Plant Physiology and Biochemistry 146 (2020) 259–268

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Functional characterization of a HD-ZIP IV transcription factor NtHDG2 in T

regulating flavonols biosynthesis in Nicotiana tabacum
Zhong Wanga, Shanshan Wangb, Yansong Xiaoc, Zefeng Lia, Mingzhu Wua, Xiaodong Xiea,
Hongguang Lic, Wenjun Mua, Feng Lia, Pingping Liua, Ran Wanga, Jun Yanga,∗
a
China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou, 450001, China
b
Xiangyang Cigarette Factory, China Tobacco Hubei Industrial Co., Ltd., Xiangyang, Hubei, 441000, China
c
Chenzhou Tobacco Company of Hunan Province, Chenzhou, Hunan, 423000, China

A R T I C LE I N FO A B S T R A C T

Keywords: The HD-ZIP Ⅳ transcription factors have been identified and functional characterized in many plant species.
HD-ZIP However, no tobacco HD-ZIP IV gene has been isolated, and it is not yet known whether HD-ZIP IV genes are
Flavonols biosynthesis involved in controlling flavonols accumulation in plants. Here, we cloned a HD ZIP gene named NtHDG2 from
Nicotiana tabacum Nicotiana tabacum, which belongs to the class IV of HD-ZIP family, and the NtHDG2-GFP fusion protein is lo-
Homeodomain glabrous
calized to the nucleus. We further observed that the flavonols contents in the NtHDG2 overexpression leaves
increase to 1.9–4.5 folds of that in WT plants, but in the NtHDG2-RNAi plants the flavonols contents reduce to
20.9%–52.7% of that in WT plants. The transcriptions of one regulatory gene NtMYB12, and three structural
genes (NtPAL, NtF3′H, NtF3GT), contributing to flavonols biosynthesis, were significantly induced by NtHDG2.
However, the transcription level of NtNAC002, a flavonols biosynthesis repressor, was also significantly up-
regulated in NtHDG2-overexpression lines, but significantly down-regulated in the RNAi lines, indicating that
HDG2 regulates the synthesis of flavonols as a complex regulatory network. Moreover, ectopic expression of
NtHDG2 gene promoted the transcription of several AP2/ERF genes, including NtERF1-5, NtERF109, NtDREB1,
and NtCIPK11, which participate in regulating root development and resistance to abiotic stresses. Our findings
reveal the new function of HD-ZIP IV transcription factors in flavonoids biosynthesis, and indicate that HD-ZIP
IV members may play an important role in plant resistance to abiotic stress. The NtHDG2 gene provides a
promising target for genetically manipulating to increase the amounts of flavonols in tobacco leaves.

1. Introduction Homeodomain-leu zipper (HD-ZIP) proteins constitute a large fa-

Flavonoids are important polyphenolic secondary metabolites in
plants, which can be further classified into several major groups, in-
cluding anthocyanins, flavonols, proanthocyanidins, flavones, flava-
nones, and so on (Winkel-Shirley, 2001). In plants, flavonols play im-
portant roles in modulating pollen fertility (Mo et al., 1992), flower
color (Gronquist et al., 2001), UV-B protection (Kusano et al., 2011),
polar transport of auxin (Kuhn et al., 2011), and ethylene signaling
(Lewis et al., 2011). Flavonols are also beneficial micronutrients to
protect human from cancer (Zhang et al., 2008), inflammation (Kang
et al., 2008) atheromatosis (Xiao et al., 2017), neurocyte injury (Du
et al., 2018), autoimmune diseases and organ transplant rejection
(Milenkovic et al., 2010; Hushmendy et al., 2009). Elevating the fla-
vonols content in crops is therefore of great biological and medicinal
significance.
mily of transcription factors in plants, which contain a HD followed by
a ZIP motif (Ruberti et al., 1991). In Arabidopsis, the HD-ZIP genes are
grouped into four classes (Sessa et al., 1998), which regulate different
physiological processes. The class Ⅰ has 17 members, which act in ABA
(abscisic acid) and sucrose signaling pathway (Henriksson et al., 2005).
The class Ⅱ contains 9 members, some of which participate in reg-
ulating leaf development and meristematic activity (Bou-Torrent et al.,
2012; Zuniga-Mayo et al., 2012). The class Ⅲ members are implicated
in vascular differentiation, meristem initiation, and lateral organ po-
larity (Prigge et al., 2005). There are 16 HD-ZIP genes in class Ⅳ of
Arabidopsis, among which GLBRA2 (GL2), FLOWERING WAGENINGEN
(FWA), ANTHOCYANINLESS2 (ANL2), PROTODERMAL FACTOR2
(PDF2), and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1)
are well characterized (Nakamura et al., 2006). GL2 is the first identi-
fied gene, so the class Ⅳ genes are also known as HD-GL2 (Nakamura

∗
Corresponding author.
E-mail address: yangjun@ztri.com.cn (J. Yang).

