Plant Science 291 (2020) 110326

Contents lists available at ScienceDirect

Plant Science
journal homepage: www.elsevier.com/locate/plantsci

The salt-induced transcription factor GmMYB84 confers salinity tolerance in T

soybean
Wenxiao Zhang1, Nan Wang1, Jingting Yang, Hui Guo, Zhenhua Liu, Xiaojian Zheng, Shuo Li*,
Fengning Xiang*
The Key Laboratory of the Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao,
Shandong, 266237, China

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

Keywords: Transcription factor activation and DNA methylation are important plant responses to abiotic stress. Here, we
GmMYB84 established that the salinity stress-induced expression of the soybean (Glycine max) transcription factor-encoding
Salinity stress gene GmMYB84 relies on DNA methylation. The level of DNA methylation at sequences 690 nt to 950 nt up-
DNA methylation stream of the GmMYB84 transcription initiation codon was markedly reduced in plants exposed to salinity stress,
Soybean
resulting in a higher abundance of transcripts. When challenged with salinity stress, plants constitutively ex-
pressing GmMYB84 outperformed untransformed plants with respect to their germination rate, primary root
elongation, proline accumulation, antioxidant enzyme activity, membrane integrity, and K+ levels. Arabidopsis
thaliana plants heterologously expressing GmMYB84 were more tolerant to salt stress and exhibited higher
germination rates than the wild type. Electrophoretic mobility shift assays revealed that GmMYB84 binds to the
cis-regulatory sequences of GmAKT1, the homolog of ARABIDOPSIS K+ TRANSPORTER 1 (AKT1). Thus, DNA
methylation modulates the salinity stress-induced expression of the soybean transcription factor-encoding gene
GmMYB84 and thereby confers salinity stress tolerance.

1. Introduction elucidated how plant roots perceive and respond to salinity in Arabi-
dopsis thaliana [9], rice [10–12], and soybean [13–15].
Soil salinity is a severe and widespread stress limiting crop pro- ARABIDOPSIS K+ TRANSPORTER 1 (AKT1), the inward-rectifying
+
ductivity. It disturbs the osmotic status of plants, represents a source of K channel, plays a crucial role in maintaining the acquisition and
toxic ions, and induces nutritional imbalances [1]. Enhancing our un- homeostasis of K+ levels under saline conditions. In Arabidopsis
derstanding of how plants perceive and respond to salinity stress will thaliana, the AKT1 potassium channel constitutes an important pathway
guide efforts to breed plants with greater salt tolerance; therefore, much for K+ acquisition in the root cells [16]. In ZxAKT1-silenced plants, the
research has focused on identifying the genetic basis of this stress re- expression levels of genes encoding several transporters/channels re-
sponse [2]. Many genes are regulated by transcription factors (TFs), lated to K+/Na+ homeostasis were reduced [17]. AKT1 expression is
including those that are stress-inducible [3]. Plant TFs are classified regulated by TFs, such as OrbHLH001 in the wild rice Oryza rufipogon,
into a number of families based on the motifs present in their amino which activates OrAKT expression and thereby controls the K+/Na+
acid sequences [4]. One of the largest TF families comprises the MYB ratio [18]. The CBL (calcineurin B-like calcium sensors)–CIPK (CBL-
proteins, many of which participate in the salinity stress response [5,6]. interacting protein kinases) – PP2C-type phosphatase network con-
Roots are critical plant organs because of their function in the up- stitutes a mechanism that controls the activity of the AKT1 channel
take of water and dissolved nutrients. Root structures have been shown [19]; however, the relationship between the MYB TFs and AKT1 in
to undergo biological changes that protect the root against salt stress plants remains elusive.
[7]; for example, the increased number of apoplastic barriers in the Epigenetic factors, notably histone modification and DNA methy-
roots of plants challenged with high salinity may be associated with a lation, can also regulate gene expression [20,21]. Particularly, DNA
reduced Na+ uptake and enhanced survival [8]. Parallel studies have methylation responds to various environmental stresses [22]. Previous

