**4. Discussion**

Acyltransferase in higher plants can catalyze transfer of acyl group to donor substrate. Acyl esters are produced as a result of the acyl group transfer from the donor substrate to the acceptor substrate [12]. Acyltransferase can be divided into two protein families, namely BAHD acyltransferase and serine carboxypeptidase-like acyltransferase, according to different donor substrates. HCT is categorized into the clade Vb of BAHD acyltransferase and characterized by its ability to catalyze various substrates.

Amino acid analysis results demonstrated that the structure and function of the CsHCT and HCT sequences of other higher plants were highly conserved (Figure 1). The amino acid domain was highly conserved in clade Vb that possessed a particularly conserved sequence SXXDL in the BAHD acyltransferase family [13]. However, whether the amino acid domain affects the catalytic function of enzymes remains to be investigated and verified. BAHD acyltransferase acts mainly in the cytoplasm of plant cells. Its substrate, acyl coenzyme A thioesters, can perform biosynthesis in various cell organs and be transferred to the cytoplasm by the transporter on the cell membrane, which facilitates the catalytic reaction of the BAHD acyltransferase [14]. After analyzing the cellular localization of CsHCT, we inferred that CsHCT is primarily located in the cytoplasm, and the amino acid sequence does not possess a signal peptide or apparent hydrophobic end (Figure 2A). Therefore, this study inferred that CsHCT primarily reacts in the cytoplasm for catalytic reaction.

Our data determined that the *CsHCT* expression level in the YS tissues of tea seedlings was higher than that in the OS and bud tissues, whereas the *CsHCT* expression level in tea plants was higher in OS and YS tissues (Figure 3). Studies on *Trifolium pretense* have shown that *HCT1* is primarily expressed in stem and flower tissues, whereas *HCT2* is mainly expressed in leaf and flower tissues, which indicates that HCT1 and HCT2 have different catalytic functions in diverse plant tissues [15]. *P. trichocarpa* possesses seven *PtrHCTs* that can be expressed in the tissues of various plant parts and exhibit differences with respect to their relative performance. In particular, *PtrHCT1* and *PtrHCT6* are primarily expressed in stem tissues, whereas *PtrHCT3* has a higher level of expression in leaf tissues [16]. In this study, nonlignified YS tissues of tea seedlings were found to contain a relatively large amount of *CsHCT* transcripts. The *CsHCT* expression level in YS tissues was eight times higher than that in OS tissues. This indicated that secondary metabolites and the expression of related biosynthesis genes in tissues vary according to the growth stages of *C. sinensis* L.

When plants are under environmental stresses, the expression of genes related to the biosynthesis of secondary metabolites is induced, which results in the generation and accumulation of compounds such as phenylpropanoid, flavonoids, and anthocyanins that can increase plants' tolerance to stresses [4]. Our results demonstrated that the *CsHCT* expression level in *C. sinensis* L. was relatively high in winter and at high altitudes (Figures 4 and 5), indicating that *CsHCT* has a high level of expression in low temperatures. The *CsHCT* expression level increased under low-temperature stress and decreased under high-temperature stress (Figure 6A). Thus, *CsHCT* expression is induced in low temperatures and may be involved in the defense pathways against low-temperature stress. Research demonstrated that HCT expression is regulated by biotic and abiotic stresses, thereby increasing stress tolerance in plants [6]. Our data indicated that *CsHCT* expression in *C. sinensis* L. can be induced with low-temperature, high-salinity, and drought stresses, and the expression level was particularly high with ABA treatment (Figure 6B).

Phytohormone ABA involved in stress tolerance in plants can be divided into ABA-dependent and ABA-independent signaling pathways. ABA-dependent pathways transmit signals through ABA, thereby activating downstream transcription factors such as the ABRE-binding factor/ABA-responsive element-binding protein, myelocytomatosis, and myeloblastosis to regulate plants' stress tolerance [17,18]. This study demonstrated that *CsHCT* expression was induced by the abiotic stresses of low temperature, high salinity, and drought as well as ABA treatment. The signals of the three stresses may be transmitted through ABA-dependent pathways and may affect the expression of transcription factors of genes associated with the regulation of secondary metabolism. In this study, our results demonstrated the relationship between CsHCT expression and hormone signaling in oolong tea plants and may help improve the quality and possible health benefits of tea in the future.
