As the most well-known and globally consumed central nervous system stimulant, caffeine is a purine alkaloid natural product usually derived from tea and coffee. Caffeine has a wide range of health benefits on the human body, and plays crucial roles in pollination, resistance to herbivore attacks, and pathogen infections in plants (Zhao et al., 2020). While caffeine biosynthetic pathways have been extensively studied in tea (Camellia sinensis L) and coffee plants, the regulation of caffeine biosynthesis is not understood (Zhao et al., 2020).

Tea Caffeine Synthase1 (TCS1) is the first N-methyltransferase gene reported in the tea plant, possessing the 1-N methyltransferase activity responsible for converting theobromine to caffeine (Kato et al., 2000). Studies on the structure–activity of TCS1 and genetic variations in the TCS1 gene of tea plant populations have supported that TCS1 is a determination enzyme for caffeine content (Jin et al., 2016). To explore the regulation of caffeine biosynthesis, 23 candidate transcription factors (TFs) from Weighted Gene Co-expression Network Analysis were screened in a luciferase reporter gene activation system driven by the TCS1 promoter (Figure S1). MYB184 (TEA029017) showed the highest TCS1 promoter activation with 4.7-fold (Figure 1a). Yeast one-hybrid assay showed that the −828 to −1670 bp region of TCS1 promoter, which contains an MYBCORE and a fused MYB1AT-MYBPLANT, was critically required for MYB184 recognition (Figure 1b). In planta promoter, trans-activation assays further confirmed the regions required for binding to and activating of the TCS1 promoter by MYB184 to be between −1596 and −1670 bp (Figure 1c). EMSA assay was performed to further validate the binding of MYB184 to the fused MYB1AT-MYBPLANT motif in vitro (Figure 1d–f).

We then examined the function of MYB184 in regulating caffeine synthesis in tea plants. An antisense oligodeoxynucleotide (asODN) interference experiment was performed with tea plant shoot tips to knock down MYB184 expression (MYB184-KD) (Figure 1g). Accordingly, caffeine contents and the expression of TCS1 were significantly reduced in MYB184-KD shoot tips compared with the senseODN control (Figure 1h–i). However, overexpression of MYB184 (MYB184OE) in tea plant transgenic hairy root lines significantly up-regulated TCS1 transcription and thereby increased the caffeine contents as compared with wild-type root controls (Figure 1j–m).

KeKecha (Camellia ptilophylla, KKC in short), belonging to the Thea section, had significantly lower caffeine but higher theobromine (Figure 1n). Although a previous study showed that TCS1 in KKC had lower NMT activity compared with TCS1 in modern tea cultivars (Jin et al., 2016), we detected a significantly lower expression level of TCS1 in KKC than in other tea cultivars (Figure 1o). To understand why TCS1 in KKC is down-regulated, we cloned and compared the promoter sequences of TCS1 from KKC and from SCZ. However, the alignment of the promoter sequences did not show critical Indels or SNPs on MYB binding sites (Figure 1p). On the other hand, transcriptome analyses showed that only MYB184 expression level was significantly lower by ~14-fold in KKC than in other tea cultivars (Figure 1q,r). We thus proposed that the lower MYB184 transcript level in KKC might be the cause of the reduced TCS1 expression level.

We further cloned the promoter of the MYB184 gene from KKC (proMYB184^KKC), and compared it with those from other tea cultivars. A 437-bp long terminal repeat (LTR) insertion was identified only in the proMYB184^KKC at the site of −982 bp, but not in these promoters from other tea cultivars, as verified with both MYB184 promoter cloning and detection with PCR primers specific for the LTR insertion (Figure 1s). In tea plants, LTR insertion in a gene usually leads to suppression of the gene expression (Xia et al., 2020), which may explain the lower expression level of MYB184 in KKC than in other tea cultivars. Indeed, GUS reporter assays showed that the proMYB184^KKC with the LTR insertion exhibited clearly lower promoter activity than four representative promoters without the LTR insertion from tea cultivars (Figure 1t). We thus concluded that the LTR insertion in the promoter of MYB184 resulted in suppressed MYB184 expression, leading to the lower TCS1 transcript level and thereby lower caffeine contents in KKC.

To expand MYB184 activation of TCS1 to other Camellia species, we examined several other wild tea relatives that are known to contain significantly lower levels of caffeine compared with modern tea cultivars (Figure 1u). They also have lower TCS1 and MYB184 expression levels compared with modern tea cultivars containing higher levels of caffeine (Figures 1v,w and S2). This further supports the indispensable role of MYB184 in activation of TCS1 gene expression and caffeine biosynthesis in C. sinensis.

In summary, we characterized MYB184 as the major activator of TCS1 and caffeine production in tea plants. An LTR insertion in the MYB184 promoter in wild tea C. ptilophylla explained its low TCS1 expression level and caffeine content. Our study may offer a molecular tool for breeding low-caffeine tea varieties to meet the market demands.

