ORIGINAL PAPER

Overexpression of a maize WRKY58 gene enhances droughtand salt tolerance in transgenic rice

Ronghao Cai • Yang Zhao • Yufu Wang • Yongxiang Lin • Xiaojian Peng •

Qian Li • Yuwei Chang • Haiyang Jiang • Yan Xiang • Beijiu Cheng

Received: 28 March 2014 / Accepted: 24 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract WRKY transcription factors (TFs) are reported

to play crucial roles in the processes of plant growth and

development, defense regulation and stress responses. In

this study, a WRKY group IId TF, designated ZmWRKY58,

was isolated from maize (Zea mays L.). Expression pattern

analysis revealed that ZmWRKY58 was induced by drought,

salt and abscisic acid treatments. Subcellular localization

experiments in onion epidermal cells showed the presence

of ZmWRKY58 in the nucleus. Overexpression of

ZmWRKY58 in rice resulted in delayed germination and

inhibited post-germination development. Further investi-

gation showed that ZmWRKY58 overexpressing transgenic

plants had higher survival rates and relative water contents,

but lower malonaldehyde contents and relative electrical

leakage compared with wild-type plants, following drought

and salt stress treatments, suggesting that overexpression of

ZmWRKY58 leads to enhanced tolerance to drought and

salt stresses in transgenic rice. Additionally, yeast two-

hybrid assay showed that ZmWRKY58 could interact with

ZmCaM2, suggesting that ZmWRKY58 may function as a

calmodulin binding protein. Taken together, these results

suggest that ZmWRKY58 may act as a positive regulator

involved in the drought and salt stress responses.

Keywords ZmWRKY58 � WRKY � Transgenic rice �Drought and salt stresses

Introduction

Drought and high salinity are two major abiotic stresses

that can adversely affect plant growth, development and

productivity. During the course of evolution, plants have

established complex mechanisms to adapt to various

adverse environments (Shinozaki and Yamaguchi-Shino-

zaki 2000). Plant stress responses are controlled by a series

of genes via intricate signal transduction networks. In these

regulatory processes, transcription factors (TFs) play

important roles in plant stress responses by regulating the

expression of their target genes through interactions with

the specific cis-acting elements present in the promoters of

these genes (Tran et al. 2004; Yamaguchi-Shinozaki and

Shinozaki 2005). Functional investigations of some TFs

such as ZmCBF3 (Xu et al. 2011), AtbZIP1 (Sun et al.

2012), ONAC045 (Zheng et al. 2009), and AtWRKY57

(Jiang et al. 2012) have demonstrated that transgenic plants

overexpressing these genes exhibited significantly stress

tolenrance.

WRKY gene family, one of the largest TF families, has

been found in numerous species, especially for higher

plants (Ulker and Somssich 2004). In the decade since the

first WRKY protein was cloned from sweet potato (Ishig-

uro and Nakamura 1994), a large number of WRKY TFs

have been reported in Arabidopsis (74) (Ulker and

Somssich 2004), rice ([100) (Ross et al. 2007), Brachyp-

odium distachyon (86) (Tripathi et al. 2012), barley ([45)

(Mangelsen et al. 2008), maize (136) (Wei et al. 2012),

soybean (197) (Schmutz et al. 2010), tomato (81) (Huang

et al. 2012), sorghum (68) (Pandey and Somssich 2009)

Ronghao Cai and Yang Zhao have contributed equally to this work.

R. Cai � Y. Zhao � Y. Wang � Y. Lin � X. Peng � Q. Li �Y. Chang � H. Jiang � Y. Xiang (&) � B. Cheng (&)

Key Laboratory of Crop Biology of Anhui Province, School of

Life Sciences, Anhui Agricultural University, Hefei 230036,

China

e-mail: xiangyanahau@sina.com

B. Cheng

e-mail: bjchengahau@163.com

123

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-014-0556-7

and Cucumis sativus (55) (Ling et al. 2011). Members in

this family are characterized by their highly conserved

DNA-binding region, known as the WRKY domain. This

domain consists of approximately 60 amino acid residues

with a conserved heptapeptide WRKYGQK motif at the

N-terminus as well as a novel C2H2 or C2HC zinc finger-

like motif at the C-terminus (Eulgem et al. 2000). Both of

these conserved elements are essential for the high binding

affinity of WRKY TFs to the W-box elements (C/TTG-

ACT/C) located in the promoter regions of their target

genes (Pandey and Somssich 2009). According to the

number of WRKY domains and the structure of their zinc

fingers, WRKY proteins can be divided into three groups

(I, II and III), and group II can be further divided into five

distinct subgroups (IIa to IIe) based on the primary amino

acid sequence (Eulgem et al. 2000).

