Molecular cloning and functional analysis of MbWRKY3 involved in improved drought tolerance in transformed tobacco

ABSTRACT Plant-specific WRKY transcription factors were involved in stress responses and ABA signaling. In the present study, a WRKY gene is isolated from Malus baccata (L.) Borkh and designated as MbWRKY3. Subcellular localization revealed that MbWRKY3 was localized onto nucleus. The MbWRKY3 expression levels were up-regulated by salinity, drought, and ABA treatments in M. baccata. When MbWRKY3 was introduced into tobaccos, it improved drought stress tolerance in transgenic plants. Compared to WT, the transgenic tobaccos had the higher levels of relative water content, and proline and chlorophyll contents, decreased levels of electrolyte leakage, MDA, and H2O2, increased activities of the reactive oxygen species-related enzymes (SOD, CAT, and POD), and greater up-regulations of the corresponding genes (NtSOD, NtCAT, and NtPOD), especially when dealt with drought stress. These results suggest that MbWRKY3 gene plays a positive regulatory role in drought stress response.


Introduction
As sessile organisms, plants are frequently exposed to variable environmental stresses, such as drought, salt, heat, chilling, pathogen attack, and nutrient deprivation, which adversely affect plant growth, development, and productivity (Gong & Liu 2013). Environmental stresses are perceived and transduced through a chain of signaling molecules that ultimately affect regulatory element of stress-inducible genes to initiate the synthesis of different classes of protein including transcription factors, enzymes, molecular chaperons, ion channels, and transporters or alter their activities (Mukhopadhyay et al. 2004). To avoid such deficiencies, plants have developed adaptable mechanisms to perceive external signaling networks and to manifest adaptive responses with appropriate physiological, cellular, and molecular changes (Liu et al. 2014). These environmental stimuli usually disrupt the cellular homeostasis of reactive oxygen species (ROS), leading to the oxidative stresses (Mittler, 2002). In these stresses, drought is the most severe threat to crop yield worldwide. Plants have evolved complex strategies to reduce potential damage of drought stress (Wang et al. 2009).
Among the numerous stress-induced genes, many transcription factors have been identified and studied, such as WRKY, NAC, and bZIP, which can interact with cis-elements present in the promoter regions of various abiotic stressrelated genes and thus regulate the expression of many genes resulting in imparting tolerance to abiotic stresses (Agarwal et al. 2006). Among TFs, the WRKY genes received much attention in past decade. The WRKY proteins, which contain one or two conserved WRKY domains consisting of 60 amino acid region with a conserved WRKYGQK motif in its N-terminus followed by a C 2 H 2 or C 2 HC zinc-finger motif, represent a large family of plant-specific TFs (Rushton et al. 2010). There are over 70 WRKY genes in Arabidopsis (Eulgem et al. 2000;Dong et al. 2003;Sáenz-Mata et al. 2014). Studies on WRKY genes predominantly point to an involvement in salicylic acid (SA) signaling and disease responses (Eulgem et al. 2000;Asai et al. 2002). In addition, WRKY genes are involved in plant responses to drought, heat, cold, salinity (Zhou et al. 2008;Liu et al. 2014;Sáenz-Mata et al. 2014;Cheng et al. 2017;Ye et al. 2017;Han et al. 2018), freezing (Huang & Duman 2002), wounding (Hara et al., 2000), and oxidative stress (Rizhsky et al. 2004;Cui et al. 2018). But these studies mostly focused on model plants or crops, and the roles of the WRKY genes in stress responses of Malus plant were less well known.
Malus baccata is widely used as an apple rootstock in northern China, but is also grown elsewhere as an ornamental tree and is highly resistant to low temperatures and drought (Xiao et al. 2008). To better understand the roles of WRKY genes involved in drought stress tolerance and to provide potentially genetic resources for the improvement of the drought resistance of Malus plant, a novel drought-responsive WRKY transcription factor was isolated from M. baccata and designated as MbWRKY3. Moreover, the over-expression of MbWRKY3 in transgenic tobacco increased the tolerance to drought stress.

