI). Samples for expression analysis of CuVOZ2 in transgenic lines were collected 40 DAI. PSM251 was the transgenic control plantwith an empty vector. Levels of detected amplicons were normalized by reference to AtTUB4. (D) Days to bolting and flowering, (E) length of the firstinflorescence, (F) no. of branches, (G) no. of siliques, (H) no. of nodes, (I) length of internodes, (J) no. of rosette leaves, and (K) no. of cauline leaves.
The transgenic plants in the third generation (T3) and the controls were grown under LD conditions (n=14). Days to bolting and flowering werecounted from the first day of incubation. All the transgenic plants showed early bolting and flowering. The length of the primary inflorescences wasmeasured at flowering and 40 DAI. The rosette and cauline leaves were counted on the day of the first flowering. Different letters at the top of eachcolumn indicate significant differences among the lines, contrasted with Tukey’s multiple comparison test (p<0.05). Values are the means±SD of theresults from three technical replicates per line.

families (Long et al. 1995; Xu et al. 2012). Similarly to
AtVOZ2, the genomic DNA of CuVOZ2 was comprisedof four exons and three introns in the coding regions(Figure 1A). To analyze the evolutionary connections of
17 putative VOZ genes from various species, including
the isolated CuVOZ2 gene from the Satsuma mandarin,a phylogenetic tree was generated based on the deduced
amino acid sequences. The phylogenetic tree revealed
that CuVOZ2 was closely clustered with the VOZ2 of the
Clementine and sweet orange and with the same clade as
AtVOZ2, although the similarities between CuVOZ2 andAtVOZ1 and between CuVOZ2 and AtVOZ2 were 61%
and 57%, respectively. Interestingly, CuVOZ2 seems to
be more closely related to VOZ1 proteins of dicots rather
than to VOZ proteins of monocots such as rice (Figure
1B). The differentiation of VOZ1 and VOZ2 transcription
factors seems to have occurred after the divergence ofmonocotyledons and dicotyledons (Shi et al. 2022). These
results indicated that CuVOZ2 might have functioned like
AtVOZ2 (Figure 1B).
Different VOZ-family members contained highly
Copyright © 2023 Japanese Society for Plant Biotechnology