https://doi.org/10.1016/j.plaphy.2019.11.033
Received 20 August 2019; Received in revised form 19 November 2019; Accepted 20 November 2019
Available online 21 November 2019
0981-9428/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Z. Wang, et al.
et al., 2006). GL2 is necessary for both root hair and trichome devel-
opment (Masucci et al., 1996; Wang et al., 2010). ANL2 promotes the
anthocyanin accumulation in the shoot subepidermal and epidermal
cells (Kubo et al., 1999). FWA is an imprinted gene identified from a
dominant late-flowering mutant (Kinoshita et al., 2004). ATML1 and
PDF2 are involved in regulating embryo development and shoot epi-
dermal cell differentiation (Abe et al., 2003). The class Ⅳ HD-ZIP genes
are also identified from other plant species. Two GaHOX genes were
isolated from Gossypium arboretum, and GaHOX1 was proved to be a
functional homolog of AtGL2 in trichome development (Guan et al.,
2008). The HD-ZIP IV transcription factor OUTER CELL LAYER4
(OCL4) is necessary for anther development and trichome patterning in
maize (Vernoud et al., 2009). The CsGL3 also play important roles in
trichome initiation in cucumber (Pan et al., 2015). In Artemisia annua,
the HD-ZIP IV protein AaHD8 regulates epidermal development by in-
teracting with a MIXTA-like protein AaMIXTA1 (Yan et al., 2018). The
RICE OUTERMOST CELL-SPECIFIC GENE 4 (Roc4) was indicated to act
as an LD (long day)-preferential flowering enhancer (Wei et al., 2016).
The wheat GL7 gene was predominantly expressed in grain, and could
activate genes encoding defensins (Kovalchuk et al., 2019).
Tobacco leaf is usually chosen as an experimental system due to its
high content of many secondary compounds such as alkaloids, terpe-
noids, and phenylpropanoids (Hahlbrock and Scheel, 1989; Saitoh
et al., 1985; Stoessl et al., 1976). For instance, tobacco was used for
investigating the effect of pathogen infection on the accumulation of
many phenylpropanoids including flavonols, quinic acid, scopolin, and
hydroxycinnamatics (Matros et al., 2006; Tanguy and Martin, 1972;
Felton et al., 1999). There are few reports of HD ZIP gene from tobacco.
A class Ⅰ gene NaHD20 was identified from N. attenuate, and proved to
play important roles in regulating ABA accumulation in leaves, ben-
zylacetone (BA) emission from flowers, and the transition time of
bolting and flowering (Re et al., 2011, 2012). However, to our knowl-
edge, no tobacco HD-ZIP IV gene has been characterized so far, and it is
not yet known whether HD-ZIP IV genes are involved in controlling
flavonols accumulation in plants.
In the present study, we cloned and characterized a HD-ZIP IV gene
(NtHDG2) from N. tabacum. The flavonoids contents in NtHDG2 over-
expressed and RNAi plants were detected to investigate its putative
functions in flavonoids accumulation. RNA sequencing (RNA-Seq) and
qRT-PCR analyses were then performed to explore the possible target
genes of NtHDG2 in regulating flavonols biosynthesis. Conclusions re-
garding the HD-ZIP IV protein NtHDG2 in influencing flavonols pro-
duction in tobacco leaves are presented.
2. Materials and methods
2.1. Plant material and growth condition
The N. tabacum (Honghua Dajinyua cultivar) and N. benthamiana
were used in the present study. Tobacco seeds kept by our lab were
germinated on MS medium after soaking and sterilizing, as described
previously (Wang et al., 2018). The sterile seedlings were either used
for transgenic manipulation, or transplanted into pots for normal
growth (28 °C/16 h light, 23 °C/8 h darkness) until the flowering stage.
Various tissues were collected from the plants for RNA extraction or
flavonoids content detection.
2.2. Gene cloning of NtHDG2 from N. tabacum
The NtHDG2 full length complementary DNA (cDNA) sequence was
used to design gene specific primers (listed in Supplementary Table S1).
Total RNA and DNA were extracted from young tobacco leaves, and the
RNA was then used to synthesize cDNA. The full length gene sequence
and coding sequence (CDS) of NtHDG2 were amplified by PCR using
DNA and cDNA as the template, separately. The amplified fragments
were cloned into the pMD19-T vector (TaKaRa Bio, Dalian, China), and
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Plant Physiology and Biochemistry 146 (2020) 259–268
then sequenced. The correct clones were chosen for further vector
construction.
2.3. Phylogenetic and sequence analysis
The NtHDG2 gene exon/intron structure was determined by
aligning the genomic DNA sequence and the CDS sequence with Clustal
X (version 1.83). Multiple sequence alignment of the HDG proteins was
performed using the DNAMAN (version 6.0) software with default gap
penalties (Jeanmougin et al., 1998). The phylogenetic tree of the
NtHDG2 and Arabidopsis HD ZIP IV proteins was constructed by MEGA
5.0 using the neighbor-joining algorithm (Tamura et al., 2011). The
Arabidopsis HD protein sequences were collected from the TAIR web-
site (https://www.arabidopsis.org/) as follows: AtPDF2 (AT4G04890);
AtML1 (AT4G21750); AtGL2 (AT1G79840); AtANL2 (AT4G00730);
AtHDG1 (At3g61150); AtHDG2 (At1g05230); AtHDG3 (At2g32370);
AtHDG4 (At4g17710); AtHDG5 (At5g46880); AtHDG6 (At4g25530);
AtHDG7 (At5g52170); AtHDG8 (At3g03260); AtHDG9 (At5g17320);
AtHDG10 (At1g34650); AtHDG11 (At1g73360); AtHDG12
(At1g17920).
2.4. Vector construction
The amplified open reading frame (ORF) sequence of NtHDG2
without the stop codon was digested with the endonucleases Spe I and
Kpn I, and then cloned into the pCAMBIA1300 vector to obtain the
fusion NtHDG2-FLAG, under the control of the CaMV35S promoter
(35S:NtHDG2–FLAG). Similarly, the digested CDS sequence was also
cloned into the pGreen-35S-GFP (green fluorescent protein) vector to
obtain the fusion NtHDG2-GFP. For the RNAi vector construction, a
CDS fragment of NtHDG2 was amplified with gene specific primers
containing attB adaptor, and the fragment was cloned into the
pHellsgate 2 vector via BP reaction. Primers for vector construction are
listed in Supplementary Table S1.
2.5. Subcellular localization of NtHDG2–GFP fusion protein
The recombinant 35S:NtHDG2-GFP plasmid was transformed into
Agrobacterium tumefaciens strain GV3101. The A. tumefaciens was cul-
tured in LB medium at 28 °C overnight, and then collected by cen-
trifugation. The transient expression of 35S:NtHDG2-GFP in tobacco
young leaves (N. benthamiana) was performed as described in previous
study (Yang et al., 2000). After agroinfiltration, the fluorescent signal
was taken with a confocal laser scanning microscope (ZEISS LSM 700,
Germany). The nuclear localized marker protein was as described in the
previous reference (Shcherbo et al., 2007).
2.6. Creation of tobacco transgenic plants
The recombinant 35S:NtHDG2-FLAG and NtHGD2-RANi plasmids
were firstly transformed into A. tumefaciens strain GV3101, and the A.
tumefaciens was used for the transgenic manipulation via leaf disks
method (Palmgren et al., 1993). The transgenic plants were selected
with hygromycin, and then confirmed by DNA and RNA analyses. Over
20 T0 positive plants for 35S:NtHDG2-FLAG and NtHGD2-RANi were
selected and identified, respectively. The T0 positive plants were then
self-pollinated twice to generated T2 transgenic progeny. 3 lines of
35S:NtHDG2-FLAG with higher NtHDG2 expression level and 3 lines of
NtHGD2-RANi with lower NtHDG2 expression level were used for the
gene expression analyses and flavonoids content analyses.
2.7. Flavonoids measurement
The determination of flavonoids content was performed as de-
scribed in our previous studies (Wang et al., 2018). In brief, 150 mg of
fresh tobacco tissue sample was grinded to powder after freeze-drying.
Z. Wang, et al. Plant Physiology and Biochemistry 146 (2020) 259–268