Plant material and growth conditions
In vitro grown seedlings of M. baccata were propagated and rooted on MS medium, and transferred to Hoagland's solution for acclimation and growing for 50 d (Han et al. 2018). When the seedlings had 8-9 mature leaves (fully expanded), they were exposed to air on filter paper with 20% relative humidity for dehydration and drought treatment. For ABA and salt treatments, seedling roots were immersed in 0.1 mM ABA or 250 mM NaCl solution, respectively (Liu et al. 2014).

Isolation, phylogenetic analysis and qRT-PCR expression analysis of MbWRKY3
Total RNA was respectively extracted from root, new leaf (partly expanded), mature leaf (fully expanded), and stem, using the CTAB method (Han et al. 2017). The samples of all control and roots of treated plants were sealed after treatments of 0, 3, 6, 9, 12 h, respectively, frozen immediately in liquid nitrogen, and then stored at −80°C for RNA extraction. First strand cDNA was synthesized with 1 μg total RNA and 1 μL super script II enzyme (Invitrogen, USA) according to the manufacturer protocol. PCR was performed to obtain a whole sequence of MbWRKY3 by using the first strand cDNA of M. baccata as a template. A pair of primers (F1, 5 ′ -ATGGCCTACTTAGAAGGGACCTTAGC-3 ′ and R1, 5 ′ -CTATGAATTTGGTCTATTTGCAGGTC-3 ′ ) was designed according to the homologous regions of Malus domestica WRKY gene 3 (MDP0000263961) to amplify the full-length cDNA sequence. The full-length cDNA of MbWRKY3 gene was isolated from M. baccata using PCR with F1 and R1 as primers. The obtained DNA fragments were gel purified and cloned into the pMD18-T vector (Takara, Dalian, China) and sequenced (Invitrogen, Beijing, China).
The expression level of MbWRKY3 to abiotic stress was analyzed by real-time (RT)-PCR method in M. baccata and conducted according to Han et al. (2015). The primers for MbWRKY3 were designed for qRT-PCR from partial sequences isolated in this study, i.e. MF, 5 ′ -TTGCAACGTTTGAGGAGAGAG-3 ′ and MR, 5 ′ -TAAGGCTGTTGTCTGTAGAATCG-3 ′ .
The thermal cycling program was one initial cycle of 93°C for 30 s, followed by 40 cycles of 93°C for 5 s, and 56°C for 30 s. As a control, the Actin gene was used as reference gene and amplified from M. baccata tissues using the following primers: ApActF1, 5 ′ -CTACAAAGTCATCGTCCAGACAT-3 ′ and ApActR1, 5 ′ -TGGGATGACATGGAGAAGATT-3 ′ . The relative gene expression data were analyzed using the 2 -ΔΔCT method (Livak & Schmittgen 2001).

Subcellular localization of the MbWRKY3 protein
The MbWRKY3 ORF was cloned into the SalI and BamHI sites of the pSAT6-GFP-N1 vector. This vector contains a modified red shifted green fluorescent protein (GFP) at SalI-BamHI sites. The MbWRKY3-GFP construct was transformed into onion epidermal cells by particle bombardment as described earlier (Han et al. 2015). The transient expression of the MbWRKY3-GFP fusion protein was observed under confocal microscopy.

