Dear Editor,Protein post-translational modifications (PTMs), such as acetylation and methylation, are established as crucial epigenetic marks in eukaryotic cells. Recent advances in mass spectrometry (MS) have led to the discovery of novel PTMs, including crotonylation, β-hydroxybutyrylation, and lactylation1. Among them, lysine β-hydroxybutyrylation (Kbhb) has been well characterized in animal cells, particularly for its influence on energy metabolism. For instance, Kbhb inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), a critical enzyme in the methionine cycle, associated with altered metabolite levels2. Notably, the detection of Kbhb in microbial species3 suggests that it may represent a conserved PTM across life forms. Recently, our study demonstrated that histone Kbhb regulates plant immunity by modulating the transcription of defense-related genes4. However, the presence and functional significance of Kbhb in plants, particularly under abiotic stress, remain largely unexplored. To address this gap, we conducted a comprehensive MS analysis of the β-hydroxybutyrylome in rice. Our results revealed that a substantial number of rice proteins were modified by Kbhb, further functional analysis demonstrated that Kbhb positively regulates drought stress responses in rice.To identify specific proteins and modification sites of Kbhb in rice, Kbhb-modified peptides were enriched from rice seedlings and analyzed by MS (Fig. 1a). The identified peptides showed mass errors < 5 ppm and lengths of 7–20 amino acids, indicating high data quality (Supplementary Fig. S1). In total, 1222 Kbhb sites on 739 proteins were identified (Supplementary Dataset S1). Immunoblotting using a validated Kbhb-specific antibody5 confirmed the MS results and demonstrated the presence of Kbhb in other plant species (Fig. 1b). Approximately 34% of Kbhb-modified proteins contained multiple modification sites, including Rubisco large subunit and Heat shock protein 81 (Fig. 1c; Supplementary Fig. S2). These proteins were predominantly localized to the chloroplast (42.1%), cytoplasm (30.4%), and nucleus (11.6%) (Fig. 1d). Functional and pathway enrichment analyses revealed significant overrepresentation of metabolic processes, gene expression, translation, photosynthesis, and ATP metabolism, consistent with protein–protein interaction network analysis (Supplementary Figs. S3–S5). Motif analysis showed enrichment of leucine (L), phenylalanine (F), or tyrosine (Y) at the +1 position and glutamic acid (E) or glycine (G) at the −1 position flanking Kbhb sites, suggesting sequence preferences for Kbhb modification (Supplementary Fig. S6). Collectively, these data provide a comprehensive Kbhb proteome in rice, revealing the widespread occurrence of Kbhb modification on proteins and its potential regulatory roles in plant.Fig. 1: Identification and functional characterization of the Kbhb proteome in rice seedlings under drought stress.a Experimental workflow for identifying Kbhb-modified proteins. b Detection of Kbhb-modified proteins in various plant species using immunoblotting. Coomassie brilliant blue (CBB) staining and ACTIN were used as loading controls. c Distribution of the number of Kbhb sites per identified protein. d Pie chart of the subcellular localization of Kbhb-modified proteins. e Immunoblot detection of Kbhb levels in rice plants under untreated (CK), heat (42 °C), PEG (20%), and salt (150 mM) treatments. f Ridge plots of β-hydroxybutyrylation levels at individual lysine sites in PEG treated and CK rice plants. g Cumulative density plots of the β-hydroxybutyrylation levels at individual lysine sites in PEG treated and CK rice seedlings. h Scatter plot of proteins with significantly upregulated and downregulated β-hydroxybutyrylation levels at individual lysine sites in PEG-treated and CK rice seedlings. i GO pathway enrichment analysis of proteins (n = 189) with elevated Kbhb levels in PEG-treated plants. j Immunoblotting detection of histone Kbhb levels in CK and PEG-treated rice plants. k Metaplots showing the differential occupancy of histone Kbhb in rice plants subjected to PEG stress compared to the CK plants. l Scatter plot of ChIP-seq data showing the differential occupancy of histone Kbhb in rice plants treated with PEG compared to CK plants. m GO-enriched pathways of genes with significantly upregulated histone Kbhb levels. n Volcano plots of differential transcript levels in rice plants treated with