conserved domains that might be closely related to theiridentical or similar regulatory functions (Koguchi etal. 2017). No apical meristem (NAM) motif (PF02365)
was identified in the VOZ-family genes during the
motif search (Figure 1C). The NAM motif was foundcommonly in both CuVOZ1 and CuVOZ2. On the other
hand, CuVOZ2 did not have a DUF4749 (PF15936)
motif (Finn et al. 2016), the characteristic feature of
CuVOZ1 (LC729264) (Figure 1C; Hasan et al. 2023).
CuVOZ2 was expressed in various tissues of theSatsuma mandarin (Figure 2). The expression was
higher in vegetative tissues (especially juvenile andadult shoot apices and the juvenile stem) than in
reproductive tissues. On the other hand, CuFT1 isexpressed exclusively in juice sacs and peels, and CuFT3
is expressed mainly in juvenile roots, adult leaves andapices, and peels (Hasan et al. 2023). According to thereport of Nishikawa et al. (2007), CiFT1, corresponding
to CuFT1, is expressed exclusively in juice sacs
and peels, and CiFT3, corresponding to CuFT3, isexpressed in vegetative tissues, such as the adult stem		and leaves. CuVOZ2 was co-expressed in the tissues
where CuFT1/CiFT1 and/or CuFT3/CiFT3 wereexpressed (Figure 2). AtVOZ2, coding a vacuolar H+pyrophosphatase (V-PPase) promoter-binding protein, isexpressed in roots, shoots, leaves, flowers, siliques, andsuspension cultures in Arabidopsis (Mitsuda et al. 2004).
The expression pattern of CuVOZ2 was similar to that of
AtVOZ2 in that both genes were expressed preferentiallyin roots, shoots, flower buds, and siliques/fruit. AtVOZ1and AtVOZ2 act as positive regulators of plant floweringby modulating and interacting with CONSTANS (CO),and they are able to bind to the cis-acting region of theA. thaliana V-PPase gene (AVP1), which regulates auxinmediated organ development and enhances NaCl anddrought tolerance (Gaxiola et al. 2001; Kumar et al. 2018;
Li et al. 2005; Prasad et al. 2018; Yasui and Kohchi 2014).
The AVP1 expression was increased by the upregulationof VOZ1 in the transgenic soybean (Han et al. 2021),
assuming that the VOZ2–FT protein complexes may
affect the activity of V-PPase and/or the function of FT
and play an additional role in flower/fruit development
and ripening.
A VOZ protein was reported for the first time tointeract with FT proteins in apple (Mimida et al. 2011).
In citrus, Hasan et al. (2023) reported that CuVOZ1interacted with CuFTs. In this study, CuVOZ2 fromthe Satsuma mandarin also exhibited protein–protein
interaction with CuFTs in the Y2H system similarly to
CuVOZ1 (Figure 3A). Strong positive interactions wereconfirmed in yeast cells with the combinations of CuFT1(AD)/CuVOZ2 (BD) and CuFT3 (AD)/CuVOZ2 (BD).
Swapping AD and BD could alter the steric force between
the proteins and affect the N-terminal region (Chen etal. 2010); hence, the opposite combination of AD and
BD may have shown weak interaction in the Y2H system
as shown in the previous study (Hasan et al. 2023). The
binding region between two proteins was investigatedusing a truncation series of CuVOZ2 as AD and fulllength CuFTs as BD constructs. The truncated constructcontaining N-terminal 400 amino acids of CuVOZ2
showed a detectable protein–protein interaction between
CuVOZ2 and CuFTs (Figure 3C, D). Based on the
results of the truncation series, it was suggested that theregion from the 1st to 400th amino acid of CuVOZ2 wasrequired, at least for the successful interaction.
Besides the Y2H, simulations predicting 3D modelsof CuVOZ2–CuFT1 and CuVOZ2–CuFT3 complexes
were performed to validate the results of the Y2H assay(Figure 4). Ten amino acid residues in the third exon of
CuVOZ2 were involved in docking with CuFT1, and six
amino acid residues in the same region were involvedin docking with CuFT3 in the CuVOZ2–CuFT1 andCuVOZ2–CuFT3 complexes, respectively (Figure 1C).
No amino acid residues were found in NAM motif
regions involved in docking in CuVOZ2–CuFT1 and