Vector construction and agrobacterium-mediated tobacco transformation
To construct an expression vector for transformation of tobacco, restriction enzyme cut sites of SmaI and SacI were added into MbWRKY3 cDNA at both 5 ′ and 3 ′ ends by PCR. To construct the pBI121-MbWRKY3 vector, the products of PCR and pBI121 were digested by SmaI and SacI, and linked together through the replacing of GUS gene. The MbWRKY3 gene driven under the CaMV 35S promoter was introduced into Nicotiana tabacum cv. Xanthi ecotype tobacco by Agrobacterium-mediated GV3101 transformation (An et al. 1986). Transformants were selected on MS medium containing 50 mg dm −3 Kanamycin. T 2 generation transgenic tobaccos were used for further analysis. Expression levels of the MbWRKY3 in tobacco transgenic lines were analyzed by semi-quantitative RT-PCR. The primers of MF and MR were used for RT-PCR detection of leaves of tobacco, with the NtUbiquitin gene (U66264.1) as reference gene (NtUbF1, 5 ′ -TTAACACATGCAAGTCGGACG-3 ′ and NtUbR1, 5 ′ -GAGACCTCAGTAGACAAAGCACATC-3 ′ ). PCR was performed for 30 and 25 amplification cycles for MbWRKY3 and NtUbiquitin, respectively.

Seeds (T 2 generation) of transgenic plants (OE-4 and OE-13)
were sown and germinated on the germination and culture matrix (nutrient soil: vermiculite ratio is 4:1) in flowerpots (diameter 10 cm) with normal management in a growth chamber at 25 ± 1°C under a 16 h light (50 µmol/m 2 /s)/8 h dark regime in parallel with WT seeds. Seedlings were grown for 3 weeks with regular irrigation prior to drought stress. Drought stress experiments were conducted by withholding water for 12 d. Then the tobaccos were rewatered for 6 d to determine the survival rate. The experiments were performed twice with three independent leaves for each treatment at each time point. During the whole growth process, all tobacco seedlings were observed and recorded with photograph on drought stress for 6 d (D6d), 12 d (D12d), and rewatered for 6 d (R6d).

Measurement the levels of relative water content and electrolyte leakage
Three-week-old transgenic tobacco (OE-4 and OE-13) and WT tobacco plants above were conducted by withholding water for 6 d, and the leaves of all samples (before drought and after drought) were collected for measurement. Leaf relative water content (RWC) was estimated according to RWC (%) = (fresh weight − dry weight)/(turgid weight − dry weight) × 100 (Wang et al. 2009). Electrolyte leakage (EL) in tobacco leaves was determined following the protocols described by Xing et al. (2011).

Measurement of the contents of proline, chlorophyll, MDA, and H 2 O 2
The leaves (including samples of before drought and after drought for 6 d) of 3-week-old transgenic and WT tobaccos above were collected for measurement. Proline contents were assayed following the method of Irigoyen et al. (1992). Chlorophyll contents were measured according to the method of Aono et al. (1993). MDA contents in tobacco leaves were measured by the method described previously (Dong et al. 2003). H 2 O 2 contents in tobacco leaves were determined by the method of Alexieva et al. (2001).

Measurement of activities of SOD, CAT, and POD
The SOD, CAT, and POD activities of all the tobacco leaf samples of before treatments and exposed to drought stresses for 6 d above were also measured. SOD (EC 1.15.1.1) activities were measured using the protocols described by Beauchamp & Fridovich (1971). CAT (EC 1.11.1.6) activities were measured following the methods used previously (Zhang et al. 2011). POD (EC 1.11.1.7) activities were measured following Ranieri et al. (2000). Relative quantification relates the enzyme activity in the transgenic lines (OE-4 and OE-13) to that of the WT.

Statistical analysis
Duncan multiple range tests were performed by using SPSS 13.0 program. Statistical differences were referred to as significant when P < .05.

Isolation of MbWRKY3 gene from M. baccata
Sequence analysis showed that the MbWRKY3 cDNA has a complete ORF of 1059 bp, the predicted MbWRKY3 comprises   352 amino acids (Figure 1) with a theoretical isoelectric point of 8.53 and a predicted molecular weight of 39.5 kDa. MbWRKY3 protein contains one WRKY domain (Figure 1(a)) and one C 2 H 2 zinc-finger motif (C-X 5 -C-X 23 -H-X-H) (Figure 1(b)).