Fig. 1. Identification of tobacco NtHDG2 gene. (A) Cloning of the full length of the NtHDG2 gene and CDS sequence. M, DNA marker; lane 1, amplification of the full
length NtHDG2 gene; lane 2, amplification of the full length NtHDG2 CDS sequence. (B) Exon/intron structure of the NtHDG2 gene. The black boxes indicate exons;
the lines indicate the introns; the numbers (bp) indicate the length of the exons and introns. (C) Phylogenetic analysis of the NtHDG2 with the 16 Arabidopsis HD-ZIP
Ⅳ proteins. MEGA5.0 was used to generate the neighbor-joining tree (Jones-Taylor-Thornton model). The statistical reliability of the tree topology was assessed by
performing a bootstrap analysis with 1000 replicates. (D) Alignment of the NtHDG2, AtPDF2, AtML1, and AtHDG2 protein sequences. The red box indicates the HD
helix-III region; Asterisks denote potential DNA sequence-specific contact residues; The lines indicate the zipper-loop-zipper motif; The green boxes indicate the
conserved Cyss implicated in redox regulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article.)

1.5 ml 80% ethanol (with 0.012 g/L vitexin as internal standard) was
added to the powder, and the mix was ultrasonicated for 30 min before
centrifuging (14,000 rpm for 10 min). The supernatant was filtered
through a 0.22 μm membrane, and then used for the flavonoids content
determination by HPLC-UV. The specific experimental parameters are
as follows: chromatographic column- ACQUITY UPLC®BEH Phenyl
(1.7 μm 2.1 mm × 150 mm, Waters); injection volume-1μl; gas tem-
perature-350 °C; flow-0.3 mL/min; wave length-230 nm, 260 nm,
360 nm, and 570 nm; mobile phase-A 100% water, B 100% acetonitrile;
gradient elution-8 min 85% A+15% B, 5 min 58% A+42% B, 0.01 min
100% B, 3 min 95% A+5% B, 18 min stop.
2.8. RNA-Seq and data analyses
RNA-Seq and data analyses were performed as described in the
previous study (Chen et al., 2017). Briefly, total RNA of tobacco leaves
was extracted with Trizol reagent (Invitrogen). The RNA integrity was
determined with an Agilent BioAnalyzer 2100, and the qualified sam-
ples were used for the construction of sequencing libraries with an Il-
lumina TruSeq RNA Sample PreKit. The sequencing was performed
using the Illumina Hiseq 2500 sequencing platform, and the FastQC
method (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) was
adapted to evaluate the quality of the mRNA-Seq reads. Low quality
(< 20) bases at the 5′ and 3’ ends and the adaptor sequence were re-
moved by Trimmomatic (v0.30) (Bolger et al., 2014). The reference
genome of N. tabacum was from ftp://ftp.solgenomics.net/genomes/
Nicotiana_tabacum/(Edwards et al., 2017), and the reads were mapped
to this genome using HISAT2 (v2.1.0) (Kim et al., 2015). Cufflinks
(v2.2.1) (Trapnell et al., 2010) was used to calculate gene expression
levels, and Cuffdiff (Trapnell et al., 2013) was adapted to identify the
differentially expressed genes (DEGs) with log2 ratios ≥2 or ≤ −2
(FDR < 0.05). All the DEGs are listed in Supplementary Table S2.
2.9. qRT-PCR analysis
Total RNA samples for qRT-PCR analysis were extracted with a
SuperPure Plantpoly RNA Kit (Gene Answer, Beijing, China). Possible
remaining genomic DNA was removed using RNase-free DNAse I. The
first strand cDNA was synthesized with Reverse Transcriptase M-MLV
(Takara Biomedical Technology, Beijing, China) and random primers.

