An evolutionarily conserved histone modification <scp>H3K37ac</scp> activates gene transcription in response to salt stress in rice

Histone acetylation is a well-known chromatin mark that is associated with gene transcriptional activation (Y. Chen et al., 2024). In plants, histone acetylation modification mainly occurs at the N-terminal lysine (Lys) residues of histone H3 (K9, K14, K18, K23, K27, K36) and H4 (K5, K8, K12, K16, K20) (Waterborg, 2011). The acetylation status of specific Lys residues on histone proteins is reversibly modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). These enzymes play essential roles in plant growth, development, and responses to abiotic stresses by regulating gene transcription. Many HDACs have been reported to be involved in modulating plants' tolerance to abiotic stresses (Ueda & Seki, 2020; Cui et al., 2023). For instance, HDAC, HDA15, interacts with MYB96 to repress negative regulators of early abscisic acid signaling and improve drought resistance in Arabidopsis (Lee & Seo, 2019). Similarly, the rice HDA706 was found to enhance salt tolerance by deacetylating the H4K5/K8ac at the promoter region of PP2C49 (Liu et al., 2023). By contrast, the Arabidopsis HDA9, responsible for H3K9ac deacetylation, was reported to negatively regulate salt and drought stress responses (Zheng et al., 2016). These studies highlight the pivotal roles of histone acetylation in modulating the transcriptional programs underlying plant abiotic stresses tolerance. However, our understanding of the full repertoire of histone Lys acetylation sites and their specific functions remains incomplete. Herein, we identified an unexplored histone Lys acetylation in rice, Lys-37 of histone H3 (H3K37ac); we found this acetylation modification is highly conserved across plant species. Notably, the level of H3K37 acetylation in rice is strongly induced by salinity stress. Furthermore, we have identified a HDAC, HDA705, that is responsible for removing the acetylation mark from H3K37, and mutation of HDA705 significantly improved the salt tolerance of rice plants. The ‘Zhonghua 11’ (ZH11) rice variety (Oryza sativa ssp. japonica) was used in this study. The hda704, hda705, hda706, hda710, hda712, and srt2 mutants (ZH11 background) used in this study were previously characterized (Xu et al., 2021, 2025). For salt stress treatment, both wild-type (WT) and mutant plants (hda705) were first hydroponically grown in liquid Yoshida's solution (Z. Chen et al., 2024). Fourteen-day-old seedlings, cultivated under 25°C with a 14 h : 10 h, light : dark cycle, were then subjected to salt stress by adding 180 mM NaCl to the nutrient solution for 3 d. Control seedlings were grown under the same temperature and light conditions but without salt stress. Following the stress treatments, plants were returned to normal conditions for 7 d, after which the survival rate was determined by calculating the percentage of viable plants (live plants/total plants × 100%). The survival rates were assessed from three groups, with each group containing 40 plants (40 replicates). For osmotic stress, 14-d-old seedlings were exposed to 20% PEG6000 in the nutrient solution for 6 h, after which leaf samples were collected for histone extraction. Heat stress was applied by incubating 14-d-old seedlings at 42°C for 6 h in a growth chamber, followed by leaf collection for histone extraction. Cold stress treatments involved incubating 14-d-old seedlings at 4°C for 6 h, with leaf samples harvested afterward for histone extraction. The histone H3K37 acetylation (H3K37ac) was identified from a global rice acetyl-proteome dataset generated by liquid chromatography tandem mass spectrometry (LC-MS/MS; Ma et al., 2023). Briefly, proteins extracted from rice panicles were digested with trypsin after reduction and alkylation. The resulting peptides were incubated with anti-acetyl-lysine beads (cat no.: PTM-104; PTM Bio, CN) for immunoaffinity enrichment. The bound acetylated peptides were eluted, vacuum-dried, and desalted using C18 ZipTips (Millipore, Billerica, MA, USA) before LC-MS/MS analysis. For LC-MS/MS analysis, the enriched acetyl-peptides were analyzed by online LC-MS/MS using an EASY-nLC 1200 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a reversed-phase column (15 cm and 75 μm i.d.) with a 60-min linear gradient from 6% to 35% solvent B (0.1% formic acid in 98% acetonitrile), followed by a step to 80% B, at a flow rate of 400 nL min−1. MS analysis was performed with a nano-electrospray ionization source at 2.0 kV. Full-scan MS spectra (m/z 350–1800) were acquired at a resolution of 70 000, followed