PEG compared to CK plants. o Cumulative density plots of histone Kbhb levels in genes significantly upregulated in n in PEG-treated plants and CK plants. p Pearson correlation analysis between expression changes and histone Kbhb changes in PEG-treated plants relative to CK plants. q Overlap between genes with hyper-Kbhb levels and significantly increased expression under PEG stress. r GO-enriched pathway analysis of the genes (n = 629) in q. s, t ChIP–qPCR (s) and RT-qPCR (t) analyses of histone Kbhb modification and gene expression levels for four selected drought-responsive genes in PEG-treated and control (CK) plants. u Analysis of histone Kbhb levels in hda705, hda706, hda710, and hda714 mutants compared to WT plants. v In vitro lysine de-β-hydroxybutyrylatase activity assay. w Phenotypes of hda710 plants under PEG stress. Seedlings were subjected to PEG stress for 3 days, followed by a 7-day recovery period under normal growth conditions (25 °C). For s, t, w significant differences between the groups were analyzed using a Student’s t-test. ACTIN or histone H3 was used as the loading control in b, e, j, u, v as indicated. For g, o the P value was calculated using a two-sample Kolmogorov–Smirnov test. TSS, transcriptional start site; TES, transcriptional end site; TE, transposable elements.Full size imagePathway analysis revealed significant enrichment of Kbhb-modified proteins in stress-response pathways (Supplementary Fig. S4), indicating a possible role of Kbhb in plant responses to abiotic stress. Consistently, immunoblotting showed a pronounced increase in global Kbhb levels in rice under polyethylene glycol (PEG)-induced drought stress (Fig. 1e), suggesting its potential role in drought stress regulation. Quantitative Kbhb proteomics, normalized to protein abundance, revealed an overall elevation of Kbhb following PEG treatment (Fig. 1f, g). Specifically, 189 Kbhb sites on 159 proteins were upregulated ( > 1.2-fold), whereas only 39 sites on 36 proteins were downregulated (Fig. 1h and Supplementary Dataset S2). Notably, three drought-induced sites were located on histones (H2AK10bhb, H2BK135bhb, and H3K56bhb), consistent with enrichment of gene expression-related pathways among proteins with increased Kbhb (Fig. 1i). Immunoblotting of histones and representative non-histone proteins further validated the dynamic changes of Kbhb proteome data under PEG treatment (Fig. 1j and Supplementary Figs. S7, S8).Given increased histone Kbhb levels under drought stress and its reported role in transcription5, we next conducted chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with a Kbhb-specific antibody to map its genomic distribution and assess its regulatory potential in rice. ChIP-seq analysis identified 27,682 Kbhb peaks corresponding to 18,351 genes in wild-type (WT) rice (Supplementary Dataset S3). ChIP–qPCR validation of regions with high and low Kbhb enrichment confirmed the reliability of the ChIP-seq data (Supplementary Fig. S9). Comparison with previously reported H3K9bhb profiles4 revealed a moderate correlation (r = 0.61), with substantial overlap between Kbhb- and H3K9bhb-marked genes (Supplementary Fig. S10). Genome-wide distribution analysis showed that Kbhb peaks were predominantly located within genic regions (Supplementary Fig. S11a). Among Kbhb-enriched genes, 91.5% were protein-coding (non-transposable element (TE)) genes, and 77% contained a single Kbhb peak (Supplementary Fig. S11b, c). Kbhb was preferentially enriched at transcription start sites (TSSs) of protein-coding genes rather than TE genes (Supplementary Fig. S11d). Correlation analyses with other histone modifications revealed that Kbhb was positively associated with active chromatin marks (H3K4ac, H3K9ac, H3K23ac, H4K5ac, and H3K4me3), but showed little or negative association with repressive marks (H3K9me2 and H3K9me3), suggesting a role in transcriptional activation (Supplementary Fig. S11e). Consistently, genes with higher expression levels exhibited stronger Kbhb enrichment (Supplementary Fig. S11f).To identify genes regulated by histone Kbhb under drought stress, we compared histone Kbhb ChIP-seq profiles between PEG-treated and control (CK) rice plants. Metaplot analysis showed a global increase in histone Kbhb deposition under drought conditions (Fig. 1k). Differential analysis identified 3770 genes with increased Kbhb deposition and 2053 genes with decreased Kbhb deposition ( > 1.5-fold, P 2-fold, P