J. Plant Biochem. Biotechnol.
DOI 10.1007/s13562-017-0396-8
ORIGINAL ARTICLE
Chalcone Isomerase-like genes in Tradescantia BNL4430:
identification, molecular characterization, and differential
expression profiles under -radiation stress
Saminathan Subburaj1 Hye-Jeong Ha1 Nuri Park1 Seo-Hee Choi1
Geung-Joo Lee1
Received: 7 May 2016 / Accepted: 5 January 2017
Society for Plant Biochemistry and Biotechnology 2017
Abstract Chalcone isomerase (CHI) is an important cat-
alytic enzyme that converts naringenin (chalcone) to (2S)-
naringenin in phenylpropanoid pathway. Here, we per-
formed the first comprehensive molecular genetic study on
Chalcone isomerase genes in Tradescantia clone BNL
4430 (designated as TrCHI). We identified seven TrCHIs
genes (five with full length and two with partial length)
through in silico analysis using available transcriptomic
resources. Phylogenetic analysis suggest that TrCHIs are
closely related to rice CHI. TrCHIs fall into three different
CHI subfamilies: (1) type I (TrCHI2 including TrCHI2a,
2b, 2c, and 2d), (2) type III (TrCHI3 including TrCHI3B
and 3C), and (3) type IV (TrCHI4). These partial or full
length TrCHI genes were 456819 bp in length with
molecular mass ranging from 23 kDa to 47 kDa. Type I
(TrCHI2) subfamily has the conserved active substrate
binding sites similar to previously reported CHIs. The
predicted tertiary structures of TrCHI2b showed structural
configurations consistent with AtCHIs, suggesting that type
I (TrCHI2) subfamily members of CHI might have active
roles in flavonoid production. Real-time quantitative
polymerase chain reaction (qRT-PCR) revealed that
TrCHIs were expressed in a tissue-specific manner. TrCHIs
in flowers under radiation treatment were up-regulated,
Saminathan Subburaj and Hye-Jeong Ha have been Contributed
equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s13562-017-0396-8) contains supplementary
material, which is available to authorized users.
& Geung-Joo Lee
gjlee@cnu.ac.kr
1
Department of Horticulture, Chungnam National University,
Daejeon 34134, South Korea
indicating their potential action against radiation stress.
Their regulation might be correlated with the presence of a
few cis elements in their promoters. Our results provided a
basis for functional study of flower pigmentation and
breeding for novel flower color. They will also help us to
elucidate radiation signal transduction pathways in
Tradescantia.
Keywords Chalcone isomerase Tradescantia
Phylogeny and evolution Gene expression -radiation
stress
Introduction
In plants, diverse biosynthetic networks of phenyl-
propanoid pathway play significant roles in the production
of a wide range of natural secondary metabolic compounds,
including lignin, hydroxyphenyl lignin, flavonoids, proan-
thocyanindins, and anthocyanins (Fraser and Chapple
2011). Among these secondary metabolic compounds,
flavonoids are water soluble polyphenolic molecules syn-
thesized through phenylpropanoid pathway when
4-coumaroyl-CoA enters flavonoid biosynthetic steps.
Flavonoids are ubiquitously present in all vascular plants.
They are responsible for the pigments in various tissues
such as root, stem, leaves, and most flowers (Kutchan
2005). In flowers, flavonoids produces yellow or red/blue
color to attract pollinators and provide protection against
UV-B filtration (Mol et al. 1998; Winkel-Shirley 2002;
Simmonds 2003). In addition, flavonoids are implicated as
signaling molecules during auxin transport and plant root
microbe symbiotic interactions (Wasson et al. 2006; Has-
san and Mathesius 2012). Flavonoids possess many
antioxidant properties, making them potential medicines
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for human. Flavonoids have been using in various phar-
maceutical applications to treat diseases such as
cardiovascular, cancers, and age related diseases such as
Alzheimers disease (Hertog et al. 1997; Knekt et al. 1997;
For review, see Kumar and Pandey 2013). Flavonoids are
generally found in all vascular plants, although leguminous
and a few non-leguminous plants appear to synthesize a
subgroup of flavonoids called isoflavonoids. It has been
reported that isoflavonoids have functions similar to fla-
vonoids (Dastmalchi and Dhaubhadel 2015).
The synthesis of flavonoids through phenylpropanoid
pathway involves various genes and enzymes which have
been well characterized in previous reports (Springob et al.
2003; Ali and McNear 2014). Among these genes, chal-
cone isomerase (CHI) (also known as chalcone flavone
isomerase) is one of the key enzymes involved in the
catalysis of either naringenin (non-legumes) or isoliquirit-
igenin chalcone (legume specific) to (2S)-naringenin or
(2S)-liquiritigenin flavanones, respectively, in the phenyl-
propanoid pathway (Springob et al. 2003). CHI has two
isozymes, namely non-leguminous type I CHIs and legu-
minous type II CHIs (Shimada et al. 2003). Depending on
variations in functional mechanism and structural evolution
such as substitution of several critical amino acid residues
in the catalytic positions, CHIs are further classified into
type-III fatty acid-binding proteins (FAPs) and type IV
CHI-like (CHIL) (Ngaki et al. 2012; Dastmalchi and
Dhaubhadel 2015). Both type III and type IV CHIs have
been suggested to be homologous to CHIs found in all
vascular plants (Ngaki et al. 2012). The first CHI was
found in green algae. Although the functions of type IV
CHIs remain unclear, type IV CHIs (FAPs) have been
implicated in fatty acid biosynthesis of developing seeds
during accumulation of lipids (Ngaki et al. 2012).
CHI genes have been identified and characterized from
various monocot and dicots, including Zea mays (Grote-
wold and Peterson 1994), Hordeum vulgare (Druka et al.
2003), Oryza sativa (Druka et al. 2003), Lotus japonicus
(Shimada et al. 2003), Ginkgo biloba (Cheng et al. 2011),
Glycine max (Dastmalchi and Dhaubhadel 2015), AraCHIs
hypogaea (Liu et al. 2015), Ipomoea batatas (Guo et al.
2015), and several horticulture flowering plants, including
Petunia hybrida (van Tunen et al. 1988), Tulipa fosteriana
(Yuan et al. 2013), and Paeonia suffruticosa (Zhao et al.
2015). CHI genes have been functionally characterized to
find out their regulatory roles in the biosynthesis of fla-
vonoids. Overexpression of Ipomoea batatas CHI gene in
Arabidopsis mutant lines has been shown to be able to
complement pigmentation phenotype in tissues of seed
coat, cotyledon, and hypocotyl (Guo et al. 2015). Similarly,
a tree peony Ps-CHI1 gene in transgenic tobacco is
reported to be able to decrease flower pigmentation, indi-
cating regulatory effects of CHI genes on flower color
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J. Plant Biochem. Biotechnol.
patterns (Zhou et al. 2014). These findings suggests that it
is possible to manipulate the metabolite content through
flavonoid biosynthetic pathways in order to obtain desir-
able agronomic traits (Sreevidya et al. 2006).
Flavonoid biosynthesis related genes can regulate
intense light such as UV-B radiations (van Tunen et al.
1988; Part et al. 2007). Radiation stress often leads to
increased flavonoid level in epidermal layers of tissues to
protect plants from radiation stress (Emiliani et al. 2013),
ultimately changing tissue colors (Park et al. 2007).
Tradescantia species is often called spiderworts. It is a
perennial plant belonging to Commelinaceae family. It is
cultivated for ornamental purposes due to their bluish or
purplish flowers (Wilson 1981). Tradescantia clone BNL
4430 has been emerged as model plant to understand
radiation induced genetic mutations that turn flower colors
from blue to pink (Ichikawa and Wushur 2000). To the best
of our knowledge, flavonoid biosynthesis genes in
Tradescantia have not been reported yet. Recently, our lab
constructed a flower cDNA library for Tradescantia clone
BNL 4430 under -radiation treatment (Unpublished raw
data, available under accession number; NN-1815-000001
at NABIC: http://nabic.rda.go.kr/). The objective of this
study was to use these transcriptomic resources to identify
flavonoid biosynthesis genes CHIs in Tradescantia.
Here, we present the first comprehensive study on the
molecular characterization of CHIs in Tradescantia
(TrCHI). Evolutionary analysis indicated that Tradescantia
genome might have seven members of CHI genes. We also
determined the expression profiles of TrCHIs in different
tissues and their response to -radiation treatment in
flower tissue. Based on expression analysis of TrCHI
genes, we suggest TrCHI2C, TrCHI3B, TrCHI3A1, and
TrCHI3A3 might have potential roles in the regulation of
flavonoid production in Tradescantia leaves, flowers, and
calyx. In addition, they might serve as important stress
response factors to provide adaptive functions against ini-
tiation of radiation stress.
Materials and methods
Plant materials and growth conditions
In vitro grown (20-day-old) Tradescantia (BNL clone
4430) seedlings were obtained from Korea Advanced
Energy Research Institute (KAERI), Seoul, Republic of
Korea. Seedlings were transferred to plastic pots (15 cm in
diameter, 3 seedlings/pot) filled with 2:1 ratio of potting
soil and commercial Perlite. Pots were placed in a green-
house with 16 h light at 25 C and 8 h dark at 20 C with
relative humidity of 7080%. Plants were subjected to
irrigation interval of 2 days. All experimental tissues were
J. Plant Biochem. Biotechnol.
collected from 60-day-old plants. Collected tissue samples
were flash frozen in liquid nitrogen and stored at 80 C for
further analyses.
Transcriptome resource, in silico identification,
and sequence analysis
Assembled transcript sequences from -radiation stress
induced flower transcriptomes of Tradescantia BNL 4430
were used for in silico BlastN analysis. Transcriptomes
(Control, 250 milligray (mGy), 500 mGy for 1 h, and
1000 mGy for 10 h) are available in the laboratory
(Unpublished data). Raw datas are accessible at National
Agricultural Biotechnology Information Center (NABIC,
http://nabic.rda.go.kr/) under accession number of NN-
