*2.4. Overexpression of CsWRKY7 A*ff*ects Flowering in Transgenic Arabidopsis*

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 *CsWRKY7* transcript levels were significantly higher in transgenic lines than in wild type. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 15

**Figure 4**. Germination rates of *CsWRKY7*-overexpressing Arabidopsis lines under different stress conditions (**A**) Expression of *CsWRKY7* in the leaves of WT and three transgenic lines (L8, L10, and L14), respectively. Data are shown as the mean ± S.E. (*n* = 3). (**B**) The germination rate of WT and transgenic lines. Their seeds grown on the 1/2 MS supplied with different concentrations of NaCl, ABA, mannitol and PEG for 4 days. Experiments were performed in five biological replicates. Fifty seeds of each WT and three transgenic lines were germinated in one replicate. Data are shown as the mean ± S.E. (**C**) Germination performance of WT and transgenic lines were taken under normal conditions, 150 mM NaCl, 0.3 μM ABA, 200 mM mannitol, and 15% PEG treatment for 7 days. **Figure 4.** Germination rates of *CsWRKY7*-overexpressing Arabidopsis lines under different stress conditions (**A**) Expression of *CsWRKY7* in the leaves of WT and three transgenic lines (L8, L10, and L14), respectively. Data are shown as the mean ± S.E. (*n* = 3). (**B**) The germination rate of WT and transgenic lines. Their seeds grown on the 1/2 MS supplied with different concentrations of NaCl, ABA, mannitol and PEG for 4 days. Experiments were performed in five biological replicates. Fifty seeds of each WT and three transgenic lines were germinated in one replicate. Data are shown as the mean ± S.E. (**C**) Germination performance of WT and transgenic lines were taken under normal conditions, 150 mM NaCl, 0.3 µM ABA, 200 mM mannitol, and 15% PEG treatment for 7 days. Asterisk indicated that the expression level is significantly different from the value of the control ('\*\*\*' *p* < 0.001).

Asterisk indicated that the expression level is significantly different from the value of the control ('\*\*\*'

*p* < 0.001). Since *CsWRKY7* responds to abiotic stress and hormone treatments, seed germination was tested with WT and homozygous 35S::*CsWRKY7* transgenic lines to determine the specific role of CsWRKY7 TF in abiotic stress. Though the germination rate of overexpressing lines was higher than that of WT (Figure 4B,C), the difference was not statistically significant under normal growth condition or under abiotic stresses, suggesting that *CsWRKY7* overexpressing lines may not be sensitive to the above induction during seed germination. No significant difference in root growth was observed between WT and overexpressing lines in the presence of different stress media (Figure 5). However, the root Since *CsWRKY7* responds to abiotic stress and hormone treatments, seed germination was tested with WT and homozygous 35S::*CsWRKY7* transgenic lines to determine the specific role of CsWRKY7 TF in abiotic stress. Though the germination rate of overexpressing lines was higher than that of WT (Figure 4B,C), the difference was not statistically significant under normal growth condition or under abiotic stresses, suggesting that *CsWRKY7* overexpressing lines may not be sensitive to the above induction during seed germination. No significant difference in root growth was observed between WT and overexpressing lines in the presence of different stress media (Figure 5). However, the root elongation of 35S::*CsWRKY7* lines were higher than that of WT with 1/2 MS medium, indicating that CsWRKY7 might promote root growth at the seedling stage under normal growth condition.

elongation of 35S::*CsWRKY7* lines were higher than that of WT with 1/2 MS medium, indicating that

CsWRKY7 might promote root growth at the seedling stage under normal growth condition.

*AP1* and *LFY*.