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Fig. 2. Characterization of tobacco NtHDG2. (A) Subcellular localization of NtHDG2-GFP fusion protein. NLS is a marker protein for nuclear localization; Merge,
merging of NLS, GFP, Chloroplast autofluorescence and Brightfield images. (B) Relative expression levels of NtHDG2 in different tobacco tissues. (C) Relative
expression levels of NtHDG2 in different parts of flowers. BP, before pollination; AP, after pollination. Values are means of three independent replicates. Error bars
denote standard deviations. The significant differences among different tissues were detected by Tucky's test.

The qRT-PCR reactions were performed with SYBR Green kit (Roche)
and an LightCycler® 96 SW 1.1 cycler (Roche, Laval, QC, Canada). The
relative gene expression levels were calculated as described in the
previous study (Livak and Schmittgen, 2001). The ubiquitously ex-
pressed NtL25 gene was chosen as the internal reference control
(Schmidt and Delaney, 2010), and three independent biological re-
plicates were performed for each gene.
NtTUBB (Tubulin beta chain, XP_016456097.1 and NP_001312648.1)
genomic fragment was used as a negative control. The primers used for
fragment amplification are listed in Supplementary Table S1.
3. Results
3.1. Gene cloning and sequence characterization of NtHDG2

2.10. ChIP-qPCR The protein sequences of AtPDF2 (AT4G04890), AtML1

The Chromatin Immunoprecipitation (ChIP) was performed with
the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (Epigentek,
New York, USA) following the manufacturers’ instructions. In brief,
tobacco seedlings were harvested and fixed with 1.0% formaldehyde
under vacuum infiltration for 10 min, and then quench cross-linked
under vacuum infiltration for an additional 5 min by adding glycine
solution (final concentration 0.125 M). The tissues were grinded in li-
quid nitrogen to fine powders, and tissue lysis was done with the lysis
buffer provided by the kit. The pellet nuclei was centrifuged, and then
re-suspended for DNA shearing by sonication. The length of sheared
DNA should be between 200 and 1000 bp. The pellet nuclei solution
was then added to the strip wells with the anti-FLAG (SAB4200071,
Sigma, Beijing, China) to pull down the NtHDG2-FLAG fusion protein,
which was binding to its putative target DNA fragment. The collected
DNA fragment was then reversed and purified for qRT-PCR analyses as
described previously (Chen et al., 2014). The enrichment of two
(AT4G21750), AtHDG2 (AT1G05230), and AtHDG3 (AT2G32370) were
used as the query sequence for searching the N. tabacum genome in the
NCBI database, respectively. The XP_016486479.1 and
XP_016466997.1 show the highest sequence similarity with the four
Arabidopsis homologous proteins, and the similarity of the amino acid
sequences between the two tobacco proteins is also very high (97.12%).
Gene specific primers were designed to amplify the tobacco HDG gene
based on the sequence information (XM_016630993.1) in the database.
The full length of the cloned NtHDG gene is about 4163 bp containing
10 exons and 9 introns, which give rise to a 2199 bp coding sequence
(Fig. 1A &B).
A phylogenetic analysis was performed to investigate the evolu-
tionary relationships between NtHDG and the 16 HD ZIP IV members in
Arabidopsis. As shown in Fig. 1C, NtHDG is most related to the four
AtHD proteins, including AtPDF2, AtML1, AtHDG2, and AtHDG3. Se-
quence alignments show that NtHDG protein share 74.83% identity
with AtPDF2, 72.81% with AtML1, 70.48% with AtHDG2, and 47.45%

