Zhang Chenyu, Ma Chunlei, Zhu Li, Yao Mingzhe
Key Laboratory of Biology, Genetics and Breeding of Special Economic Animals and Plants, Ministry of Agriculture and Rural Affairs, Tea Research Institute of the Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China.
Plant Methods. 2023 Mar 30;19(1):34. doi: 10.1186/s13007-023-01008-y.
Insertion of Mg into protoporphyrin IX (PPIX) to produce magnesium-protoporphyrin IX (Mg-PPIX) was the first step toward chlorophyll biosynthesis, which not only imparts plants green pigmentation but underpins photosynthesis. Plants that blocked the conversion of PPIX to Mg-PPIX displayed yellowish or albino-lethal phenotypes. However, the lack of systematic study of the detection method and the metabolic difference between species have caused the research on chloroplast retrograde signaling controversial for a long time.
An advanced and sensitive UPLC-MS/MS strategy for determining PPIX and Mg-PPIX was established in two metabolic different plants, Arabidopsis thaliana (Columbia-0) and Camellia sinensis var. sinensis. Two metabolites could be extracted by 80% acetone (v/v) and 20% 0.1 M NHOH (v/v) without hexane washing. Since the Mg-PPIX could be substantially de-metalized into PPIX in acidic conditions, analysis was carried out by UPLC-MS/MS with 0.1% ammonia (v/v) and 0.1% ammonium acetonitrile (v/v) as mobile phases using negative ion multiple reaction monitoring modes. Interestingly, it could be easier to monitor these two compounds in dehydrated samples rather than in fresh samples. Validation was performed in spiked samples and mean recoveries ranged from 70.5 to 916%, and the intra-day and inter-day variations were less than 7.5 and 10.9%, respectively. The limit of detection was 0.01 mg·kg and the limit of quantification was 0.05 mg·kg. The contents of PPIX (1.67 ± 0.12 mg·kg) and Mg-PPIX (3.37 ± 0.10 mg·kg) in tea were significantly higher than in Arabidopsis (PPIX: 0.05 ± 0.02 mg·kg; Mg-PPIX: 0.08 ± 0.01 mg·kg) and they were only detected in the leaf.
Our study establishes a universal and reliable method for determining PPIX and Mg-PPIX in two plants using UPLC-MS/MS. This procedure will facilitate studying chlorophyll metabolism and natural chlorophyll production.
镁插入原卟啉IX（PPIX）以生成镁原卟啉IX（Mg-PPIX）是叶绿素生物合成的第一步，这不仅赋予植物绿色色素沉着，还支撑着光合作用。阻断PPIX向Mg-PPIX转化的植物表现出淡黄色或白化致死表型。然而，由于缺乏对检测方法和物种间代谢差异的系统研究，叶绿体逆行信号转导的研究长期以来存在争议。
在两种代谢不同的植物拟南芥（哥伦比亚-0）和茶树中建立了一种先进且灵敏的用于测定PPIX和Mg-PPIX的超高效液相色谱-串联质谱（UPLC-MS/MS）策略。两种代谢物可用80%丙酮（v/v）和20% 0.1 M NHOH（v/v）提取，无需己烷洗涤。由于Mg-PPIX在酸性条件下可大量脱金属化为PPIX，因此采用0.1%氨水（v/v）和0.1%乙酸铵乙腈（v/v）作为流动相，在负离子多反应监测模式下通过UPLC-MS/MS进行分析。有趣的是，在脱水样品中监测这两种化合物比在新鲜样品中更容易。在加标样品中进行了验证，平均回收率在70.5%至916%之间，日内和日间变化分别小于7.5%和10.9%。检测限为0.01 mg·kg，定量限为0.05 mg·kg。茶叶中PPIX（1.67±0.12 mg·kg）和Mg-PPIX（3.37±0.10 mg·kg）的含量显著高于拟南芥（PPIX：0.05±0.02 mg·kg；Mg-PPIX：0.08±0.01 mg·kg），且仅在叶片中检测到。
我们的研究建立了一种使用UPLC-MS/MS测定两种植物中PPIX和Mg-PPIX的通用且可靠的方法。该方法将有助于研究叶绿素代谢和天然叶绿素的产生。
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