University of Turku Plant Biology - ACONITASE 3

ACONITASE 3 is part of the ANAC017 transcription factor-dependent mitochondrial dysfunction response
Data License: CC BY 4.0 | ProteomeXchange: PXD018881
  • Organism: Arabidopsis thaliana
  • Instrument: Q Exactive,Q Exactive HF
  • SpikeIn: No
  • Keywords: aconitase, mitochondrial dysfunction response, phosphorylation, post-translational regulation
  • Lab head: Saijaliisa Kangasjärvi Submitter: Jesús Pascual
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). Post-translational 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 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.
Experiment Description
Protein from four weeks old Arabidopsis rosettes was isolated by grinding the leaf material in 50 mM HEPES KOH pH 7.8, 10 mM MgCl2 supplemented with protease (Complete-Mini, Roche) and phosphatase (PhosSTOP, Roche) inhibitor cocktails. Protein was quantified with Protein Assay Dye Reagent (no. 5000006, Bio-Rad, Hercules, CA, USA). The individual ACONITASE 1 (ACO1, AT4G35830), ACONITASE 2 (ACO2, AT4G26970) and ACONITASE 3 (ACO3, AT2G05710) (www.arabidopsis.org) isoforms were quantified by parallel reaction monitoring (PRM). Samples consisting of 25 µg of protein were prepared for MS analysis as described in Trotta et al., 2019. The trypsin-digested samples were first spiked with iRT peptides (Biognosys) according to the manufacturer´s instructions and analyzed in Data Dependent Acquisition (DDA) mode, selecting the top 20 most intense precursors in each scan (m/z 300 – 2000) for higher-energy collisional dissociation (HCD) fragmentation with an exclusion window of 12 seconds. The analysis was performed in a nanoflow HPLC system (EasyNanoLC1000, Thermo Fisher Scientific) equipped with a 20 x 0.1 mm i.d. pre-column combined with a 150 mm x 75 µm i.d. analytical column, both packed with 5 µm Reprosil C18-bonded silica (Dr Maisch GmbH), and injection to an electrospray ionization (ESI) source coupled to a Q‐Exactive (Thermo Fisher Scientific) mass spectrometer. The mobile phase consisted of water/acetonitrile (ACN) (98:2 (v/v)) with 0.1% formic acid (FA) (v/v) (solvent A) or ACN/water (80:20 (v/v)) with 0.1% FA (v/v) (solvent B) at a flow rate of 300 nL min−1. The peptides were separated by a three-step elution gradient: from 3% to 43% solvent B in 45 minutes, followed by an increase to 100% in 5 minutes and 10 minutes of 100% solvent B. In parallel, foliar extracts consisting of 100 µg of protein were separated on a 10% SDS-PAGE gel and a protein band migrating at the Arabidopsis aconitases molecular weight (100 kDa) was excised and processed and analyzed by MS in the same way. The resulting tandem MS spectra were searched in Proteome Discoverer 2.3 (Thermo Scientific) with a non-redundant Arabidopsis proteome (TAIR10, www.arabidopsis.org) appended with the most common contaminants using Mascot (v. 2.7; Matrix Science). Monoisotopic mass, a maximum of two missed cleavages, 10 ppm precursor mass tolerance, 0.02 Da fragment mass tolerance and charge ≥ 2 + were used as settings for the searches. Additionally, methionine oxidation and serine, threonine and tyrosine phosphorylation were considered as variable modifications and cysteine carbamidomethylation as fixed modifications. Identification confidence was validated through PhosphoRS filter and Decoy Database Search using 0.01% (strict) and 0.05% (relaxed) false discovery rate (FDR) confidence thresholds. These data were used for construction of a reference library for the performance of label-free relative quantification of ACO1, ACO2 and ACO3 by PRM. Three proteotypic peptides per protein were selected from the reference library, avoiding peptides with potential post-translational modifications (PTMs). PRM analysis was performed in a Q-Exactive mass spectrometer (Thermo Fisher Scientific) using the same elution gradient described above. PRM data were analyzed with Skyline (Maclean et al., 2010). Comparability between samples was ensured by injecting an equal amount of digested protein (100 µg). In addition, in stress experiments, the abundance of RUBISCO LARGE SUBUNIT (RBCL; ATCG00490), RUBISCO SMALL