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with AtHDG3. The characteristic domain or residues for HD ZIP IV
members including the HD helix-III region, potential DNA sequence-
specific contact residues, zipper-loop-zipper motif, and the conserved
Cyss, are highly conserved in NtHDG (Fig. 1D). These analyses indicate
that the clone NtHDG gene belongs to the HD ZIP IV group, and is
closely related to AtPDF2, AtML1, and AtHDG2 gene. So we named it as
NtHDG2 gene.
3.2. Subcellular localization and expression pattern of NtHDG2
To investigate the subcellular localization of the NtHDG2 protein,
the fusion protein NtHDG2-GFP was transiently expressed in N. ben-
thamiana leaves. The GFP fluorescence signal and a known nuclear lo-
calized marker protein signal (NLS) were detected with laser scanning
confocal microscopy. As shown in Fig. 2A, the location of NtHDG2-GFP
is complete match with that of the NLS marker protein, suggesting the
cloned NtHDG2 is a nuclear protein, possibly functioning as a tran-
scription factor.
To explore the possible roles of NtHDG2, its expression pattern was
detected by qRT-PCR. The transcripts of NtHDG2 could be detected in
all the tissues examined, but were most abundant in flowers and ax-
illary buds (Fig. 2B). We further detected the NtHDG2 expression level
in different parts of flowers, and found the calyx and corolla had high
expression level of NtHDG2 (Fig. 2C). The NtHDG2 expression level was
low in stamen and pistil before pollination, but was obviously induced
in the pollinated pistil (Fig. 2C). Moreover, the leaves and buds also had
high expression level of NtHDG2 (Fig. 2B).
3.3. NtHDG2 promotes flavonols accumulation in tobacco leaves
To investigate the possible role of NtHDG2 in flavonoids accumu-
lation, the over-expression (OE) and RNAi lines of NtHDG2 gene was
generated. The positive transgenic plants were selected with antibiotic,
and then confirmed by PCR method with vector specific primers
(Supplementary Fig. S1). The expression level of NtHDG2 gene was
significantly up regulated in the positive OE lines, but significantly
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Plant Physiology and Biochemistry 146 (2020) 259–268
Fig. 3. Quantitative assay of flavonoids content in
tobacco leaves. (A) Chromatogram of flavonoids
standards. N1, Keracyanin chloride; N2, Chlorogenic
acid; N3, Delphinidin chloride; N4, Scopoletin; N5,
Cyanidin chloride; N6, Procyanidin B2; N7,
Kaempferol rutinoside; N8, Quercetin rutinoside; N9,
Kaempferol; N10, Quercetin. (B) Flavonols contents
in fresh tobacco leaves. WT, wild type; OE, NtHDG2
overexpressed lines; RNAi, NtHDG2 RNAi lines. (C)
Chlorogenic acid contents in fresh tobacco leaves. (D)
Anthocyanins contents in fresh tobacco leaves.
Asterisks represent statistically significant differences
between WT and transgenic plants determined by
two-tailed paired Student's t-test (**, P < 0.01; *,
P < 0.05).
down regulated in the RNAi lines. There was no obvious phenotypic
defect in these positive transgenic seedlings (Supplementary Fig. S1).
Since leaf is a main tissue for secondary metabolites synthesis in to-
bacco, we then measured the flavonoids levels in the leaves of trans-
genic plants. The HPLC-UV method was adapted to separate and detect
the metabolites. The contents of chlorogenic acid and major flavonoids
in tobacco leaves were determined (Fig. 3A, Supplementary Table S2).
The flavonols contents in three independent OE lines were all sig-
nificantly higher than that in the WT plants, while the flavonols con-
tents it the RNAi lines were significantly lower than that in the WT
plants (Fig. 3B). In detail, the flavonols content in WT leaves was
16.38 μg/mg, and that in the OE lines was 58.31–139.08 μg/mg, which
was 1.9 folds–4.5 folds of that in WT plants. The flavonols content in
RNAi lines was 3.43–16.38 μg/mg, which was 20.9%–52.7% of that in
the WT plants (Fig. 3B). The chlorogenic acid contents in two OE lines
were significantly higher than that of WT plants, but showed no sig-
nificant change in the RNAi lines (Fig. 3C). The anthocyanins contents
in all the transgenic plants were similar to that of the WT plant
(Fig. 3D). These results suggested that NtHDG2 promotes the flavonols
accumulation in tobacco leaves.
3.4. NtHDG2 promotes the transcription of several key genes contributing to
flavonols biosynthesis
To elucidate the regulatory networks underlying NtHDG2-mediated
flavonols biosynthesis in tobacco, RNA-seq analysis of the OE-#3 and
WT leaves was performed. The analysis revealed 970 DEGs (differen-
tially expressed genes), of which 123 genes were expressed at lower
levels (down-regulated) and 847 genes at higher levels (up-regulated)
in the OE leaves (Supplementary Table S2). Based on the functional
annotations, 13 genes are related to flavonoids biosynthesis, of which
12 genes are up-regulated and 1 gene is down-regulated (Table 1). The
12 up-regulated genes encode 2 transcription factors (MYB12 and
MYB4) and 7 catalytic enzymes, including phenylalanine ammonia-
lyase (PAL), flavonoid 3′-hydroxylase (F3′H), anthocyanidin 5-O-glu-
cosyltransferase (5 GT), flavonoid 3-O-glucosyltransferase (F3GT),
Z. Wang, et al. Plant Physiology and Biochemistry 146 (2020) 259–268

Differentially expressed genes (DEGs) related to flavonoids biosynthesis in the WT and OE tobacco leaves. DEGs with FDR < 0.05 and |log2 ratios|≥2.00 are listed here. EBGs, early biosynthesis genes in flavonoids
anthocyanin 3′-O-beta-glucosyltransferase (3′GT) (Fukuchi-Mizutani

