DISCUSSION
Plants encounter natural seasonal changes in temperature and respond by optimizing their growth and development. For tea, an unsuitable growth temperature seriously affects yield and quality, causing enormous economic losses. Adaption to low temperature and frost have been extensively reported in tea plants mainly through increasing the enzymatic activity responsible for free radical scavenging and accumulating high levels of indole and sucrose (Hao et al., 2018a,b; Wang et al., 2013; Shen et al., 2015; Zhou et al., 2020). With the presently climbing global temperature, the mechanism by which HT affects tea quality has gradually become of considerable interest. Therefore, we investigated the physiological response of tea plants to heat stress and explored ways by which their heat tolerance might be improved. Previous work has shown that anthocyanin-related genes and anthocyanin contents are down-regulated under HT (Shen et al., 2019). In this study, we focused on the influence of extreme temperature (40 ℃) on metabolites in tea leaves, and found that 11 of 15 detected flavonoids were significantly reduced after HT treatment. HPLC further verified that HT inhibited the accumulation of catechins in tea leaves. Finally, in parallel with the metabolic changes in tea flavonoids, expression levels of flavonoid-related genes were repressed by HT.
The effect of HT on secondary metabolism remains largely unexplored in tea leaves, including the effect on tea’s characteristic compounds, catechins. Increasing evidence supports that HSFs play a critical role in heat stress tolerance. HSFs are divided into three main classes, HSFA, HSFB, and HSFC, and plants have multiple different HSFs: there are 21 total in Arabidopsis , 25 in rice, 28 in Populus trichocarpa , and 25 in tea, (Scharf et al., 2011; Zhang et al., 2015; Muthuramalingam et al., 2020; Zhang et al., 2020a). AHA motifs are usually present in HSFA subfamily members and confer transcriptional activator functionality. Within this subfamily, HSFA1 proteins function as “master regulators” by activating HSR genes and enhancing thermotolerance, while HSFA2 proteins have been identified as coactivators of HSFA1-targeted transcription (Busch et al., 2005; Charng et al., 2007).
As we observed levels of CsHSFA1b and CsHSFA2 to be more responsive to HT treatment than those of other transcripts, we further investigated the effect of CsHSFA1b and CsHSFA2 on the HSR in tea. Although the heat-stress-activated transcriptional network that regulates thermo-responsive gene expression has been characterized in many species (Liu et al., 2013), evidence concerning the interaction between metabolites and CsHSF-involved heat stress responses is yet lacking in tea. Here, we transiently silenced CsHSFA1b andCsHSFA2 in tea plants, and found that catechin content was much higher than in controls; in addition, flavonoid-related genes were up-regulated. We concluded that CsHSFA1b and CsHSFA2 could negatively regulate expression levels of flavonoid-biosynthesis-related genes and ultimately impair flavonoid production.
As JA signaling is known to regulate HT-mediated flavonoid synthesis, we further investigated this pathway as a prospective molecular mechanism and explored the involved components. In tea plants, a series of CsMYBs has been identified to play key roles in flavonoid biosynthesis, including CsAN1, CsMYB1, CsMYB2, CsMYB26, CsMYB5a, CsMYB5e, and CsMYB184 (Li et al., 2022; Wang et al., 2018; Wei et al., 2019; Jiang et al., 2018; Wei et al., 2018; Sun et al., 2016). Among those, CsAN1 in particular has been demonstrated to regulate catechin accumulation by forming the MBW complex through interaction with bHLH transcription factors (CsGL3 and CsEGL3) and recruitment of a WD-repeat protein CsTTG1 (Zhao et al., 2013; Sun et al., 2016). Herein, our findings further highlight CsJAZ6 as influencing formation of the MBW complex through competing with CsEGL3 and CsTTG1, and thereby inhibiting catechin biosynthesis. Our previous study also demonstrated CsJAZ6 to interact with CsMYC2 (Chen et al., 2021), the core mediator of the JA pathway. Thus, it is also possible that CsJAZ6 could indirectly regulate catechin biosynthesis through repressing the CsMYC2-mediated JA pathway.
