Jia Xinxin, Zhang Xiaoliang, Chen Xueli, Fernie Alisdair R, Wen Weiwei
National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Hubei Hongshan Lab, Huazhong Agricultural University, Wuhan, 430070, China.
Max-Planck-Institute of Molecular Plant Physiology, Am Muehlenberg 1, Potsdam-Golm, 14476, Germany.
J Integr Plant Biol. 2025 Jul 3. doi: 10.1111/jipb.13957.
Purine-related metabolites are central to primary metabolic pathways in plants and serve as precursors for purine alkaloid biosynthesis in caffeinated species such as tea plants (Camellia sinensis). In this study, metabolite profiling of two tissues (young and mature leaves) was performed across 183 genetically diverse tea accessions, identifying and quantifying 10 purine alkaloid-related metabolites. Metabolite genome-wide association studies revealed 17 significant loci associated with these metabolites, including both known loci such as caffeine synthase and 16 novel loci (P < 1.05 × 10). Through functional annotation and in vitro enzymatic assay, we characterized 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (CsMTAN) as the causal gene underlying natural variation in adenosine and adenine content. CsMTAN can catalyze the degradation of both 5'-methylthioadenosine and S-adenosylhomocysteine to release adenine. The T → A nucleotide substitution at SNP55151898, which leads to a phenylalanine → tyrosine substitution at residue 179 (F179Y), resulted in a significant alteration of enzyme activity in vitro, as evidenced by an approximately 50% reduction in adenine abundance (P < 0.05). Transient overexpression of CsMTAN-A and CsMTAN-T in Nicotiana benthamiana both significantly increased adenine content and dramatically decreased adenosine content, providing direct evidence for the functional involvement of CsMTAN in plant purine metabolism. CsMTAN-T overexpression resulted in significantly lower adenosine level than CsMTAN-A (P < 0.05). Phylogenetic analysis across 115 species and protein structural modeling revealed a distinct evolutionary divergence between plant MTAN evolution and species phylogeny, strongly suggesting the occurrence of horizontal gene transfer events in the evolutionary history of plant MTANs. This study thus furthered our understanding of the genetics and molecular mechanisms regulating purine metabolism and purine alkaloid biosynthesis in tea plants and provided novel targets for molecular breeding and synthetic biology applications.
嘌呤相关代谢物是植物初级代谢途径的核心，并且是茶树（Camellia sinensis）等含咖啡因物种中嘌呤生物碱生物合成的前体。在本研究中，对183个遗传多样性不同的茶树品种的两个组织（幼叶和成熟叶）进行了代谢物谱分析，鉴定并定量了10种与嘌呤生物碱相关的代谢物。代谢物全基因组关联研究揭示了17个与这些代谢物相关的显著位点，包括咖啡因合酶等已知位点和16个新位点（P < 1.05 × 10）。通过功能注释和体外酶活性测定，我们将5'-甲硫基腺苷/S-腺苷同型半胱氨酸核苷酶（CsMTAN）鉴定为腺苷和腺嘌呤含量自然变异的因果基因。CsMTAN可以催化5'-甲硫基腺苷和S-腺苷同型半胱氨酸的降解以释放腺嘌呤。SNP55151898处的T→A核苷酸替换导致第179位残基处的苯丙氨酸→酪氨酸替换（F179Y），导致体外酶活性发生显著改变，腺嘌呤丰度降低约50%证明了这一点（P < 0.05）。在本氏烟草中瞬时过表达CsMTAN-A和CsMTAN-T均显著增加了腺嘌呤含量并显著降低了腺苷含量，为CsMTAN参与植物嘌呤代谢提供了直接证据。CsMTAN-T过表达导致腺苷水平显著低于CsMTAN-A（P < 0.05）。对115个物种的系统发育分析和蛋白质结构建模揭示了植物MTAN进化与物种系统发育之间存在明显的进化分歧，强烈表明植物MTANs的进化历史中发生了水平基因转移事件。因此，本研究进一步加深了我们对茶树中嘌呤代谢和嘌呤生物碱生物合成调控的遗传学和分子机制的理解，并为分子育种和合成生物学应用提供了新的靶点。