**Figure 5.** Root growth of *CsWRKY7*-overexpressing Arabidopsis lines under different stress conditions. Seeds were germinated for 4 days on 1/2 MS medium, and the seedlings were then transferred to 1/2 MS medium with or without different treatment for 10 days. (**A**) Normal condition, (**B**) 150 mM NaCl treatment, (**C**) 0.3 μM ABA treatment, (**D**) 200 mM mannitol treatment, and (**E**) 15%PEG6000 treatment, and (**F**) root length were measured at 10 d after the transfer, each line included three seedlings, experiments were performed in four biological replicates. Data are represented as the mean ± SE of 12 seedlings. The significant level is presented by the asterisks (\* *p* < **Figure 5.** Root growth of *CsWRKY7*-overexpressing Arabidopsis lines under different stress conditions. Seeds were germinated for 4 days on 1/2 MS medium, and the seedlings were then transferred to 1/2 MS medium with or without different treatment for 10 days. (**A**) Normal condition, (**B**) 150 mM NaCl treatment, (**C**) 0.3 µM ABA treatment, (**D**) 200 mM mannitol treatment, and (**E**) 15%PEG6000 treatment, and (**F**) root length were measured at 10 d after the transfer, each line included three seedlings, experiments were performed in four biological replicates. Data are represented as the mean ± SE of 12 seedlings. The significant level is presented by the asterisks (\* *p* < 0.05).

#### 0.05). *2.5. CsWRKY7 Overexpressing Lines Exhibit the Phenotype of Delayed Flowering*

*2.5. CsWRKY7 Overexpressing Lines Exhibit the Phenotype of Delayed Flowering*  In order to investigate whether CsWRKY7 TF participated in plant growth and development, the phenotype of *CsWRKY7*-overexpressing lines and WT was observed during plant growth process. As shown in Figure 6A, B, WT plants bolt earlier than transgenic lines after 25-day growth. When wide-type plants were in silique stage, *CsWRKY7* overexpressing plants were in bolting stage or vegetative growth stage after 35-day growth. To elucidate the regulation mechanism of *CsWRKY7* in flowering, several flowering-related genes was analyzed (Figure 6C). *SUPPRESSOR OF CONSTANS 1* (*SCO1*), a floral integrator, was significantly induced in 35S::*CsWRKY7* lines compared to WT. No noticeable difference in the relative expression level of *CONSTANS* (*CO*) gene that the central regulators in the photoperiod pathway was observed between WT and transgenic lines. As a longdistance transport signal, *FLOWERING LOCUS T* (*FT*) was significantly downregulated in transgenic lines. Simultaneously, two important genes related to inflorescence meristem—*APETALA1* (*AP1*) and LEAFY (*LFY*)—were significantly suppressed in Line 8. The expression levels of *AP1* and *LFY* were downregulated by 64% and 60%, respectively. However, a flowering inhibitor, *FLOWERING LOCUS C (FLC)*, was significantly downregulated in overexpressing lines, while its homologous gene *FLOWERING LOCUS M (FLM)* was upregulated in 35S::*CsWRKY7* lines, compared to WT. Therefore, we speculate that CsWRKY7 delay flowering time might through inhibiting the transcription level of In order to investigate whether CsWRKY7 TF participated in plant growth and development, the phenotype of *CsWRKY7*-overexpressing lines and WT was observed during plant growth process. As shown in Figure 6A, B, WT plants bolt earlier than transgenic lines after 25-day growth. When wide-type plants were in silique stage, *CsWRKY7* overexpressing plants were in bolting stage or vegetative growth stage after 35-day growth. To elucidate the regulation mechanism of *CsWRKY7* in flowering, several flowering-related genes was analyzed (Figure 6C). *SUPPRESSOR OF CONSTANS 1* (*SCO1*), a floral integrator, was significantly induced in 35S::*CsWRKY7* lines compared to WT. No noticeable difference in the relative expression level of *CONSTANS* (*CO*) gene that the central regulators in the photoperiod pathway was observed between WT and transgenic lines. As a long-distance transport signal, *FLOWERING LOCUS T* (*FT*) was significantly downregulated in transgenic lines. Simultaneously, two important genes related to inflorescence meristem—*APETALA1* (*AP1*) and LEAFY (*LFY*)—were significantly suppressed in Line 8. The expression levels of *AP1* and *LFY* were downregulated by 64% and 60%, respectively. However, a flowering inhibitor, *FLOWERING LOCUS C (FLC)*, was significantly downregulated in overexpressing lines, while its homologous gene *FLOWERING LOCUS M (FLM)* was upregulated in 35S::*CsWRKY7* lines, compared to WT. Therefore, we speculate that CsWRKY7 delay flowering time might through inhibiting the transcription level of *AP1* and *LFY*.

