Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum

WRKY transcription factors play important roles in plant growth development, resistance and substance metabolism regulation. However, the exact function of the response to salt stress in plants with specific WRKY transcription factors remains unclear. In this research, we isolated a new WRKY transcription factor DgWRKY5 from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C2H2 zinc fingers. The expression of DgWRKY5 in chrysanthemum was up-regulated under various treatments. Meanwhile, we observed higher expression levels in the leaves contrasted with other tissues. Under salt stress, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes in transgenic chrysanthemum were significantly higher than those in WT, whereas the accumulation of H2O2, O2− and malondialdehyde (MDA) was reduced in transgenic chrysanthemum. Several parameters including root length, root length, fresh weight, chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress. Moreover, the expression of stress-related genes DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT. These results suggested that DgWRKY5 could function as a positive regulator of salt stress in chrysanthemum.

Salt tolerance analysis of DgWRKY5 in transgenic chrysanthemum. To investigate whether overexpression of DgWRKY5 enhances salt tolerance, transgenic chrysanthemum lines overexpressing DgWRKY5 were generated, and DgWRKY5 transcription levels in eight transgenic lines were detected by qRT-PCR (Fig. S4a-c). DgWRKY5 transcription levels were substantially (P < 0.05) higher in OE-17 and OE-56 than in WT (Fig. 2a). Therefore, we selected OE-17 and OE-56 lines plants for further salt tolerance evaluation. Under normal conditions, the phenotype of transgenic plants and WT plants had no significant difference. However, leaves of WT plants were evident wilting and yellowing compared with transgenic lines OE-17 and OE-56 under salt stress (Fig. 2c,d). Two weeks recovery after salinity, the survival rate in WT was 35%, while the survival rates in transgenic lines OE-17 and OE-56 were 88% and 78%, respectively, which were two times as many as that in WT (Fig. 2b). and MDA between wild-type and transgenic plants had no significant (P < 0.05) difference ( Fig. 3a-c). Under salt stress, the H 2 O 2 , O 2 − and MDA content of all lines increased slightly after exposed to salinity. However, the accumulation of H 2 O 2 , O 2 − and MDA in transgenic lines was much smaller than WT in response to salt stress ( Fig. 3a-c). To intuitively understand the oxidation status of chrysanthemum, the accumulation of H 2 O 2 and O 2 − , two major reactive oxygen species, was detected with 3, 3′-diaminovenzidine (DAB) staining and nitroblue tetazolium (NBT) staining. The consequence of research indicated that the accumulation of H 2 O 2 and O 2 in leaves of transgenic lines was significantly (P < 0.05) lower, compared with WT plants (Fig. 3d, e). These data suggested that WT plants suffered more severe membrane damage than DgWRKY5 transgenic plants. And it indicated that overexpression of DgWRKY5 conferred transgenic chrysanthemum greater tolerance to the oxidative stress associated with salt stress.

Accumulation of osmotic regulators in DgWRKY5 transformed chrysanthemum plants under salt stress.
In order to examine the effect of osmotic adjustment on the salt tolerance between WT and transgenic plants, we determined the contents of soluble protein, soluble sugar and proline in transgenic lines and wild-type plants (Fig. 4a-c). These three osmotic regulators had no significant (P < 0.05) difference between transgenic lines and wild-type plants under non-stress conditions. With the increase of salinity, the proline content of wild type and transgenic lines increased rapidly, and the accumulation in transgenic lines was significantly (P < 0.05) higher than in wild type (Fig. 4a). In addition, after 5 days of salt processing, the content of soluble protein and soluble sugar in wild-type and transgenic lines was changed rarely, then with the increase of salinity, the content of transgenic plants in the following 10 days increased by nearly twice as much as WT (Fig. 4a-c). These osmotic adjustment substances were positively correlated with the salt tolerance of plants. These results indicated that overexpression of DgWRKY5 plants increased salt tolerance by enhancing osmotic regulators.  (Fig. 4d-f). However, with the increase of salinity, the activities of SOD, POD and CAT in transgenic plants rapidly raised and remained high levels, which were markedly (P < 0.05) higher than WT plants ( Fig. 4d-f). These results suggested that salt tolerance in transgenic chrysanthemum was enhanced by the boost of antioxidant enzyme activities.