1815-000001. Previously reported Chalcone isomerase-
like genes from Arabidopsis, rice, and soybean (Dast-
malchi and Dhaubhadel 2015; Przysiecka et al. 2015)
were used as queries to search against Tradescantia
transcriptome using BlastN. Candidate transcript sequen-
ces were simultaneously queried against PFAM (http://
pfam.xfam.org/search) and CDD (http://www.ncbi.nlm.
nih.gov/Structure/cdd/wrpsb.cgi) databases to predict their
putative protein domain signatures. Open reading frames
of putative candidate Tradescantia Chalcone isomerase
(TrCHI) gene sequences were obtained using NCBIs
ORF finder. Subcellular location of predicted TrCHI-like
proteins were identified using TargetP 1.1 and SignalP 4.1
(Emanuelsson et al. 2000). Theoretical pI/molecular
weight of protein was estimated using Compute pI/Mw
tool in ExPASy platform (http://web.expasy. org/com-
pute_pi/).
Multiple sequence alignment, tertiary structure
prediction, conserved motifs, and promoter analysis
of TrCHI-like genes
Multiple sequence alignments of nucleotide and deduced
amino acid sequences of identified TrCHI-like genes along
with other plant species genes were performed using
BioEdit 7.0 (Therapeutics, Carlsbad, CA, USA) and
MEGA5.1 software (Tamura et al. 2011). Tertiary struc-
tures of TrCHI-like proteins were predicted using
i-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-
TASSER/) (Zhang 2008). Conserved protein signature
motifs among CHI-like family members were obtained
using MEME tool (http://meme.nbcr.net/) (Bailey et al.
2009) with the following parameters: zero or one motif per
sequence, 6 and 50 amino acids as the minimum and the
maximum size of motifs, respectively, and 26 optimum
sites for each motif. To find cis-acting regulatory elements
in promoters of TrCHI genes, available upstream regions
were searched against Plant CARE database (http://bioin
formatics.psb.ugent.be/webtools/plantcare/html/).
Phylogenetic analysis and divergent time calculation
To find the evolutionary relationships among CHI-like
gene family members from different plant species, a phy-
logenetic tree was built using data set of CHI-like genes
containing 70 deduced amino acid sequences, including
those from Tradescantia in this study and those from other
plant species reported in previous studies (Dastmalchi and
Dhaubhadel 2015; Przysiecka et al. 2015). Alignments of
amino acid sequences were done using MEGA5 (Tamura
et al. 2011) of MUSCLE program (multiple sequence
comparison by log-expectation) (Edgar 2004) with the
following parameters: Gap open, 1; Hydrophobicity, 1.2;
maximum iterations, 1000. Alignments were imported into
MEGA5 for phylogenetic analysis. After building a maxi-
mum likelihood (ML) tree based on Jones-Taylor-Thornton
(JTT) model with pairwise deletion option and 1000
replicates of bootstrap, the evolutionary relationships
among CHI-like gene family members were inferred.
mRNA isolation and qRT-PCR
Total mRNAs were isolated from various tissues such as
mature leaves, young leaves, stem, calyx, bud, flower petal,
and root, including flower petals from -radiation treated
Tradescantia BNL 4430) using Hybrid-R TotalRNA isola-
tion kit (GeneAll, Daejeon, Korea) according to the
manufacturers instructions. Extracted total mRNAs were
subjected to cDNA synthesis using PrimeSrcript RT reagent
with gDNA eraser kit (Takara Korea Biomedical, Seoul,
Republic of Korea). qRT-PCR primer pairs specific for TrCHI-
like genes and actin (as reference) were designed using Primer
Express (version 3.0, Applied Biosystems). Primers used in
this study are listed in Supplementary Table S2. A reaction
mixture in 20 l volume containing 2 l of reverse transcribed
cDNA, 2 9 quantispeed SYBR Green mix (PhileKorea,
Daejeon, Korea), and 0.5 M of gene-specific forward and
reverse oligonucleotide primers was used to estimate the rel-
ative mRNA expression level using SYBR Green-based qRT-
PCR method [Eco Real Time PCR System (Illumina, Seoul,
Republic of Korea]. PCR reactions were performed with the
following parameters: one cycle at 95 C for 3 min, 39 cycles of
95 C for 15 s, 60 C for 20 s, 72 C for 15 s, a cycle at 65 C for
5 s, and a final cycle of 95 C for 2 s to detect primer specificity
based on melt curve analysis. For each gene, PCR reactions
with three biological replicates were analyzed. The expression
levels of target genes were normalized against the level of
housekeeping actin (KX173444) gene using the 2CT
method (Livak and Schmittgen 2001).
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Results
In silico identification of putative Tradescantia CHI
genes
Recently, we have investigated the transcriptomes of
Tradescantia clone BNL 4430 flower in response to -radi-
ation treatment (raw data available at NABIC: http://nabic.
rda.go.kr/). The present study used these transcriptomic data
and de novo assembled transcript sequences to find Trades-
cantia CHI (TrCHI) gene family. Conserved domain
sequences from previously identified and classified CHI gene
family members from Arabidopsis, rice, and soybean were
used as queries to search against Tradescantia transcript
databases using in silico BlastN. When an E value cutoff of
1e5 was applied, 15 transcript sequences were identified.
The sequence length of these transcripts ranged from 479 to
1794 base pairs (bp), indicating that some of them might
contain the 5 or the 3 regions or the coding regions either
partially or in full length (Supplementary Table S1). Various
numbers of ORFs in all six possible reading frames were noted
for all transcripts after running the NCBIs ORF finder pro-
gram. Only ORFs with the maximum nucleotide length were
selected and subjected to structural and functional analysis by
searching for conserved domains. PFAM analysis revealed
that all transcripts contained either chalcone-flavone iso-
merase or chalcone isomerase-like domains. CDD analysis
using these sequences as queries also showed similar predic-
tions. ORF sequences with the lowest E values during domain
search in PFAM were chosen as putative candidate Trades-
cantia Chalcone isomerase (TrCHI) genes. Furthermore,
PFAM search showed that the minimum length and maximum
nucleotide length of domains in these sequences were 206 bp
and 611 bp, respectively (Supplementary Table S1). After
removing redundant sequences, a total of nine non-redundant
sequences with the most conserved domains were selected as
TrCHI genes, including 5 full-length and 4 partial length ORFs
encoding CHI in Tradescantia. Their corresponding coding
sequence lengths and domain characteristics of these TrCHI
genes are shown in Supplementary Table S1. The maximum
and minimum length of coding sequences (CDS) of TrCHI
genes were 1290 bp and 456 bp, respectively. All TrCHI genes
predicted and identified in this study were deposited at NCBI
database with GenBank accession numbers: KX034650
KX034658.
TrCHI protein nomenclature, sequence, and tertiary
structure analysis
According to previous reports, CHI gene family could be
subdivided into type I, type II, type III, and type IV
subfamilies (Dastmalchi and Dhaubhadel 2015). In order
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J. Plant Biochem. Biotechnol.
to classify these putative TrCHI genes identified in this
study, multiple alignment (Fig. 1) and phylogenetic
(Fig. 3) analysis were performed based on deduced amino
acid sequences of these putative TrCHIs genes along with
previously characterized CHI gene sequences from
Alfalfa, Arabidopsis, rice, soybean, and maize. Among
nine TrCHI proteins, two (Accession numbers: KX034652
and KX034653) showed inconsistencies during phyloge-
netic classification. This was also reflected in conserved
motif analysis due to their partial CDS nature and
therefore these TrCHI proteins were excluded from fur-
ther analysis. Phylogenetic classification and conserved
motif analysis clearly distinguished 3 types of CHI sub-
families in Tradescantia, including type I (TrCHI2a, 2b,
2c, 2d), type III (TrCHI3B, 3C), and type IV (TrCHI4)
(Fig. 1; Table 1). The molecular weights of partial and
full length TrCHI proteins ranged from 23 kilodalton
(kDa) to 47 kDa. Among full length CDS, type III
TrCHI3C was the largest CHI protein with molecular
weight of 47 kDa, whereas type IV TrCHI4 was the
smallest CHI protein with molecular weight of 23 kDa
(Table 1). TargetP program indicated that the subcellular
location of these TrCHI proteins were mostly unknown. A
few of these proteins were predicted to be involved in
secretary pathways (TrCHI2c and TrCHI1) or associated
with chloroplast (TrCHI3B) (Table 1). TrCHI3B was also
found to contain signal peptide at the extended N-terminal
regions. However, the rest of TrCHI proteins had no
signal peptides. Detailed information of seven TrCHIs,
including their nomenclature, CDS length, deduced amino
acid length, molecular mass, and TargetP locations is
shown in Table 1.
Based on multiple sequence alignment (Fig. 1), type I
CHI2 subfamily (TrCHI2a, 2b, 2c, and 2d) contained the
necessary catalytically active site and critical site between
263 and 276 amino acid (AA) and 435436 AA, respec-
tively, except that partial coding sequences (TrCHI2c and
2d) were unable to show the active sites. Some members of
this subfamily had substitutions of Tyr Phe at 342 AA
(TrCHI2d) and Thr Ser at 435 AA (TrCHI2a, 2b, 2c,
and 2d). However, type III (TrCHI3B and 3C) and type IV
(TrCHI4) subfamilies did not retain any catalytically active
site (Fig. 1). Instead, they had more substitutions at almost
all critical sites. Sequence identities among TrCHI family
members were estimated using pairwise multiple align-
ment. The identities among TrCHIs ranged from 19 to 58%
in AA sequences and 3768% in nucleotide sequences
(Table 2). Within type I (TrCHI2) family members, the
shared identities ranged from 45 to 58% in AA sequences
and from 62 to 68% in nucleotide sequences. Among type
III (TrCHI3) members, the maximum identities shared
were 35% in AA sequences and 55% in nucleotide
sequences (Table 2). The maximum identities among
J. Plant Biochem. Biotechnol.