It has been well demonstrated that WRKY proteins are

involved in plant responses to various biotic stresses (Dong

et al. 2003; Hu et al. 2012; Jing et al. 2009; Peng et al.

2012; Ren et al. 2010) as well as many developmental

processes such as trichome and seed coat development

(Johnson et al. 2002), embryo morphogenesis (Lagace and

Matton 2004), leaf senescence (Robatzek and Somssich

2002), dormancy (Pnueli et al. 2002), some biosynthetic

pathways and hormone signaling (Song et al. 2010; Sun

et al. 2003; Zhang et al. 2004). Currently, increasing

researchers have focused on the functional analysis of

WRKY proteins in plant responses to abiotic stress. For

example, GsWRKY20, a WRKY gene isolated from Gly-

cine soja, was found to be a positive regulator of drought

adaptation in transgenic Arabidopsis (Luo et al. 2013).

Constitutive expression of barley HvWRKY38 resulted in

increasing tolerance to drought in Paspalum notatum Flu-

gge (Xiong et al. 2010). TcWRKY53 from Thlaspi cae-

rulescens acts as a negative regulator in the osmotic stress

tolerance of transgenic tobacco (Wei et al. 2008). In

Arabidopsis, AtWRKY22 participates in dark-induced leaf

senescence (Zhou et al. 2011). Male gametophyte-specific

AtWRKY34 mediates the cold sensitivity of mature pollen

(Zou et al. 2010). In addition, WRKY25, WRKY26, and

WRKY33 positively regulate the cooperation between the

ethylene-activated and heat shock proteins-related signal-

ing pathways that function in plant response to heat stress

(Li et al. 2011b).

Based on the information offered by previous studies,

we noted that a maize WRKY gene appeared to be highly

expressed among all tissues (Sekhon et al. 2011; Wei et al.

2012). Microarray data showed that this gene was induced

under drought treatment (Zheng et al. 2010). Further

expression pattern analysis revealed that expression of this

gene was significantly up-regulated under various abiotic

stresses, including PEG, salt and abscisic acid (ABA). To

further understand the biological function, we isolated the

novel maize WRKY gene and designated it as ZmWRKY58.

Abiotic stress tolerance assays indicated that overexpres-

sion of ZmWRKY58 in rice significantly increased tolerance

to drought and salt stresses. These results provide a primary

role and regulator mechanism for ZmWRKY58 in the

response to abiotic stress, and also provides a candidate

gene which is potential useful for engineering stress tol-

erant crops.

Materials and methods

Plant materials, growth conditions and stress treatments

Seeds of the maize inbred line B73 were sown in pots

containing mixed soil (soil/vermiculite/perlite, 4:1:1, v/v/v)

in a greenhouse and grown under a 16 h light/8 h dark

cycle at 28 �C. Seedlings at the three-leaf stage were

exposed to various stress treatments. For ABA treatments,

the leaves of developed seedlings were sprayed with

100 lM ABA. For salt and drought treatments, the seed-

lings were irrigated with solutions containing 200 mM

NaCl or 20 % PEG-6000, respectively. Leaves were col-

lected at 0, 12, 24 and 48 h after treatment, immediately

frozen into liquid nitrogen and stored at -80 �C for RNA

extraction.

To study the spatial expression of ZmWRKY58 at dif-

ferent developmental stages in maize, the roots, stalks and

leaves of seedlings at the three-leaf stage, tassels at the pre-

flowering stage and silks at the flowering stage were

sampled for RNA isolation.

Expression patterns of ZmWRKY58 in maize

To determine the expression patterns of ZmWRKY58, semi-

quantitative RT-PCR was performed. The primers DF 50-AGGAAGTGGAGGAGGCGAACA-30 and DR 50-GGAT

GGCTTGCGCTTGC-30 were designed to detect the

expression of ZmWRKY58 under different stress treatments.

The maize GAPDH gene was used as an internal control

and amplified by primers GF 50-CAACGACCCCTTCATCA

CCAC-30 and GR 50-ATACTCAGCGCCAGCCTCACC-30.The PCR thermal cycling conditions were as follows:

denaturation at 95 �C for 5 min followed by 30 cycles of

94 �C for 30 s, 60 �C for 30 s, 72 �C for 90 s and a final

extension at 72 �C for 10 min. The products of PCR were

photographed after electrophoresis on 1.0 % agarose gel

and the band densities were quantified by image densi-

tometry using image analysis software (Eastman Kodak

Co., Rochester, NY, USA). The sample quantities were

adjusted to make the band density in each GAPDH group

stay at a relatively identical level and then the ratios of

ZmWRKY58 to GAPDH between groups were compared.