**Figure 6.** Identification of the *CsWRKY7* in transgenic Arabidopsis. (**A**,**B**) Overexpression of *CsWRKY7* delayed Arabidopsis flowering in different developmental stages: (**A**) Representative photographs of 25-day-old-plant of WT and transgenic lines (L8, L10, and L14) growing in normal conditions. (**B**) 35-day-old-plant. Each line included four seedlings. Experiments were performed in five biological replicates. (**C**) Expression patterns of flowering-related genes in *CsWRKY7* overexpressing and wild type Arabidopsis. Leaf samples were harvested from 25-day-old transgenic lines and WT. The error bars indicate the means ± S.E. (*n* = 5), \* indicates that the differences are significant (*p* < 0.05), \*\* indicate that the differences are highly significant (*p* < 0.01). **Figure 6.** Identification of the *CsWRKY7* in transgenic Arabidopsis. (**A**,**B**) Overexpression of *CsWRKY7* delayed Arabidopsis flowering in different developmental stages: (**A**) Representative photographs of 25-day-old-plant of WT and transgenic lines (L8, L10, and L14) growing in normal conditions. (**B**) 35-day-old-plant. Each line included four seedlings. Experiments were performed in five biological replicates. (**C**) Expression patterns of flowering-related genes in *CsWRKY7*-overexpressing and wild type Arabidopsis. Leaf samples were harvested from 25-day-old transgenic lines and WT. The error bars indicate the means ± S.E. (*n* = 5), \* indicates that the differences are significant (*p* < 0.05), \*\* indicate that the differences are highly significant (*p* < 0.01).

#### **3. Discussion 3. Discussion**

WRKY TFs widely participate in plant stress responses and plant growth and development. This study indicated that *CsWRKY7* is involved in abiotic stresses. Constitutive overexpression of *CsWRKY7* not only promotes root growth, but also delays the flowering time in transgenic WRKY TFs widely participate in plant stress responses and plant growth and development. This study indicated that *CsWRKY7* is involved in abiotic stresses. Constitutive overexpression of *CsWRKY7* not only promotes root growth, but also delays the flowering time in transgenic Arabidopsis plants by suppressing flowering-related genes.