Fig. 1 Deduced amino acid sequence comparison of CHI proteins.
Multiple sequence alignment of TrCHI protein family along with
various types of CHI proteins (I, II, III, and IV) from other plant
species. Identity, similarity, and deletions among CHI alignments are
shown by black shades, grey shades, and hyphens, respectively. Green
stars and red boxes indicate the critical active sites (substrate binding
catalytic residues) and other active site residues, respectively (Jez
et al. 2002; Dastmalchi and Dhaubhadel 2015). Substitutions in
critical active site residues are indicated by pink bold letters (color
figure online)

Table 1 Molecular sequence characteristics of Chalcone isomerase gene family in Tradescantia

Nomenclature NCBI CDS length Residue length ORF Theoretical Predicted Type
accession (bp) (aa) type pI/Mw TargetP location of CHI/FAP

TrCHI2a KX034654 684 227 Full 5.85/24713.77 Any other Type I

TrCHI2b KX034655 684 227 Full 7.84/25007.56 Any other Type I
TrCHI2c KX034651 585 194 Partial 4.77/21042.93 Secretory pathway Type I
TrCHI2d KX034650 456 151 Partial 5.37/16454.58 Any other Type I
TrCHI3B KX034656 819 272 Full 9.45/29710.26 Chloroplast Type III/FAPa1
TrCHI3C KX034657 1290 429 Full 8.15/47264.21 Any other Type III/FAPa2
TrCHI4 KX034658 630 209 Full 4.84/23687.14 Any other Type IV

TrCHI subfamilies were also analyzed. Type I and type III
TrCHI subfamilies shared maximum identities of 22 and
48% in amino acid sequences and nucleotide sequences,
respectively. Type I and type IV TrCHI subfamilies shared
maximum identities of 29 and 51% in amino acid
sequences and nucleotide sequences, respectively. Simi-
larly, type III and type IV TrCHI subfamilies shared
maximum identities of 19 and 44% in amino acid
sequences and nucleotide sequences, respectively
(Table 2).

