INTRODUCTION
Temperature is one of the most important environmental factors affecting the seasonal growth and geographic distribution of plants (Li et al., 2018). Tea (Camellia sinensis ) is an economic crop with wide popularity around the world for its stimulating health effects and mellow flavor, which are attributable to the remarkable composition of flavonoids, caffeine, and theanine it contains. Nowadays, tea-growing countries include 64 countries spread across five continents of the world spanning tropical and subtropical as well as temperate regions (Wei et al., 2018). In China, the cultivated area occupied by ​​tea trees comprises 3.059 million hectares sited in 22 of the country’s 35 provinces. Notably, the temperature range suitable for tea-growing is 20 °C to 30 °C, thus the increased prevalence and frequency of high temperature (HT) conditions around the world have seriously affected the yield and quality of tea.
The catechin biosynthetic pathway has been identified as producing the primary characteristic flavonoid components of tea. The enzymes of this pathway include chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), which sequentially catalyze chalcone to produce the intermediate naringenin. Subsequently, dihydroflavonol 4-reductase (DFR), leucoanthocyanidin 4-reductase (LAR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR) are responsible for the formation of final products under the control of the MBW complex (Baudry et al. 2004; Zhao et al. 2013; Li. 2014; Huang et al. 2016). This complex consists of R2R3 MYB transcriptional factors (MYB21, MYB75, MYB90, MYB113, or MYB114) (Borevitz et al., 2000; Zimmermann et al., 2004; Stracke et al., 2007; Allan et al., 2008; Gonzalez et al., 2008; Rowan et al., 2009; Shan et al., 2020; Zhang et al., 2021), and basic-helix-loop-helix (bHLH) transcription factors (Toledo-Ortiz et al., 2003) such as enhancer of glabra3 (EGL3) (Zhang et al., 2003) and the WD-repeat protein transparent testa glabra1 (TTG1) (Walker et al., 1999). Investigation of the MBW complex has suggested that additional regulators which influence its formation and function would also participate in catechin biosynthesis.
HT conditions refer to the ambient temperature rising above the optimum temperature for a period, which causes reversible damage to plant growth and development (Alcázar and Parker. 2011). Recent studies have demonstrated that various signaling pathways are integrated to regulate the plant heat stress response, including the heat shock transcription factor-heat shock protein pathway, calcium ion-calmodulin (Ca2+-CaM) pathway, reactive oxygen pathway, and hormone pathways (Mittler et al., 2012). As the terminal components of heat shock signal transmission, heat shock factors (HSFs) directly regulate thermo-responsive gene expression after perception of ambient HT and as such play a central role in the heat shock response process. Most HSFs recognize heat shock elements (HSEs; nGAAnnTTCn or nTTCnnGAAn) in the promoters of heat-inducible genes (Xiao et al., 1988; Ohama et al., 2017). HSF proteins are divided into A, B, and C classes, among which members of the A class (HSFA) act as master regulators in the heat stress response (HSR); these mainly include HSFA1 and HSFA2 (Scharf et al., 2011). Based on recent research, HSFA1 can activate 65% of heat stress-inducible genes (Liu et al., 2011), while HSFA2 has been identified to act downstream of HSFA1 and regulate a subset of genes involved in thermotolerance (Charng et al., 2007, Yoshida et al., 2011; Liu et al., 2018; Friedrich et al., 2021). Heterologous overexpression of HSFA2 from Lilium longiflorum , Zea mays, andOryza sativa in Arabidopsis has been shown to confer heat tolerance (Xin et al., 2010; Yokotani et al., 2008; Gu et al., 2019). While several CsHSF genes in the tea genome have been predicted based on bioinformatics studies, the involvement of CsHSFs in the regulation of secondary metabolism in tea plants under HT yet remains unclear (Zhang et al., 2020b).
JA is an oxylipin phytohormone that acts as a defensive signal to protect plants from biotic and abiotic stress, and in this role regulates most secondary metabolites (Zhai et al., 2015; Mao et al., 2017; Wasternack et a., 2019). Recently, the JA pathway and its components have been identified. In the absence of bioactive JA, JAZ proteins interact with MYC transcription factors to block downstream processes (Katsir et al., 2008; Wasternack et al., 2013). JA transduces external signals to reprogram metabolic pathways that initiate the production of defense compounds against biotic and abiotic stress (Mao et al., 2017; Chen et al., 2019; Jing et al., 2021). Furthermore, JA can induce flavonoid accumulation by increasing the expression of members of the MBW complex (Qi et al., 2011; An et al., 2015; Wang et al., 2019). However, whether JA-mediated flavonoid metabolites are involved in the adaption of tea plants to HT stress has not been examined.
In this study, we explored the HT-regulatory networks of tea metabolism and found that HT decreases both JA biosynthesis and catechin accumulation in tea leaves. Based on the involvement of JA in HT-regulated catechin biosynthesis, we further demonstrated that CsJAZ6, through integrating HT signal and JA pathway, acts as an important negative regulator in catechin accumulation. Using a combination of biochemical and genetic analysis, we determined HT to activate CsHSFA1b and CsHSFA2 and those proteins to directly bind the HSEcis -element in the promoter of CsJAZ6 , thereby up-regulating CsJAZ6 transcription. More importantly, our data further suggested that CsJAZ6 could repress catechin accumulation through direct interaction with components of the catechin biosynthetic regulator complex, CsEGL3 and CsTTG1. Altogether, our work builds a bridge between plant internal hormones and the external environment to offer a good guide for tea cultivation.