Plant Cell Tiss Organ Cult

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All experiments were carried out with two biological

repeats and three technical trials.

Isolation of ZmWRKY58 and sequence analysis

Total RNA was extracted from maize leaves at the three-

leaf stage using Trizol Reagents (TianGen, Beijing, China)

according to the manufacturer’s instructions. The RNA was

sequentially treated with DNase I (TaKaRa) to eliminate

genomic DNA contamination. Then, cDNA was synthe-

sized using AMV Reverse Transcriptase (Promega, USA).

According to the clone sequence (GenBank Accession

Number: BT083614.1), the full-length cDNA of

ZmWRKY58 was amplified by PCR with gene-specific

primers CL-F 50-AGATGAGGAAGTGGAGGAGGCGAA

CAG-30 and CL-R 50- CACCTGTGCTGCTGCTGCT

GCTGACT-30. The PCR product was purified and cloned

into the pMD18-T vector (TaKaRa) for sequencing.

Database searches were performed by NCBI/Blast.

Nucleotide and amino acid sequences were analyzed using

DNAMAN software. MEGA 4.0 was used to construct a

phylogenetic tree of ZmWRKY58 and other WRKY

members.

Subcellular localization of ZmWRKY58

The full-length open reading frame (ORF) of ZmWRKY58

without the termination codon was amplified by PCR using

specific primers RH-F 50-ATATCCATGGTAGATGAG

GAAGTGGAG-30 (NcoI site underlined) and RH-R 50-GGACTAGTCACCTGTGCTGCTGCTGC-30 (SpeI site

underlined). After verification by sequencing, the PCR

products were inserted into NcoI and SpeI-digested

pCAMBIA1302-GFP vector under the control of the cau-

liflower mosaic virus 35S (CaMV 35S) promoter. The

p1302-GFP-ZmWRKY58 construct and the control vector

(p1302-GFP) were transformed into onion epidermal cells

by particle bombardment using a PDS-1000/He system

(Bio-Rad, USA). Transformed onion epidermal cells were

cultured on MS medium under dark conditions for 18–36 h

at 28 �C. GFP signals were detected by a confocal

microscopy (Olympus, Japan).

Vector construction and genetic transformation

The full-length coding sequence of ZmWRKY58 was

amplified from maize with primers GL-F 50-TTCTCT-

AGATAGATGAGGAAGTGGAG-30 (BamHI site in bold

italics) and GL-R 50-TTAGGATCCCACCTGTGCTGCTG

CTGC-30 (XbaI site in bold italics). The sequencing-con-

firmed PCR products were ligated into the plant overex-

pression vector pCAMBIA1301. The construct

pCAMBIA1301-ZmWRKY58 was transformed into

Zhonghua11 (ZH11) rice (Oryza sativa L. ssp. Japonica)

using Agrobacterium-mediated transformation (Lin and

Zhang 2005). ZmWRKY58 transgenic rice plants were

selected for hygromycin resistance and confirmed by PCR

using specific primers (Hyg-F 50-ACTCACCGCGACGT

CTGT-30 and Hyg-R 50-TTTCTTTGCCCTCGGACG-30).

Southern blot analysis

Genomic DNA was extracted from young leaves of T0

transgenic plants using a DNeasy Plant Mini Kit (Qiagen

GmbH, Hilden, Germany), and 10 lg of total DNA was

digested with HindIII. The ZmWRKY58 overexpressing

vector was used as a positive control, and genomic DNA

from wild-type (WT) rice was used as a negative control.

The digested genomic DNA was separated in a 0.7 %

agarose gel and transferred to a nylon membrane (Hybond-

N?; Amersham Pharmacia Biotech). Hybridization was

carried out using a DIG-High Prime DNA Labeling and

Detection Starter Kit II (Roche, Germany).

Germination assay

Seeds of WT and transgenic rice plants were surface ster-

ilized with 75 % (v/v) ethanol and washed with sterile

distilled water. Sterile seeds were grown hydroponically

with nutrient solution in a plant growth chamber (28 �C,

80 % relative humidity under a 14/10 h day/night photo-

period) for germination. The germination rate was scored at

2, 3, 4 and 5 days, respectively. Root length and shoot

height were monitored at 9 days after germination.

Drought and salt tolerance assay

Three independent T2 transgenic homozygous lines and the

WT plants were planted for the stress tolerance experi-

ments. For the drought tolerance test, four-week-old

seedlings were subjected to progressive drought for

15 days by withholding water, followed by rehydration and

recovery for 7 days. For the salt tolerance assay, grown

seedlings of WT and transgenic lines were irrigate with a

solution containing 200 mM NaCl for 14 days, followed by

an additional 7 days of recovery with watering. All stress

tolerance experiments were repeated three times. After

stress treatments and recovery, the survival rates of trans-

genic lines and the WT plants were recorded.

