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Backgrounds: Insertion 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. Results: 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 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·kg− 1 and the limit of quantification was 0.05 mg·kg− 1. The contents of PPIX (1.67 ± 0.12 mg·kg− 1) and Mg-PPIX (3.37 ± 0.10 mg·kg− 1) in tea were significantly higher than in Arabidopsis (PPIX: 0.05 ± 0.02 mg·kg− 1; Mg-PPIX: 0.08 ± 0.01 mg·kg− 1) and they were only detected in the leaf. Conclusions: 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. [ABSTRACT FROM AUTHOR]

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement. [ABSTRACT FROM AUTHOR]