Analysis of antioxidant enzyme activity in
Effect of salt stress on growth, chlorophyll content and photosynthesis in DgWRKY5 transformed chrysanthemum plants. In order to study the effects of salt stress on growth and Photosynthesis of transgenic Chrysanthemum, we measured the root length, fresh weight, chlorophyll content and leaf gas exchange parameters under salt stress. Salt stress inhibited the growth and development of chrysanthemum, WT and transgenic plants both showed root atrophy and fresh weight reduction, but the root length, fresh weight of transgenic plants decreased less, compared to WT (Fig. 5a,b). As shown in Fig. 5c, under different salt concentrations, the chlorophyll increased first and then decreased, and the transgenic plants decreased slightly than WT. With the increase of NaCl concentration, the net photosynthetic rate, stomatal conductance and transpiration rate of WT and transgenic plants both decreased, and the reduction range of transgenic plants was less than WT ( Fig. 5d,e,g). On the contrary, intercellular CO 2 concentration increased with salinity under salt stress, and the amplitude of WT was higher than that of transgenic plants (Fig. 5f). These results demonstrate that salt stress hindered the growth and photosynthesis of chrysanthemum, while the DgWRKY5 transgenic plants had stronger salt tolerance compared with WT.

Expression of abiotic stress-related genes in DgWRKY5 transformed chrysanthemum plants.
To reveal the underlying regulatory mechanisms of DgWRKY5 transgenic lines in response to salinity stress, we investigated the relative expression level of eight stress-related genes through qRT-PCR, including DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2. The expression levels of these genes were no obvious difference in wild type and transgenic lines during normal conditions ( Fig. 6a-h). After salt stress, relative expression level of DgAPX and DgCAT genes which encoded ROS scavenging enzymes were significantly (P < 0.05) up-regulated in DgWRKY5 transgenic lines, comparing with WT plants increased by about two and three times, respectively (Fig. 6a,b). And the expression of ABA-responsive genes DgNCED3A, DgNCED3B and DgCuZnSOD had also greatly raised in transgenic lines compared to WT under salt treatment ( Fig. 6c-e). Moreover, other abiotic stress-response genes, such as DgP5CS, which functions in osmotic adjustment, DgCSD1, and DgCSD2 were all significantly (P < 0.05) increased in transgenic plants than WT under salinity condition (Fig. 6f-h). Therefore, DgWRKY5 may enhance salt tolerance of transgenic chrysanthemum by up-regulating expression levels of stress-related genes.

