Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis . Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis , of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.

Summary: It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin‐affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α‐tubulin, associates with α/β‐tubulin and is retained on GTP/taxol‐stabilized microtubules, at least in part, by direct association with the PP2A‐A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin‐Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α‐tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A‐subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.