作为最知名和全球消费的中枢神经系统兴奋剂，咖啡因是一种嘌呤生物碱天然产物，通常来源于茶和咖啡。咖啡因对人体有广泛的健康益处，并且在植物的授粉、抗食草动物攻击和病原体感染方面发挥着至关重要的作用（Zhao et al ., 2020）。虽然已经在茶（ Camellia sinensis L）和咖啡植物中广泛研究了咖啡因生物合成途径，但对咖啡因生物合成的调控尚不清楚（Zhao et al ., 2020）。

茶咖啡因合成酶1 ( TCS1 ) 是第一个在茶树中报道的N-甲基转移酶基因，具有负责将可可碱转化为咖啡因的 1- N甲基转移酶活性 (Kato et al ., 2000 )。对茶树种群TCS1结构-活性和TCS1基因遗传变异的研究支持TCS1是咖啡因含量的测定酶(Jin et al ., 2016 )）。为了探索咖啡因生物合成的调控，在由TCS1启动子驱动的荧光素酶报告基因激活系统中筛选了来自加权基因共表达网络分析的 23 个候选转录因子（TF）（图 S1）。MYB184（TEA029017）显示出最高的TCS1启动子激活，达到 4.7 倍（图 1a）。酵母单杂交试验显示 TCS1 启动子的 -828 至 -1670 bp 区域（包含MYBCORE 和融合的 MYB1AT-MYBPLANT）是识别 MYB184 的关键（图 1b）。在植物启动子中，反式激活分析进一步证实了与TCS1结合和激活所需的区域MYB184 的启动子在 -1596 和 -1670 bp 之间（图 1c）。进行 EMSA 测定以进一步验证 MYB184在体外与融合的 MYB1AT-MYBPLANT 基序的结合（图 1d-f）。

然后，我们检查了 MYB184 在调节茶树中咖啡因合成中的功能。用茶树梢尖进行反义寡脱氧核苷酸 ( asODN ) 干扰实验以敲低MYB184表达 ( MYB184-KD ) (图 1g)。因此，与senseODN对照相比， MYB184-KD芽尖中的咖啡因含量和 TCS1 的表达显着降低（图 1h-i）。然而，在茶树转基因毛根系中过表达MYB184 ( MYB184OE ) 显着上调TCS1与野生型根对照相比，转录并因此增加了咖啡因含量（图1j-m）。

KeKecha（Camellia ptilophylla，简称KKC​​）属于Thea科，其咖啡因含量显着降低，但可可碱含量较高（图1n）。尽管先前的一项研究表明，与现代茶品种中的 TCS1 相比，KKC 中的 TCS1 具有较低的 NMT 活性（Jin等，2016 ），但我们检测到 KKC 中TCS1的表达水平明显低于其他茶品种（图 1o）。为了了解为什么KKC中的 TCS1被下调，我们克隆并比较了TCS1的启动子序列来自 KKC 和 SCZ。然而，启动子序列的比对未显示 MYB 结合位点上的关键 Indel 或 SNP（图 1p）。另一方面，转录组分析显示，KKC 中只有MYB184 的表达水平比其他茶品种显着降低约 14 倍（图 1q，r）。因此，我们提出 KKC 中较低的MYB184转录水平可能是 TCS1 表达水平降低的原因。

我们进一步从KKC（ proMYB184 ^KKC ）中克隆了MYB184基因的启动子，并将其与其他茶树品种的启动子进行了比较。仅在proMYB184 ^KKC的 -982 bp 位点发现了一个 437 bp 长的末端重复 (LTR) 插入，但在来自其他茶品种的这些启动子中没有发现，正如MYB184启动子克隆和用特异性 PCR 引物检测所证实的那样LTR 插入（图 1s）。在茶树中，基因中的 LTR 插入通常会导致基因表达受到抑制（Xia et al ., 2020 ），这可能解释了 KKC 中MYB184的表达水平低于其他茶品种。的确，GUS报告基因分析表明，具有 LTR 插入的proMYB184 ^KKC的启动子活性明显低于来自茶品种的四个没有 LTR 插入的代表性启动子（图 1t）。因此，我们得出结论，在 MYB184 的启动子中插入 LTR导致MYB184 表达受到抑制，导致TCS1转录水平降低，从而降低 KKC 中的咖啡因含量。

为了将 TCS1 的 MYB184 激活扩展到其他山茶属物种，我们检查了其他几种野生茶近缘植物，这些近缘植物已知与现代茶品种相比含有显着较低水平的咖啡因（图 1u）。与含有较高水平咖啡因的现代茶品种相比，它们还具有较低的TCS1和MYB184表达水平（图 1v、w 和 S2）。这进一步支持了 MYB184 在 C. sinensis 中 TCS1 基因表达和咖啡因生物合成的激活中不可或缺的作用。

总之，我们将 MYB184 定性为茶树中 TCS1 和咖啡因生产的主要激活剂。野生茶C中MYB184启动子中的 LTR 插入。ptilophylla解释了它的低TCS1表达水平和咖啡因含量。我们的研究可能为培育低咖啡因茶品种以满足市场需求提供分子工具。