Volume 9, Issue 7, Pages 1018-1027 (July 2016) Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL  Xiang Gao, Hui Hong, Wei-Chao Li, Lili Yang, Jirong Huang, You-Li Xiao, Xiao-Ya Chen, Gen-Yun Chen  Molecular Plant  Volume 9, Issue 7, Pages 1018-1027 (July 2016) DOI: 10.1016/j.molp.2016.03.012 Copyright © 2016 The Author Terms and Conditions

Figure 1 Light Increases Rubisco Activity and Decreases RbcL Acetylation in Plants. (A) Arabidopsis Rubisco large subunit (RbcL) is acetylated. Purified Rubisco proteins, isolated from leaves at day (light) or night (dark), were resolved on 12% SDS–PAGE and stained with Coomassie brilliant blue (CBB, top), and acetylated proteins were detected by immunoblotting with the antibody against acetyllysine (Anti-AcK, bottom). RbcS, Rubisco small subunit. (B) Activities of Rubisco purified from leaves at day (light) and night (dark). Right: Rubisco carboxylase activity determined by incorporation of 14CO2 into acid-stable products. Values are mean ± SD, n = 4, two-tailed Student's t-test, **P ≤ 0.01. (C) Examination of acetylation of RbcL isolated from C3 plants of rice (Oryza sativa L. cv. japonica), spinach (Spinacia oleracea), and A. thaliana, C4 plant of maize (Zea mays), cyanobacteria of Synechocystis sp. PCC 6803 (6803), and Anabaena sp. PCC 7120 (7120). Total soluble proteins were isolated from leaves or cyanobacteria under light, and acetylated proteins were detected with antibody against RbcL or Anti-AcK. (D) Tandem mass tag (TMT) data. Acetylation level of RbcL Lys201 is relatively high at night (dark) and low during the day (light). Total soluble proteins were resolved on SDS–PAGE, and RbcL bands were sliced and digested to peptides. Synthesized peptides, corresponding to the acetylated and unacetylated RbcL fragments, were used as internal controls. Samples and internal control peptides were labeled by TMT labeling reagents, respectively, and combined before loading to LC–MS/MS. Quantification was based on the relative intensity of the TMT tags. (E) Alleviated RbcL acetylation under higher light. Left: One-month-old plants, grown in normal light (80 μmol/m2/s), were treated with higher light (250 μmol/m2/s). Right: protoplasts isolated from rosette leaves were treated with DCMU (in protoplast buffer) or nigericin (in ethanol) for 0.5 h. Molecular Plant 2016 9, 1018-1027DOI: (10.1016/j.molp.2016.03.012) Copyright © 2016 The Author Terms and Conditions

Figure 2 Acetylation of RbcL by 7-acetoxy-4-methylcoumarin. (A) Arabidopsis RbcL was acetylated by 7-acetoxy-4-methylcoumarin (AMC) but not other O-acetyl chemicals tested. Rubisco was incubated with AMC or other chemicals as indicated for 2 h at 30°C. Acetylated level of RbcL was detected with Anti-AcK. (B) Acetylation of Arabidopsis phosphoglycerate kinase (PGK) by AMC. Prokaryotic expressed His-tagged PGK (0.05 μg/μl) was incubated with 0.3 mM AMC for 30 min at 30°C, and the acetylation level was detected by immunoblotting (Anti-AcK). (C and D) Acetylation of RbcL by AMC is dependent on the reaction time and AMC concentrations. Rubisco was incubated with 0.3 mM AMC for different times (C), or with different concentrations of AMC for 30 min (D). (E) Denatured Rubisco could be acetylated by AMC but to a lesser extent. Rubisco was firstly denatured at 98°C for 10 min, then incubated with 0.3 mM AMC at 30°C for 30 min. (F) Pretreatment with RuBP inhibits RbcL acetylation. Rubisco was firstly incubated with RuBP at 30°C for 10 min, then AMC was added and incubation continued for another 30 min. (G) Pre-activation of Rubisco increases RbcL acetylation. Rubisco was firstly incubated with chemicals as indicated at 30°C for 10 min, then AMC was added and incubation continued for another 30 min. Molecular Plant 2016 9, 1018-1027DOI: (10.1016/j.molp.2016.03.012) Copyright © 2016 The Author Terms and Conditions

Figure 3 Identification of AMC-Acetylated Lysines in RbcL. (A and B) Mass spectrum of acetylated lysine residues in RbcL. Rubisco was incubated with 0.6 mM D3-AMC at 37°C for 2 h, followed by digestion and loading to LC–MS/MS. (A) Acetylated Lys201. (B) Acetylated Lys334. The fragment sequence is shown in each figure. Molecular Plant 2016 9, 1018-1027DOI: (10.1016/j.molp.2016.03.012) Copyright © 2016 The Author Terms and Conditions

Figure 4 Acetylation of Lysine in Synthesized Peptides by AMC. Synthesized RbcL peptides (0.25 μg/μl) were incubated with 0.3 mM AMC at 30°C for 2 h, and the products analyzed by LC–MS. (A and B) Lysine in the peptide of K201 (A) and K334 (B) was acetylated by AMC. The acetylated K334 peptides were used as a positive control. (C) High AMC concentrations promote lysine acetylation of K201. Plot (right) shows the ratio of acetylated (AcK201) to unacetylated (K201) peptides after the treatment with different concentrations of AMC, calculated from the LC chromatogram (left). (D) Substitution of lysine with glutamine (K201Q) abolished the AMC-mediated peptide acetylation. Molecular Plant 2016 9, 1018-1027DOI: (10.1016/j.molp.2016.03.012) Copyright © 2016 The Author Terms and Conditions

Figure 5 AMC and Analogs Modify RbcL Lysine Residues and Affect Rubisco Activity. (A) Acetylation by AMC decreases Rubisco activity. Rubisco was treated with AMC or 4-methylumbelliferone for 2 h. The total activity was determined after full activation of Rubisco through incubation in a buffer containing 10 mM NaHCO3 and 10 mM Mg2+ at 30°C for 10 min. Values are mean ± SD, n = 4, Student's t-test, *P ≤ 0.05, ***P ≤ 0.001. (B) RbcL modifications by AMC analogs and the effects on Rubisco activity. Rubisco was treated with 0.1 mM ClMC or BMC at 30°C for 1 h, respectively, and the initial activity was measured immediately without pre-activation. Values are mean ± SD, n = 3, Student's t-test, **P ≤ 0.01, ***P < 0.001. (C and D) Modification of K201 peptide by AMC analogs. K201 peptide (0.25 μg/μl) was incubated with 0.1 mM ClMC (C) or BMC (D) at 30°C for 2 h, and the products were analyzed by LC–MS. Molecular Plant 2016 9, 1018-1027DOI: (10.1016/j.molp.2016.03.012) Copyright © 2016 The Author Terms and Conditions