Phylogenetic relationship of MbWRKY3 with other WRKY transcription factors
As shown in Figure 2, all the deduced amino acid sequence of WRKY TFs include a WRKY domain (WRKYGQK, Figure 2 (a)) and one conserved C 2 H 2 zinc-finger motif (Figure 2(b)). Comparing the amino acid sequences of MbWRKY3 with other WRKY TFs, we found that MbWRKY3 has a high identity to the WRKY TFs.
Additionally, a phylogenetic tree (neighbor-joining) was constructed with the amino acid residues (Figure 3) by MEGA program (v.4.1). Phylogenetic analysis demonstrated that the majority of 24 isolated proteins belong to 7 different subgroups of II-a, II-b, II-d, II-c, I, II-e, and III. MbWRKY3 was clustered into II-a subgroup, and more closely related to the VpWRKY3 and BhWRKY1.

MbWRKY3 was localized in nucleus
As shown in Figure 4, the MbWRKY3-GFP fusion protein is targeted into nucleus with 4 ′ ,6-diamidino-2-phenylindole (DAPI) staining, whereas the control GFP alone is distributed throughout the cytoplasm. These results showed that the MbWRKY3 is a nucleus localization protein.

Expression patterns of MbWRKY3 under abiotic stresses in M. baccata
The spatial-specific expression of MbWRKY3 in different tissues of M. baccata was determined by qRT-PCR. The result shows that MbWRKY3 mRNA is more abundant in root and new leaf than in stem and mature leaf ( Figure 5(a)).
The results showed that MbWRKY3 increased in root under high salinity, drought, and ABA treatments ( Figure 5(b-d)) by the qRT-PCR method. For high salinity stress, the expression level of MbWRKY3 increased rapidly and reached the maximum at 9 h and maintained the stable high level for 12 h (Figure 5(b)). Under drought stress, the expression levels of MbWRKY3 began to increase after 3 h dehydration treatment and increased gradually until the experiment was concluded at 12 h ( Figure 5(c)). The expression level of MbWRKY3 increased rapidly and peaked at 9 h, and then decreased at 12 h in response to ABA treatment ( Figure 5(d)).

Over-expression of MbWRKY3 confers enhanced tolerance to drought stress
In order to investigate the role of MbWRKY3 in response to drought stress in plants, we generated transgenic tobacco with over-expression of MbWRKY3 under the control of the CaMV 35S promoter. Among 25 transformed lines, 6 of them (OE-3, OE-4, OE-9, OE-13, OE-17, and OE-23) were confirmed by using RT-PCR analysis with WT as control (Figure 6(a)).
Over-expression of MbWRKY3 in transformed tobaccos conferred higher levels of RWC, proline, and chlorophyll under drought stress In order to study the reasons why the transgenic tobaccos had the better appearances under the drought stress, the RWC, proline, and chlorophyll levels of all lines (both transgenic tobaccos OE-4, OE-13, and WT line) under normal irrigation and drought stress were measured. When dealt with drought stress for 6 d, the RWC, Pro and Chl levels in leaves of transgenic tobaccos were significantly higher relative to WT (Figure 7(a-c)). However, these are no significant difference in the levels of RWC, Pro, and Chl between the MbWRKY3-over-expression tobaccos and WT line under normal conditions.
MbWRKY3-over-expression tobaccos accumulated less EL, MDA, and H 2 O 2 under drought stress We also measured the EL, MDA, and H 2 O 2 levels of all lines (OE-4, OE-13, and WT) under normal irrigation and drought stress. The EL, MDA, and H 2 O 2 levels in leaves of transgenic tobaccos were significantly lower relative to WT, especially when dealt with drought stress for 6 d (Figure 7(d-f)). When dealt with drought stress, the levels of EL, MDA, and H 2 O 2 in WT tobaccos were about respectively 1.6-, 1.7-, and 1.8-fold than transgenic lines, which indicated that the WT line had more severe membrane damage. These results indicate that the over-expression of MbWRKY3 in transgenic tobaccos confers greater tolerance to the oxidative stress associated with drought stress.

