Ethylene Receptors, CTRs and EIN2 Target Protein Identification and Quantification Through Parallel Reaction Monitoring During Tomato Fruit Ripening

Tomato (Solanum lycopersicum) fruit ripening is a complex genetic trait correlating with notable fruit phenotypic, physiologic, and biochemical changes. Transcription factors (TFs) play crucial roles during this process. LeHB-1, an HD-zip homeobox protein, is a ripening-related TF and acts as an important regulator of fruit ripening. However, the detailed biochemical and molecular basis of LeHB-1 on tomato fruit ripening is unclear. In the current study, the biologic functions of LeHB-1 were determined by a potato virus X (PVX)-mediated gene-silencing approach. The results indicate that PVX-induced LeHB-1 silencing in tomato could decrease pigment accumulation and delay fruit ripening. Compared with controls, nonripening flesh retains a greater pH value and a lesser anthocyanin content. By evaluating expression levels of genes related to tomato fruit ripening, we inferred that LeHB-1 located at the downstream of LeMADS-RIN-mediated regulatory network. In addition, LeHB-1 silencing mainly disturbed phytoene desaturation and isomerization, and led to a decrease in trans-lycopene accumulation, but did not influence flavonoid biosynthesis directly in tomato fruit. The findings provide a theoretical foundation for illustrating the biologic functions of LeHB-1 in tomato fruit ripening and quality.

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Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures

PeerJ ◽

10.7717/peerj.9941 ◽

2020 ◽

Vol 8 ◽

pp. e9941

Author(s):

Chuan He ◽

Simiao Hu ◽

Wanxing Zhou

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Culture Supernatant ◽

Reaction Monitoring ◽

Ang Ii ◽

Parallel Reaction ◽

Parallel Reaction Monitoring ◽

Relative Standard ◽

Ang Iv ◽

Nanoflow Liquid Chromatography

Background This study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant. Methods HUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism. Results The limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated. Conclusion The nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.

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