N. Hasan et al.

CuVOZ2–CuFT3 complexes. Four amino acids involved
in binding with CuFTs, Leu201, Ile203, His205, andPhe238, were common in the third exon of CuVOZ2.
The difference in the position of associated aminoacids between CuFT1 and CuFT3 might be explainedby the difference in their 3D structures, leading to
the functional divergence of CuFT1 and CuFT3. The
AtVOZ proteins have two distinct conserved regions,N-terminal Domain A, including a stretch of aminoacids, and a C-terminal Domain B, also known as theVOZ domain. The third exon of AtVOZ1 and AtVOZ2contains the zinc-coordination motif with threeconserved cysteines and one histidine residue that isrequired for the DNA-binding and dimerization of VOZ
family proteins (Mitsuda et al. 2004). In CuVOZ2, theZn2+ would stabilize the fold by binding with four aminoacid residues, Cys216, Cys221, Cys235, and His239, inthe zinc-coordination motif with predicted moleculardistances of 2.11–2.63 Å (Figure 4H). The predicted
residues involved in the interaction between CuVOZ2and CuFTs were found in the VOZ domain region of
CuVOZ2 (Figures 1C, 3C, D, 4A–D), suggesting that
the CuVOZ2–CuFT complexes might affect the DNA
binding function in CuVOZ2.
The amino acid residues of CuFTs involved in theinteractions were found mainly in the PBP motif region.
The distance between the residues was predicted to be
1.09–4.37 Å, suggesting that the interaction forces between
CuVOZ2 and CuFTs or AtFT in the CuVOZ2–CuFT or
CuVOZ2–AtFT complexes were weak Van der Waals forces(Figures 1C, D, 4A–G). A hydrogen bond with a distanceof 2.96 Å was found between CuVOZ2 Ser200 and CuFT3
Pro113 in CuVOZ2–CuFT3 complexes (Figures 1C, D,
red marked residues; 4E) unlike in the CuVOZ1–CuFT
complexes. Based on this prediction, the interaction between
CuVOZ2 and CuFT3 might be stronger than those in othercomplexes. In contrast with CuVOZ1–CuFT complexes(Hasan et al. 2023), CuVOZ2–CuFT complexes did not
limit the interacting residues to the fourth exon of CuFTs(Figure 1D). The simulation of the interaction between
CuVOZ2 and AtFT also demonstrated that the interacting
residues dispersed more than those in CuVOZ1–AtFT
complex (Figure 1D). These results imply that the sites of theinteraction between CuVOZ2 and CuFTs were not restricted
to the fourth exon of CuFTs, where the segments B and C
defined by Ahn et al. (2006) are included.
In our previous study, the 35SΩ:CuVOZ1 transgeniclines showed early bolting, precocious flowering,increased numbers of branches, siliques, nodes, rosetteand cauline leaves, and elongated flower stalks withadventitious fertile flowers (Hasan et al. 2023). On theother hand, the 35SΩ:CuVOZ2 transgenic lines showed
early bolting and flowering with an elongated stem withfertile flowers and increased numbers of siliques but noadventitious flower buds (Figure 5). It is noteworthy
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Molecular characterization of Satsuma mandarin CuVOZ2that the internodes of the 35SΩ:CuVOZ2 transgeniclines were extended unlike those of the 35SΩ:CuVOZ1transgenic lines (Figure 5I). The differences in thephenotype between 35SΩ:CuVOZ2 and 35SΩ:CuVOZ1transgenic lines might be due to the presence or absenceof DUF-4749 motif in the N-terminal region and/or low
identity/similarity in amino acid sequences between
them. The 3D protein structures of CuVOZ1 andCuVOZ2 were different in the coordination of α-helices
and β-pleated sheets. The number of α-helices andβ-pleated sheets in CuVOZ1 was ten and five, whereas
CuVOZ2 had 11 and six, respectively (Figure 4I). Thisdifference in protein structure might also be a cause of
the differences in phenotypes.
The role of AtVOZs in flowering, concerning CO, FLC,
MAF, and PHYTOCHROME B (PHYB), was previously
explained in the studies conducted with a double mutant(voz1 voz2) in Arabidopsis (Celesnik et al. 2013; Kumar
et al. 2018; Yasui et al. 2012). The early flowering andother specific phenotypes of 35SΩ:CuVOZ2 transgeniclines might result from the alteration of FT expression
because CuVOZ2 showed protein–protein interaction
with AtFT in simulation (Figure 4F, G). AtVOZ1 andAtVOZ2 suppress FLC and act together with CO byinteracting physically to control flowering (Kumar etal. 2018; Yasui et al. 2012). The downregulation of FLC
and/or the stabilization of CO in transgenic Arabidopsis
could be another reason for early flowering. VOZ1
genes in the soybean showed a significant alteration in
expression levels in response to dehydration and salt
stress treatments, whereas VOZ2 genes showed a weakchange (Li et al. 2020). Light is a key environmental
factor affecting flowering time (Zeevaart 1976). The
presence of cis-acting binding elements, such as LTR,
MYB, MBS, ABRE, the CGT​CA motif, and the W-box,
including a light-related element, Box-4, was reported in
the promoter region of VOZ1/2 from soybeans (Glycine
max and G. soja) and tef (Eragrostis tef) (Luo et al. 2020;
Mulat and Sinha 2022; Rehman et al. 2021). These studies
indicate that VOZ TFs might be required to regulate
flower induction through some factors, such as abiotic
stresses and light. There is a possibility that the stabilized
CuVOZ2 functions as one of the triggers inducing
flowering, elongating the inflorescence and internode,and increasing the number of siliques with a consistent
result of expression analysis (Figure 5).
The VOZs are also shown to have a function
in seed germination. A recent study showed that
PHYTOCHROME INTERACTING FACTOR1 (PIF1)
induces the expression of AtVOZ1 and AtVOZ2 in thedark (Luo et al. 2020). Although both AtVOZ1 andAtVOZ2 were reported to interact with PHYB andpromote flowering (Yasui et al. 2012), AtVOZ2 wasfound to bind to the promoter of a GA biosynthetic gene,
GIBBERELLIN 3-OXIDASE1 (GA3ox1) in vitro and
Copyright © 2023 Japanese Society for Plant Biotechnology