⁎
Corresponding authors at: The Key Laboratory of the Plant Cell Engineering and Germplasm Innovation, School of Life Sciences, Shandong University, Qingdao,
266237, Shandong, China.
E-mail addresses: [email protected] (S. Li), [email protected] (F. Xiang).
1
Contributed equally.

https://doi.org/10.1016/j.plantsci.2019.110326
Received 16 June 2019; Received in revised form 19 October 2019; Accepted 21 October 2019
Available online 05 November 2019
0168-9452/ © 2019 Elsevier B.V. All rights reserved.
W. Zhang, et al. Plant Science 291 (2020) 110326

studies have demonstrated that DNA methylation and/or histone reference sequence was AtTUB (TUBULIN BETA-5 CHAIN)
modifications play critical roles in activating or repressing genes en- (AT1G20010). All relevant primer sequences are provided in Table S3.
coding various TFs affecting salinity stress tolerance in soybean [23].
Although salinity stress alters the DNA methylation levels of the cis- 2.3. DNA methylation analysis
regulatory sequences of some TFs [24,25], the effect of this methylation
on the expression levels of many TFs is yet to be elucidated. Genomic DNA was isolated from the leaves of three-week-old
Soybean is a major source of plant oil and protein [26]; however, its seedlings using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany).
productivity is severely compromised by salinity stress. The soybean Bisulfite sequencing was carried out using a template of 80–100 ng DNA
genome harbors at least 244 MYB genes [27], few of which have been treated with sodium bisulfite, which was purified using a BisulFlash
functionally characterized. GmMYB84 encodes a MYB TF that influ- DNA Modification kit (EpiGentek, Farmingdale, NY, USA). The con-
ences plant responses to drought stress [28]. Here, we report that its version efficiency was > 98% for each bisulphite-treated sample. The
expression is induced by salinity stress in a DNA demethylation-de- DNA was PCR-amplified based on the EasyTaq enzyme (TransGen
pendent manner, and that it functions as a positive regulator of salinity Biotech Co. Ltd., Beijing, China). Amplicons were inserted into the
stress tolerance in soybean. pEASY-T5 plasmid (TransGen Biotech Co. Ltd.) for sequencing. At least
30 clones were sequenced per sample. All relevant primer sequences are
2. Materials and methods provided in Table S3.

