Volume 10, Issue 12, Pages 1510-1522 (December 2017) Histone Deacetylase AtSRT1 Links Metabolic Flux and Stress Response in Arabidopsis  Xiaoyun Liu, Wei Wei, Wenjun Zhu, Lufang Su, Zeyang Xiong, Man Zhou, Yu Zheng, Dao-Xiu Zhou  Molecular Plant  Volume 10, Issue 12, Pages 1510-1522 (December 2017) DOI: 10.1016/j.molp.2017.10.010 Copyright © 2017 The Author Terms and Conditions

Figure 1 AtSRT1 Expression Levels Affect Seedling Sensitivities to ABA. (A) Images of wild-type (WT) (Col-0), RNAi, srt1 mutant, and 35S::AtSRT1-GFP (OX-AtSRT1) lines grown for 7 days on MS medium with or without 0.5 μM ABA. (B) Germination rates of wild-type and AtSRT1 mutant and transgenic seedlings. Seeds were germinated on MS medium (with or without 0.5 μM ABA) and germination rate was counted 4 days after germination. Error bars represent means and SD from three replications (*p < 0.05; **p < 0.01). Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 2 AtSRT1 Interacts with AtMBP-1. (A) Diagrams of LOS2/ENO2 protein and the alternatively translated form (AtMBP-1). Red bars represent positions of longest clones isolated from yeast two-hybrid screening. (B) AtSRT1 interacts with AtMBP-1, but not with LOS2/ENO2 in a yeast two-hybrid assay. Each cDNA was cloned to pGADT7 and pGBKT7 vectors. Yeast Gold cells were co-transformed with a combination of the indicated plasmids. Yeast cells were plated on selective media SD/-L-T or SD/-L-T-H-A + X-gel and incubated for 5 days. (C) CoIP assays of AtSRT1 and AtMBP-1 interaction in vivo. Total proteins extracted from 35S::At SRT1-GFP/35::AtMBP-1-FLAG and 35::At SRT1-GFP/35::FLAG transgenic plants were incubated or not (input) with anti-GFP agarose. The agarose-bound proteins were eluted and analyzed by immunoblotting with anti-GFP or anti-FLAG antibodies. (D) BiFC analysis of AtSRT1 and AtMBP-1 interaction in N. benthamiana leaf cell nuclei using Agrobacterium-mediated transient expression of the indicated vectors. Images were taken under confocal microscopy. Red blobs indicate protein interactions occurred. Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 3 Analysis of ABA Sensitivity of Plants Overexpressing both AtSRT1 and AtMBP-1. (A) Comparison of germination rates of the single (OX-AtSRT1-1) and double (OX-AtMBP-1-1, OX-AtSRT1/OX-AtMBP-1-2) overexpression plants with wild-type. Seeds were germinated on MS medium with 0.5 μM ABA. Germination rates were counted 4 days after germination. Error bars represent means ± SD from three biological replicates. (B) Phenotypes of the 7-day-old wild-type and the transgenic seedlings grown on MS medium with or without 0.5 μM ABA. Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 4 AtSRT1 Directly Binds to the AtMBP1-1 Targets STZ/ZAT10 and LOS2/ENO2, Reduces H3K9ac from the Loci, and Represses Transcription of the Genes. (A) STZ/ZAT10, LOS2/ENO2, and two additional stress-responsive genes RD29A and RD29B were upregulated in srt1 mutant and downregulated in the AtSRT1 overexpression (OX-AtSRT1-1) seedlings grown under normal conditions. (B) H3K9ac levels detected at the promoter regions of ZAT10, LOS2, RD29A, and RD29B in wild-type and AtSRT1 mutant and overexpression lines. (C) Direct association of AtSRT1 protein with ZAT10, LOS2/ENO2, and RD29A promoters. ChIP assays were performed with anti-AtSRT1 and immunoglobulin G (IgG) (as control) and analyzed with the same primer sets as in (B).The relative positions of P1 and P2 regions in the genes are indicated in the bottom. Error bars represent means ± SD from three biological replicates. Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 5 AtSRT1 Deacetylates and Enhances AtMBP-1 Protein Stability In Vivo. (A) AtMBP-1 is lysine-acetylated and the acetylation is regulated by AtSRT1 deacetylase activity. Protein extracts of transgenic plants expressing both AtSRT1-GFP and AtMBP-1-FLAG or AtMBP-1-FLAG alone grown in the presence (+) or absence (−) of 25 μM sirtinol, a sirtuin deacetylase inhibitor, were analyzed by immunoblotting with antibodies of pan lysine acetylation (anti-Lys) and anti-FLAG as loading controls. Left and right panels are two repetitions of the experiments. (B) AtSRT1 enhances AtMBP-1 stability. Upper: 12-day-old 35S::AtMBP-1-FLAG transgenic seedlings were treated with 100 M cycloheximide (CHX) plus or minus MG132, harvested at the indicated time points, and analyzed by immunoblotting with anti-FLAG and anti-actin as loading controls. Lower: 35S::AtMBP-1-FLAG single and 35S::AtSRT1-GFP/35S::AtMBP-1-FLAG double-overexpression plants were treated with 100 μM CHX and harvested at the indicated time points. (C) Effect of the deacetylase inhibitor sirtinol on AtMBP-1 stability. The double-overexpression plants grown in the presence or absence of sirtinol were analyzed as (A) and (B). Relative quantifications of the western blot bands are indicated. Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 6 HK, PK, PFK, and Enolase Activity in AtSRT1 and AtMBP-1 Transgenic Plants Compared with the Wild Type. Enzymatic activities were measures using commercial kits (see Methods) in rosette leaves of 4-week-old plants grown in short-day conditions. Error bars represent means ± SD from three biological repeats. Significance of differences (Student's t-tests) compared with wild-type are indicated: *p < 0.05, **p < 0.01). Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 7 Respiration Rates in AtSRT1 and AtMBP-1 Transgenic Lines and srt1 Mutant Compared with Wild-Type. Respiration rates were measured by Licor “LI-6400XT portable photosynthesis system” in leaves of 7-week-old plants grown under short days (8 h light/16 h dark, at 20°C). The absolute values of the net respiration rate were CO2 productions. Left: plants after 3 h in light. Right: plants after 2 days in complete darkness. For each transgenic genotype, two lines were measured; for each line, three individual plants were measured; for each plant, five leaves were measured. The means and SD were calculated from 15 measures. Significance of differences compared with wild-type are indicated: *p < 0.05, **p < 0.01) (Student’s t-tests). Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions

Figure 8 Proposed Model of the Regulation of Glycolysis, Mitochondrial Respiration (TCA), and Stress Tolerance by AtSRT1 and AtMBP-1 Interaction. Activation is indicated by arrows and repression by bars. Dashed lines are hypothetical in plants. Molecular Plant 2017 10, 1510-1522DOI: (10.1016/j.molp.2017.10.010) Copyright © 2017 The Author Terms and Conditions