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Literature Sharing | Divergent MYB paralogs determine spatial distribution of linalool mediated by JA and DNA demethylation participating in aroma formation and cold tolerance of tea plants
Release time:
2025-03-19
Linalool not only is one of characteristic flavour volatiles of tea, contributing to floral aroma, but also a kind of defensive compounds, playing essential roles in resistance against biotic/abiotic stresses. Although the linalool synthases have been identified, much is unknown about the regulation mechanism in tea plants. We identified two pairs of MYB paralogs as linalool biosynthesis activators, in which one pair (CsMYB148/CsMYB193) specifically expressed in flowers, and another (CsMYB68/CsMYB147) highly expressed in flowers, leaves, fruits and roots. These activators interacted with CsMYC2 to form MYC2-MYB complexes to regulate linalool synthase. While Jasmonate ZIM-domain (JAZ) proteins served as the linalool biosynthesis repressors by interfering MYC2-MYB complex. Further, we found that the transcripts of CsMYB68/CsMYB147 were significantly upregulated by jasmonic acid (JA) to improve linalool products during tea processing and that linalool pathway may as one of the downstream pathways of JA signalling and DNA methylation processes to participate in cold resistance. Under cold stress, JA signalling was activated to elevate the abundance of MYC-MYB complexes; meanwhile, DNA demethylation was also activated, leading to declining methylation levels and increasing transcripts of CsMYB68/CsMYB147. Our study provides a new insight into synergistically improving tea quality and tea plant resistance.
In tea plants, linalool biosynthesis pathway has been well studied, in which the linalool synthases are the determining components of linalool concentration in flowers and leaves . Recently, numerous studies have showed that MYB21-type transcription factors were the crucial regulators of linalool biosynthesis in plants, such as in Arabidopsis and Freesia hybrida . To dig linalool biosynthesis regulators in tea plants, the MYB21 homologues, including CsMYB148 and CsMYB193, were identified from tea genome (Figure 1a; Figure S1). Expression analysis showed that the two MYB21-type homologues were specifically expressed in tea flowers, but not in leaves (Figure 1b; Table S1). However, metabolic analysis showed that linalool mainly accumulated in tea flowers and young leaves (Figure 1c). Additionally, the linalool synthase gene Terpene Synthase76 (CsTPS76) was highly expressed in both flowers and leaves (Figure 1b; Figure S2; Table S1).
Figure 1 Character of candidate MYB transcription factors involved in regulating linalool biosynthesis in tea plants
For exploring how these MYB transcription factors regulate linalool biosynthesis, yeast one hybrid (Y1H) assay was performed to check the interaction between MYB proteins and CsTPS76 promoter (approximately 2 kb). The data indicated that all of MYB proteins could bind to the CsTPS76 promoter region (Figure 2a). Further, four MYB binding sites were detected in the CsTPS76 promoter region, namely P1, P2, P3 and P4 (Figure 2b). In order to confirm the binding sites of MYB proteins, we performed the Electrophoretic Mobility Shift Assay (EMSA). EMSA data showed that CsMYB68 could bind to the four sites, and CsMYB193 could bind to two of them, including P3 and P4. While CsMYB147 and CsMYB148 only could bind to the P3 site (Figure 2c–f).
Figure 2 CsMYB68, CsMYB147, CsMYB148, CsMYB193 and CsMYC2 directly regulate CsTPS76 in tea plants.
Given both four MYB transcription factors and CsMYC2 participating in linalool biosynthesis in tea plants, the relationship between CsMYBs and CsMYC2 was investigated. Yeast two hybrid (Y2H) assay indicated the interactions between CsMYC2 with CsMYB68, CsMYB147 and CsMYB193, but not with CsMYB148 (Figure 3a). To validate the interactions, Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) pull-down assays were performed. BiFC and pull-down assays further indicated that CsMYC2 could interact with CsMYB68, CsMYB147, CsMYB148 and CsMYB193 to form divergent MYC-MYB complexes respectively (Figure 3b–f; Figure S5). To explore the functions of MYC2-MYB complexes in regulating linalool biosynthesis, we created a transient transactivation system with CsTPS76 promoter (Figure 3g). In Arabidopsis protoplasts, we found that the CsTPS76 promoter was slightly activated when MYB or MYC2 were separately expressed, while significantly activated by MYC2-MYB complexes (Figure 3h).
Figure 3 MYC2-MYB complexes activate CsTPS76 promoter.
CsMYC2 is not only a positive regulator of linalool biosynthesis, but also the key JA signal regulator in tea plants, implying the crucial role of JA in linalool biosynthesis. To comprehensively explore the function of JA on linalool biosynthesis, we focused on the JAZ proteins, which serve as the repressors of JA signal transduction. In tea genome, at least 13 JAZ members were detected, with dramatic tissue-specific expression patterns . In which, only four JAZ genes, including CsJAZ1, CsJAZ8, CsJAZ12 and CsJAZ13, were highly expressed in young leaves and in developing flowers (Figure 4a,b; Table S4). In order to investigate whether JAZ proteins regulated linalool biosynthesis together with other linalool biosynthesis regulators, CsJAZ1, CsJAZ8, CsJAZ12 and CsJAZ13 were introduced into Y2H system to be screened by the MYB transcription factors. The results indicated that only CsJAZ12 could interact with all four MYB transcription factors (Figure 4c). Further, both BiFC and GST pull-down assays were performed to verify the interactions between CsJAZ12 and CsMYBs in vivo and in vitro respectively (Figure 4d–h; Figure S5). In addition, we also detected the strict conserved JAZ-MYC2 complex mediated by CsMYC2 and CsJAZ12 with Y2H and BiFC assays (Figure S6). These data imply that CsJAZ12 maybe is the major JAZ protein in regulating linalool biosynthesis via interfering the formation of MYC2-MYB complexes.