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 J. Plant Biochem. Biotechnol.

Table 2 Identity (%) of Nucleotide

nucleotide and amino acid
sequence between the Chalcone Type I Type III Type IV
isomerase gene family members
in Tradescantia TrCHI2a TrCHI2b TrCHI2c TrCHI2d TrCHI3B TrCHI3C TrCHI4

Type I TrCHI2a 68.72 63.33 61.81 41.11 48.39 50.98

TrCHI2b 58.41 62.46 68.64 42.76 47.8 49.56
TrCHI2c 51.06 53.72 63.17 43.86 48.78 51.05
TrCHI2d 45.7 53.64 55.33 37.32 46.02 44.58
Type III TrCHI3B 18.45 20.87 20.35 18.31 55.79 43.54
TrCHI3C 20.67 22.6 21.3 19.01 35.23 44.11
Type IV TrCHI4 24.64 25 29.14 26.85 19.49 19.1
Amino Acid
Calculated identity % matrix between the 7 members of Chalcone isomerase family in Tradescantia.
nucleotide and deduced amino acid identity levels shown in top and bottom triangles, respectively.
Sequences were aligned by Clustal Omega and identities were estimated from the pairwise multiple
sequence alignment

Tertiary structure and catalytic activity of CHI proteins
are unique and highly conserved among plant species (Jez
et al. 2000; Ngaki et al. 2012). In order to further
understand the structural characteristics of TrCHIs,
homology modeling of the three dimensional (3D) struc-
ture of typical TrCHI proteins were built using
i-TAASER (Zhang 2008). The predicted 3D structures of
TrCHI2b (KX034655) and TrCHI4 (KX034658) along
with AtCHI (CAB94981) and AtCHIL (NP_568154)
suggested that the basic structure of TrCHI proteins
(Fig. 2b, d) were consistent with CHI proteins of Ara-
bidopsis (Fig. 2a, c). Moreover, the distribution of several
critical amino acid sites in TrCHIs was also highly similar
to the structural characteristic of AtCHIs. The predicted
3D models of both TrCHI2a and TrCHI4 revealed 6
strands, 7 helices, and 14 coils. Some of the active sites
were distributed on the helices (106 Y, 109 K, 110 V,
113 N, 191 S, and 192 I sites in TrCHI2b and 100 Y, 103
Q, 104 L, 107 A, 183 W, and 184 Y in TrCHI4). Only
one active site was in coil (101 L in TrCHI2b and 95 I in
TrCHI4). The rest sites were distributed on the sheets
(Fig. 2b, d). The predicted 3D structures showed that the
five substrate binding catalytic active site (Jez et al. 2000;
Ngaki et al. 2012) distributions between TrCHI
(TrCHI2b: 36 R, 48 T, 106 Y, 113 N and 191 S; TrCHI4:
31 T, 43 N, 100 Y, 107 A, 183 W) and AtCHI (AtCHI:
47 R, 59 T, 117 Y, 124 N and 201 S; AtCHIL: 31 T, 43
T, 101 Y, 108 T, 184 W) proteins were highly identical
and conserved (Fig. 3a, b). These findings suggested that
these substrate binding sites might determine the func-
tional importance of these regions in TrCHI proteins.
However, detailed further studies are required to under-
stand the functions of these TrCHI proteins identified in
this study.
Phylogenetic analysis of TrCHI members
In order to determine the molecular evolutionary relation-
ships between TrCHIs and CHIs from other species, a
phylogenetic ML tree was built by using a set of CHIs
reported in previous studies as reference (Dastmalchi and
Dhaubhadel 2015; Przysiecka et al. 2015). As shown in
Fig. 3, the tree had six different clades in which CHIs were
classified into six subfamilies, including type I (CHI2),
type II (CHI1), type III (CHI3), and type IV (CHI4).
TrCHI3C were clustered with fatty acid binding proteins
(FAPs) from O. sativa and A. thaliana in CHI3C subfamily
(type III). It had the farthest phylogenetic distance from
other CHI subfamilies, suggesting that CHI3 might be
ancestral to the rest of CHI subfamilies according to pre-
vious studies (Dastmalchi and Dhaubhadel 2015).
Similarly, TrCHI3B fell into a clade with AtFAP1 from
A. thaliana in CHI3C subfamily (type III). Although sub-
family CHI3A has been reported to be derived from their
ancestral CHI3C through CHI3B during evolution, no
TrCHI in CHI3A (type III) clade was found. Subfamily
CHI3A (type III) might have further diverged to produce
CHI-like sequences such as subfamily CHIL (type IV)
(Fig. 3). In this clade, TrCHI4 was clustered closely with
CHIL from O. sativa (NP_001065587). Subfamily CHI2
(type I) clade consisted of four members of TrCHI
homologs (TrCHI2a, 2b, 2c, and 2d) in a monocot specific
class including CHIs from O. sativa (NP_001051714) and
Z. mays (Q08704). CHI2 (type I) also contained a dicot
specific class including CHIs from A. thaliana
(CAB94981) and G. max (ABA86742.1) with a Thr Ser
substitution (Fig. 1). This substitution was also noted in
TrCHI2a, 2b, 2c, and 2d members, confirming that these
TrCHIs belong to the known type I CHIs (Fig. 1, 3).

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Fig. 2 Predicted three dimensional structures of Arabidopsis and
Tradescantia CHI proteins. a AtCHI, b TrCHI2b, c AtCHIL and
d TrCHI4 structures were obtained using i-TASSER (Zhang 2008).
The amino (NH2) and carboxyl (COOH) terminals are shown in light
To further validate the phylogenetic classification of
CHI members and to understand structural evolution, a
conserved protein motif analysis among CHI subfamily
members were conducted using Multiple Em for Motif
Elicitation (MEME) search (Bailey et al. 2009). All CHI
family members possessed seven different kinds of motifs
as shown in Fig. 3. Motif analysis revealed that CHI
proteins of the same subfamily had similar motif types
and orders. However, between CHI subfamilies, motif
types and orders were different. For example, both type
III CHI3C and CHI3B subfamilies had motif 4, 6, and 2,
indicating that they were ancestors and highly different
from other CHI subfamilies. Although type III CHI3A
was derived from their ancestor CHI3C, it conserved only
motif 2 with two new motifs (5 and 1). Afterwards, in the
course of evolution, these motifs (1, 2, and 5) were found
to be highly conserved in all CHI subfamilies (type IV:
CHI4, type I:CHI2, and type II:CHI1) with a new motif 3
(type I:CHI2 and type II:CHI1) or motif 7 (type IV:CHI4)
but without motif 2 in type IV (CHI4). In corresponding
blue. In these structures, -helices, -strands, and coils are shown in
green, yellow, and red, respectively. Distribution of substrate binding
critical active sites (red) and other active sites (light blue) on the
structural regions is indicated (color figure online)
CHI subfamilies, all TrCHI members had highly con-
served motif types and orders, similar to CHI members in
other species except TrCHI2c and TrCHI2d (type I:CHI2)
that had no motif 5 due to their partial CDS length. A
highly different motif organization was also noted in two
TrCHI proteins (Accession numbers: KX034652 and
KX034653) due to their partial CDS lengths. This might
explain why they formed a nonspecific out-groups in
clusters of type IV:CHI4 and type II:CHI1 subfamily
(Fig. 3). Therefore, these two TrCHI members (Accession
numbers: KX034652 and KX034653) were excluded from
further analyses due to their inconsistency in motif
organization and undefined cluster in the tree. These
results demonstrated that the seven CHIs identified from
Tradescantia belonged to type I: TrCHI2, type III:
TrCHI3, and type IV: TrCHI4 containing 4 (TrCHI2a, 2b,
2c, and 2d), 2 (TrCHI3B, and 3C), and 1 (TrCHI4) CHI
member, respectively. They are closely related to those
from rice, suggesting that Tradescantia is closely related
to monocotyledons (Fig. 3).
123
 J. Plant Biochem. Biotechnol.