Arabidopsis plants by suppressing flowering-related genes. CsWRKY7, homologous to AtWRKY7 and AtWRKY15, belongs to subgroup IId of WRKY family. AtWRKY7 transcription factor not only acted as a negative regulator in PAMP-mediated immune response but also participated in leaf senescence [34–36]. *CsWRKY7* was abundant in old leaves and roots in tea plant, which was somewhat similar to the expression patterns of *AtWRKY7* in Arabidopsis leaves, thus it could be speculated that CsWRKY7 might be involved in tea growth and development. Besides, *CsWRKY7* gene expression was induced by various osmotic stresses and hormones exposure (Figure 3). Sequence analysis identified several potential stress-responsive elements in the promoter region including WBOXHVISO1, CCAATBOX1, GT1GMSCAM4, and MYB1AT and pollen-specific cis-regulatory elements (GTGANTG10), which participated in regulating the response to sugar, heat, NaCl, and drought stresses, respectively (Table 1). Some previous studies have reported that these cis-regulatory elements were stress-related. For example, MYB1AT existed in the promoter region of the Arabidopsis dehydration gene *RD22*, and GT1GMSCAM4 was present in the promoter region of the pathogen and salt-inducible gene *SCaM-4*, which consisted with the cis-regulatory elements in the promoter of *CsWRKY7*, suggesting that *CsWRKY7* participated in various stress responses. (Figure 3) [37,38]. Though two pollen-specific ciselements present in the promoter, the expression level of *CsWRKY7* in flower was not significantly different from that in bud, which might be probably due to the complicated regulation mechanisms of gene expression. Besides, two hormone response elements—SA and auxin-response elements are also found in this region. The hormone induction experiment showed that *CsWRKY7* gene was responded to gibberellin and auxin treatment (NAA and 2,4-D), indicating that CsWRKY7 might also CsWRKY7, homologous to AtWRKY7 and AtWRKY15, belongs to subgroup IId of WRKY family. AtWRKY7 transcription factor not only acted as a negative regulator in PAMP-mediated immune response but also participated in leaf senescence [34–36]. *CsWRKY7* was abundant in old leaves and roots in tea plant, which was somewhat similar to the expression patterns of *AtWRKY7* in Arabidopsis leaves, thus it could be speculated that CsWRKY7 might be involved in tea growth and development. Besides, *CsWRKY7* gene expression was induced by various osmotic stresses and hormones exposure (Figure 3). Sequence analysis identified several potential stress-responsive elements in the promoter region including WBOXHVISO1, CCAATBOX1, GT1GMSCAM4, and MYB1AT and pollen-specific cis-regulatory elements (GTGANTG10), which participated in regulating the response to sugar, heat, NaCl, and drought stresses, respectively (Table 1). Some previous studies have reported that these cis-regulatory elements were stress-related. For example, MYB1AT existed in the promoter region of the Arabidopsis dehydration gene *RD22*, and GT1GMSCAM4 was present in the promoter region of the pathogen and salt-inducible gene *SCaM-4*, which consisted with the cis-regulatory elements in the promoter of *CsWRKY7*, suggesting that *CsWRKY7* participated in various stress responses. (Figure 3) [37,38]. Though two pollen-specific cis-elements present in the promoter, the expression level of *CsWRKY7* in flower was not significantly different from that in bud, which might be probably due to the complicated regulation mechanisms of gene expression. Besides, two hormone response elements—SA and auxin-response elements—are also found in this region. The hormone induction experiment showed that *CsWRKY7* gene was responded to gibberellin and auxin treatment (NAA and 2,4-D), indicating that CsWRKY7 might also be involved in GA- and auxin- and abiotic stress-mediated signaling, but the corresponding mechanism remains be further explored.

be involved in GA- and auxin- and abiotic stress-mediated signaling, but the corresponding mechanism remains be further explored. Plant growth such as flowering, is a sophisticated regulated process that can be affected by Plant growth such as flowering, is a sophisticated regulated process that can be affected by diverse environmental stimuli. Previous research has reported that several WRKY members participate widely

diverse environmental stimuli. Previous research has reported that several WRKY members participate widely in plant growth and development. AtWRKY44 was reported to be involved in in plant growth and development. AtWRKY44 was reported to be involved in trichome and seed coat development in Arabidopsis [15]. AtWRKY4/-6/-7/-11/-57 were involved in leaf senescence [4]. WRKY transcription factors were involved in the regulation of plants flowering. For example, AtWRKY75 directly bound to the promoter region of *FT* gene, thereby positively regulating Arabidopsis flowering [27]. Both AtWRKY12 and AtWRKY13 transcription factors regulated Arabidopsis flowering time under short day [26]. Additionally, mango MlWRKY12 [24], soybean GsWRKY20 [23], and rice OsWRKY11 [25] also had a regulatory role in flowering. However, few studies of WRKY TFs in tea plant were reported, especially their functions related to plant development. In this study, its ORF was overexpressed in Arabidopsis to determine whether or not CsWRKY7 involved in plant growth. Transgenic analysis indicated that the overexpression of CsWRKY7 altered growth and flowering time of transgenic Arabidopsis (Figures 5 and 6). Gene analysis showed that two meristem identity genes including *AP1* and *LFY* were downregulated in transgenic Arabidopsis, compared with WT (Figure 6). Interestingly, previous studies have reported that there existed one or more W-boxes in the promoter region of *AP1* and *LFY* [39]. As noted earlier, WRKY TFs could specifically bind to W-box [4,5,11–13]. Therefore, the reason why *CsWRKY7* overexpressing lines delayed flowering may lie in that *CsWRKY7* gene directly bound to the promoter regions of *AP1* and *LFY* and inhibited their transcription levels.

