*2.3. Subcellular Localization of CsWRKY7*

To examine the localization of CsWRKY7, the full-length cDNA of *CsWRKY7* was fused to enhanced green fluorescent protein (eGFP). As shown in Figure 2A, *CsWRKY7*-eGEP was localized to the nucleus when it was transiently expressed in *Nicotiana benthamiana* leaves. The green fluorescence was observed both in the cell membrane and nucleus in the GFP control vector. Similarly, green fluorescence was also observed in the nucleus of transgenic Arabidopsis seedlings (Figure 2B). These results showed that CsWRKY7 was targeted to the nucleus.

The qRT-PCR result indicated that *CsWRKY7* was expressed in all detected tissues. The expression level in old leaves was approximately 4.4 times as high as that in buds (Figure 3A). To investigate the role of CsWRKY7 TF in abiotic stresses, we analyzed the expression patterns of *CsWRKY7*, when it was exposed to temperature, sodium chloride (NaCl), sucrose (Suc), polyethylene glycol (PEG), and mannitol (Man) stresses. As shown in Figure 3B,C, in leaves, *CsWRKY7* was downregulated under high temperature stress, especially at 8 h, its expression level decreased by 65%. When exposed to low temperature, the expression level of *CsWRKY7* was not significantly changed at 4 h or 12 h. However, the expression level of *CsWRKY7* was upregulated under the treatments of NaCl, Man, and PEG. For example, its expression level increased gradually after NaCl and PEG treatment for 3 h, reaching a peak of 3.26- and 6.23- fold at 72 h, respectively. Though two sugar-responsive elements existed in the promoter region, no significant difference in the expression level of *CsWRKY7* after sucrose treatment. In order to investigate whether *CsWRKY7* expression was regulated by phytohormones, two-year-old tea plants were exposed to the major plant hormones including indolyl-3-acetic acid (IAA), naphthalene-1-acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), and gibberellins (GA). As presented in Figure 3D, the expression level of *CsWRKY7* was elevated under these hormone treatments except IAA. These results implied that *CsWRKY7* might be involved in the regulation of abiotic stress and hormones networks.

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**Figure 2** Subcellular localization analysis of CsWRKY7 protein: (**A**) GFP alone (upper panel) and *CsWRKY7*-eGFP (middle panel) were transiently expressed in tobacco epidermal cells. Representative images from left to right in each panel were taken under fluorescence, chlorophyll, transmitted light and an overlay of both channels. Scale bar = 20 μm. (**B**) The roots of *CsWRKY7*-eGFP (bottom panel) in transgenic Arabidopsis was used for observation of GFP fluorescence. Representative images from left to right were taken under fluorescence, bright field and an overlay of both channels. Scale bar = 100 μm. **Figure 2.** Subcellular localization analysis of CsWRKY7 protein: (**A**) GFP alone (upper panel) and *CsWRKY7*-eGFP (middle panel) were transiently expressed in tobacco epidermal cells. Representative images from left to right in each panel were taken under fluorescence, chlorophyll, transmitted light and an overlay of both channels. Scale bar = 20 µm. (**B**) The roots of *CsWRKY7*-eGFP (bottom panel) in transgenic Arabidopsis was used for observation of GFP fluorescence. Representative images from left to right were taken under fluorescence, bright field and an overlay of both channels. Scale bar = 100 µm.

The qRT-PCR result indicated that *CsWRKY7* was expressed in all detected tissues. The

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 6 of 15

treatments except IAA. These results implied that *CsWRKY7* might be involved in the regulation of abiotic stress and hormones networks. **Figure 3** Relative expression levels of *CsWRKY7* in tea plants (**A**) Tissue expression profiles of *CsWRKY7* in 'Longjing 43'. Different tissues include bud, 1st leaf, 2nd leaf, 3rd leaf, old leaf, flower, stem, and root. The expression levels of *CsWRKY7* in different tissues were compared with the bud. (**B**) The transcript levels of *CsWRKY7* under cold (4 °C) and heat (38 °C). *CsWRKY7* expression levels are detected at four different time points (0, 4, 8 and 12 h) post-temperature stress treatment. (**C**) Relative expression levels of *CsWRKY7* under different abiotic stress. Two-year-old tea seedlings were treated with 150 mM NaCl, 90 mM sucrose (Suc), 10% (*w*/*v*) PEG4000 (PEG) and 90 mM mannitol (Man), and samples were harvested at the time intervals indicated. (**D**) The transcript levels of *CsWRKY7* under various phytohormone, including 100 μM indolyl-3-acetic acid (IAA), 5 μM naphthalene-1-acetic acid (NAA), 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 100 μM abscisic acid (ABA), 50 μM methyl jasmonate (MeJA), 5 mM salicylic acid (SA), and 100 μM gibberellins (GA), which were added to the culture solution, the functional leaves were harvested at 0, 3, 6, or 24 h posttreatment. Treated samples at 0 h served as controls. Error bars represent ± S.E. for three independent experiments. The significant level is presented by the asterisks (\* *p* < 0.05, \*\* *p* < 0.01). *2.4. Overexpression of CsWRKY7 Affects Flowering in Transgenic Arabidopsis*  **Figure 3.** Relative expression levels of *CsWRKY7* in tea plants (**A**) Tissue expression profiles of *CsWRKY7* in 'Longjing 43'. Different tissues include bud, 1st leaf, 2nd leaf, 3rd leaf, old leaf, flower, stem, and root. The expression levels of *CsWRKY7* in different tissues were compared with the bud. (**B**) The transcript levels of *CsWRKY7* under cold (4 ◦C) and heat (38 ◦C). *CsWRKY7* expression levels are detected at four different time points (0, 4, 8 and 12 h) post-temperature stress treatment. (**C**) Relative expression levels of *CsWRKY7* under different abiotic stress. Two-year-old tea seedlings were treated with 150 mM NaCl, 90 mM sucrose (Suc), 10% (*w*/*v*) PEG4000 (PEG) and 90 mM mannitol (Man), and samples were harvested at the time intervals indicated. (**D**) The transcript levels of *CsWRKY7* under various phytohormone, including 100 µM indolyl-3-acetic acid (IAA), 5 µM naphthalene-1-acetic acid (NAA), 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 100 µM abscisic acid (ABA), 50 µM methyl jasmonate (MeJA), 5 mM salicylic acid (SA), and 100 µM gibberellins (GA), which were added to the culture solution, the functional leaves were harvested at 0, 3, 6, or 24 h post-treatment. Treated samples at 0 h served as controls. Error bars represent ± S.E. for three independent experiments. The significant level is presented by the asterisks (\* *p* < 0.05, \*\* *p* < 0.01).

Seed Germination and Root Growth in Transgenic Plants under Abiotic Stresses

To assess the function of CsWRKY7 TF, an expression construct pH7FGW2.0-*CsWRKY7* was transformed into *A. thaliana*. Three homozygous transgenic lines—L8, L10, and L14—were confirmed by real-time PCR with *Actin-2* gene serving as an internal reference. As shown in Figure 4A, the
