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.