Over-expression of MbWRKY3 confers to enhanced antioxidant enzyme activities
Aim to research the reason why WT tobaccos had more severe membrane damage relative to transgenic lines, the activities of ROS-scavenging enzymes such as SOD, CAT, and POD in both transgenic tobaccos (OE-4 and OE-13) and WT line before and after drought stress were also measured. Under normal water management, the activities of SOD, CAT, and POD in the transgenic lines were 1.4-, 1.2-, and 1.3-fold higher than WT line, respectively. When dealt with drought stress, the activities of SOD, CAT, and POD increased in all lines approximately 1.7-, 1.6-, and 1.8fold than WT, respectively (Figure 7(g-i)). The change tendency of activities of ROS-scavenging enzymes between the WT and transgenic lines remained more or less the same. Compared to WT line, the transgenic tobaccos had the higher ROS-scavenging enzymes activities so that they can remove more reactive oxygen radicals and protect integrity of the membrane.

Over-expression of MbWRKY3 confers to enhanced expression levels of ROS-related genes
In order to research what caused the higher antioxidant enzyme activities (SOD, CAT, and POD) in transgenic tobaccos (OE-4 and OE-13) than in WT line, the expression levels of NtSOD, NtCAT, and NtPOD in all lines were also measured by qRT-PCR method. The result showed that the expression levels of three genes were 2.4-, 1.9-, and 2.5-fold higher than the WT under normal water management. Under drought stress, the expression levels of NtSOD, NtCAT, and NtPOD increased in all lines, which in the transgenic tobaccos were about 2.8-, 3.6-, and 4.8-fold than WT line, respectively (Figure 8(a-c)).

