ABSTRACT
Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Posttranslational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B'γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against ultraviolet B (UV-B) or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.
Subject(s)
Aconitate Hydratase/genetics , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Mitochondria/metabolism , Transcription Factors/metabolism , Aconitate Hydratase/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/metabolismABSTRACT
This paper describes a novel mass spectrometry based analytical method for analyzing thyroid hormones (THs). Thyroid hormones play a critical role in the regulation of many biological processes such as growth, metabolism and development. Several analytical methods using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) have previously been developed to measure THs, especially in humans. For biomedical and toxicological research using small animal models, and in ecophysiological research using wild species where sample volume is limiting, sensitive methods are needed. In this study, we developed a nano-LC-MS/MS method enabling quantification of low concentrations of two key THs, thyroxine (T4) and 3,3',5-triiodothyronine (T3). The method was tested with egg yolk samples. We used a low flow rate (300â¯nl/min) to obtain maximal sensitivity of the method. The limit of quantitation was 10.6â¯amol for T4 and 17.9â¯amol for T3. The method shows good linearity (râ¯>â¯0.99), repeatability and reproducibility (CVs <10%). We also reanalyzed yolk samples with radioimmunoassay for a comparison of the newly developed and previously used methods. Finally, we applied the methodology to measure hormones in egg yolk extracts in multiple avian species, and report interesting variation in maternal TH deposition. The newly developed nano-LC-MS/MS method is thus suitable for measuring THs in low concentrations and across species.
Subject(s)
Chromatography, Liquid/methods , Nanotechnology/methods , Tandem Mass Spectrometry/methods , Thyroid Hormones/analysis , Animals , Egg Yolk/chemistry , Linear Models , Quail , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A bicyclononyne-based prosthetic group has been developed for (18)F-labeling of anti-microRNA-21, an oligonucleotide, in a near-stoichiometric manner.