Fig. 3 Phylogenetic tree of CHI

family proteins based on
deduced amino acid sequences
of CHIs or CHI-like proteins
from Tradescantia along with
other plant CHIs. A maximum
likelihood (ML) tree was
constructed using seventy-one
amino acid sequences divide
into six families (CHI1, CHI2,
CHI3A, CHI3B, CHI3C, and
CHI4) and four functional
catalytic groups (type I, II, III,
and IV). CHI proteins from
Tradescantia are indicated with
a dot symbol. On the right side
panel, conserved protein motif
analysis using MEME shows
motif types and orders in
colored boxes corresponding to
CHI sequences in the
phylogenetic tree (color
figure online)

123
J. Plant Biochem. Biotechnol.
Differential tissue expression analysis of TrCHI gene
family
CHIs have been reported to be ubiquitously expressed in a
wide range of tissues (Dastmalchi and Dhaubhadel 2015).
In order to determine whether TrCHI family members in
Tradescantia had tissue-specific expressions, the expres-
sion levels of the seven TrCHIs were in different tissues
(mature and young leaves, stem, root, calyx, bud, and
petals) were determined by qRT-PCR. The designed qRT-
PCR primers for TrCHI members (Supplementary
Table S2) showed unique and distinct melt curves (Tm),
confirming target-specific amplifications during qRT-PCR
analysis. These results also suggested that the TrCHI
members identified in this study were indeed expressed in
the genome of Tradescantia clone BNL 4430. qRT-PCR
analysis revealed that almost all TrCHI members were
ubiquitously expressed in all tissues (Fig. 4a). It was noted
that the mRNA expression of all TrCHI members except
TrCHI2a were strong and conspicuous in leaves (mature
and young) and this would appears that Tradescantia leaves
undergo rapid changes in the composition of secondary
metabolites such as anthocyanins during the developmental
process. With the inadequate knowledge about molecular
controls of CHI genes in Tradescantia, the present study
used the expression level of each TrCHIs in mature leaves
Fig. 4 Differential tissue expression analysis of seven TrCHI genes.
a Transcriptional levels of TrCHI genes were accessed in seven
different tissues using qRT-PCR analysis. Transcriptional levels in
mature leaves were set as control (the value of 1) to calculate the
expression levels in other tissues. The relative expression levels of
TrCHI genes were normalized to actin reference gene. b Heat map of
as a control to measure the relative expression fold changes
in other tissues by using actin as reference (Fig. 4a). In the
present study, TrCHI2a showed a very low or unde-
tectable expression levels in leaves, stem and root.
However, TrCHI2a had the maximum expression level in
flower petals compared to the rest of TrCHI members
(Fig. 4a). Both TrCHI2c and TrCHI3C were highly
upregulated in calyx compared to other TrCHI members.
These results indicated that different TrCHI members had
distinct expression patterns in various tissues (Fig. 4a). To
evaluate organ-specific expression of TrCHIs, a heat map
expression profile was constructed by using qRT-PCR
expression results in fold change. As shown in Fig. 4b, heat
map contained various expression clusters. Each cluster
could describe tissue specific expression patterns of TrCHI
members (Fig. 4b). Subfamily type II TrCHI2d and
TrCHI2b were found in the same cluster of high expression
in mature leaves, although TrCHI2b was abundant in both
mature and young leaves. Transcript of TrCHI3C was
accumulated more in mature leaves and calyx compared to
that in other tissues where it showed down-regulation only.
TrCHI2c was expressed more in mature leaves similar to its
subfamily members TrCHI2d and TrCHI2b. TrCHI2c was
also upregulated in calyx, similar to TrCHI3C that had high
expression in both mature leaves and calyx (Fig. 4b).
Subfamily TrCHI3B (type III) and TrCHI4 (type IV) were
tissue specific expression profile of TrCHI gene family. The heat map
was built by using qRT-PCR fold change values as described in
Materials and methods. A tree cluster is used to show tissue specific
expression of TrCHI genes. A gradient color bar scale is used to
describe either up- (red) or down- (green) regulation of TrCHI genes
(color figure online)
123