Catalyzing the hydroxylation of dihydrokaempferol to dihydroquercetin (Dalman et al., 2017)

et al., 2003), anthocyanin 5-aromatic acyltransferase (Fujiwara et al.,
1998), and flavonol 3-sulfotransferase (Varin and Ibrahim, 1992)
Involved in anthocyanin biosynthesis (Chen et al., 2014Fukuchi-Mizutani et al., 2003)

Catalyzing the glucosylation of both anthocyanins and flavonols (Kamata et al., 2013)
The entry-point enzyme of the general phenylpropanoid pathway (Iida et al., 2019)
(Table 1). The down-regulated gene encodes dihydroflavonol-4-re-
ductase (DFR), a key enzyme involved in anthocyanin biosynthesis.
qRT-PCR was then performed to verify the DEGs contributing to

Catalyzing the key step of anthocyanin biosynthesis (Dalman et al., 2017)

flavonoids accumulation in both the OE and RNAi lines. As shown in
Fig. 4, the expression levels of NtMYB12, NtF3′H, NtF3GT, and NtPAL
were significantly higher in the OE lines compared with the wild type

Controlling the balance of FVBP production (Wang et al., 2017)

plants, which was consistent with the RNA-seq results. Moreover, the

Promoting the transcription of the EBGs (Wang et al., 2016)

Involved in anthocyanin biosynthesis (Kamata et al., 2013)

expression levels of NtMYB12, NtF3′H, and NtF3GT were significantly

Involved in flavonol biosynthesis (Fujiwara et al., 1998)

lower in the RNAi lines than those of the WT plants. By contrast, the
expression level of NtDFR gene was significantly down-regulated in the
OE lines, but significantly up-regulated in the RNAi lines (Fig. 4). The
expression levels of NtMYB4, Nt5GT, and Nt3′GT genes showed no
significant changes in both the OE and RNAi lines (Fig. 4). Taken to-
gether, these results suggested that NtHDG2 promotes flavonols accu-
mulation through inducing the transcripts of these regulatory and
structural genes in tobacco leaves.
To further explore the molecular mechanism of NtHDG2 in reg-
Function & References

ulating flavonols biosynthesis in tobacco, ChIP assays were performed

to test whether NtHDG2 could directly bind to the promoter regions of
NtMYB12, NtF3′H, NtF3GT, and NtPAL genes. The Arabidopsis homolog
ATML1 and AtPDF2 could bind to the conserved elements, which was
an 8-bp 5′-TAAATG(C/T)A-3′ sequence (Abe et al., 2001; Rombola-
Caldentey et al., 2014). We thus searched this element in the 3 kb se-
quences upstream the start code of the 4 target genes, and found 1
element for NtMYB12 gene, 2 for NtF3′H gene, 1 for NtF3GT gene, 3 for
Anthocyanin 3′-O-beta-glucosyltransferase

NtPAL gene (Fig. 5A). After immunoenrichment, one fragment of

Anthocyanin 5-aromatic acyltransferase

Anthocyanidin 5-O-glucosyltransferase

NtF3′H gene containing a 5′-TAAATGTA-3′ sequence was significantly

Flavonoid 3-O-glucosyltransferase

enriched, while one fragment of NtPAL gene with two 5′-TAAATGTA-3′

Phenylalanine ammonia-lyase

sequences was also significantly enriched (Fig. 5B). These data sug-
Dihydroflavonol-4-reductase
Transcription factor MYB12
Flavonol 3-sulfotransferase

Myb-related protein Myb4

Flavonoid 3′-hydroxylase

gested NtHDG2 might directly bind to the conserved element to reg-

ulate the expression levels of NtF3′H and NtF3GT gene.

3.5. RNA-seq analysis indicates the multifunction of NtHDG2 in tobacco

Annotation

In addition to the DEGs involved in flavonoids biosynthesis, the

RNA-seq analysis also found many DEGs participating in various phy-
siological processes, including meristem and embryonic development,
plant hormone signaling, stress resistance, protein phosphorylation,
9.50E-04
8.00E-04
1.78E-02
1.50E-04
2.00E-04
1.85E-03
5.50E-04
2.05E-02
4.85E-03
2.05E-02
2.65E-03
3.00E-03
1.27E-02
p_value

and so on (Fig. 6A, Supplementary Table S2). Besides, the DEGs also
contain many transcription factor genes, which encode AP2/ERF,
WRKY, MYB proteins (Fig. 6A, Supplementary Table S2). The AP2/ERF
genes have been proved to enhance plant resistance to several biotic
log2 (OE/WT)

stresses (Zhu et al., 2013; Pan et al., 2012; Yamasaki and Randall, 2016;
Kidokoro et al., 2015). Therefore, expression levels of the 8 AP2/ERF
−2.25

genes were confirmed in the OE and RNAi lines by qRT-PCR. As shown

2.26
2.92
2.09
2.30
2.06
3.76
3.61
2.98
2.86
3.31
4.90
2.11
pathway; FVBP, floral volatile benzenoid/phenylpropanoid.

in Fig. 6B, the expression levels of NtERF1, NtERF2, NtERF3, NtERF4,

NtERF5, NtERF109, NtDREB1, and NtCIPK11 genes are significant
OE RPKM

higher in the OE lines compared with the WT plants, but significant

106.60
30.52

20.47
21.52
11.56

19.75

lower in the RNAi lines. These results indicate the putative roles of
5.95

8.75
2.94
3.43
6.38

9.82
1.54

NtHDG2 in tobacco resistance to stresses.

