**1. Introduction**

Plants suffering from diverse abiotic stresses in the developmental process have evolved and obtained a series of mechanisms to combat with these environmental stresses. Majority of transcription factors (TFs) participate in these adaptive mechanisms [1]. WRKY TFs—a large family of transcription factors in plants—are involved in response to multiple stresses and external stimuli [2–4].

WRKY transcription factor is named after the WRKY domain, which contains one or two highly conserved WRKYGQK motifs and one zinc-finger motif [5,6]. According to the number of WRKY domains and the type of zinc finger motif, WRKY TFs can be divided into three types: Group I, containing two WRKY domains and one C2H2 zinc-finger structure. Group II, containing one WRKY domain, and sharing the same zinc finger structure with group I. In addition, based on the amino acid sequence of the DNA-binding domain, Group II can be further divided into IIa, IIb, IIc, IId, and IIe subgroups; Group III also contains one WRKY domain, but its zinc finger structure is C2HC [4,7].

SWEET POTATO FACTOR1 (SPF1) specifically binding to W-box, the first defined WRKY TF, negatively regulates the expression of storage proteins and β-amylase in sweet potato [8]. Since then, considerable efforts have been made to unveil the role of WRKY TFs in plant response to biotic and abiotic stresses. In *Arabidopsis thaliana*, it has been demonstrated that WRKY TFs contribute to pathogen resistance, salinity, heat, and drought stress responses [7,9–11]. W-box (TTGACT/C) was present in the promoters of several stress-associated genes. Since WRKY proteins bind to W-box, they induce the expressions of these stress-associated genes [4,5,11–13]. Many studies have shown that WRKY proteins participate in secondary metabolism (phenylpropane, alkaloids, and terpenes.), and hormone signaling [10]. Moreover, WRKY proteins have been demonstrated to involve in plant growth processes, such as leaf senescence, shoot branching, trichome, and flowering. For instance, potato ScWRKY1 [14], Arabidopsis TTG2 [15], and MINISEED3 are involved in seed growth [16]; Arabidopsis AtWRKY6, -45, -53, -57, and -70 are involved in senescence regulation [17–21]; Arabidopsis AtWRKY12, -13, -75, -34, mango MIWRKY12, soybean GsWRKY20, and rice DIf1 have been reported to be involved in plant flowering regulation or pollen development [22–27]. However, few studies have reported WRKY TFs in tea plant.

Tea plant (*Camellia sinensis* L.) is an important commercial woody crop. As a nonalcoholic beverage, tea is processed from tea leaves and is widely consumed worldwide. Early blooming in tea plant and adverse environmental factors affect the quality and yields of tea. Increasing studies have demonstrated that WRKY TFs play pivotal roles in plant growth and abiotic stresses. In one previous study, two WRKY TFs—CsWRKY31 and CsWRKY48—were reported to participate in O-methylated catechin biosynthesis in tea plant (*Camellia sinensis*) [28]. Several *CsWRKY* genes were induced by diverse stresses such as temperature, ABA, and NaCl [29–31]. However, the roles of CsWRKYs in plant growth and development remain unclear. The current study aims to provide a functional characterization of *CsWRKY7*, a member of the group IId WRKY family in tea plant. CsWRKY7 was a close homolog of Arabidopsis *AtWRKY7* and *AtWRKY15*, two well-characterized Group IId WRKY proteins with important roles in plant defense, leaf senescence, and abiotic stress responses [32–34]. When it is exposed to diverse stresses, *CsWRKY7* is upregulated and localized to the nuclei in both tobacco leaves and Arabidopsis roots. *CsWRKY7*-overexpressing Arabidopsis did not respond to abiotic stresses. However, the overexpression of *CsWRKY7* delayed flowering. Gene analysis revealed the downregulation of several flowering-related genes in *CsWRKY7* overexpression lines. A better understanding of the flowering mechanisms is conducive to breeding late-blooming tea plants.

#### **2. Results**

#### *2.1. Isolation and Characterization of CsWRKY7 from C. sinensis*

*CsWRKY7*, one *WRKY* gene, was amplified from tea leaves cDNA by RT-PCR. This amplified *CsWRKY7* contained the complete open reading frame (ORF) of 972 bp encoding 323 amino acids. CsWRKY7 protein had a predicted molecular mass of 35.37 kDa and an isoelectric point of 9.47. Sequence analysis showed that CsWRKY7 in tea plant shared a high similarity (71.94%) to AcWRKY15 in kiwifruit (Genbank: PSS21265). Additionally, CsWRKY7 was found to have a nuclear localization signal (NLS) at 227–230 amino acid region, and have two motifs, namely, HARF structure and a shorter conserved structural motif (C-region which is known as a Ca2+-dependent calmodulin-binding domain) (Figure 1A). Thus, CsWRKY7 was assigned to group IId subfamily. Phylogenetic analysis showed that CsWRKY7 was closely related to AcWRKY15, PtrWRKY7, VvWRKY7, AtWRKY7, and AtWRKY15 (Figure 1B). AtWRKY7 and AtWRKY15 TFs have been reported to be involved in plant defense response to bacterial pathogens, leaf senescence, or mitochondrial-mediated osmotic stress [32–34],

their homologous genes *CsWRKY7* was predicted to be a TF that may participate in plant development and respond to stress treatment. mediated osmotic stress [32–34], their homologous genes *CsWRKY7* was predicted to be a TF that may participate in plant development and respond to stress treatment.

