ChIP-MS in Plant Systems: Mapping the H3K27ac Proteome During the Greening Process

We have established a method for chromatin immunoprecipitation coupled to mass spectrometry (ChIP-MS) in Arabidopsis thaliana. We demonstrate its utility by investigating proteins associated with histone H3 lysine 27 acetylation (H3K27ac), a key epigenetic mark regulating photosynthesis-associated nuclear genes (PhANGs) during chloroplast development and establishment of photosynthesis. Purification of chromatin-associated proteins from light-grown Arabidopsis cell cultures identified 66 proteins associated with H3K27ac that met the selection criteria in the two replicate experiments: (i) 2-fold change in relation to IgG, (ii) at least two unique peptides, and (iii) relevant biological annotations. The identified proteins included chromatin remodelers, chromatin regulators and transcription factors with potential roles in H3K27ac deposition. To evaluate the physiological role of the candidates associated with the H3K27ac mark, we developed a rapid and reproducible phenotyping method based on controlled light scanning to determine chlorophyll accumulation in mutant seedlings. We complemented with pigment quantification and analysis of photosynthesis-associated nuclear genes (PhANGs) expression. Several mutants displayed altered greening, pigment accumulation, or affected photosynthetic gene expression consistent with a role during chloroplast development. Notably, chr11, chr17, and atpds5a mutants showed impaired pigment accumulation and reduced expression of PhANGs, whereas hmgb4 and mbd10 mutants exhibited increased greening and induction of PhANGs. Together, these findings establish ChIP-MS as a robust approach to identify histone mark-associated proteins in plants and provide a first set of candidate regulators of H3K27ac during chloroplast biogenesis. This technical advance opens new possibilities to discover chromatin-based regulation of plant development and environmental responses.