2.1. Plant materials and growth conditions 2.4. Proline and soluble sugar content measurements

The soybean (Glycine max) cultivars Shengdou No. 9 and Wei6823 The proline content of the leaf was measured as previously de-
were used for the gene cloning and phenotypic assays, respectively. For scribed [29], and that of the soluble sugars was determined following
the root growth analysis, the soybean seeds were sown on moistened another previously published protocol [30].
filter paper and incubated for 2 d at 25 °C. Seedlings of a uniform size
were transferred to a 16-h photoperiod regime (light/dark temperature 2.5. Characterization of the GmMYB84 promoter
28 °C/20 °C) under 950 μmol m–2 s–1 illumination and a relative hu-
midity of 60%, and were grown hydroponically in half-strength The promoter sequence of GmMYB84 represented in the cultivar
Hoagland solution. Williams 82 was acquired from the Phytozome v12.1 database (http://
For the soybean seed germination assays, abiotic stress was applied phytozome.jgi.doe.gov/pz/portal.html) and was amplified from a
to seeds by adding 150 or 200 mM NaCl or 10 or 20 μM abscisic acid template of soybean genomic DNA using TransStart FastPfu DNA
(ABA) to the wet filter paper. Germination (emergence of radicles) was Polymerase (TransGen Biotech Co. Ltd.). A promoter-GUS construct was
scored at the indicated time points. Each independent experiment in- transformed into Agrobacterium tumefaciens strain GV3101, and from
cluded three biological replicates, each containing 45 seeds. there into A. thaliana using the floral dip method. The relevant primer
A. thaliana (ecotype Col-0) represented the WT Arabidopsis genotype sequences are listed in Table S3. The GmMYB84 promoter sequence was
and the genetic background for all the transgenic plants. Mature analyzed in silico using PLACE (www.dna.affrc.go.jp) and PlantCARE
Arabidopsis seeds were stored in a dehumidifier for at least two months (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) soft-
beforegermination. The seeds were laid on agar plates containing 1% ware.
(v/v) sucrose at 4 °C for two days, before being transferred to a cabinet
at 23 ± 1 °C. 2.6. Transcriptional activation activity assay
For the germination assay, at least 100 seeds of each Arabidopsis
genotype were surface-sterilized and sown on a solidified half-strength The GmMYB84 coding sequence was transferred into the Gateway
Murashige and Skoog (MS) medium with or without NaCl (0, 50, 100, pDONR221 vector via the BP reaction (Invitrogen, Carlsbad, CA, USA)
150, 200 mM) or ABA (0, 0.1, 0.2, 0.5, 1 μM). The seeds were inspected and then into the pDEST32 vector (Thermo Fisher Scientific) via the LR
daily for one week, with successful germination being determined by reaction. The resulting constructs were transformed into the yeast strain
the emergence of the radicle tip. The mean germination scores were MaV203. The transformed yeast cells were cultured on yeast nitrogen
based on at least three independent experiments. base plates, either containing or lacking additional Leu, His, and Ade
(Sigma-Aldrich, St. Louis, MO, USA). The protocol recommended by the
2.2. RNA isolation, cDNA synthesis, and qRT-PCR supplier of the ProQuest Two-Hybrid System (Thermo Fisher Scientific)
was followed.
Total RNA was isolated from plants using TRIzol reagent (Thermo
Fisher Scientific, Waltham, MA, USA). Subsequently, an 18-μg aliquot 2.7. Expression of the GmMYB84 protein and EMSA
of total RNA was digested using Turbo DNA-free DNaseI (Thermo Fisher
Scientific) in a 50-μL reaction, following the manufacturer’s instruc- The full-length GmMYB84 protein fused with the GST-tag was ex-
tions, and the RNA concentration was determined using a NanoDrop pressed in the pGEX-4T-2 vector in the Escherichia coli strain BL21. The
spectrometer (Thermo Fisher Scientific). The first cDNA strand was protein extraction and subsequent EMSA were performed as previously
synthesized from a 1-μg aliquot of the DNase-treated total RNA using an described [31].
iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) in
a 20-μL reaction. The cDNA was used as the template to profile the 2.8. GUS activity analysis
transcription of nine stress-inducible genes: AtCLCa (CHLORIDE
CHANNEL a) (AT5G40890), AtAKT1 (AT2G26650), AtABF3 (ABSCISIC The GmMYB84 promoter fragment, including 2000 bp directly up-
ACID RESPONSIVE ELEMENTS-BINDING FACTOR 3) (AT4G34000), stream of the start codon, was amplified from the genomic DNA of
AtABF4 (ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR 4) soybean cultivar Williams 82. The promoter fragment was transferred
(AT3G19290), AtABI5 (ABA INSENSITIVE 5) (AT2G36270), AtSOS1 into the pDONR221 vector via the BP reaction and was then transferred
(ARABIDOPSIS SALT OVERLY SENSITIVE 1) (AT2G01980), AtSOS2 into the pBGWFS7 vector [32] via the LR reaction. This expression
(ARABIDOPSIS SALT OVERLY SENSITIVE 2) (AT5G35410), AtSOS3 vector was introduced into Agrobacterium strain GV3101, which was
(ARABIDOPSIS SALT OVERLY SENSITIVE 3) (AT5G24270), and AtABA3 then transformed into Arabidopsis Col-0 plants using the floral dip
(ABA DEFICIENT 3) (AT1G16540)) using qRT-PCR. The chosen method. The GmMYB84 promoter was used to drive the expression of a

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W. Zhang, et al. Plant Science 291 (2020) 110326

Fig. 1. The effect of exposure to salinity on the activity of the full-length and truncated versions of the GmMYB84 promoter.
(A) The structure of the truncated GmMYB84 promoter-GUS fusion transgenes. (B) The phenotypes of salt-stressed transgenic A. thaliana plants expressing the various
truncated GmMYB84-GUS fusions. Two-week-old seedlings were exposed to 50 mM NaCl for either 2 h or 6 h, and then stained for GUS activity. P: the full 2000-nt
upstream sequence; P1: bases –950 to 0; P2: bases –1300 to –950; P3: bases –690 to 0; P4: bases –950 to –690; and P5: bases –1849 to –1649.