also slightly upregulated in mature leaves and calyx,
although both of them reached their highest expression
level in young leaves. The mRNA accumulation of
TrCHI2a was quite different from other TrCHI members. It
shown the highest expression level only in petals. In the
rest of tissues, it was either significantly down-regulated or
undetectable (Fig. 4b). Taken together, these results sug-
gested that TrCHI members had tissue-specific expression
patterns. In leaves (both mature and young), TrCHI2d and
TrCHI2b were expressed. In mature leaves and calyx,
TrCHI3B and TrCHI4 were expressed. In leaves (mature
and young) and calyx, TrCHI3B and TrCHI4 were
expressed. In flower petals, TrCHI2a was expressed
(Fig. 4a, b).
Expression of TrCHI genes is induced in flowers
upon radiation treatment
Flavonoids have light absorbing characteristics that are
particularly influenced by ultra-violet (UV) light stress (Li
et al. 1993). The expression levels of CHI genes have been
found to be regulated by UV light to protect plant under
heavy light stress, resulting in enhanced levels of CHI
expression in plant tissues including flower (van Tunen
et al. 1989; Li et al. 1993; Cheng et al. 2011). Since fla-
vonoids play significant roles in pigmentation of plant
tissues including flowers and in the protection against light
stress, it is important to understand TrCHI expression
patterns under radiation conditions to shed light on their
regulatory effects on flowers of Tradescantia clone BNL
4430. Therefore, flower samples from radiation treat-
ments were collected for qRT-PCR analysis of TrCHI
members. radiation treatments included 50, 250, and
500 mGy for 1 h and 1000 mGy for 10 h. As shown in
Fig. 5a, the transcriptional levels of all TrCHI genes were
induced upon radiation treatment. Subfamily TrCHI2 (type
I) members such as TrCHI2a and TrCHI2d showed a down-
regulated pattern at mild (50 mGy for 1 h) and medium
radiation doses (50, 250, and 500 mGy for 1 h). However,
they were up-regulated by radiation at 1000 mGy for 1 h.
TrCHI2b was up-regulated only by high radiation doses
(500 and 1000 mGy). Under mild radiation conditions, it
maintained its transcript levels similar to that in the control
(0 mGy). Transcriptional level of TrCHI2c was decreased
by all radiation doses except 50 mGy which slightly
upregulated its level compared to the control. Regarding
TrCHI3 members, TrCHI3B was mostly down-regulated by
mild radiation doses (50500 mGy). However, its expres-
sion level was steeply increased to the highest level by
1000 mGy. Unlike TrCHI3B, TrCHI3C showed a gradually
increasing expression pattern throughout radiation treat-
ment. It reached its maximum expression level (8-fold)
when treated by 500 and 1000 mGy doses. However,
123
J. Plant Biochem. Biotechnol.
TrCHI4 was down-regulated by almost all radiation doses
except 50 mGy which up-regulated its level compared to
the control (0 mGy). In order to find radiation specific
expression of TrCHI members, a heat map was built. As
shown in Fig. 5b, all TrCHI genes were highly induced by
radiation stress with distinct radiation-specific expression
patterns. For example, TrCHI2c and TrCHI4 were induc-
tion higher by 50 mGy than the rest of radiation doses,
indicating that they are TrCHI members in response to mild
radiation stress. However, TrCHI2b and TrCHI3C were
abundantly expressed under radiation treatments with both
500 mGy and 1000 mGy (Fig. 5b), suggesting that they are
TrCHI genes in response to higher radiation stress. Simi-
larly, TrCHI2d and TrCHI3B were also highly induced by
radiation at 1000 mGy. However, TrCHI2a was different
from rest TrCHI genes. Its transcriptional levels after
radiation treatments were not significantly different from
those in the control. Taken together, these results suggest
that TrCHI genes are differentially regulated in flowers
under radiation stress.
Discussion
Flavonoids are essential for floral pigmentation patterns in
ornamental crops. In flowers, these flavonoids generally
produce colors ranging from orange/red to violet/blue
(Kutchan 2005). In the flavonoid biosynthetic pathway,
Chalcone isomerase or chalcone-avanone isomerase is a
catalytic enzyme responsible for cyclization of chalcone to
(2S)-naringenin. Since Tradescantia is an ornamental
flowering plant with flavonoids influencing flower colors, it
is important to understand the basic structure and expres-
sion characteristics of flavonoid biosynthetic pathway
genes in this plant for future flower color manipulation
studies. Molecular characterization studies of CHI genes
from various plant species including several horticultural
flowers such as Petunia, tulip, and Luoyang Hong (van
Tunen et al. 1988; Yuan et al. 2013; Zhao et al. 2015) have
been performed. However, a comprehensive study about
CHIs in Tradescantia has not been reported yet. For the
first time, this study identified CHI genes in Tradescantia.
By using transcriptomic resources, we performed in silico
search and identified nine members of TrCHI genes (Sup-
plementary Table S1). Through phylogenetic analysis of
TrCHI members compared to previously reported CHI
genes (Dastmalchi and Dhaubhadel 2015; Przysiecka et al.
2015), we were able to classify these TrCHI members into
three subfamilies: type I: TrCHI2 (TrCHI2a, 2b, 2c, and
2d), type III: TrCHI3 (TrCHI3B and 3C), and type IV:
TrCHI4 (TrCHI4) (Fig. 2, Table 1).
In the present study, the classification of two typical
TrCHI members (Accession numbers: KX034652 and
J. Plant Biochem. Biotechnol.
Fig. 5 Relative mRNA expression levels of TrCHI genes under
radiation treatment with different doses (50, 250, 500 mGy for 1 h,
and 1000 mGy for 10 h). a Expression levels of TrCHI genes were
normalized against actin reference gene. b A heat map was
KX034653) based on phylogenetic tree was impossible due
to their partial CDS nature. Similar kind of difficulty in the
classification of GbCHI genes based on phylogeny analysis
has also been noted in a previous study (Cheng et al. 2011).
A highly different motif pattern and the presence of several
conserved substrate binding active sites (Jez et al. 2000)
during a typical multiple sequence alignment suggest that
these two TrCHI members might belong to type I:CHI2
(non-leguminous) and type III:CHI3 (data not shown).
Based on multiple sequence alignment (Fig. 1) and phy-
logenetic classification (Fig. 3), TrCHI3C and TrCHI3B
subfamilies belong to an ancestral clade of CHI (Dast-
malchi and Dhaubhadel 2015). They clustered among
FAP2 and FAP1 proteins, respectively. The next clade
contained the other five TrCHI subfamilies: TrCHI4,
TrCHI2a, TrCHI2b, TrCHI2c, and TrCHI2d. These seven
TrCHI genes were subjected to further analysis. All seven
TrCHI proteins were found to be closely clustered with
CHIs of monocot species such as rice and maize during
phylogenetic analysis, indicating that Tradescantia has a
closer relationship with monocotyledons (Croxdale 1998).
The predicted molecular weight of full length CDS of
TrCHIs (47 kDa and 23 kDa for TrCHI3C and TrCHI4,
respectively) were in accordance with previously reported
CHI proteins from Arabidopsis and soybean (Dastmalchi
and Dhaubhadel 2015; Ngaki et al. 2012). TrCHI3B clus-
tered with AtFAP2 in type III CHI3 subfamily in the
phylogenetic tree (Fig. 3). It possessed the N-terminal
signal peptide with subcellular location in chloroplast
(Table 1). These features suggest that TrCHI3B might have
constructed as described in Materials and methods to find radiation
dose-specific expression patterns of TrCHI genes. A gradient from
dark or green (low) to red (high) is used to show the expression levels
of TrCHI genes at different radiation doses (color figure online)
similar function as AtFAPs or GmFAPs (Ngaki et al. 2012;
Dastmalchi and Dhaubhadel 2015). Future functional study
is merited to determine the role of TrCHI3B compared to
FAPs. Pairwise identity matrix among TrCHI members
showed that they only shared 19 to 58% identities in AA
sequence and 37 to 68% identities in nucleotide sequences
(Table 2). Some leguminous plants such as G. max and L.
japonicus have been found to share more than 80% of
identities among their CHIs (Dastmalchi and Dhaubhadel
2015; Shimada et al. 2003). However, in Arabidopsis, 10 to
63% of identities have been noted among the domains of
AtCHIs (Ngaki et al. 2014). The present study also noted
1862% identities among the domain regions of TrCHI
members (Supplementary Table S3). Therefore, legume-
specific CHIs might share higher sequence identities
among their CHIs compared to non-leguminous CHIs as
reported in previous studies (Dastmalchi and Dhaubhadel
2015; Shimada et al. 2003).
According to Jez et al. (2000), several conserved critical
active site amino acids are important for the catalytic
action of CHI enzymes in all plants. After aligning the
deduced amino acid sequences from the seven TrCHI
identified in this study with typical type I-IV CHIs reported
previously, TrCHI members in type I subfamily (TrCHI2a,
2b, 2c, and 2d) had all the catalytic active sites along with a
substitution of Thr Ser at 435 AA (all TrCHI2) as in rice
(NP_001051714) and Arabidopsis CHIs (CAB94981). In
addition, Tyr Phe at 342 AA in TrCHI2d was found
when compared to M. sativa CHI (Jez et al. 2000). The
remaining TrCHI members in type III and IV subfamilies
123

