Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Sanat Nagar, Srinagar, Jammu and Kashmir, 190005, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, 201002, India.
Planta. 2024 May 12;259(6):152. doi: 10.1007/s00425-024-04433-y.
Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-L-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC-MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content.
青蒿茉莉酸羧基甲基转移酶（AaJMT）的过表达导致青蒿中青蒿素含量增加。基于青蒿素的联合疗法仍然是对抗致命疾病疟疾的唯一手段，而青蒿仍然是青蒿素的唯一天然来源。在这项研究中，从青蒿中鉴定出了 1101bp 的基因 S-腺苷-L-蛋氨酸（SAM）：青蒿茉莉酸羧基甲基转移酶（AaJMT），它将茉莉酸（JA）转化为甲基茉莉酸（MeJA）。从系统发育分析中，我们证实 AaJMT 与拟南芥、日本油菜和茶树的 JMT 具有共同的祖先，并且与茶树的 JMT 具有密切的同源性。此外，Clustal Omega 表明，AaJMT 存在分别结合 SAM 和 JA 的保守基序 I、基序 III 和基序 SSSS（丝氨酸）。AaJMT 的相对表达受创伤、MeJA 和水杨酸（SA）处理诱导。此外，我们发现重组 AaJMT 蛋白能以 37.16µM 的 Km 值催化 JA 合成 MeJA。此外，将 motif SSSS 中的丝氨酸-151突变为酪氨酸、天冬酰胺-10 突变为苏氨酸和谷氨酰胺-25 突变为组氨酸，使 AaJMT 的酶活性丧失，这表明它们在 JA 底物结合中起决定性作用。GC-MS 分析证实，AaJMT 的突变蛋白无法将 JA 转化为 MeJA。最后，AaJMT 过表达转基因系中青蒿素生物合成和毛状体发育基因上调，从而增加了青蒿素的含量。