Measurements of RWC, MDA contents and REL

Following drought and salt treatments, leaves of similar

developmental stages from transgenic and WT plants were

collected at pre-determined times. For Relative water

contents (RWC) detection, fresh weight (FW), dry weight

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(DW) and turgid weight (TW) were used to calculate the

RWC according to the formula: RWC = [(FW - DW)/

(TW - DW)] 9 100 %. Malonaldehyde (MDA) content

was measured following the method of Zhang et al. (Zhang

et al. 2010). The relative electrolyte leakage (REL) was

measured according to a previously described method (Li

et al. 2011a) with some modifications.

Yeast two-hybrid assay

Previous studies have shown that WRKY group IId pro-

teins in Arabidopsis interact with calmodulin (CaM) in

yeast (Park et al. 2005). To test whether ZmWRKY58 can

bind to the CaM, the yeast two-hybrid assay was performed

using the Matchmaker GAL4 two-hybrid system (Clon-

tech, Palo Alto, CA). The full length of ZmWRKY58 cDNA

cloned into the pGBKT7 bait vector. The full length of

ZmCaM2 cDNA (GeneBank Accession Number:

X77397.1) was amplified from maize library plasmid using

the primers SZ-F 50-CGGAATTCATGGCGGACCAGCT-

CACCGACG-30 and SZ-R 50-CCGCTCGAGTCACTTGG

CCATCATCACCTTC-30 and then cloned into the

pGADT7 prey vector. The bait and prey plasmids were co-

transformed into yeast strain Y2HGold. The transformed

yeast cells was examined on SD/-Leu/-Trp and SD/-Trp/-

Leu/X-a-Gal/AureobasidinA plates at 30 �C for 3–5 days

to determine the protein–protein interaction. The interac-

tion between the BD-p53 and the AD-SV40 large T-antigen

as the positive control, and yeast cells co-transformed with

the human lamin C and the AD-SV40 large T-antigen as

the negative control.

Results

Expression patterns of ZmWRKY58 in maize

We performed a semi-quantitative RT-PCR to examine the

tissue expression pattern of ZmWRKY58 in different tis-

sues. As shown in Fig. 1a, e, the highest expression level

was observed in roots, with weak expression observed in

leaves and silks and marginally expression observed in

stems and tassels.

In addition, the expression patterns of ZmWRKY58

under various stress treatments were also investigated.

Under drought (20 % PEG6000) treatment, a significantly

induced-expression of ZmWRKY58 was observed after 12 h

of treatment, while the expression of this gene decreased at

24 h and then reached its highest level at 48 h (Fig. 1b, f).

Under NaCl stress, a slight accumulation of ZmWRKY58

expression was observed at 12 h, which declined at 24 h

and reached a maximum peak at 48 h (Fig. 1c, f). Under

ABA treatment, the expression level of ZmWRKY58 was

peaked at 12 h and declined thereafter (Fig. 1d, f).

Cloning and characterization of ZmWRKY58

The full-length cDNA of ZmWRKY58 was isolated

(Accession No: BT083614.1) from total RNA extracted

from drought-stressed maize leaves using RT-PCR. The

nucleotide sequence of ZmWRKY58 gene is 1,635 bp in

length, consisting of a 252 bp 50 untranslated region

(UTR), an 1,110 bp ORF and a 273 bp 30 UTR. The ORF

Fig. 1 Expression patterns of

ZmWRKY58 in maize. a Tissue-

specific expression of

ZmWRKY58 in roots (R), stems

(S), leaves (L), silks (SK) and

tassels (T). b, c and d represent

the expression patterns of

ZmWRKY58 under drought, salt

and ABA treatments,

respectively. The GAPDH gene

was used as the internal control

for normalization. e and

f represent the relative

expression levels of

ZmWRKY58 in different organs

and different treatments by

determining the band image

densitometry. Error bars are

standard deviations of three

technical repeats and two

biological repeats

Plant Cell Tiss Organ Cult

123

encodes a putative protein of 369 amino acids with a cal-

culated molecular mass of 39.4 kDa and a predicted pI of

9.81. Sequence alignments between ZmWRKY58 and

other plant WRKY proteins indicated that the amino acid

sequences of these proteins are highly similar, with a

homology of 72.85 % to TaWRKY16 (ACD80360.1),

73.17 % to BdWRKY31 (XP_003562250.1), 60.10 % to

HvWRKY8 (BAK05943.1) and 44.65 % to AtWRKY74

(NP_198217.1). Multiple sequence alignment analysis

revealed that ZmWRKY58 contains one putative WRKY

domain followed by a C2H2-type zinc finger motif, a

putative nuclear localization signal (NLS) and a short

conserved structural motif (C-motif), indicating that

ZmWRKY58 belongs to the group IId of the WRKY

family (Fig. 2).