GUS reporter gene in 14-day-old transgenic A. thaliana seedlings. GUS 3.2. Induction of GmMYB84 expression by salinity stress is accompanied by
activity assays were performed as previously described [33]. demethylation of its promoter sequence

An inspection of the GmMYB84 promoter region revealed that it had

3. Results a high level of DNA methylation under normal growth conditions. A
qRT-PCR assay was used to compare the transcript abundance of
3.1. Isolation and characterization of the salinity-induced GmMYB84 GmMYB84 in soybean plants treated with either 0 or 100 μM 5-azacy-
promoter tidine (an inhibitor of DNA methylation), which were then exposed to
150 mM NaCl for 12 h. A ∼2.5-fold increase was observed in the
An in silico analysis of the 2000-nt sequence upstream of the abundance of the GmMYB84 transcripts in the control plants exposed to
GmMYB84 transcription initiation codon resulted in the identification salt stress, while this increase was much more modest in the 5-azacy-
of eight cis-acting elements previously associated with the abiotic stress tidine-treated plants (Fig. S2). Bisulphite sequencing of the GmMYB84
response (Table S2). The various segments of the GmMYB84 promoter promoter region revealed that the level of DNA methylation in the se-
sequence were fused to the glucuronidase (GUS) reporter gene uidA (P: quence 690 nt upstream of the GmMYB84 transcription initiation codon
the full 2000 nt, P1: bases –950 to 0, P2: bases –1300 to –950, P3: bases was markedly reduced when the WT plants were exposed to salinity
–690 to 0, P4: bases –950 to –690, and P5: bases –1849 to –1649), with stress (Fig. S3), with around a 40% reduction in some CHH sites (Fig.
each fragment harboring one or more of the cis-acting elements (Figs. S4).
S1, 1 A). The gene fusions were transformed into Arabidopsis, and the
level of GUS activity was monitored in the leaves and roots following a 3.3. The GmMYB84 protein contains a transcriptional activation domain
treatment with 50 mM NaCl for 2 h or 6 h). The various segments of the
GmMYB84 promoter sequence were more inducible by salinity than was To identify the transcriptional activation region of GmMYB84, the
the WT control. The transgene containing the P1 fragment was the most sequences encoding either the full-length protein, the N-terminal end
strongly induced of all suggesting that it was a key salt-inducible seg- (residues 1–121), the central 100 residues (121–221), or the C-terminal
ment (Fig. 1B). end (221–317) were fused to the GAL4 DNA-binding domain, resulting

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W. Zhang, et al. Plant Science 291 (2020) 110326

E). Real-time quantitative PCR analysis was used to reveal that

GmMYB84, GmAKT1, and GmABA3 (molybdenum cofactor sulfur-
transferase) (Glyma.09G002300) were induced to a higher level in the
GmMYB84-OE lines exposed to a 2-h to 24-h salt treatment than in WT
plants under these conditions (Fig. 5A–C). We also analyzed the effect
of heterologously expressing GmMYB84 on the transcript levels of a set
of nine Arabidopsis genes known to be induced by abiotic stress. The
transcripts of all nine genes were more abundant in the GmMYB84-
expressing transgenic plants than in the non-transformed plants (Fig.
S6A–I). We concluded that GmMYB84 functioned as a positive regulator
of abiotic stress by upregulating these genes.