also had mutations shown in other plant CHIs. In Ara-
bidopsis, AtCHI (CAB94981) has been found to be a bona
de CHI protein (Ngaki et al. 2012). Therefore, TrCHI
members TrCHI2a, TrCHI2b, and TrCHI2c appeared to be
bona de CHI proteins in Tradescantia. The conserved 3D
structure reflects the conserved catalytic action of CHI
proteins in all plant species (Jez et al. 2000; Ngaki et al.
2012). Therefore, we chose two typical TrCHIs (type I:
TrCHI2b (KX034655) and type III:TrCHI4 (KX034658))
along with AtCHI (CAB94981) and AtCHIL (NP_568154)
to describe bona de and non-bona de CHIs, respectively,
to predicted their tertiary structure using homology based
model (Fig. 3a-d). The predicted 3D structures revealed
that the structural configurations (6 strands, 7 helices, and
14 coils) along with substrate binding catalytic active site
distributions between TrCHI2b (36 R, 48T, 106 Y, 113 N
and 191 S) and AtCHI proteins (47 R, 59 T, 117 Y, 124 N,
201 S) (Fig. 3a, b) were identical, indicating that type I
(TrCHI2a, 2b, and 2c) TrCHIs may have non-leguminous
CHI activity (Ngaki et al. 2012) such as converting 6-
hydroxynchalcone to 5-hydroxyflavanone (Shimada et al.
2003). CHI actually catalyses the production of flavone (5-
hydroxyflavanone or (2S)-naringenin) from chalcone (6-
hydroxynchalcone or naringenin chalcone) through a
stereospecific isomerization reaction called Michael-type
addition reaction (Jez et al. 2000, 2002; Jez and Noel
2002). This happens by the interaction of five substrate
binding active amino acid side chains of CHI protein with
the chalcone. Each substrate binding amino acids found to
have different roles in isomerization reaction comprising of
deprotonation of water molecules to produce negatively
charged nucleophilic 2 oxyanions (117 T), electrostatic
stabilization of the oxyanions (R 47), positioning of sub-
strate in specific place happens from a hydrogen-bond
network between two active sites (S 201 and N 124), and a
constant drive of reaction by favoring the displacement of
charge from the 2 oxyanion (T 59) (Jez et al. 2002; Jez and
Noel 2002). The catalytic activity of CHI on isomerization
reaction mainly depends upon the active site residues and
these were mostly found to conserved. However in alfalfa
CHI, a substitution of Y (Tyr) F (Phe) at 106 AA was
shown to retained its catalytic activity even the presence of
mutation. Similar kind of substitution (Tyr Phe at 342
AA) was observed in TrCHI2d of Tradescantia (Fig. 1)
supporting that TrCHI2d still could involves in isomer-
ization reaction (Gensheimer and Mushegian 2004). Result
analysis from tertiary structure (Fig. 2) suggesting that type
I TrCHI members (TrCHI2a, 2b, 2c, and 2d) could catal-
yses the in vitro production of flavone and thus play
significant roles in the production of flavonoids in
Tradescantia. However, functional characterization of
these TrCHI proteins is needed to understand their bio-
chemical actions.
123
J. Plant Biochem. Biotechnol.
Transcription levels of CHI genes have been investi-
gated in pigmented and non-pigmented tissues of plants
(Shoeva et al. 2014). The accumulation of CHI transcripts
sometimes correlates with the amount of anthocyanins in
target tissues (Shoeva et al. 2014; Cheng et al. 2011).
However, temporal expression studies of CHI gene in
Tradescantia have not been reported yet. Therefore, the
transcript levels of TrCHI genes in different parts of
Tradescantia were determined. As shown in Fig. 4, all
TrCHI members were actively transcribed in all tissues.
However, their expressions were distinct, showing tissue-
specific expression patterns. Of the seven TrCHIs, some
(TrCHI2b and TrCHI2d) showed higher expression only in
mature leaves, some (TrCHI2c and TrCHI3C) showed high
expression in mature leaves and calyx, some (TrCHI3B and
TrCHI4) had high expression in young leaves and calyx,
while others (TrCHI2a) showed high expression only in
flowers (Fig. 4a, b). These results indicate that all TrCHI
genes are actively transcribed in different parts of
Tradescantia. In herbaceous peony, most flavonoid
biosynthetic genes including CHIs have been reported to be
expressed in flowers as well as in leaves and bark tissues
(Zhao et al. 2015). A high level of accumulation of type I:
CHI1 transcripts in leaves of Ginkgo biloba has been
reported, in which GbCHI1 has higher expression in leaves
than in other tissues (Cheng et al. 2011). Similarly, in soy
bean, some type III:GmCHI3 genes have been found to be
expressed more in leaves (Dastmalchi and Dhaubhadel
2015). This suggests that leaf- specific TrCHI genes might
play active roles in phenylpropanoid metabolism of
Tradescantia.
All TrCHI transcripts showed relatively very low
expression in stem and roots. Similar expression pattern
has been observed in herbaceous peony flowers (Zhao et al.
2012), indicating that TrCHIs are non-leguminous CHIs.
Compare to leaves (mature and young), the expression
profiles of all the TrCHI genes remained low levels in
stems. This is consistent with a expression study investi-
gated in herbaceous peony on various flavonoid
biosynthetic genes including CHI gene (Zhao et al. 2015).
Some of the TrCHI members had a certain degree in
transcript accumulations (TrCHI2b, TrCHI3B, and
TrCHI3C) or accumulated extremely low (TrCHI2c,
TrCHI2d, and TrCHI4) in roots. This is suggesting that
these TrCHI members could possibly responsible for
anthocyanin and proanthocyanidin syntheses as mentioned
in a recent study (Jiang et al. 2016). CHIs are implicated in
various plant microbe interactions. In leguminous plants,
only CHI genes expressed more in roots have been sug-
gested to have roles as signaling molecules during root-
nodule development (Lambais and Mehdy 1993; Przy-
siecka et al. 2015). In the present study, we also noted a
abundant accumulation of several TrCHI (TrCHI2c,
J. Plant Biochem. Biotechnol.
TrCHI3B, TrCHI3C, and TrCH4I) transcripts in calyx tis-
sues (Fig. 4b) which is consistent with the high level
expression of anthocyanin biosynthetic pathway genes
(CHS and DFR) in sepals (calyx) of an orchid called
P. schilleriana (Ma et al. 2009). Several TrCHI genes
(TrCHI2c, TrCHI2d, and TrCHI3B, TrCHI4) noted to be
expressed slightly higher in bud than the flower tissues
(Fig. 4b). Temporal expression patterns of several different
flavonoid biosynthesis-related genes have been already
investigated in ornamental flowering plants of Matthiola
incana and Gentiana triora (Dangelmayr et al. 1983;
Nakatsuka et al. 2005). During the flower developmental
stages, it was found that both CHS and CHI genes were
accumulated higher in earlier bud (S1) than late fully
opened flowers (S4) (Nakatsuka et al. 2005). This
demonstrate that transcript expression of these TrCHI
members may possibly regulate the pigment accumulation
during Tradescantia flower development. Of all TrCHI
genes identified in this study, only TrCHI2a was accumu-
lated at relatively high level in flower tissues (Fig. 4a, b),
suggesting that TrCHI2a gene might be an active TrCHI
member responsible for flavonoid production in Trades-
cantia flowers. Accumulation of major phenylpropanoid
metabolism genes including CHIs in flowers has already
been reported in Chrysanthemum, tree peony, and tulips
(Chen et al. 2012; Yuan et al. 2013; Zhao et al. 2015).
Differential expression patterns of CHS and CHI genes
were found to responsible for the petal color variations in
petunia and a high level expression of these genes shown to
confers red petals (Akhar et al. 2016). In an another study,
suppression of a CHI gene through RNA interference in
tobacco was found to resulted in yellow coloration in petals
suggests that expression of CHI plays major role in
determination of flower color by conversion of chalcone to
flavone (Nishihara et al. 2005). Apart from type I TrCHI
members, some of the type III and type IV TrCHI members
also found to expressed in flowers at very low level in the
present study (Fig. 4). A type IV CHI protein called
enhancer of flavonoid production (EFP) was identified to
implicates in flower colors of Japanese morning glory
(Ipomoea nil). RNAi mediated gene silencing of these type
IV CHI homologs in petunia and torenia (Torenia hybrida)
resulted in pale flowers indicates that type IV CHI cold also
contributes in flavonoid biosynthetic pathway (Morita et al.
2014). Since Tradescantia is a flowering plant, expression
profiles of all TrCHI genes under flower developmental
stages will be needed to understand their functional roles in
floral pigmentations. Expression profiles of TrCHIs
obtained in this study will contribute to further study on the
isoflavonoid biosynthesis pathway in Tradescantia in the
future.
Flavonoids such as Chalcone isomerase and Chalcone
synthase have ultraviolet absorbing characteristics,
suggesting that they play significant roles against UV
induced damage in plants (van Tunen et al. 1988; Li et al.
1993). Apart from UV-B light, certain ionization radiation
such as radiations have also been reported to be able to
stimulate the expression of flavonoid pigments to protect
plants against irradiation stress (Kovacs and Keresztes
2002). Since flavonoids affect flower colors (Falcone et al.
2012; Nishihara et al. 2005), the present study investigated
the regulatory effect of radiation (50, 250, 500 mGy for
1 h, and 1000 mGy for 10 h) on TrCHIs mRNA levels in
flower tissues of Tradescantia. As shown in Fig. 5b, TrCHI
genes had radiation-dose specific expression patterns,
indicating that TrCHIs are more actively transcribed under
radiation stress compared to that under normal conditions.
Several TrCHI genes had a higher expression level under
50 of mGy low (TrCHI2c, and TrCHI4) and high doses of
either 500 and 1000 mGy (TrCHI2b, and TrCHI3C) or only
1000 mGy (TrCHI2a, TrCHI2d, TrCHI3B) radiation
(Fig. 5b). Increased CHI transcript levels have already been
reported in plants after exposure to UV-B irradiation (van
Tunen et al. 1989; Park et al. 2007; Cheng et al. 2011).
Centella asiatica plantlets also possess greater amount of
total flavonoid contents after a radiation treatment
(Moghaddam et al. 2011). Flavonoids are UV-B absorbing
compound and reported to involves in protection of plants
against UV-B irradiations (Graham 1998). In Arabidopsis,
a flavonoid-less mutant tt5 (CHI gene) was shown to
hypersensitive to UV-B irradiation (Li et al. 1993). The
UV-B irradiation usually results in production of reactive
oxygen species (ROS) and nitric oxide (NO) signalling
molecules in plants(Tossi et al. 2011, Zhang et al. 2011).
These signals were found to induce the expression of
polyphenylpropanoid genes such as CHS and CHI in vac-
uoles of epidermal layers to minimize the UV-B induced
cellular damage in mesophyll cells (Tossi et al. 2011,
Zhang et al. 2011). So the high level expression of TrCHI
genes may involves in protection of in Tradescantia tissues
under irradiation stress conditions. Apart from the irradi-
ation stress, other abiotic stresses also were reported to
induce the transcript accumulation of phenylpropanoid
biosynthesis genes in plants. In A. mongolicusto, all of
flavonoid biosynthesis genes including CHI have been
found to significantly up-regulated under drought and cold
stress conditions (Wu et al. 2014). Similarly in rice, genes
encoding for enzymes including CHI, CHS and Phe
ammonia lyase 1 (PAL1) from flavonoid biosynthetic
pathway were noted to up-regulated in response to salinity
stress (Walia et al. 2005). These studies indicating that the
high level mRNA expression of genes encoding for fla-
vonoid biosynthesis pathway including CHIs may have
significant role in abiotic stress adaptation of plants.
Transcriptional induction of phenylpropanoid biosynthesis
genes is sometimes regulated by several significant cis-
123