We also performed a phylogenetic analysis to inves-

tigate the evolutionary relationships among ZmWRKY58

and other WRKY proteins. As shown in Fig. 3, ZmWR

KY58 shown a close relationship with AtWRKY15 in

Arabidopsis, GmWRKY13 in soybean and GhWRKY39-

1 in Gossypium hirsutum. These proteins have been

demonstrated to participate in plant response to abiotic

stresses (Shi et al. 2014; Vanderauwera et al. 2012; Zhou

et al. 2008). Thus, the result suggested that these pro-

teins, with high homology among different species, may

share some similar functions.

Fig. 2 Alignment of the putative amino acid sequence of

ZmWRKY58 with sequences from Arabidopsis (NP_198217.1),

Triticum aestivum (ACD80360.1), Brachypodium distachyon

(XP_003562250.1) and Hordeum vulgare (BAK05943.1). Identical

amino acids are shaded in black. Approximately 60 amino acid of the

WRKY domain and the cysteine and histidine residues of the putative

zinc finger motif are marked by a two-headed arrow and red arrow,

respectively. The putative nucleus localization signal (NLS) and the

highly conserved amino acid sequence WRKYGQK in the WRKY

domain are enclosed by black boxes. (Color figure online)

Fig. 3 Phylogenetic analysis of ZmWRKY58 and closely related

WRKY transcription factors from other species. The accession

numbers of selected WRKYs are as follows: ZmWRKY58

(BT083614.1), ZmWRKY6 (NP_001147091.1) from Zea mays,

BdWRKY31 (XP_003562250.1) and BdWRKY74

(XP_003559879.1) from Brachypodium distachyon, TaWRKY16

(ACD80360.1) from Triticum Aestivum, HvWRKY8 (BAK05943.1)

and HvWRKY9 (ABI13375.1) from Hordeum vulgare, AtWRKY7

(NP_194155.1), AtWRKY15 (NP_179913) and AtWRKY74

(NP_198217.1) from A. thaliana, BnWRKY39 (ACN89259.1) from

Brassica napus, GhWRKY6 (AFH01344.1) and GhWRKY39-1

(KF220642) from G. hirsutum, GmWRKY13 (DQ322694) from

soybean

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123

Subcellular localization of ZmWRKY58

To determine the subcellular localization of ZmWRKY58,

the ORF of ZmWRKY58 without the termination codon was

fused to the 50 end of the GFP reporter gene under the

control of the CaMV 35S promoter. The recombinant

construct and the GFP vector alone were introduced into

onion epidermal cells through particle bombardment.

Confocal imaging showed that the GFP-ZmWRKY58

fusion protein was exclusively localized in the nucleus. By

contrast, onion cells transformed with GFP vector alone

displayed fluorescence throughout the entire cell, demon-

strating that ZmWRKY58 is a nuclear localized protein

(Fig. 4).

Generation of transgenic plants

The full-length cDNA of ZmWRKY58 under the control of

the CaMV 35S promoter was transformed into ZH11 rice

(O. sativa L. ssp. Japonica). Of the ten independent T0

transgenic plants generated, seven were positive transfor-

mants, as detected by PCR analysis of the hygromycin

resistance gene. Southern blot analysis revealed that one

copy of ZmWRKY58 was integrated into the genomes of

three T0 transgenic lines, including L1, L2 and L4 (Fig. 5).

Overexpression of ZmWRKY58 delayed germination

and inhibited post-germination development in rice

To investigate the effects of ZmWRKY58 overexpression in

plant growth and development, WT plants and T2 progeny

of the three transgenic lines L1-2, L2-3 and L4-5 were

selected for germination assays. As shown in Fig. 6a, we

observed a great difference in seed germination between

transgenic lines and WT plants under normal conditions.