3.6. Soybean lines overexpressing GmMYB84 accumulate more proline and

soluble sugars, and exhibit an increased level of antioxidant enzyme activity

Fig. 2. The transactivational ability of the full-length and truncated The physiological performance of the WT and GmMYB84-OE soy-
GmMYB84 proteins in yeast. bean lines were compared when subjected to salinity stress. A 24-h
An empty pDEST32 (pBD) vector was used as the negative control and GAL4 was exposure to 200 mM NaCl increased the leaf proline and soluble sugar
used as the positive control. The numbers shown indicate the position and contents of both the WT and the transgenic plants, but the proline
length of the truncated GmMYB84 proteins. content increased more in the GmMYB84-OE plants (Fig. 6A, B). Simi-
larly, the activity levels of three antioxidant enzymes, peroxidase
in the constructs pDEST32-1-317, pDEST32-1-121, pDEST32-121-221, (POD), superoxide dismutase (SOD), and catalase (CAT), were higher in
pDEST32-122-317, and pDEST32-221-317, respectively. These trans- the transgenic plants than in the WT (Fig. 6C–E). Consistently, the
genes were introduced into yeast strain MaV203, and the resulting yeast malondialdehyde (MDA) content of the transgenic leaves was less under
transformants were tested for their ability to transactivate a reporter salt stress than in the WT plants. This indicated that the cell membrane
gene and promote cell growth on specific media. Transformants con- system in transgenic plants was less damaged than in WT plants
taining an empty vector were used as the negative control. All of the (Fig. 6F).
transformants grew well on medium lacking leucine, while those har-
boring either pDEST32-1-317, pDEST32-122-317, or pDEST32-121-221 3.7. The constitutive expression of GmMYB84 lowers Na+ uptake
were able to grow well on the selection medium (Fig. 2). On the other
hand, those harboring either the empty vector, pDEST32-1-121, or Measurements of the Na+ and K+ tissue concentrations in the roots,
pDEST32-221-317 did not grow. This suggests that GmMYB84 contains stems, and leaves revealed that they did not differ between the WT and
a transcriptional activation domain between amino acid residues 121 GmMYB84-overexpressing soybean plants grown in the absence of
and 221. salinity stress. In plants exposed to 150 mM NaCl, the [Na+] was lower
and the [K+]/[Na+] ratio was higher in the transgenic soybean plants
than in the WT, which further demonstrated the higher salt stress tol-
3.4. GmMYB84 overexpression supports germination and root growth in the
erance of transgenic plants compared to WT plants (Fig. 7).
presence of salinity or ABA
4. Discussion
To assess the role of GmMYB84 in ABA and salt stress tolerance, WT
and GmMYB84-OE transgenic soybean seeds were treated with NaCl
Soil salinity affects a broad spectrum of physiological and devel-
and ABA. The GmMYB84-OE transgenic lines had higher seed germi-
opmental events throughout a plant’s lifecycle [34]. It imposes both
nation rates than the WT in the presence of either salinity (100 or
ionic and osmotic stress [35], the former being most prominent during
150 mM NaCl) or ABA (10 or 20 μM) (Fig. 3A, B), suggesting that the
the early period of exposure, while the latter sets in later. ABA is a key
constitutive expression of GmMYB84 alleviated the negative effects of
regulator of the abiotic stress response, as reflected by the rise in en-
salinity and ABA on seed germination. The seed germination pheno-
dogenous ABA levels induced by various stresses [36,37]. The salinity
types of two Arabidopsis lines heterologously expressing the GmMYB84-
stress response involves both ABA-dependent and ABA-independent
OE transgenes were similar to those of the soybean seeds over-
pathways [38]. The soybean gene GmMYB84, a member of one of the
expressing GmMYB84 (Fig. S5).
largest plant TF families [39], was previously shown to be upregulated
We also investigated the salinity tolerance of transgenic soybeans in
by exposure to salinity or exogenous ABA treatments [40]. The con-
root growth. The GmMYB84-OE transgenic soybean lines produced a
stitutive expression of GmMYB84 in soybean and Arabidopsis lessened
longer primary root than the WT after a 10-day growth period in the
the inhibitory effect of both salinity and exogenous ABA on the ability
presence of 150 mM NaCl (Fig. 4A, B). These results indicate that
of seeds to germinate (Figs. 3, S5), suggesting that this TF may act
GmMYB84 improved the salinity stress tolerance of soybean seeds and
through an ABA-dependent signal transduction pathway. A number of
seedlings. 3,3-Diaminobenzidine (DAB) treatment revealed that the
Arabidopsis genes associated with a positive effect on salinity tolerance
H2O2 levels were reduced in the salt stressed transgenic plants com-
were also previously shown to act via an ABA-dependent signaling
pared to WT plants, which suggested that the overexpression of
pathway [41–44], and all of these were found to be upregulated by the
GmMYB84 can reduce the oxidative damage under salt treatment
constitutive expression of GmMYB84 in the present study, even in the
(Fig. 4C, D).
absence of salinity stress (Fig. S6A–I). Some MYB TFs were previously
shown to bind to the MBSI cis-element TAACTG [45,46], which we
3.5. GmMYB84 directly binds to the GmAKT1 promoter revealed was also true for GmMYB84 (Fig. 5D, E). The enhanced salinity
tolerance induced by the overexpression of GmMYB84 may therefore
We next explored whether the salt tolerance of the transgenic plants have resulted from its upregulation of a number of stress response-as-
was associated with ionic stress. An electrophoretic mobility shift assay sociated genes.
(EMSA) indicated that GmMYB84 specifically binds to the MYB binding Several physiological traits, including soluble sugar and proline
site I (MBSI) in the GmAKT1 (Glyma.05G010600) promoter (Fig. 5D, tissue contents, the activity of certain antioxidant enzymes, the

