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Article

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

View ORCID ProfileMarkus Hartl, Magdalena Füßl, Paul J Boersema, Jan‐Oliver Jost, Katharina Kramer, Ahmet Bakirbas, View ORCID ProfileJulia Sindlinger, Magdalena Plöchinger, Dario Leister, Glen Uhrig, View ORCID ProfileGreg BG Moorhead, Jürgen Cox, Michael E Salvucci, View ORCID ProfileDirk Schwarzer, View ORCID ProfileMatthias Mann, View ORCID ProfileIris Finkemeier
DOI 10.15252/msb.20177819 | Published online 23.10.2017
Molecular Systems Biology (2017) 13, 949
Markus Hartl
Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPlant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, GermanyMass Spectrometry Facility, Max F. Perutz Laboratories (MFPL), Vienna Biocenter (VBC), University of Vienna, Vienna, Austria
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Magdalena Füßl
Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPlant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, GermanyPlant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany
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Paul J Boersema
Proteomics and Signal Transduction, Max‐Planck Institute of Biochemistry, Martinsried, Germany
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Jan‐Oliver Jost
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
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Katharina Kramer
Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, Germany
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Ahmet Bakirbas
Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPlant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany
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Julia Sindlinger
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
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Magdalena Plöchinger
Plant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, Germany
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Dario Leister
Plant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, Germany
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Glen Uhrig
Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
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Greg BG Moorhead
Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
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Jürgen Cox
Proteomics and Signal Transduction, Max‐Planck Institute of Biochemistry, Martinsried, Germany
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Michael E Salvucci
US Department of Agriculture, Agricultural Research Service, Arid‐Land Agricultural Research Center, Maricopa, AZ, USA
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Dirk Schwarzer
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
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Matthias Mann
Proteomics and Signal Transduction, Max‐Planck Institute of Biochemistry, Martinsried, Germany
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Iris Finkemeier
Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPlant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, GermanyPlant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany
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Author Affiliations

  1. Markus Hartl1,2,3,†,
  2. Magdalena Füßl1,2,4,†,
  3. Paul J Boersema5,9,
  4. Jan‐Oliver Jost6,10,
  5. Katharina Kramer1,
  6. Ahmet Bakirbas1,4,11,
  7. Julia Sindlinger6,
  8. Magdalena Plöchinger2,
  9. Dario Leister2,
  10. Glen Uhrig7,
  11. Greg BG Moorhead7,
  12. Jürgen Cox5,
  13. Michael E Salvucci8,
  14. Dirk Schwarzer6,
  15. Matthias Mann5 and
  16. Iris Finkemeier (iris.finkemeier{at}uni-muenster.de)*,1,2,4
  1. 1Plant Proteomics, Max Planck Institute for Plant Breeding Research, Cologne, Germany
  2. 2Plant Molecular Biology, Department Biology I, Ludwig‐Maximilians‐University Munich, Martinsried, Germany
  3. 3Mass Spectrometry Facility, Max F. Perutz Laboratories (MFPL), Vienna Biocenter (VBC), University of Vienna, Vienna, Austria
  4. 4Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany
  5. 5Proteomics and Signal Transduction, Max‐Planck Institute of Biochemistry, Martinsried, Germany
  6. 6Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
  7. 7Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
  8. 8US Department of Agriculture, Agricultural Research Service, Arid‐Land Agricultural Research Center, Maricopa, AZ, USA
  9. 9Present Address: Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
  10. 10Present Address: Leibniz‐Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP), Berlin, Germany
  11. 11Present Address: Plant Biology Graduate Program University of Massachusetts Amherst, Amherst, USA
  1. ↵*Corresponding author. Tel: +49 251 8323805; E‐mail: iris.finkemeier{at}uni-muenster.de
  1. ↵† These authors contributed equally to this work

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  • Figure 1.
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    Figure 1. Proteome‐wide identification and classification of the Arabidopsis thaliana lysine acetylome

    • A. Experimental overview.

    • B, C Functional classification and subcellular localization of identified lysine‐acetylated proteins. Lysine‐acetylated proteins identified over all experiments were classified according to MapMan categories and SUBA4 localization information, respectively. Over‐ or underrepresentation of categories was determined using a Fisher's exact test with all proteins identified at 1% FDR as background population. Blue and red arrows mark categories significantly enriched at 5% FDR (Benjamini–Hochberg) and a 1.5‐fold‐change cut‐off.

    • D. Sequence logos for all lysine acetylation sites with all proteins identified as background population (sequence logos were generated using iceLogo, Maddelein et al, 2015).

  • Figure 2.
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    Figure 2. Overview of lysine‐acetylated proteins in the light reactions (A) and the Calvin–Benson cycle (B) identified in this study in Arabidopsis

    • A, B The classification of proteins into functional bins was performed using MapMan (Thimm et al, 2004). Color code: proteins not identified in the LC‐MS/MS analyses (white), proteins without identified lysine‐acetylated sites (gray), and proteins with one (yellow), two (orange), three (dark orange), or four or more acetylation sites (red). For the Calvin–Benson cycle, each box indicates a separate Arabidopsis AGI identifier as indicated in Dataset EV6. Cytb6f, cytochrome b6f; FBPase, fructose‐1,6‐bisphosphatase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PGK, phosphoglycerate kinase; PPE, phosphopentose epimerase; PPI, phosphopentose isomerase; PRK, phosphoribulokinase; PSII, photosystem II; PSI, photosystem I; RuBisCO, ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase; SBPAse, seduheptulose‐1,7‐bisphosphatase; TPI, triose phosphate isomerase; TK, transketolase. A template of the light‐reaction schematic was kindly provided by Jon Nield and modified.