Only 64.2–84.4 % of the transgenic seeds germinated at

5 day, whereas 97.5 % of WT seeds germinated. This

result suggested that overexpression of ZmWRKY58 in rice

obviously delays seed germination. In addition, the seed-

lings derived from transgenic seeds exhibited apparent

growth retardation during their post-germination

Fig. 4 Nuclear localization of

ZmWRKY58. The

ZmWRKY58-GFP fusion

protein and GFP alone, driven

by the CaMV 35S promoter,

were transiently expressed in

onion epidermal cells and

visualized by fluorescence

microscopy, respectively

Fig. 5 Southern blot analysis of transgenic plants using the hygro-

mycin resistance gene as a probe. M, DNA molecular weight marker;

?, plasmid pCAMBIA1301-ZmWRKY58; Lanes 1–3 transgenic lines

L1, L2 and L4; -, WT plants

Plant Cell Tiss Organ Cult

123

development stage. We measured root length and shoot

height at 9 day after germination. As shown in Fig. 6b, c,

the WT seedlings exhibited a more rapid growth trend than

those of the transgenic lines. These results indicated that

ZmWRKY58 can clearly delay both the time of seed ger-

mination and post-germination development in rice.

Overexpression of ZmWRKY58 enhances tolerance

to drought and salt stresses

To examine the function of ZmWRKY58 in stress response,

ZmWRKY58 overexpressing construct driven by the CaMV

35S promoter was transformed into ZH11 rice and seven

transgenic lines were generated. According to the result of

southern blotting, three homozygous T2 progeny of the

transgenic lines (L1-2, L2-3 and L4-5) were chosen for the

stress tolerance assay. After continuously withholding

water, most transgenic seedlings remained green and dis-

played continuous growth. However, WT seedlings showed

severe leaf wilting or rolling (Fig. 7a). After recovery with

water for 7 days, more than 82 % of L1-2, 76 % of L2-3

and 89 % of L4-5 seedlings remained vigor. By contrast,

only approximately 43 % of WT seedlings survived

(Fig. 7b). Similar results were observed after treatment

with 200 mM NaCl for 14 days, followed by recovery for

7 days. As shown in Fig. 8a, b, the survival rates of L1-2,

L2-3 and L4-5 were more than 48 %, which were signifi-

cantly higher than that of WT plants (17 %). Taken toge-

ther, these results indicated that overexpression of

ZmWRKY58 significantly improves drought and salt stress

tolerance in transgenic rice.

Changes in physiological traits under stress conditions

We assessed water loss in plants by measuring the RWC of

detached leaves from the three ZmWRKY58 overexpression

transgenic lines and WT plants at designated time pionts.

As shown in Fig. 7c, there was no significant difference in

RWC between transgenic and control plants under normal

growth conditions. However, after treatment with drought

and salt stresses, the transgenic lines showed remarkably

higher levels of RWC than that of WT plants. In addition,

osmotic stress-induced cell membrane damage in plants

can be estimated by measuring REL and lipid peroxidation,

which is evaluated by determining MDA levels (Li et al.

2010). Under normal conditions, no obvious differences in

MDA levels or REL were detected between the transgenic

lines and WT plants. Osmotic stress resulted in increased

levels of MDA and REL in both of the WT and transgenic

lines, but these increases occurred at different rates. During

Fig. 6 Comparison of seed germination and growth of transgenic and

WT plants under normal conditions. a Germination rate of transgenic

and WT plants at 5 days. b The growth phenotype of transgenic and

WT plants at 9 days after germination. c The root length and shoot

height of transgenic and WT plants measured at 9 days after

germination. The error bars indicate the SD of three independent

experiments performed

Plant Cell Tiss Organ Cult

123

drought and salt stress treatments, ZmWRKY58 transgenic

lines exhibited lower MDA contents (Figs. 7d, 8d) and

REL (Figs. 7e, 8e) than WT plants. These findings indi-

cated that ZmWRKY58 can reduce membrane damage

caused by drought and salt stresses.

Protein interaction analysis of ZmWRKY58

with ZmCaM2

In Arabidopsis, all WRKY group IId members can interact

with CaM2, and the CaM-binding domain (CaMBD) of this

group is a conserved structural motif (C-motif) (Park et al.

2005). To determine whether the interaction also exist in

maize, yeast two-hybrid assay was performed to examine

the interaction of ZmWRKY58 with ZmCaM2. The full

length of ZmCaM2 cDNA was fused to the yeast GAL4-

AD vector and co-transformed into yeast cells with the

ZmWRKY58 bait vector. As shown in Fig. 9, all the

transformants containing positive control, negative control

and experimental group grow well on SD/-Leu/-Trp med-

ium. When transferred onto SD/-Trp/-Leu/X-a-Gal/Au-

reobasidinA plates for 5 days, the yeast cells of positive

control and experimental group turned blue, whereas the

negative control exhibited no a-galactosidase activity. The

results indicated that ZmWRKY58 could interact with

ZmCaM2.

