Simultaneous determination of protoporphyrin IX and magnesium protoporphyrin IX in Arabidopsis thaliana and Camellia sinensis using UPLC-MS/MS

文献类型： 外文期刊

第一作者： Zhang, Chenyu

作者： Zhang, Chenyu;Ma, Chunlei;Zhu, Li;Yao, Mingzhe

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关键词： Mg-protoporphyrin IX; Protoporphyrin IX; UPLC-MS; MS; Chlorophyll biosynthesis; Camellia sinensis; Arabidopsis thaliana

期刊名称：PLANT METHODS （ 影响因子：5.1； 五年影响因子：6.1 ）

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年卷期： 2023 年 19 卷 1 期

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收录情况： SCI

摘要： BackgroundsInsertion of Mg2+ 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.ResultsAn 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 NH4OH (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 center dot kg(- 1) and the limit of quantification was 0.05 mg center dot kg(- 1). The contents of PPIX (1.67 +/- 0.12 mg center dot kg(- 1)) and Mg-PPIX (3.37 +/- 0.10 mg center dot kg(- 1)) in tea were significantly higher than in Arabidopsis (PPIX: 0.05 +/- 0.02 mg center dot kg(- 1); Mg-PPIX: 0.08 +/- 0.01 mg center dot kg(- 1)) and they were only detected in the leaf.ConclusionsOur 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.

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