WT RPKM

4. Discussion
22.30
4.04
1.40
4.15
5.18
0.85
0.72
0.37
0.47
0.64
0.66
2.27
7.35

The characterization of anthocyanin biosynthesis in plant tissues is

much more detail than that of flavonols. HDG2 belongs to the class IV
HD-ZIP family. The members in this class have been shown to partici-
Nitab4.5_0002740g0110
Nitab4.5_0001256g0060
Nitab4.5_0012950g0010
Nitab4.5_0000101g0270
Nitab4.5_0005320g0020
Nitab4.5_0001574g0010
Nitab4.5_0000403g0080
Nitab4.5_0016068g0010
Nitab4.5_0012974g0030
Nitab4.5_0003061g0020
Nitab4.5_0000116g0020
Nitab4.5_0002898g0020
Nitab4.5_0009344g0030

pate in root hairless cell specification (Kubo et al., 1999; Ohashi et al.,
2003), trichome differentiation (Ohashi et al., 2002), anthocyanin
biosynthesis (Kubo et al., 1999; Wang et al., 2015), seed oil accumu-
lation (Shi et al., 2012), flowering time (Wei et al., 2016), embryo
development and SAM maintenance (Abe et al., 2003; Iida et al., 2019).
Table 1

Gene

Previous study revealed that HDG2 works redundantly with PDF2 in

regulating epidermal and flower development (Kamata et al., 2013).