JA is an emerging player in alleviating the deleterious effects of adverse heat stress conditions on plants (Balfagón et al., 2019). Although there remains much to be elucidated regarding the function of JA in the HSR, a series of findings give us hints that JA could contribute to plant adaption to HT. In Arabidopsis , thecpr5-1 mutant, which exhibits constitutive activation of the JA signaling pathway, displays enhanced heat tolerance, whereas thecoi1 mutant, which is deficient in JA biosynthesis, is more sensitive to HT (Clarke et al., 2009). Warm temperature has also been shown to increase levels of 12HSO4-JA and consequently alter levels of bioactive JA-Ile; this leads to the stabilization of JAZ proteins, which in turn facilitates plant growth (Zhu et al., 2021). In tomato and rice, HT also inhibits JA accumulation (Pan et al., 2019; Wu et al., 2022), with the rice protein OsHTG3 conferring heat tolerance through activating expression of two JAZ genes (Wu et al., 2022). Exogenous application of JA has proved effective in improving plant stress tolerance under adverse temperature conditions, whether low or high (Du et al., 2013; Pan et al., 2019). Here, we observed that HT reduced JA content in tea leaves as well as the expression levels of JA biosynthesis and responsive genes. Meanwhile, we noticed thatCsJAZ6 , the expression of which was activated, showed an opposite pattern in responding to HT. Importantly, we further identified that CsHSFA1b and CsHSFA2 positively regulate the expression of CsJAZ6by directly binding to the HSE1 cis -element in its promoter. Specifically, CsHSFA1b could bind all three HSEs in the promoter of CsJAZ6, whereas CsHSFA2 could only bind the HSE1 element. In addition, we noticed that HSE1 had a gap of two bases between TTC and GAA, whereas HSE2 and HSE3 had a gap of three bases between two corecis -elements. Thus, our data suggest that CsHSFAs may recognize HSE elements in the form of “nGAAnnTTCn”, which is consistent with previous reports (Xiao et al., 1988; Ogawa et al., 2007). Altogether, our data indicate the involvement of JAZ proteins in the heat stress response, namely by acting as inhibitors of JA signaling.
Flavonoid biosynthesis is affected by multiple environment factors and regulated by plant hormones (Liang et al., 2020; Zhou et al., 2020). JA, classified as a defensive hormone, plays important roles in the regulation of flavonoid biosynthesis when plants face adversities (An et al., 2021; Ding et al., 2022). The metabolic profiling in this study revealed MeJA content to be reduced under HT and encouraged us to investigate whether JA is involved in HT-induced regulation of flavonoid accumulation. Our results suggest that JA treatment could increase catechin content in tea leaves under HT conditions to levels paralleling those in plants grown under normal conditions. Conversely, treatment with ibuprofen, a JA synthesis inhibitor, repressed flavonoid accumulation under normal conditions. However, the promotive effect of JA was not enhanced under normal conditions, nor was the inhibitory effect of ibuprofen enhanced under heat stress. To further distinguish the effect of JA biosynthesis and JA signaling on HT-regulated flavonoid production, we treated tea plants with JA plus MG132, a specific inhibitor of JA signal transduction. We found that the addition of MG132 could abolish the effect of JA on flavonoid synthesis under HT condition. Altogether, our findings highlight impaired JA signaling, not JA biosynthesis, as a key hub in HT-regulated flavonoid synthesis. In parallel, JA treatment was able to partially alleviate the inhibitory effect of HT on anthocyanin synthesis in Arabidopsis . Based on these findings, we further developed a mechanistic model of JA-mediated HT modulation of flavonoid metabolite biosynthesis (Figure 9). Comprehensively, CsHSFAs mediate the JA pathway in regulating secondary metabolism under HT conditions, and this mechanism is conserved inArabidopsi s and tea plants.