Discussion
The WRKY transcription factors have been proved to play important functions in the regulation of transcriptional reprogramming related to plant biotic and abiotic stress responses ). In the present study, a new WRKY gene was isolated from M. baccata and designated as MbWRKY3. Sequence analysis showed that MbWRKY3 transcription factor contains one WRKY domain and one C 2 H 2 zinc-finger motif (C-X 5 -C-X 23 -H-X-H) (Figure 1).
All the WRKY TFs include a WRKY domain (WRKYGQK) and one conserved C 2 H 2 or C 2 HC zincfinger motif (Liu et al. 2014). These results showed that the WRKY TFs family was highly conserved during evolution (Rinerson et al. 2015). Previous reports have indicated that WRKY TFs genes are widely distributed in Arabidopsis, rice, soybean, maize, cotton, strawberry, orange, grape, and peach, which are known to be involved in abiotic (Ramamoorthy et al. 2008;Han et al. 2018) and biotic stress responses (Pandey & Somssich 2009), seed development   were respectively shown the WT and transgenic tobacco seedlings growth on culture matrix. T 2 seeds were grown for 3 weeks in the growth chamber with normal water management (Unt: untreatment), then withheld irrigation for 6 d (D6d), 12 d (D12d), and rewatered for 6 d (R6d). Scale bars 5 cm. and germination (Johnson et al. 2002), and leaf senescence (Miao et al. 2004).
The MbWRKY3 was structurally similar to VpWRKY3 and BhWRKY1, which were isolated from Vitis pseudoreticulata (Zhu et al. 2012) and Boea hygrometrica (Wang et al. 2009) under high-drought stress. Phylogenetic analysis demonstrated that MbWRKY3 was clustered into II-a subgroup and more closely related to the VpWRKY3 and BhWRKY1. These results indicate that MbWRKY3 is a novel member of the WRKY transcription factor family ( Figure 3).
Certain WRKY TFs were reported to localize also in nucleus (Miao et al. 2004;Ramamoorthy et al. 2008;Liu et al. 2014). Subcellular localization experiment has revealed that MbWRKY3 is preferentially localized in nucleus ( Figure  4), which is consistent with previous research.
The expression of MbWRKY3 was much enriched in root and new leaf than in stem and mature leaf (Figure 5(a)),  (a-c) The relative transcript levels of NtSOD, NtCAT, and NtPOD under normal water management or withheld irrigation for 6 d in WT and transgenic tobaccos, respectively. Data represent means and standard errors of three replicates. Different letters above columns indicate (P < .05) significance using Duncan's multiple range test differences between treatments. which indicated that MbWRKY3 may play an important role in active organs. When treated with salt and drought stresses, the transcript levels of MbWRKY3 in roots were markedly increased by drought and salt stress ( Figure 5(b,c)). The expression of MbWRKY3 was markedly affected by ABA treatment, which increased in the first stage, and then decreased ( Figure 5(d)). ABA has been proved to mediate many stress responses through regulating the expression levels of the stress defense genes (Chinnusamy et al. 2004). In this study, the expression analysis showed that MbWRKY3 substantially induced by the treatment of salt, drought, and ABA, may be involved in the abiotic stress response via the ABA-dependent signaling pathway. These results above suggested that MbWRKY3 gene had participated in stress responses in M. baccata.
The 35S:MbWRKY3 transgenic tobacco exhibited a markedly increased tolerance to drought. Over-expression of BhWRKY1 also enhanced the tolerance to dehydration stress in transgenic tobacco (Wang et al. 2009). The over-expression of stress-responsive WRKY genes TaWRKY2 and TaWRKY19 in Arabidopsis have been reported to enhance the tolerance to drought and salt (Niu et al. 2012). The chlorophyll content and RWC were usually used as markers for severity of drought stress (Paknejad et al. 2007;Wang et al. 2009). Proline has been associated with the general stress response (Toka et al. 2010), which may also be cryoprotective, since proline overproducers display an enhanced drought tolerance (Van Rensburg et al. 1993). When dealt with drought treatment, the 35S:MbWRKY3 transgenic tobaccos had the higher RWC level and contents of proline and chlorophyll than WT. EL reflects membrane injury severity after abiotic stresses (Xing et al. 2011). MDA is the organic compound with the formula CH 2 (CHO) 2 and usually used as a marker for lipid peroxidation (Caradonna & Mauro 2016). Under drought stress, the MbWRKY3-over-expression transgenic tobaccos had the lower EL levels and generated smaller amounts of MDA and H 2 O 2 , and then in WT (Figure 7(d-f)).
Drought stress can cause the accumulation of ROS and induce lipid peroxidation, which can damage cytomembrane structure and lead to oxidative stress (Huda et al. 2013). In this study, it was found that MbWRKY3-over-expression transgenic plants possess higher activities of ROS-scavenging enzymes (SOD, CAT, and POD) in comparison with WT line under normal water management and drought stress ( Figure  7(g-i)). These results suggest that over-expression of MbWRKY3 in transgenic tobaccos results in the higher activities of ROS-scavenging enzymes. Consequently, higher protective enzyme activities lead to the suppression of ROS accumulation in order to suffer less from oxidative damage under drought stress.
The results suggest that over-expression of MbWRKY3 in transgenic tobaccos stimulates the enhanced expressions of the ROS-scavenging enzyme genes, including NtSOD, NtCAT, and NtPOD (Figure 8(a-c)), and results in the higher activities of ROS-scavenging enzymes. Consequently, higher protective enzyme activities lead to the suppression of ROS accumulation in order to suffer less from oxidative damage under drought stress. These were the reasons why overexpression of MbWRKY3 in transgenic tobaccos confer enhanced tolerance ( Figure 6) and have the higher survival rate to drought stress.
In conclusion, a WKRY transcription factor encoding MbWRKY3, which was induced by salt and drought stresses, and ABA treatment, was isolated from M. baccata. Overexpression of MbWRKY3 in tobacco resulted in enhanced tolerance to drought stress. This was partially correlated with the activation of ROS-related antioxidant genes/enzymes, leading to less accumulation of ROS under drought stress. More importantly, over-expression of MbWRKY3 in tobacco was achieved without affecting their phenotypes under normal conditions. Therefore, MbWRKY3 provides a potentially excellent genetic resource for improving drought tolerance in plants.

Disclosure statement
No potential conflict of interest was reported by the authors.