Discussion
Previous studies have shown that WRKY transcription factors play an important role in regulating the response of plants to abiotic stress 15,16 . In this research, we isolated and identified a transcription factor, DgWRKY5, from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C2H2 zinc-fingers in sequence analysis (Fig. S2). The phylogenetic analysis showed that DgWRKY5 is clustered with AtWRKY26 and AtWRKY25 from Arabidopsis thaliana, TaWRKY2 from Triticicum aestivum L. and OsWRKY30 from rice, all these genes belong to WRKY family group I (Fig. S3).
DgWRKY5 was significantly induced by salt stress, which is similar with AtWRKY25, AtWRKY33 in Arabidopsis thaliana and TaWRKY2 in Triticicum aestivum L 10,17 . Moreover, the DgWRKY5 transgenic chrysanthemum was more tolerant to salt stress which is similar to other WRKY transcription factors from same subgroup, including AtWRKY26, AtWRKY25, OsWRKY30 and TaWRKY2, which also strengthen transgenic plants' resistance to abiotic stress as described in the previous section. In addition, it is possible that the same subgroup transcription factors have analogous effects on abiotic stress. In addition, DgWRKY5 is also strongly induced by multiple stresses, including ABA, H 2 O 2 and GA. These results indicate that DgWRKY5 is a novel transcription factor of WRKY family, which may participate in response to abiotic stress.
In response to salt stress, plant cells often tend to accumulate some organic molecules, such as proline, soluble sugar and soluble protein in the cytoplasm, to maintain a high osmotic pressure and ensure the plant can be still absorb moisture from the soil. The accumulation of proline is a protective measure taken by plants in order to fight against salt stress 18 . And it has been reported that plants with higher proline and soluble sugar had better resistant to stress 19 . In our research, we found that the contents of osmotic adjustment substances in transgenic plants were noticeably higher than WT plants. These data indicated that DgWRKY5-overexpression plants may enhance the ability to withstand salt stress in plants by increasing the content of osmotic adjustment substances.
High salinity leads to reactive oxygen species (ROS) accumulation, which may injure cytomembrane, cause mediate lipid peroxidation and more oxidative damage in plants 20 . It was reported that abiotic stress can causes lipid peroxidation, leading to accumulation of MDA 21 . The content of MDA can reflect the degree of harm brought about by salt stress on plants 22 . In this experiment, DgWRKY5 transgenic plants produced a small amount Salt stress can cause plant growth and development to be seriously hindered, root length decreased and fresh weight reduction 24 . In our study, the higher the salinity was, the more inhibited the root length and fresh weight of chrysanthemum, but the inhibition of salinity was less on transgenic plants, compared with WT. Chlorophyll is the basic pigment for photosynthesis in plants, and its changes directly affect the photosynthesis of plants 25 . Salt stress increased chlorophyllase activity and promoted chlorophyll degradation, which caused chlorophyll content to decrease 26 . However, some studies have shown that salt stress can increase chlorophyll content significantly, it is considered that the binding between chlorophyll and chloroplast proteins in leaves is relaxed under salt stress, which makes chlorophyll easy to extract 27 . Our data revealed that DgWRKY5 transgenic plants had higher chlorophyll content than WT, and the content was both increased under 100 mM NaCl stress. Plant photosynthesis is the basic process of material accumulation and physiological metabolism in plant production process 28 . It is also an important means to analyze environmental affecting plant growth and metabolism. In this study, the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) decreased, while intercellular CO 2 concentration (Ci) increased under high salinity stress, it showed that the photosynthetic ability of mesophyll cells was further decreased, and the utilization of CO 2 was lower, leading to CO 2 excess and a corresponding decrease in photosynthetic product, so plants are more seriously damaged. However, these parameters of leaf gas exchange in DgWRKY5 transgenic plants showed that it was more photosynthetic than WT and had stronger salt resistance. These results suggest that DgWRKY5 transgenic plants may be resistant to salt stress by slowing salt damage to roots and enhancing leaf photosynthesis.
Under various stresses, transcription factors play important roles by modulating the target genes expression to strengthen the ability of plants stress tolerance 29 . P5CS gene, as a kind of osmotic protective agent, helps the plant to resist the change of osmotic imbalance under salt stress 30 . CSD1 and CSD2, which are very effective on the detoxification of superoxide radicals 31,32 . NCED is another important enzyme in the synthesis of ABA and has been determined to function in drought and salt stress 6 . In addition, CuZnSOD and APX have essential roles in enhancing the salt tolerance of sweet potato 33 . In this study, relative expression levels of stress responsive genes which were participated in oxidative stress-response (DgCAT and DgAPX), osmotic adjustment membrane protection (DgP5CS), and others mentioned above, were markedly up-regulated in DgWRKY5 transgenic lines compared with wild-type under salt stress. The results suggested DgWRKY5 may function as a constructive potential regulator of salt stress response pathway by controlling the expression of stress responsive genes.
In conclusion, DgWRKY5, a new WRKY transcription factor, was isolated from chrysanthemum and induced by abiotic stress. DgWRKY5-overexpression chrysanthemum showed enhanced salt tolerance compared with WT plants. In this study, we explored the physiological and molecular mechanism of DgWRKY5, and revealed that DgWRKY5 played roles as a positive regulator in salt stress-response through regulating ROS scavenging and osmotic adjustment system as well as expression levels of stress-related genes.