However, *FLC,* a suppressor in flowering, was significantly downregulated in transgenic lines, while its homologous gene *FLM* was upregulated, indicating that *FLM* and *FLC* regulated flowering through different pathways. This finding was consistent with that of Katia (2003) [40]. *CO* gene encoding a B-box protein activated the transcription of *FT*. This study indicated that no difference in the expression level of *CO* was observed between transgenic lines and wild type, whereas the downstream gene *FT* was significantly inhibited, indicating that *FT* gene might be inhibited by other complexes such as PRC2, LHP1, SMZ, and TEM [41,42]. Hence, it could be speculated that *CsWRKY7* gene may be involved in flowering regulation independent of the autonomous and vernalization pathway. Generally, these data support our hypothesis that *CsWRKY7* delays flowering. The potential role of *CsWRKY7* as a negative regulator in flowering sheds new light on the development of tea plants at transcription level, but its regulation mechanism should be further explored.

Although *CsWRKY7* was induced by some abiotic stresses in *C. sinensis*, no significant difference in seed germination rate and seedling root growth was observed between *CsWRKY7* overexpressing Arabidopsis lines and WT under abiotic stresses (salt, mannitol, PEG, and exogenous ABA) (Figures 4 and 5), which might be possibly due to the lack of correlation between the levels of mRNA and protein encoded by CsWRKY7, or due to the effect of the inserted transgene on the phenotype. However, the function of *CsWRKY7* gene in terms of osmotic stress response remains to be further characterized. The results of our study not only reveal an important role of CsWRKY7 in plant development, but also provide a foundation for breeding late-blooming tea plants.

To summarize, this study determined the response of *CsWRKY7* to various abiotic stresses and hormones treatments. Our data revealed that CsWRKY7 delayed flowering and promoted root growth in Arabidopsis. Nevertheless, it is necessary to investigate further the pathways through which CsWRKY7 regulates both the development and stress response of tea plant.

#### **4. Materials and Methods**

### *4.1. Plant Materials, Growth Conditions, and Stress Treatments*

Two-year-old tea seedlings (*Camellia sinensis* cv. 'Longjing 430 ) were grown in the greenhouse of Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRICAAS).

The buds, tender stem, flowers, and roots, and leaves at different developmental stages were collected for tissue-specific analysis. The methods of abiotic stresses were essentially the same with those described in our previous research [43] and the materials were collected for further analysis. *N. benthamiana* was used for protein subcellular localization, and was grown in a climate chamber (24 ± 2 ◦C, 70% relative humidity, and 12 h/12 h light–dark photoperiod). *A. thaliana* ecotype, Columbia-0 (Col-0), was used as the background material and experiment control. The seeds of wild type and transgenic lines were surface sterilized with 75% ethanol and 0.01% (*v*/*v*) Tween-20 for 8–10 min, and then were washed by distilled water 3−4 times. The seeds were placed on 1/2 MS medium plate at 4 ◦C in darkness for 48 h, then grown in a climate incubator under a 16 h day/8 h night cycle, respectively at 22 ◦C/20 ◦C.