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W. Zhang, et al. Plant Science 291 (2020) 110326

Fig. 3. The germination of soybean seeds constitutively expressing GmMYB84 was less affected by exposure to salinity or an ABA treatment than the wild-
type (WT) seeds.
(A) The effect of salinity on the germination of the WT and two independent transgenic lines (GmMYB84 OE-1 and OE-2). (B) The effect of exogenous ABA on
germination. Three biological replicates were performed, each consisting of 45 seeds. Error bars represent the mean ± SE (n = 3 biological repeats). *: significant
difference (Student’s t-test *** P < 0.001, ** P < 0.01 and * P < 0.1) between the WT and GmMYB84 OE plants.

accumulation of MDA, and the [K+]/[Na+] ratio in the tissues, were plants is the restriction of Na+ accumulation in the cytoplasm
previously proposed as indicators diagnostics of stress tolerance [47]. [45,51,52]. The measurement of [Na+] in the WT and GmMYB84-
The leaf proline content, used by plants for the purpose of osmotic overexpressing plants showed that, when challenged with 150 mM
adjustment [48], was higher in the GmMYB84-OE plants than in the WT NaCl, the former were less able to limit the accumulation of this ion,
when exposed to salinity stress (Fig. 6B). POD, SOD, and CAT activity resulting in a higher [K+]/[Na+] ratio in the transgenic plants (Fig. 7).
were higher in the salinity-challenged GmMYB84-OE plants than in the Gene promoters are responsible for the regulation of gene expres-
WT (Fig. 6C–E). Similarly, a lower MDA content (indicative of a less sion; therefore, their activity is key for understanding gene function.
severe level of damage to membranes) was observed in the transgenic Typical promoters harbor various cis-acting elements that interact with
plants than in the WT (Fig. 6F). Apart from its negative effect on the TFs to trigger or suppress transcription [53]. According to an in silico-
plant’s osmotic status and its cytotoxicity, Na+ uptake results in com- based analysis, the GmMYB84 promoter sequence harbors eight puta-
petition at protein sites that rely on the binding of K+ to be metabo- tive CAREs (Cis-Acting Regulatory Element) (Figs. S1, 1 A). When the
lically active [49,50]. Thus, a major salt tolerance strategy adopted by activities of various truncated versions of the GmMYB84 promoter were

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W. Zhang, et al. Plant Science 291 (2020) 110326

Fig. 4. Increased salt tolerance of

GmMYB84-OE plants.
(A) Analysis of the salt stress response in wild-
type (WT) and GmMYB84-OE plants. The WT
and OE lines were treated with 0 mM or
150 mM NaCl for 10 days after the second leaf
stage. Scale bar =5 cm. (B) The primary root
length of the plants in (A) All data are given as
mean ± SE (n = 30). *: significant difference
(Student’s t-test *** P < 0.001, ** P < 0.01,
and * P < 0.1) between the WT and GmMYB84
OE plants. (C, D) 3,3-Diaminobenzidine (DAB)
staining in the leaves and primary root tips of
15-day-old GmMYB84-OE and WT seedlings
treated with either 0 mM (control) or 150 mM
NaCl for 10 days. The strength of the color in-
dicates the concentration of H2O2 in the leaves
(scale bar =3 mm) and roots (scale bar
=1 mm).