  • Figure 3.
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    Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment

    • A–D Vacuum infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C) or trichostatin A (B, D) versus a buffer control for 4 h leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B) or protein ratios (C, D) for inhibitor treatment versus control, with P‐values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected P‐values < 5% or Benjamini–Hochberg corrected FDR < 5%. A missing line indicates that the significance threshold was not reached by any of the data points.

  • Figure 4.
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    Figure 4. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, and its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors

    1. GFP localization (green) of the HDA14‐GFP fusion constructs in Arabidopsis protoplasts (35S:HDA14:GFP) from stable transformants. The mitochondrial marker TMRM is depicted in purple. GFP+TMRM shows the overlay image of 35S:HDA14:GFP and TMRM, AF indicates the chlorophyll autofluorescence and BF the bright‐field image of the protoplast. GFP+AF+TMRM represents the overlay image of the three fluorescence channels. Scale bar: 10 μm.

    2. Deacetylase activity of the recombinant 6xHis‐HDA14 protein using a colorimetric assay. Co2+ and Zn2+ were used as cofactors, apicidin (100 μM) and trichostatin A (5 μM) as deacetylase inhibitors (n = 5, ± SD).

  • Figure 5.
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    Figure 5. Differential lysine acetylation and protein expression in hda14 versus wild‐type leaves under normal light (A, D), in isolated thylakoids (B, E), and under low‐light conditions (C, F)

    • A–F Volcano plots depict lysine acetylation site ratios (A–C, top row) or protein ratios (D–F, bottom row) for mutant versus control, with P‐values determined using the LIMMA package. Orange, protein with nuclear localization; green, protein with plastidial localization; purple triangles, proteins of the Calvin–Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected P‐values < 5% or Benjamini–Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.

  • Figure 6.
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    Figure 6. RuBisCO activity and RuBisCO activation state are increased in the hda14 mutant under low‐light conditions

    1. RuBisCO initial and total activity in WT and hda14 in low‐light‐treated plants. Initial activity was measured directly upon extraction. For the total activity, samples were incubated with H2CO3 for 3 min to fully carbamylate the active site of RuBisCO (n = 10, *P < 0.05, t‐test).

    2. RuBisCO activation state (P < 0.05, t‐test).

    3. ATPase activity of recombinant 6x‐HisRCAβ1 WT, K438Q and K438R with ATP and ADP/ATP = 0.11, respectively (n = 3, *P < 0.05, +P < 0.1, t‐test). Percentage values on top indicate percent ADP inhibition.

    Data information: Boxes indicate lower and upper quartiles of data and whiskers indicate highest and lowest values. Small circles represent outliers. The bars across boxes indicate median values.

Tables

  • Figures
  • Supplementary Materials
  • Table 1. Summary of identified features
    Whole proteome analysisAcetyllysine‐containing
    ExperimentDescriptionProtein groupsPeptidesProtein groupsPeptidesSites
    1Apicidin versus Ctrl2,38411,1885381,0641,041
    2TSA versus Ctrl5,10732,8094931,002930
    3hda14 versus WT2,88913,7555451,133920
    4hda14 versus WT low‐light4,13827,835367756700
    5hda14 versus WT thylakoids2,90415,064237592546
    Total6,67247,3381,0222,4052,152
    • Filters applied: 1% FDR at PSM and protein level, score for modified peptides ≥ 35, delta score for modified peptides ≥ 6, acetyllysine site localization probability ≥ 0.75; contaminants removed.

  • Table 2. KDAC pull‐down with mini‐AsuHd probe
    Majority protein IDsNamePeptidesMS/MS countLog2‐LFQ CP AsuHdLog2‐LFQ CP LysLog2 enrichment CPLog2‐LFQ LF AsuHdLog2‐LFQ LF LysLog2 enrichment LF
    AT5G61060.1/.2HDA5611n.d.n.d.n.d.24.50 ± 0.12n.d.> 7a
    AT4G33470.1HDA1472224.74 ± 1.8319.54 ± 0.055.226.20 ± 0.221.12 ± 0.495.1
    AT3G18520.1/2HDA1522n.d.n.d.n.d.20.84 ± 0.08n.d.> 3a
    ATCG00490.1RBCL2854533.65 ± 0.3633.73 ± 0.13−0.133.91 ± 0.0933.75 ± 0.030.2
    • Selected proteins identified and quantified in pull‐downs by LC‐MS/MS analysis. Protein abundances are expressed as label free quantification (LFQ) values. Numbers indicate mean log2‐transformed LFQ values from two biological replicates of Arabidopsis leaves (LF) and isolated chloroplasts (CP). Mini‐Lys probes were used as pull‐down controls to calculate relative enrichments of proteins. LFQ values for RuBisCO are indicated in all samples as background control.

    • ↵a Estimated enrichment factor assuming a minimum Log2‐LFQ threshold of 17.

Supplementary Materials

  • Figures
  • Tables
  • Appendix [msb177819-sup-0001-Appendix.pdf]

  • Dataset EV1 [msb177819-sup-0002-DatasetEV1.zip]

  • Dataset EV2 [msb177819-sup-0003-DatasetEV2.zip]

  • Dataset EV3 [msb177819-sup-0004-DatasetEV3.zip]

  • Dataset EV4 [msb177819-sup-0005-DatasetEV4.zip]

  • Dataset EV5 [msb177819-sup-0006-DatasetEV5.zip]

  • Dataset EV6 [msb177819-sup-0007-DatasetEV6.zip]

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Volume 13, Issue 10
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