Methods
Plant materials and stress treatments. The experimental plant material is wild-type chrysanthemum variety "Jinba" (Dendronthema grandiform). Chrysanthemum seedlings were grown in a greenhouse at 25 °C/16 h lights, 22°C/8 h dark cycle (light intensity of 200 μmol m-2s-1; relative humidity of 70%). For salinity and ABA treatments, chrysanthemum plants at the 6-7 leaves stage were used to culture in 200 mM NaCl or in 100 μM ABA media, respectively. For the H 2 O 2 and GA treatments, the seedings were sprayed with 10 mM H 2 O 2 or 5 μM GA, respectively. Untreated plants were used as controls. Samples of all the treatments were harvested at 0, 1, 3, 6, 12, and 24 h, frozen in liquid nitrogen promptly and stored at −80 °C for RNA extraction. For tissue-specific expression analyses, roots, stems and leaves of the same untreated seedlings were likewise collected. Each experiment contained three biological repeats.
Cloning of DgWRKY5 gene. On the basis of transcriptions data in chrysanthemum seedlings under non-stress conditions and salt stress conditions applying 454 high throughout sequencing technique, a large amount of salt-induced transcription was appraised. DgWRKY5, a novel transcription factor, was obviously brought about by salt stress. To obtain the total RNA sequence of DgWRKY5, leaves of seedlings at the 6-7 leaves stage were collected after 24 h under 400 mM NaCl treatment. Total RNA from chrysanthemum leaves was extracted with TRIzol reagent (Mylab, Beijing). The full-length cDNA of DgWRKY5 sequence was obtained by PCR (polymerase chain reaction) and utilizing the gene specific primers (Table S1).
Expression of DgWRKY5 under salt treatment. We isolated total RNA from the chrysanthemum plants under salt stress treatment using TRIzol reagent (Mylab, Beijing) according to manufacturer's protocol. Then RNA was used for first-strand cDNA synthesis with reverse transcriptase (Transgen, Beijing) according to the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) was performed using SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA, USA) and Bio-Rad CFX96TM detection system. The gene Elongation Factor 1α (EF1α) was used as a reference for quantitative expression analysis. A final 20 μL qPCR reaction mixture contained 10 uL SsoFast EvaGreen supermix, 2 uL diluted cDNA sample, and 300 nM primers. Then the reactions were incubated under the following program: 1 cycle of 95 °C for 30 s, 40 cycles of 15 s at 95 °C and 30 s at 60 °C, and a single melt cycle from 65 to 95 °C. Each reaction had repeated at least three times and negative controls without templates were detected in case of contamination. Relative expression levels were calculated by the 2 −ΔΔCT method 34 .

Generation of DgWRKY5 transgenic Chrysanthemum plants. In order to get the generation of
DgWRKY5 transgenic Chrysanthemum, DgWRKY5 full-length cDNA was inserted into the expression vector pCAMBIA 2300 under the control of CaMV (cauliflower mosaic virus) 35 S promoter by replacing the gus gene. The obtained vector was transformed into chrysanthemum at leaf disk via Agrobacterium tumefaciens (strain LBA4404) according to An et al. 35 . The obtained DgWRKY5 transgenic chrysanthemum lines (OE-17 and OE-56) were employed in subsequent experiments.
Salt treatment of transgenic chrysanthemum. For the salinity tolerance experiment, three-week-old WT and DgWRKY5 transgenic chrysanthemum seedlings (OE-17 and OE-56) were used. Chrysanthemum seedlings were watering with an increasing concentration of NaCl every fifth day over the following days: 100 mM on day 1-5, 200 mM on day 6-10, 400 mM on day 11-15, based on referring to the suggestion of Chen et al. 36 .Root length and fresh weight were measured at 0, 5, 10 and 15 days. The survival rate of the seedlings was calculated after 2 weeks recovery processes.
Physiological changes in transgenic chrysanthemum. Leaves of the above WT and transgenic plants were harvested for biochemical analysis after 0 days, 5 days and 10 days of salt treatment. Accumulation of H 2 O 2 was assayed as described by Alexieva et al. 37 . Content of MDA was mensurate following the method of Zhang et al. 38 . Accumulation of proline was estimated according to Liu et al. 39 . Accumulation of soluble protein and soluble sugar was measured following Wang et al. 40 . Activities of SOD and POD were measured following Pan et al. 41 . And CAT activities were assayed following Zhang et al. 42 . The chlorophyll content was determined following Qin et al. 43 . Leaf gas exchange parameters were measured following Khaled et al. 26 , setting the endogenous light intensity is 600μmol·m −2 ·S −1 , the concentration of CO 2 is 360 μL·L −1 , the temperature is 25 °C. Expression of abiotic stress response genes in DgWRKY5 transgenic chrysanthemum. In order to valuate the expression of abiotic stress-related genes, the RNA of WT and transgenic lines were extracted for reverse transcription to generate cDNA as described above. Then relative expression levels of several abiotic stress-related genes in DgWRKY5 transgenic chrysanthemum were detected by qRT-PCR with EF1α served as the internal reference gene. Abiotic stress-response genes monitored were DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2. All relevant primers in the article are listed in Table S1.