analyzed in Arabidopsis plants subjected to salinity stress, it was found [23]. Here, the level of DNA methylation at sequences 690 nt upstream
that the first 950 nt upstream of the transcription initiation codon were of the GmMYB84 transcription initiation codon was markedly reduced
the most important for its activation (Fig. 1B). by salinity stress (Fig. S4), while the activity of the P1 promoter frag-
The regulation of gene expression by DNA methylation is an im- ment containing this 690-nt sequence was induced under salinity stress
portant pathway in the abiotic stress response in plants [54]. De novo (Figs. S1, 1), suggesting that methylation is negatively correlated with
DNA methylation is mediated by methyltransferase [55,56], which GmMYB84 expression in this stress response. Notably, it remains un-
shows significant site specificity and spatiotemporal dynamics [57,58]. clear whether the demethylation in the MYB promoter enhances salinity
It was previously reported that DNA demethylation may play a key role tolerance. We found that salinity stress induced cytosine demethylation
in inducing MYB expression in response to salinity stress in soybean in the GmMYB84 promoter (Fig. S4), and that the induction of

Fig. 5. Transcription profiling of

GmMYB84, GmAKT1, and GmABA3
in the wild-type (WT) and
GmMYB84-OE plants using a qRT-
PCR assay, along with an EMSA of
the binding of GmMYB84 to the
GmAKT1 promoter.
(A, B, C) Expression of GmMYB84,
GmAKT1, and GmABA3 analyzed using
qRT-PCR after treatment with 200 mM
NaCl for 2, 6, 12, and 24 h. The data
represent the average of three biolo-
gical replicates, with 20 plants per re-
plicate. Error bars represent standard
deviations. (D, E) EMSA demonstrating
the binding of GmMYB84 to the MBSI
sites in the GmAKT1 promoter. (D) The
position of the MBSI cis-elements in the
GmAKT1 promoter is shown by red
lines, the 5′-UTR is indicated by an
empty box, and the ATG transcription
initiation codon is shown as a filled
box. (E) EMSA profiles. Lane 1: free
probe (labeled probe with no protein
added); lane 2: labeled probe with GST
protein; lane 3: labeled probe with
GmMYB84-GST protein; lanes 4 and 5:
GmMYB84-GST binding to the labeled
probe competing with either 50× or
200× unlabeled WT probe, respec-
tively; and lanes 6 and 7: GmMYB84-
GST binding to the labeled probe com-
peting with either 50× or 200× un-
labeled mutated probe sequence, re-
spectively. Arrows indicate
protein–probe complexes.

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W. Zhang, et al. Plant Science 291 (2020) 110326

Fig. 6. The effect of abiotic stress on

the accumulation of soluble sugars
and proline, the activity of known
antioxidant enzymes, and the MDA
content of wild-type (WT) and
GmMYB84-OE soybean.
The leaf contents of (A) soluble sugars
and (B) proline were measured fol-
lowing the exposure of the plants to
200 mM NaCl for 24 h. The activity of
(C) POD, (D) SOD, and (E) CAT fol-
lowing the exposure of the plants to
200 mM NaCl for 24 h. (F) The leaf
MDA content following the exposure of
the plants to 200 mM NaCl for 24 h.
Data represent the mean ± SE (n = 3).
**, *: significant difference (P < 0.01
and < 0.05, respectively) between the
WT and the GmMYB84-OE plants. FW:
fresh weight.

Fig. 7. GmMYB84-OE transgenic soybean lines have limited Na+ uptake when exposed to salinity stress.
The (A, D, G) [K+], (B, E, H) [Na+], and (C, F, I) [K+]/[Na+] ratios were measured in the (A–C) roots, (C–F) stems, and (G–I) leaves of the wild-type (WT) and
GmMYB84-OE-1 transgenic plants grown in the presence of either 0 mM or 150 mM NaCl. DW: dry weight. Data represent the mean ± SE (n = 3). *: significant
difference (t-test; *** P < 0.001, ** P < 0.01 and * P < 0.1) between the WT and GmMYB84-OE-1 plants.