264
Z. Wang, et al.
Fig. 4. Relative expression levels of flavonoids-related DEGs revealed by RNA-seq in the WT, NtHDG2 OE lines, and RNAi lines. The expression levels were measured
by qRT-PCR, and normalized to the expression of the internal reference gene, NtL25. Values are the means of three replicates, and the error bars indicate standard
deviations. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student's t-test (**, P < 0.01;
*, P < 0.05).
However, the role of HDG2 in regulation of flavonoids accumulation is
not well understood. In the present study, we demonstrated that
NtHDG2 increase flavonols accumulation in tobacco leaves via pro-
moting the transcription of key genes in flavonoids pathway.
The NtHDG2 gene showed highest expression levels in tobacco
flowers and axillary buds, which was consistent with its known func-
tions in regulating meristem and flower development. Tobacco leaves
also had high expression level of NtHDG2. RNA-seq and qRT-PCR
analyses revealed that three structural genes (NtPAL, NtF3′H, NtF3GT)
and one regulatory gene (NtMYB12) were considerably up-regulated in
the OE lines, but significantly down-regulated in the RNAi lines
(Table 1, Fig. 4, Supplementary Table S3). PAL directs the carbon flow
from the shikimate pathway to the various branches of the general
phenylpropanoid pathway (Vogt, 2010). F3′H converts dihy-
drokaempferol into dihydroquercetin, while DFR catalyzes the common
265
Plant Physiology and Biochemistry 146 (2020) 259–268
Fig. 5. ChIP enrichment test showing the
binding of NtHDG2-FLAG to the NtF3′H and
NtPAL promoters. (A) Distribution of the
conserved binding element and the detecting
primer position in the promoter regions of
NtMYB12, NtF3′H, NtF3GT, and NtPAL genes.
(B) Enrichment of the fragments containing
the conserved binding element. Asterisks re-
present statistically significant differences
between target gene and control gene de-
termined by two-tailed paired Student's t-test
(**, P < 0.01).
stem toward anthocyanins and PAs (Routaboul et al., 2012). F3GT
catalyzes the glucosylation of both cyanidin and flavonols at the 3-
position, thereby promoting the accumulation of anthocyanin and fla-
vonols (Tohge et al., 2005). MYB12 has been shown to promote fla-
vonoids accumulation in several plant species by activating the early
biosynthesis genes (EBGs) in the pathway, including CHS, CHI, F3H,
and FLS (Wang et al., 2016, 2017; Pandey et al., 2015). Based on these
information, we propose that NtHDG2 elevates the expression of
NtMYB12, NtPAL, NtF3′H, and NtF3GT genes, leading to the higher
flavonols accumulation in tobacco leaves. We observed no differences
among the anthocyanin contents of WT, OE, and RNAi plants. This
might be caused by different regulation of the NtDFR expression levels
(Table 1, Fig. 4, Supplementary Table S3). There was no difference in
the transcript levels of NtCHS, NtCHI, NtF3H, and NtFLS genes in the OE
lines compared with the WT plants (Supplementary Table S3). These
Z. Wang, et al.
genes supposed to be regulated by NtMYB12, are likely regulated by
unknown regulatory genes or repressed by NtHDG2, but the underlying
regulatory mechanism has yet to be determined.
We further found that NtHDG2 could bind to the conserved element
of NtF3′H and NtPAL gene by ChIP assay, suggesting a direct regulation
of NtHDG2 on these two genes. The enrichment of NtMYB12 and
NtF3GT fragments were not detected in our analysis, but we can't rule
out the possibility that NtHDG2 directly regulates these two genes. In
the present study, we only searched the conserved elements within the
3 kb sequences upstream the start code of target genes, but there might
be more conserved elements upstream the 3 kb sequences or within the
genes. Moreover, the conserved element was identified in other plant
species. The NtHDG2 might have different binding element in tobacco.
Therefore, more studies need to be done to reveal the molecular me-
chanism of NtHDG2 in regulating flavonols biosynthesis.
Initially, it's confusing for us that a known transcription factor in-
volved in meristem and embryo development, participate in regulating
flavonols biosynthesis in tobacco. Then we noticed a close relationship
between these two traits. It was reported that the accumulation of di-
hydroflavonols in seed coat inhibited embryo growth in Petunia hybrid
(Richard et al., 2002). However, the reduction of flavonols content in
Norway spruce overexpressed a PaNAC03 gene also showed aberrant
embryo development (Dalman et al., 2017). The flavonols have been
linked to auxin homeostasis and polar auxin transport in plants (Buer
et al., 2013). Therefore, it seems possible that appropriate flavonols
content is essential for plant embryonic development, and HDG2 might
promote the normal development of plant embryos by regulating the
synthesis of flavonols. In the NtHDG2 OE plants,NtHDG2 induces the
transcription of NtMYB12 and NtF3′H gene, but represses the NtDFR
expression level, leading to the accumulation of flavonols (Figs. 3 and 4,
Table 1). However, no obvious embryonic defect was found in the OE
lines, which contained significant more flavonols than WT plants. This
might be due to the up-regulation of NtNAC002 gene in the OE lines
(Supplementary Table S2, Fig. S2). NtNAC002 is the closest homolog of
AtAF1 (At1G01720, ANAC002) in tobacco, while AtAF1 is the closest
homolog of PaNAC03 (Dalman et al., 2017). Overexpression of
PaNAC03 repressed the expression of PaHB2 (homologous genes of
NtHDG2) and flavonols accumulation in Norway spruce. Based on this
information, we propose that ectopic expression of NtHDG2 lead to
higher content of flavonols in tobacco, which might be harmful for
plant development. Meanwhile, the transcription of NtNAC002 is in-
duced by NtHDG2, and NtNAC002 can feedback inhibit NtHDG2 gene
266
Plant Physiology and Biochemistry 146 (2020) 259–268
Fig. 6. NtHDG2 promoted the tran-
scription of several AP2/ERF genes. (A)
GO annotation of the DEGs between WT
and OE plants revealed by RNA-seq. (B)
Relative expression levels of the ERF
genes in the individual transgenic line.
The expression levels were measured by
qRT-PCR, and normalized to the ex-
pression of the internal reference gene,
NtL25. Values are the means of three
replicates, and the error bars indicate
standard deviations. Asterisks represent
statistically significant differences be-
tween WT and transgenic plants de-
termined by two-tailed paired Student's
t-test (**, P < 0.01; *, P < 0.05).
expression and flavonols. These interesting deductions need to be fur-
ther investigated.
Ectopic expression of NtHDG2 gene promoted the transcription of
several AP2/ERF genes, including NtERF1-5, NtERF109, NtDREB1, and
NtCIPK11. ERF1 has been shown to affect primary root elongation by
mediating the cross-talk between auxin and ethylene biosynthesis in
Arabidopsis (Mao et al., 2016). ERF3 regulates the genes involved in
cytokinin signaling, and then promotes crown root development in rice
(Zhao et al., 2015). ERF5 enhances plant adaptation to drought and salt
tolerance in tomato (Pan et al., 2012), while DREB1 is involved in
regulating heat, drought, and cold stress-responsive gene expression in
soybean (Yamasaki and Randall, 2016; Kidokoro et al., 2015). All these
suggest the multifunction of NtHDG2 in root development and re-
sistance to various biotic stresses in tobacco.
In summary, we conclude that a HD ZIP IV gene, NtHDG2, promotes
flavonols accumulation in tobacco leaves by controlling the expression
of one regulatory gene (NtMYB12) and three structural genes (NtPAL,
NtF3′H, NtF3GT) involved in flavonoids biosynthesis (Fig. 4 and
Table 1). In addition, we found that NtHDG2 proteins activated the
expression of several AP2/ERF genes, indicating an important role of
NtHDG2 in regulating plant root development and resisting abiotic
stresses.
Contributions
Zhong Wang and Jun Yang conceived the project and designed the
experiment plans; Zhong Wang, Shanshan Wang, Yansong Xiao, Zefeng
Li, Mingzhu, Wu, Xiaodong Xie, Hongguang Li, Wenjun Mu, Feng Li,
Pingping Liu, and Ran Wang conducted the experiments; Zhong Wang,
Zefeng Li, and Jun Yang analyzed the data and wrote the article.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by Tobacco Genome Projects of CNTC
(Grant No. 110201901020 (JY-07)), Presidential Foundation of ZTRI
(Grant No. 902017CA0220), and National Science Foundation for
Z. Wang, et al.
Young Scientists of China (Grant No. 31801770 and No. 31701347).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.plaphy.2019.11.033.
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