SummaryPost-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phasespecific manner, and is important for progression through mitosis.

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ORCID IDs: 0000-0002-7279-0246 (Y.L.); 0000-0001-6202-5826 (Z.A.)Deg proteases are involved in protein quality control in prokaryotes. Of the three Arabidopsis (Arabidopsis thaliana) homologs, Deg1, Deg5, and Deg8, located in the thylakoid lumen, Deg1 forms a homohexamer, whereas Deg5 and Deg8 form a heterocomplex. Both Deg1 and Deg5-Deg8 were shown separately to degrade photosynthetic proteins during photoinhibition. To investigate whether Deg1 and Deg5-Deg8 are redundant, a full set of Arabidopsis Deg knockout mutants were generated and their phenotypes were compared. Under all conditions tested, deg1 mutants were affected more than the wild type and deg5 and deg8 mutants. Moreover, overexpression of Deg5-Deg8 could only partially compensate for the loss of Deg1. Comparative proteomics of deg1 mutants revealed moderate up-regulation of thylakoid proteins involved in photoprotection, assembly, repair, and housekeeping and down-regulation of those that form photosynthetic complexes. Quantification of protein levels in the wild type revealed that Deg1 was 2-fold more abundant than Deg5-Deg8. Moreover, recombinant Deg1 displayed higher in vitro proteolytic activity. Affinity enrichment assays revealed that Deg1 was precipitated with very few interacting proteins, whereas Deg5-Deg8 was associated with a number of thylakoid proteins, including D1, OECs, LHCBs, Cyt b 6 f, and NDH subunits, thus implying that Deg5-Deg8 is capable of binding substrates but is unable to degrade them efficiently. This work suggests that differences in protein abundance and proteolytic activity underlie the differential importance of Deg1 and Deg5-Deg8 protease complexes observed in vivo.

HighlightFIE, a WD-40 subunit of the Arabidopsis PRC2 complexes known to take part in H3K27 methylation of nuclear chromatin, is also localized in cytoplasmic complexes.