by data-dependent MS/MS scans of the top 20 precursors (NCE: 28; resolution: 17500). Dynamic exclusion was set to 15 s. The raw MS/MS data were processed with MaxQuant (v.1.6.6.0) against the Oryza sativa protein database (http://rice.hzau.edu.cn/rice_rs2/). Search parameters included: trypsin/P as the cleavage enzyme, allowing up to two miscleavages; peptides with a minimum length of seven amino acids and a maximum of five modifications; precursor mass tolerance of 5 ppm (main search) and fragment mass tolerance of 0.04 Da; fixed modification of carbamidomethylation (C); variable modifications of oxidation (M), acetylation (K), and N-terminal acetylation. The false discovery rate (FDR) threshold for proteins, peptides, and modified sites was set to <0.01, and the minimum score for modified peptides was set to >40. Total histone-enriched fractions were extracted from Arabidopsis, tobacco, maize, and rice seedlings using an EpiQuik Total Histone Extraction Kit (OP-0006-100; Epigentek, Farmingdale, NY, USA). Before immunoblotting, half of the volume of 2 × SDS loading buffer (120 mM Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 0.04% bromophenol blue, and 10% β-mercaptoethanol) was added to the supernatant of extracted. The mixture was thoroughly combined and heated at 95°C for 10 min. The histone proteins were then subjected to immunoblot analysis using anti-H3 (1 : 1000, ab1791; Abcam, Cambridge, UK) and anti-H3K37ac (1 : 1000, PTM-128; PTM, CN) antibodies. The specificity of the H3K37ac antibody was examined by dot blot and peptide competition assays. For the dot blot assay, 1, 5, or 10 ng of each synthetic peptide (H3K37ac, H3K36ac, and H3K27ac) was spotted onto a nitrocellulose membrane and air-dried. The membrane was blocked with 5% nonfat milk in Tris Buffered Saline with Tween-20 (TBST) for 1 h and then incubated with the H3K37ac antibody (1 : 1000 dilution) overnight at 4°C. Signals were detected using an horseradish peroxide (HRP)-conjugated secondary antibody and chemiluminescence. For the peptide competition assay, the H3K37ac antibody (1 μg) was preincubated with synthetic H3K37ac peptide (0, 1, 2, 5, or 10 μg) in 10 ml of TBST at room temperature for 1 h before being used to probe histones separated by 15% Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were subsequently incubated with HRP-conjugated secondary antibodies, and signals were visualized using ECL detection reagents. The full-length coding sequences of SRT2 and HDA705 were cloned into the PGEX-6P-1 expression vector for protein production. These plasmids were used to express the respective proteins in Escherichia coli BL21 (DE3) cells. The GST-tagged proteins GST, SRT2-GST, and HDA705-GST were then purified using GST beads (17–5132-01; GE Healthcare, Chicago, IL, USA). Nuclear proteins were extracted from WT rice leaves and used for the in vitro de-Kac activity assay. Purified GST-tagged HDACs were incubated with the extracted nuclear proteins in a reaction buffer containing 50 mmol l−1 Tris–HCl (pH 8.5), 2.7 mmol l−1 KCl, 1 mmol l−1 MgCl2, 137 mmol l−1 NaCl, and 1 mmol l−1 dithiothreitol at 37°C for 4 h. The reaction products were analyzed by immunoblotting with anti-H3K37ac antibodies (1 : 1000, PTM-128; PTM, CN). For the HDAC activity assay, constructs expressing HDA705-3 × FLAG, SRT2-3 × FLAG, H3-6 × His, and H3K37A-6 × His were introduced into Nicotiana benthamiana leaf epidermal cells. After 48 h of infiltration, the transformed leaves were harvested and frozen in liquid nitrogen for tissue disruption. Total proteins were extracted using a lysis buffer (10 mM Tris–HCl, pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.5% Nonidet™ P40 Substitute), purified with Ni-NTA Magnetic Agarose Beads (36113; QIAGEN, Hilden, Germany), and analyzed by western blotting. One microgram of total RNA was reverse-transcribed in a 20 μl reaction volume using DNase and reverse transcriptase (R233-01; Vazyme, Nanjing, China) according to the manufacturer's instructions to generate cDNA. RT-qPCR was performed on an ABI PRISM 7500 device. Three biological replicates were conducted for each gene. The rice ACTIN1 gene was used as the internal control. Chromatin immunoprecipitation (ChIP) protocol followed a previously established method (Xu et al., 2023). Two grams of 14-d-old rice seedlings were cross-linked by 1% (v/v) formaldehyde and used for chromatin extraction. Chromatin was fragmented to c. 200 bp by sonication and then incubated with antibody-coated beads (10001D; Invitrogen/Life Technologies, Carlsbad, CA, USA) overnight. After extensive washing, immunoprecipitated chromatin was