Discussion

Increasing evidence has indicated that WRKY TFs play

important roles in plant responses to biotic and abiotic

stresses. In this study, we isolated the ZmWRKY58 gene

from the maize inbred line B73. Previous research showed

that the expression of this gene was induced under drought

treatment (Zheng et al. 2010). However, the function of

ZmWRKY58 under environmental stress has not been fully

investigated. The current study are important in elucidating

maize WRKY protein-regulated responses to abiotic stress.

Based on the high similarity between ZmWRKY58 pro-

tein and other WRKY proteins obtained from Triticum

aestivum L., Arabidopsis, Hordeum vulgare and B. dis-

tachyon, we affirmed that the gene that we isolated from

maize is a WRKY gene. The results of subcellular locali-

zation of ZmWRKY58-GFP show that the GFP signal was

located in the nucleus, which suggestes that ZmWRKY58

Fig. 7 Enhanced drought tolerance of ZmWRKY58 overexpressing

transgenic rice lines. a Performance of WT plants and ZmWRKY58-

overexpression lines after 15 days of drought stress and 7 days of

recovery. b Survival rates of the WT plants and transgenic rice lines

after recovery for 7 days following drought stress. RWC content (c),

MDA content (d) and REL (e) were measured in WT plants and

transgenic lines after drought treatment. The data represent the means

of three replicates. The error bars indicate the SD. Significant

differences between the transgenic lines and WT control are indicated

as **P \ 0.01 using the Student’s t test

Plant Cell Tiss Organ Cult

123

might function in the nucleus. Transcriptional activation

assay suggested that ZmWRKY58 showed no transcrip-

tional activation ability (data not shown). Similar results

were observed in previous reports. For example, four

soybean WRKY proteins (GmWRKY13, GmWRKY27,

GmWRKY40, GmWRKY54) do not possess transcrip-

tional activation activity in the yeast assay system (Zhou

et al. 2008). Qin et al. demonstrated that TaWRKY71-1

protein also had no transcriptional activation activity (Qin

et al. 2013). Further investigation should be performed to

examine whether ZmWRKY58 protein can function as

activators or repressors of transcription in plants.

Previous studies have shown that overexpression of

WRKY TF genes improved abiotic stress tolerance (Li et al.

2010; Wang et al. 2012, 2013). However, most functional

analysis of WRKY genes have focused on model plants such

as Arabidopsis, tobacco and rice, and few WRKY family

members from maize have been reported. To our knowledge,

only one WRKY gene, ZmWRKY33, has been studied in

maize. Overexpression of ZmWRKY33 was shown to confer

enhanced salt stress tolerance in Arabidopsis (Li et al. 2013).

Fig. 8 Enhanced salt tolerance of ZmWRKY58 overexpressing trans-

genic rice lines. a Performance of WT plants and ZmWRKY58-

overexpression lines after 14 days of salt stress treatment and 7 days

of recovery. b Survival rates of the WT control and transgenic rice

lines. RWC content (c), MDA content (d) and REL (e) were measured

in WT plants and transgenic lines after salt treatment. The data

represent the means of three replicates. The error bars indicate the

SD. Significant differences between the transgenic lines and WT

control are indicated as **P \ 0.01 using the Student’s t-test

Fig. 9 Interaction between ZmWRKY58 and ZmCaM2. The BD-

WRKY58 and AD-CaM2 plasmids were co-transformed into yeast

strain Y2HGold and grew on SD/-Trp/-Leu medium and the selective

medium SD/-Trp/-Leu/X-a-Gal/AureobasidinA at 30 �C for 5 days. 1

positive control (pGBKT7-53 ? pGADT7-T); 2 negative control

(pGBTKT7-Lam ? pGADT7-T); 3 and 4 two different colonies

containing positive transformants of pGBKT7-ZmWRKY58 ? p-

GADT7-ZmCaM2

Plant Cell Tiss Organ Cult

123

In the current study, to evaluate the effects of constitutive

overexpression of ZmWRKY58 in the response to abiotic

stresses, we generated ZmWRKY58 overexpressing rice

plants. Maize and rice shared a common ancestor and their

genomes are highly syntenic (Wei et al. 2009), which pro-

vide a reasonable basis of heterologous expression of maize

gene in rice plant. The former researcher had achieved some

meaningful results using this method. For instance, expres-

sion of the maize ZmGF14-6 gene in rice confers tolerance to

drought stress as well as enhancs susceptibility to pathogen

infection (Campo et al. 2012). Overexpression of ZmCBF3

in transgenic rice enhanced the tolerance to drought, salt and

low-temperature stresses (Xu et al. 2011). Phenotype anal-

yses showed that the transgenic plants displayed delayed

germination and inhibited post-germination development

compared with the WT plants under normal condition.

