Downregulation of N-terminal initiator methionine imprinting by NatB impacts growth and the response to osmotic stress in Arabidopsis

N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes and is catalyzed in humans by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the characterization of the plant Nat machinery and its biological relevance is still in its infancy, although NTA has gained recognition as key regulator of crucial processes like protein turnover, protein-protein interaction and protein targeting. In this study we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics and physiological assays to characterize the Arabidopsis NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, naa25-1) decreased growth to 50% of wild-type level. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, while yeast NAA20 failed to complement naa20-1. Quantitative N-terminomics of approximately 2000 peptides identified 29 bona fide substrates of the plant NatB. In vivo, NatB preferentially acetylated N-termini starting with the initiator methionine followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. The global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. In agreement, we revealed the specific impact of NatB on the resistance of plants to osmotic or high-salt stress. Remarkably, depletion of NatA did not affect these resistances. Comparison of the wild-type Arabidopsis thaliana accession Col-0 (Control, N=3) with the naa20-1 mutant (N=3) that is depleted of the catalytically active subunit of the Nat B complex.