de-cross-linked and retrieved, and un-immunoprecipitated chromatin was used as input. The precipitated and input DNA samples were analyzed by real-time PCR with gene-specific primers (Supporting Information Table S1). The raw reads of RNA-seq were processed to remove low-quality sequence using FastP (v.0.232). Clean reads were then aligned to the rice genome sequence (MSU v.7.0) using the Subjunc aligner with default settings. Differential gene expression analysis was carried out using DESeq2 (v.1.36.0), with genes exhibiting a fold change (FC) > 2 and a P-adjusted value of < 0.05 considered as differentially expressed. CUT&Tag experiments were conducted as described previously (Xu et al., 2023). Briefly, rice nuclei were isolated, washed with ice-cold wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, and 0.5 mM spermidine), and immobilized to concanavalin A-coated beads (TD902; Vazyme) by incubation at room temperature for 10 min. The bead-bound nuclei were incubated overnight at 4°C in 200 μl of primary antibody buffer containing anti-H3K37ac (PTM-128; PTM, CN) antibody with a 1 : 50 dilution. After removing the primary antibody buffer, nuclei were washed three times with 800 μl of dig-wash buffer (wash buffer with 1% bovine serum albumin (BSA) and 0.05% digitonin). The nuclei were then resuspended in 200 μl dig-wash buffer with a dilution secondary antibody (TD902; Vazyme) and incubated at room temperature for 1 h. Following a brief dig-wash buffer wash, nuclei were resuspended in 200 μl dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1% BSA, and 0.01% digitonin) and incubated for 1 h at room temperature. pA-Tn5-bound nuclei were washed three times with 800 μl of dig-300 buffer and then tagmentated at 37°C for 1 h. Tagmentation was terminated and proteins were digested by adding 15 mM EDTA, 500 μg/ml of proteinase K, and 0.1% SDS. Genomic DNA was extracted and purified with VAHTS DNA Clean Beads (N411-01; Vazyme). Purified genomic DNA was amplified using Vazyme Master Mix (TD902; Vazyme) with universal i5 primer and barcoded i7 primer (TD202; Vazyme). The library PCR products were purified with Agencourt AMPure XP beads (A63881; Beckman Coulter, Brea, CA, USA) and sequenced on Illumina HiSeq 2500. For CUT&Tag-seq data analysis, low-quality reads were removed using Fastp (v.0.232). The filtered reads were aligned to the rice genome (MSU v.7.0) using Bowtie2 (v.2.2.5), allowing for up to two mismatches. To eliminate potential PCR duplicates, Samtools (v.0.1.19) was employed. Peak calling for histone modifications was performed using MACS (v.2.2.7.1) with default settings (bandwidth of 300-bp, model fold of 10 to 30, and P-value cutoff of 1.00e−5). The wiggle files generated with deepTools (v.2.5.3) were normalized using BPM and visualized with the Integrative Genomics Viewer (IGV, v.2.3.88). Differentially modified histone peaks were detected using DiffBind (v.3.5), and peak annotation was completed using Homer's annotatePeaks.pl (v.4.11). Gene Ontology (GO) enrichment was conducted with the clusterProfiler package (v.4.10.0). Visualization of ridge plots, scatter plots, and heatmaps was done using TBtools (v.0.6; Chen et al., 2020) and R (v.3.5). We identified a novel lysine acetylation site K37ac in the rice histone protein H3 using LC-MS/MS technology (Fig. 1a). To determine whether this lysine residue is conserved across different species, we performed sequence alignment of histone H3 from various organisms, including mammals (human (Homo sapiens) and mice (Mus musculus)), Physcomitrium patens, Saccharomyces cerevisiae, and higher plants (such as Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), maize (Zea mays), cotton (Gossypium hirsutum), and rice (Oryza sativa)). The analysis showed that the K37 in histone H3 is evolutionarily conserved across these species (Fig. S1). To functionally study H3K37ac in plants, we first obtained a commercial H3K37ac antibody and verified its specificity using dot blot and peptide competition assays (Figs S2, S3). Furthermore, we generated a point mutation of H3 (H3K37A) and expressed both WT and mutant H3 in tobacco cells. Western blot analysis with the H3K37ac antibody showed that the H3K37A mutation abolished the signal (Fig. S4a). Collectively, these results confirmed the specificity of the H3K37ac antibody. Next, to investigate whether the H3K37ac modification is conserved across different plant species, we performed immunoblotting assays using the specific H3K37ac antibody in Arabidopsis, rice, maize, and tobacco. The results indicated that H3K37ac modification is a conserved chromatin mark in plants (Fig. 