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W. Zhang, et al. Plant Science 291 (2020) 110326

GmMYB84 expression in response to NaCl was eliminated in the pre- [16] F. Rubio, F. Alemán, M. Nievescordones, V. Martínez, Studies on Arabidopsis
sence of a methylation inhibitor (Fig. S2), suggesting that a reversible athak5, atakt1 double mutants disclose the range of concentrations at which
AtHAK5, AtAKT1 and unknown systems mediate K+ uptake, Physiol. Plant. 139
epigenetic modification contributed to the salinity response of (2010) 220–228.
GmMYB84. Moreover, overexpression of GmMYB84 improved the salt [17] Q. Ma, J. Hu, X.R. Zhou, H.J. Yuan, T. Kumar, S. Luan, S.M. Wang, ZxAKT1 is
tolerance of soybean (Fig. 4). These results indicate that salinity stress essential for K(+) uptake and K(+) /Na(+) homeostasis in the succulent xerophyte
Zygophyllum xanthoxylum, Plant J. 90 (2017) 48–60.
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X.F.N. and L.S. supervised the experiments; Z.W.X. and W.N. per-
[21] R. Xu, Y. Wang, H. Zheng, W. Lu, C. Wu, J. Huang, K. Yan, G. Yang, C. Zheng, Salt-
formed most of the experiments; X.F.N., L.S., and L.Z.H. provided induced transcription factor MYB74 is regulated by the RNA-directed DNA me-
technical assistance to Z.W.X., W.N., Y.J.T., G.H. and Z.X.J. performed thylation pathway in Arabidopsis, J. Exp. Bot. 66 (2015) 5997–6008.
part of the experiments. X.F.N. and L.S. supervised and complemented [22] V. Chinnusamy, J.K. Zhu, Epigenetic regulation of stress responses in plants, Curr.
Opin. Plant Biol. 12 (2009) 133–139.
the writing. [23] Y. Song, D. Ji, S. Li, P. Wang, Q. Li, F. Xiang, The dynamic changes of DNA me-
thylation and histone modifications of salt responsive transcription factor genes in
Funding soybean, PLoS One 7 (2012) e41274.
[24] M. Wang, L. Qin, C. Xie, W. Li, J. Yuan, L. Kong, W. Yu, G. Xia, S. Liu, Induced and
constitutive DNA methylation in a salinity-tolerant wheat introgression line, Plant
This research was supported by the National Transgenic Project of Cell Physiol. 55 (2014) 1354–1365.
China (grant no. 2018ZX08009-14B, 2016ZX08010002-009), the Major [25] L.J. Ferreira, V. Azevedo, J. Maroco, M.M. Oliveira, A.P. Santos, Salt tolerant and
sensitive rice varieties display differential methylome flexibility under salt stress,
Program of Shandong Province Natural Science Foundation (grant no. PLoS One 10 (2015) e0124060.
ZR2018ZC0334), the National Key Research and Development Program [26] Y. Liao, H.F. Zou, H.W. Wang, W.K. Zhang, B. Ma, J.S. Zhang, S.Y. Chen, Soybean
of China (grant no. 2016YFD0101902), the National Natural Science GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic
Arabidopsis plants, Cell Res. 18 (2008) 1047–1060.
Foundation (grant nos. 31770317, 31471515, 31270328, 31500232). [27] X.W. Li, Y. Wang, F. Yan, J.W. Li, Y. Zhao, X. Zhao, Y. Zhai, Q.Y. Wang,
Overexpression of soybean R2R3-MYB transcription factor, GmMYB12B2, and tol-
Declaration of Competing Interest erance to UV radiation and salt stress in transgenic Arabidopsis, Genet. Mol. Res. 15
(2016).
[28] N. Wang, W. Zhang, M. Qin, S. Li, M. Qiao, Z. Liu, F. Xiang, Drought tolerance
The authors declare no conflicts of interest. conferred in soybean (Glycine max. L) by GmMYB84, a novel R2R3-MYB tran-
scription factor, Plant Cell Physiol. 58 (2017) 1764–1776.
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