Article Figures & Data
Figures
- 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. 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. 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. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, and its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors
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.
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. 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. RuBisCO activity and RuBisCO activation state are increased in the hda14 mutant under low‐light conditions
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).
RuBisCO activation state (P < 0.05, t‐test).
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.
Tables
- Table 1. Summary of identified features
Whole proteome analysis Acetyllysine‐containing Experiment Description Protein groups Peptides Protein groups Peptides Sites 1 Apicidin versus Ctrl 2,384 11,188 538 1,064 1,041 2 TSA versus Ctrl 5,107 32,809 493 1,002 930 3 hda14 versus WT 2,889 13,755 545 1,133 920 4 hda14 versus WT low‐light 4,138 27,835 367 756 700 5 hda14 versus WT thylakoids 2,904 15,064 237 592 546 Total 6,672 47,338 1,022 2,405 2,152 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 IDs Name Peptides MS/MS count Log2‐LFQ CP AsuHd Log2‐LFQ CP Lys Log2 enrichment CP Log2‐LFQ LF AsuHd Log2‐LFQ LF Lys Log2 enrichment LF AT5G61060.1/.2 HDA5 6 11 n.d. n.d. n.d. 24.50 ± 0.12 n.d. > 7a AT4G33470.1 HDA14 7 22 24.74 ± 1.83 19.54 ± 0.05 5.2 26.20 ± 0.2 21.12 ± 0.49 5.1 AT3G18520.1/2 HDA15 2 2 n.d. n.d. n.d. 20.84 ± 0.08 n.d. > 3a ATCG00490.1 RBCL 28 545 33.65 ± 0.36 33.73 ± 0.13 −0.1 33.91 ± 0.09 33.75 ± 0.03 0.2 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.
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]