1b). To investigate the chromatin function of the H3K37ac modification in the rice genome, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) analysis on WT rice seedlings, with two biological replicates (Fig. S5a). A total of 23 578 peaks (16 888 marked genes) were identified (Table S2), with most of (81.15%) the H3K37ac marked genes containing only one peak (Fig. 1c). The majority of H3K37ac peaks were located in genic regions (Fig. 1d), and the modification was highly enriched at the transcription start site (TSS) and within the exons of protein-coding genes (Figs 1d, S5b), whereas its levels were markedly lower in transposable elements (TEs) and TE-related genes (Figs 1e, S6). GO pathway enrichment analysis showed that H3K37ac-marked genes were associated with various processes, including cell communication, signaling, and gene expression regulation, with the strongest enrichment in response to stimulus, suggesting a role in the regulation of stress response (Fig. S7). Correlation analysis between H3K37ac and other histone modifications revealed that H3K37ac is positively correlated with the active markers, such as H3K9ac, H3K23ac, H4K8ac, and H4K16ac, while showing a negative correlation with repressive markers like H3K9me3 and H3K27me3 (Fig. 1f), suggesting that H3K37ac functions as a marker of active genes in rice. is by the correlation between H3K37ac levels and gene expression levels (Figs To the biological function of H3K37ac modification in rice, we assessed its levels in 14-d-old rice seedlings under various abiotic including osmotic stress and salinity mM The H3K37ac modification levels were under these abiotic for salt stress (Fig. To investigate the underlying response to salt stress, we performed CUT&Tag for H3K37ac using WT seedlings with salt mM NaCl for 6 Two biological replicates were performed for normal or salt conditions (Fig. and the of the data was by the (Fig. CUT&Tag data analysis revealed that the modification levels of H3K37ac in salt plants were higher that under normal conditions (Fig. with the immunoblot with the the modification levels of peaks to > < were significantly while of peaks > < were under salt stress (Fig. Table S3). analysis of genes verified the CUT&Tag results (Fig. GO analysis revealed that the genes with H3K37ac were mainly enriched in regulation of gene response to stimulus, and response (Fig. To H3K37ac modification is involved in modulating salt stress we analyzed the of rice seedlings under salt stress same as for with three biological replicates for both and In we identified genes < Table and genes < Table By CUT&Tag we found that genes H3K37ac enrichment at their salinity genes (Fig. analysis of and CUT&Tag data revealed that genes both transcription levels and H3K37ac modification levels under salt stress (Fig. Table GO enrichment analysis revealed that these genes mainly involved in response to stimulus, response to stress (Fig. genes previously such as and et al., et al., To we performed real-time and to the transcription and H3K37ac modification levels of five and with the analysis, the expression levels and H3K37ac modification levels of the five genes were significantly under salt stress (Fig. Collectively, these results that H3K37ac modification is involved in modulating salt stress response in rice. The correlation between H3K37ac levels and the expression of genes that this novel chromatin mark an role in the transcriptional regulation of the stress To the enzymes responsible for H3K37ac we analyzed the modification levels in different rice HDACs assays showed that H3K37ac level was in mutant and was in HDAC mutant srt2 (Fig. To their we histone H3 with SRT2 or HDA705 in tobacco purified the and performed western with the H3K37ac antibody. of the enzymes significantly H3K37ac their to histone H3 at the K37 (Fig. Furthermore, in vitro HDAC activity assays that HDA705 activity (Fig. To investigate whether HDA705 salt stress responses H3K37ac regulation in rice, WT plants and two mutant and were subjected to salt stress The results that the mutants with a higher survival rate the WT after from salt stress mM NaCl; and revealed that the expression levels and H3K37ac levels of genes in mutants were significantly to WT (Fig. that the of H3K37ac on the genes their expression and salt tolerance in mutant plants. these data that HDA705 is the for H3K37ac and that this a role in modulating rice salt tolerance. In this study identified H3K37ac as a previously histone acetylation mark that is associated with transcriptional activation and to salt stress in rice. 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