regulatory elements such as transcription factor binding
sites (MYB/MYC) and light responsive elements (H-
Box (CCTACC) and G-Box (CACGTG)) often located in
the promoters (Cheng et al. 2011; Chen et al. 2015).
Therefore, we searched for the presence of the above
mentioned cis-regulatory elements in the available pro-
moter regions of TrCHI genes. Our results revealed that the
promoters of three TrCHI (TrCHI2c, TrCHI3C, and
TrCHI4) genes consisted of some interesting light respon-
sive cis-elements such as 3-AF1 binding site, Box-4
(ATTAAT), CATT-motif (GCATTC), TC-rich motif, and
G-Box (CACGTT) (data not shown), demonstrating that
TrCHI members are highly inducible by light stress as
reported in previous studies (van Tunen et al. 1989; Park
et al. 2007; Cheng et al. 2011). Flavonoids are usually
distributed on the epidermal cell layers of tissues such as
leaves, apical meristem, pollen, and flowers that are highly
vulnerable to light stress. After gamma irradiation treat-
ment, chrysanthemum is reported to have higher level of
anthocyanins in ray florets than in disc florets (Lee et al.
2008), indicating that floral tissues are highly susceptible to
abiotic stresses. In the present study, Tradescantia showed
a rapid accumulation of TrCHI transcripts in flowers under
gamma radiation stress. This might result in increased
TrCHI enzyme activity and increased level of flavonoids in
florets to protect plant cells against damages caused by
radiation stress.
In conclusion, this is the first attempt to identify flavo-
noid biosynthetic related genes in Tradescantia using
molecular approaches. Through in silico analysis, we
identified and characterized seven TrCHIs in Tradescantia.
TrCHI2a, TrCHI2b, TrCHI2c, and TrCHI2d are considered
to be bona de TrCHIs based on predicted 3D models.
TrCHI3C is similar to FAP2 with chloroplast localization.
TrCHI3C and TrCHI4 are likely FAP1 and CHI-like iso-
forms. Further studies are merited to determine the
functions of these TrCHI members in flavonoid biosyn-
thetic pathway of Tradescantia. Differential expression
analysis revealed that these TrCHI members had tissue
specific variations at transcriptional level. The expression
of TrCHI in flower was highly up-regulated by radiation
in a dose-specific manner to possibly reduce irradiation
damage. These results were in accordance with cis ele-
ments in promoter regions of some TrCHI genes. The
regulatory effects of irradiation on TrCHIs further help
elucidate radiation signal transduction pathways in
plants. The findings from this study will improve our
understanding on the molecular mechanisms of flower
pigmentation through flavonoid biosynthetic pathways in
Tradescantia. This study also provides a basis for flower
color manipulation studies for Tradescantia through
molecular breeding approaches in the future.
123
J. Plant Biochem. Biotechnol.
Acknowledgements This work was supported by a grant (NRF-
2013M2A2A6043621) of Radiation Technology R&D program
through the National Research Foundation of Korea funded by the
Ministry of Science, ICT & Future Planning, Republic of Korea.
Author contribution SS carried out bioinformatics analysis and
drafted the manuscript. H-JH participated in the study and helped
drafting the manuscript. NP and S-HC assisted in the drafting of the
manuscript. G-JL supervised the study and assisted in the drafting of
the manuscript. All authors have read and approved the final
manuscript.
Compliance with ethical standards
Conflicts of interest The authors declare no competing financial
interests.
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