Figure 4 Identification of JAZ proteins in regulating linalool biosynthesis.
To verify the function of CsJAZ12 on linalool biosynthesis, we generated the stable transgenic Arabidopsis overexpressing CsJAZ12. Expression analysis showed that compared with the WT, the expression levels of several monoterpene synthases, such as AtTPS11, AtTPS14 and AtTPS21, were significantly suppressed in CsJAZ12-overexpression lines (Figure 5a). Then asODN assay was performed to interfere the CsJAZ12 expression. qRT-PCR assay validated the suppression of CsJAZ12 expression in CsJAZ12-KD compared with Sense ODN (Figure 5b). Metabolic analysis showed that linalool concentration was significantly increased in CsJAZ12-KD compared with Sense ODN, which was consistent with the elevation of CsTPS76 expression level (Figure 5b,c).
Figure 5 CsJAZ12 is a repressor of linalool biosynthesis in tea plants
Most aroma compounds were generated largely during tea processing, particularly the terpenes. For example, in processing of white tea, green tea, black tea and oolong tea, the withering or spreading technologies were required to improve the floral aroma of tea. Linalool is the characterized volatile of several types of tea, such as white tea and green tea. The linalool, with a low level in fresh leaves, mostly was generated during tea processing, but the mechanism has not been unveiled. For exploring the mechanism, we collected tea samples at 0, 12, 24 and 48 h during withering (Figure 6a), which was the key process of white tea processing. GC–MS analysis showed that withering significantly increased the linalool content, particularly at 24 h (Figure 6b). Consistently, we found that the expression levels of CsTPS76, CsMYB68 and CsMYB147 also increased during withering stage, but CsMYB148 or CsMYB193 not (Figure 6c,d), suggesting that CsMYB68 and CsMYB147 participated in linalool biosynthesis during tea withering.
Figure 6 JA regulating linalool biosynthesis in tea processing via MYC2-MYB complexes.
Previous studies have showed that linalool plays a crucial role in priming cold tolerance of tea plants via CBF-dependent pathway; however, the regulation mechanism of linalool biosynthesis under cold stress is largely unknown in plants. Here, we also found that the linalool biosynthesis was significantly activated in tea leaves by cold treatment (Figure 7a,b). Consistently, the expression levels of CsTPS76, CsMYB68, CsMYB147 and CsMYC2 were upregulated by cold treatment (Figure 7c). In tea plants, JA plays a positive role in cold tolerance, which acts on the ICE-CBF pathway . It was not surprised that JA signalling participated in linalool biosynthesis regulation under cold stress, for the expression elevation of linalool regulator CsMYC2 under cold stress. Expectedly, the JA biosynthesis and signal transduction pathways were significantly activated in tea leaves under cold stress (Figure 7d), as well as the concentrations of JA and JA-Ile (Figure 7e). Inversely, according to the chlorophyll fluorescence imaging, Fv/Fm (variable fluorescence/maximal fluorescence) values and nitroblue tetrazolium (NBT) staining, DIECA treatment aggravated the cold damage of tea leaves compared with control (Figure 7f–h). These data indicate that linalool pathway may as one of the downstream pathways of JA signalling to participate in cold resistance of tea plants. To validate our hypothesis, we turned back to investigate the cold tolerance of tea plants with silencing the CsMYBs, CsMYC2 or CsJAZ12, respectively. Both Fv/Fm values and the indicators of hydrogen peroxide indicated that CsMYB68, CsMYB147 and CsMYC2 served as the positive regulators of cold tolerance (Figure 7i,j), while CsJAZ12 as the negative regulator (Figure 7k), which were consistent with their regulatory functions on linalool biosynthesis in tea leaves.
Figure 7 CsMYBs-CsMYC2-CsJAZ12 complex regulating cold tolerance in tea plants.
Our previous study has showed that demethylation process played key roles in cold tolerance of tea plants by reducing methylation levels of a series of cold-related genes to improve their expression. To know the function of DNA methylation on linalool biosynthesis under cold stress in tea plans, weighted methylation levels of the genes (including gene body region and 2 kb upstream and downstream of gene body) were analysed in tea leaves under cold stress. Interestingly, we found that the methylation levels of CsMYB68 and CsMYB147 were strongly decreased at 6 and 12 h after cold treatment, however, the methylation levels of their patterners, such as CsMYB148, CsMYB193, CsMYC2 and CsTPS76, showed no significant inhibition under cold stress (Figure 8a). Then we further analysed the expression patterns of demethylase genes in tea leaves under cold stress. Heatmap analysis showed that the demethylase family genes, such as REPRESSOR OF SILENCING 1 (ROS1), were significantly activated by cold treatment (Figure 8b), indicating that the demethylation process induced by cold stress may directly act on CsMYB68 and CsMYB147 genes to upregulate their expression and the linalool biosynthesis for improving cold tolerance of tea plants.
Figure 8 Cold stress triggers the demethylation process of CsMYB68 and CsMYB147 in tea leaves.
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