involved in plant defense response to bacterial pathogens, leaf senescence, or mitochondrial-

**Figure 1** Protein sequence alignment and phylogenetic relationship of CsWRKY7 (**A**) Sequence alignment of the deduced CsWRKY7 protein with other Group IId WRKY homologs. Black lines highlight the conserved region of WRKY. Blue box and arrows highlight the WRKY domain and the zinc-finger motif, respectively. The conserved primary sequences—HARF motif, C-region, and putative NLS—are boxed in red. (**B**) The phylogenetic tree of CsWRKY7 and 17 other group IId WRKY subfamily members. Accession number for group IId WRKY members: AtWRKY7 (AAK28440), AtWRKY11 (AAK96194), AtWRKY15 (AF224704), AtWRKY17 (AAL13049), AtWRKY21 (AAK28441), AtWRKY39 (AAK96198), AtWRKY74 (AAL35291), AcWRKY7 (PSS36058.1), AcWRKY15 (PSS21265.1), VvWRKY7 (RVX23377.1), PtrWRKY7 (XP\_006380693.1), PtrWRKY15 (XP-002310122), GhWRKY17 (HQ651068), TcWRKY7 (EOX91521), TcWRKY15 (XP-007047365), CmWRKY17 (AJF11725), and DzWRKY15 (XP-022740807). **Figure 1.** Protein sequence alignment and phylogenetic relationship of CsWRKY7 (**A**) Sequence alignment of the deduced CsWRKY7 protein with other Group IId WRKY homologs. Black lines highlight the conserved region of WRKY. Blue box and arrows highlight the WRKY domain and the zinc-finger motif, respectively. The conserved primary sequences—HARF motif, C-region, and putative NLS—are boxed in red. (**B**) The phylogenetic tree of CsWRKY7 and 17 other group IId WRKY subfamily members. Accession number for group IId WRKY members: AtWRKY7 (AAK28440), AtWRKY11 (AAK96194), AtWRKY15 (AF224704), AtWRKY17 (AAL13049), AtWRKY21 (AAK28441), AtWRKY39 (AAK96198), AtWRKY74 (AAL35291), AcWRKY7 (PSS36058.1), AcWRKY15 (PSS21265.1), VvWRKY7 (RVX23377.1), PtrWRKY7 (XP\_006380693.1), PtrWRKY15 (XP-002310122), GhWRKY17 (HQ651068), TcWRKY7 (EOX91521), TcWRKY15 (XP-007047365), CmWRKY17 (AJF11725), and DzWRKY15 (XP-022740807).

#### *2.2. Sequence Analysis of CsWRKY7 Promoter 2.2. Sequence Analysis of CsWRKY7 Promoter*

According to 'Shuchazao' tea genome data, the promoter sequence of *CsWRKY7* was amplified by PCR. A 1680 bp *CsWRKY7* promoter sequence was cloned and the putative cis-elements were predicted through the PlantCARE database. Two functional elements—TATA-box and CAAT-box were widely distributed in the promoter region. Additionally, a group of elements which respond to such environmental stresses as phytohormone stress (salicylic acid and auxin), light, plant growth (pollen), and abiotic stresses (anaerobic, sugar, wounding, NaCl, dehydration, and heat) were found (Table 1). Interestingly, many MYB-recognition sites were present in the promoter region of *CsWRKY7*, and these motifs also existed in dehydration-responsive gene *RD22A*, indicating that *CsWRKY7* might be involved in dehydration stress and be modulated by MYB members. In addition, *CsWRKY7* promoter region existed two W-boxes, which specially bind to WRKY TFs. These prediction results indicated that CsWRKY7 TF may play a vital role in stress responses and plant growth through multiple signal transduction pathways. According to 'Shuchazao' tea genome data, the promoter sequence of *CsWRKY7* was amplified by PCR. A 1680 bp *CsWRKY7* promoter sequence was cloned and the putative cis-elements were predicted through the PlantCARE database. Two functional elements—TATA-box and CAAT-box—were widely distributed in the promoter region. Additionally, a group of elements which respond to such environmental stresses as phytohormone stress (salicylic acid and auxin), light, plant growth (pollen), and abiotic stresses (anaerobic, sugar, wounding, NaCl, dehydration, and heat) were found (Table 1). Interestingly, many MYB-recognition sites were present in the promoter region of *CsWRKY7*, and these motifs also existed in dehydration-responsive gene *RD22A*, indicating that *CsWRKY7* might be involved in dehydration stress and be modulated by MYB members. In addition, *CsWRKY7* promoter region existed two W-boxes, which specially bind to WRKY TFs. These prediction results indicated that CsWRKY7 TF may play a vital role in stress responses and plant growth through multiple signal transduction pathways.


**Table 1.** Hormone-, light-, and stress-responsive elements in the 1680 bp 50 -flanking sequence of CsWRKY7 TF as predicted by the PlantCARE website.