SUBUNIT 1A (RBCS1A; AT1G67090) and GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE A SUBUNIT (GAPA; AT3G26650) was monitored in parallel with the aconitase isoforms. Relative protein abundance was calculated as the sum of the intensity of the three proteotypic peptides targeted for each protein and calculated as the sum of the integrated peak area of the three most intense fragments with the following formula: A=(f11+f12+f13)+(f21+f22+f23)+(f31+f32+f33) (1) where A is the relative protein abundance, f11, f12… are the integrated areas of the most intense ion fragments of peptide 1; f21, f22… the integrated areas of the most intense ion fragments of peptide 2, and so on. Relative protein abundance values were expressed relative to wild type. The quantification of ACO3 Ser91 phosphorylation was performed by targeted selected ion monitoring coupled to parallel reaction monitoring (tSIM-PRM). The spectral libraries generated for the PRM analysis of ACO3 in this study and the libraries reported by Konert et al., 2015 were used as a reference. Foliar extracts consisting of 150 µg of protein were first separated on a 10% SDS-PAGE gel. The protein band migrating at 100 kDa and containing the Arabidopsis aconitase isoforms was excised and prepared for MS analysis as described in Trotta et al (2019). The analysis was performed in a nano HPLC system (EasyNanoLC1000, Thermo Fisher Scientific) coupled to a Q-Exactive HF (Thermo Fisher Scientific). The peptides were separated using the same elution gradient described above. ACO3 relative phosphorylation levels, i.e., the stoichiometry of ACO3-Ser91 phosphopeptide, were calculated as in Trotta et al., 2019 and Secher et al., 2016 as the percentage of the intensity of the phosphopeptide with respect to the sum of the intensities of the nonphosphorylated peptide plus the phosphopeptide. The following formula was used: P=((pt1+pt2+pt3))/(((t1+t2+t3)+(pt1+pt2+pt3)) ) x 100 where P is the relative level of phosphorylation, pt1, pt2… are the most intense peptide fragments of the phosphorylated form, and t1, t2… are the most intense transitions of the nonphosphorylated form. The percentages of phosphorylation in all the mutants and experimental conditions were compared using this calculation. ACO3 relative phosphorylation levels were expressed relative to wild type.
Sample Description
Protein samples from 4-week-old Arabidopsis rosettes growth in 8/16 hour light/dark period under 120 μmol photons m-2s-1 at 23oC and 50% relative humidity untreated or treated with UV-B with Philips Broadband ultraviolet-B lamp (TL20W/12 RS, Philips) at an irradiance of 1.5 W m-2 for 45 minutes coinciding with the light period midday (i.e. 4 hours into the light period) and collected 24 hours after. Protein was isolated by grinding the leaf material in 50 mM HEPES KOH pH 7.8, 10 mM MgCl2 supplemented with protease (Complete-Mini, Roche) and phosphatase (PhosSTOP, Roche) inhibitor cocktails.
Created on 2/25/21, 10:11 AM
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13-tSIM-PRM_ACO3-Ser91_UV-B_11d_Fig8D.sky.zip2022-03-14 21:11:161231096
12-tSIM-PRM_ACO3-Ser91_aco3-ox-3_UV-B-24h_Fig6C.sky.zip2022-03-14 21:11:151236711
10-tSIM-PRM_ACO3-Ser91_CHX_UV-B-24h_Fig5B.sky.zip2022-03-14 21:11:141238611
6-PRM_ACOs_WT_anac017-1_AA_Fig4B_S3B.sky.zip2021-02-25 10:10:506192723012
5-PRM_ACOs_WT_anac017-1_UV-B-24h_Fig4A_S3A.sky.zip2021-02-25 10:10:506192723012
3-PRM_ACOs_aco3 phosphomutants_R3_Fig3B.sky.zip2021-02-25 10:10:49399976
7-tSIM-PRM_ACO3-Ser91_WT_anac017-1_UV-B-24h_Fig4C.sky.zip2021-02-25 10:10:491237812
11-PRM_ACOs_HKs_aco3-ox-3_UV-B-24h_Fig6B.sky.zip2021-02-25 10:10:49619273256
9-PRM_ACOs_HKs_CHX_UV-B-24h_Fig5A_S3C.sky.zip2021-02-25 10:10:496192732312
1-PRM_ACOs_aco3 phosphomutants_R1_Fig3B.sky.zip2021-02-25 10:10:49399976
2-PRM_ACOs_aco3 phosphomutants_R2_Fig3B.sky.zip2021-02-25 10:10:49399996
4-PRM_ACOs_aco3-ox-3_Fig3C.sky.zip2021-02-25 10:10:49399956
8-tSIM-PRM_ACO3-Ser91_WT_anac017-1_AA_Fig4D.sky.zip2021-02-25 10:10:491238012
14-PRM_ACOs_aco3_phosphomutants_second_set_mutant_lines_FigS2A.sky.zip2021-02-25 10:10:493997818
tSIM-PRM_ACO3-Ser91_WT_vs_aco3.sky.zip2021-02-25 10:10:491231062