Similar phenomenon was also observed in several previous

functional studies of TFs. Transgenic rice plants over-

expressing OsTZF1 exhibited delayed seed germination and

growth retardation at the seedling stage (Jan et al. 2013).

Overexpression of OsWRKY72 in Arabidopsis can obviously

inhibit seed germination (Song et al. 2010). Our results

suggested that ZmWRKY58 may play a negative regulatory

role during seed germination and seedling growth. Previous

studies indicated that the regulation of seed germination are

associated with the balance of ABA:GA levels and sensi-

tivity (Finkelstein et al. 2008), which implying that the

delayed-germination phenotype may be attributed to the

destruction of this balance caused by overexpression of

ZmWRKY58. Since ABA/GA metabolic or signaling-related

genes were not detected in our analysis, further experiments

need to verify the above possibility.

In the current study, overexpression of ZmWRKY58 in

rice led to enhanced drought and salt stress tolerance,

which was mainly demonstrated by the higher survival

rates, higher RWC, lower MDA contents and REL of the

transgenic plants under stress conditions. Abiotic stress can

cause lipid peroxidation, resulting in MDA accumulation

(Kong et al. 2011). The MDA contents of plants reflect the

degree of oxidization of cell membrane lipids. REL, an

important parameter related to membrane injury, was lower

from ZmWRKY58 overexpressing transgenic plants than

from WT plants, which implies that the ZmWRKY58

protein may protect the cell membrane integrity of plants

under drought and salt stress conditions.

Although notable progress has been made in determining

the involvement of WRKY TFs in regulating plant responses

to abiotic stress, far less information is available about the

mechanisms underlying the signaling and transcriptional

reprogramming controlled by these proteins. Recent studies

have demonstrated that WRKY TFs are key nodes in ABA-

responsive signaling networks (Rushton et al. 2012). In

Arabidopsis, a series of experiments has led to the

identification of AtWRKY40 as a protein that interacts with

ABA receptors (Shang et al. 2010). Furthermore, ChIP

experiments have shown that AtWRKY40 directly targets a

number of known ABA-responsive genes, including ABI4,

ABI5, ABF4, MYB2, DREB1A and RAB18 (Shang et al.

2010). In the current study, semi-quantitative RT-PCR

demonstrated that expression of ZmWRKY58 was signifi-

cantly induced under drought, salt and ABA treatments,

implying that ZmWRKY58 may function via the ABA-

dependent signaling pathway in the process of stress

responses. Meanwhile, this result also demonstrated that a

single TF may function in several seemingly disparate sig-

naling pathways. In addition, research over the last several

years has demonstrated that WRKY TFs physically interact

with a wide range of proteins with roles in signaling, tran-

scription, and chromatin remodeling (Chi et al. 2013). The

action mode of WRKY TFs with other proteins includes

WRKY–WRKY (Chen et al. 2009), WRKY-VQ (Hu et al.

2013), WRKY-MAPK (Pitzschke et al. 2009), WRKY-

chromatin remodeling proteins (Zhu et al. 2010), WRKY-

14-3-3 proteins (Chang et al. 2009) and WRKY-R proteins

(Shen et al. 2007). In this study, ZmWRKY58, a group IId

WRKY protein, has been demonstrated to interact with CaM

just like its Arabidopsis thaliana homolog (Park et al. 2005),

suggesting that ZmWRKY58 may function as a CaM-

binding protein (CaMBP) in plant stress response. CaM is

one of the best characterized Ca2? responsive proteins in

eukaryotic cells (Snedden and Fromm 2001). Increasing

evidence indicated that CaM has important roles in plant

response to abiotic stresses (Liu et al. 2003; Olsson et al.

2004; Taybi and Cushman 1999; Townley and Knight 2002).

The discovery of WRKY proteins’ interacting partners

facilitates the reconstruction of signaling pathways that

involve WRKY proteins.

In conclusion, a maize group IId WRKY gene,

ZmWRKY58, were isolated and characterized. Our results

clearly suggested that ZmWRKY58 is a stress-inducible TF.

Overexpression of ZmWRKY58 in rice significantly affects

seed germination and post-germination growth, and enhances

drought and salt stress tolerance. Yeast two-hybrid assay

showed that ZmWRKY58 could interact with ZmCaM2.

However, the detailed regulatory mechanisms of ZmWRKY58

remains to be further investigated.

Acknowledgments This work was supported by the National Natural

Science Foundation of China (31071423, 31201217, 31301324),

National Key Technology R&D Program of China (2012BAD20B02)

and the Key Science and Technology Program of Anhui Province

(1206c0805032).

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