*3.3. CsHCT Transcription Levels in Oolong Tea Plants during Four Growing Seasons and at Different Altitudes*

Temperature affects gene expression related to the secondary metabolism of plants, which regulates the generation of secondary metabolites and enables plants to adapt to environmental changes. To evaluate the influence of temperature on *CsHCT* expression, we used qRT-PCR to analyze its transcription levels in one-tip-two-leaf tissues of oolong tea plants during four seasons and compared their *CsHCT* expression. Specifically, the tea samples were collected from Zhushan, Nantou County, Taiwan in April, June, September, and December. The results showed that the *CsHCT* expression level of tea samples from December was the highest, and that of the tea samples from June was the lowest (Figure 4A). According to the average monthly temperature in Zhushan between 2015 and 2016 released by the Central Weather Bureau, *CsHCT* expression levels were negatively correlated with temperature (Figure 4B). *CsHCT* expression levels were high in low-temperature seasons.

**Figure 4.** *CsHCT* transcription levels in four growing seasons. (**A**) The qRT-PCR analysis of CsHCT transcription levels in four growing seasons in the field. (**B**) Monthly average temperature in the Zhushan area. Total RNA was isolated from tissues of handpicked one-tip-two-leaf oolong tea samples collected in April, June, September, and December. Transcript levels of *CsHCT* were calculated and normalized to that of *18S rRNA*. Values represent means ± SD from five biologically independent experiments. Values with different letters are significantly different at *p* < 0.05, according to a post hoc least significant difference (LSD) test.

Altitude influences temperature, humidity, and sunlight intensity. Temperature decreases as altitude increases. To further verify *CsHCT* expression, the tea samples collected from the Alishan area of Nantou County from altitudes of 700, 1000, and 1300 m and bud tissues were analyzed using qRT-PCR to determine CsHCT expression. The results demonstrated that the *CsHCT* expression level was the highest at 1300 m and decreased as the altitude decreased (Figure 5).

**Figure 5.** *CsHCT* expression levels at various altitudes; qRT-PCR analysis of *CsHCT* transcription levels in the Alishan area of Nantou County. Total RNA was isolated from the tissues of handpicked one-tip-two-leaf oolong tea samples collected at altitudes of 700, 1000, and 1300 m. Transcription levels of *CsHCT* were calculated and normalized to that of *18S rRNA*. Values represent means ± SD from three biologically independent experiments. Values with different letters are significantly different at *p* < 0.05, according to a post hoc LSD test.