in vivo and to directly repress GA3ox1 in Arabidopsis,
thereby negatively regulating PHYB-mediated seedgermination (Luo et al. 2020). The ga3ox1 Arabidopsis
mutant has a semi-dwarf phenotype (Mitchum et al.
2006), opposite to the phenotype of 35SΩ:CuVOZ2.
It can be assumed that CuVOZ2 did not repress but
increased active GAs in transgenic Arabidopsis. The
relationship between GA biosynthesis and CuVOZ2
remains to be studied.

Conclusion
The protein–protein interactions between CuVOZ2and CuFTs were confirmed in this study through a
Y2H system. The N-terminal 1st to 400th region of
CuVOZ2 was suggested via a truncation experiment
to be necessary for the interaction with CuFTs.
Furthermore, the 3D structures of the proteins, thenature of interactions, the specific residues involved indocking, and the Zn2+ binding site were predicted byvirtual simulations. Interestingly, the amino acid residues
associated with binding to CuVOZ2 were not exactly thesame between CuFT1 and CuFT3. The phenotypes oftransgenic Arabidopsis with 35SΩ:CuVOZ2 was similar
to those of 35SΩ:CuVOZ1, but they differed in thatthe 35SΩ:CuVOZ2 lines showed extended internodes
and did not produce adventitious flower buds on theelongated flower stalks, whereas 35SΩ:CuVOZ1 lines
showed more branching. CuVOZ2 might trigger the
early flowering and elongation of inflorescence like
CuVOZ1, but the difference in the phenotypes between
the transgenic lines with each gene might partly be dueto the variation in the mode of interaction between
CuVOZs and other proteins such as FTs. These findings
would provide us with an understanding not only of
the function of CuVOZ2 but also of the functional
divergence of CuFT1 and CuFT3 or CuVOZ1 andCuVOZ2 in citrus. The relationship between the protein
complexes and the phytohormones, such as abscisic acidand GAs, and the response of the protein complexes
to environmental stimuli and/or stresses remains to bestudied further.
Acknowledgements
We are grateful to the staff of Analytical Research Center for
Experimental Sciences, Saga University, for technical assistance.
We also thank Dr. H. Ichikawa for providing the binary vectors,
pSMAK251 and pSMAK193E, and Dr. E. E. Hood for providing
Agrobacterium tumefaciens EHA101.

Author contribution
NH, NT, and TN did experiments, NH and NK designed
experiments, NH and NK wrote the paper, and NK acquired
research funds.

		

Funding
This study was supported by a Grant-in-Aid for scientific researchfrom the Ministry of Education, Culture, Sports, Science andTechnology (16K07598, 20K06018).

Conflict of interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared toinfluence the work reported in this paper.

Description of Supplementary Files
Supplementary Table S1. Primer sets used in gene cloning, Y2H,
quantitative real-time RT-PCR analysis, and vector construction.

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