Description:
<jats:title>Summary</jats:title><jats:p>Protein phosphorylation and acetylation are the two most abundant post‐translational modifications (<jats:styled-content style="fixed-case">PTM</jats:styled-content>s) that regulate protein functions in eukaryotes. In plants, these <jats:styled-content style="fixed-case">PTM</jats:styled-content>s have been investigated individually; however, their co‐occurrence and dynamics on proteins is currently unknown. Using <jats:italic>Arabidopsis thaliana</jats:italic>, we quantified changes in protein phosphorylation, acetylation and protein abundance in leaf rosettes, roots, flowers, siliques and seedlings at the end of day (<jats:styled-content style="fixed-case">ED</jats:styled-content>) and at the end of night (<jats:styled-content style="fixed-case">EN</jats:styled-content>). This identified 2549 phosphorylated and 909 acetylated proteins, of which 1724 phosphorylated and 536 acetylated proteins were also quantified for changes in <jats:styled-content style="fixed-case">PTM</jats:styled-content> abundance between <jats:styled-content style="fixed-case">ED</jats:styled-content> and <jats:styled-content style="fixed-case">EN</jats:styled-content>. Using a sequential dual‐<jats:styled-content style="fixed-case">PTM</jats:styled-content> workflow, we identified significant <jats:styled-content style="fixed-case">PTM</jats:styled-content> changes and intersections in these organs and plant developmental stages. In particular, cellular process‐, pathway‐ and protein‐level analyses reveal that the phosphoproteome and acetylome predominantly intersect at the pathway‐ and cellular process‐level at <jats:styled-content style="fixed-case">ED</jats:styled-content> versus <jats:styled-content style="fixed-case">EN</jats:styled-content>. We found 134 proteins involved in core plant cell processes, such as light harvesting and photosynthesis, translation, metabolism and cellular transport, that were both phosphorylated and acetylated. Our results establish connections between <jats:styled-content style="fixed-case">PTM</jats:styled-content> motifs, <jats:styled-content style="fixed-case">PTM</jats:styled-content> catalyzing enzymes and putative substrate networks. We also identified <jats:styled-content style="fixed-case">PTM</jats:styled-content> motifs for further characterization of the regulatory mechanisms that control cellular processes during the diurnal cycle in different Arabidopsis organs and seedlings. The sequential dual‐<jats:styled-content style="fixed-case">PTM</jats:styled-content> analysis expands our understanding of diurnal plant cell regulation by <jats:styled-content style="fixed-case">PTM</jats:styled-content>s and provides a useful resource for future analyses, while emphasizing the importance of analyzing multiple <jats:styled-content style="fixed-case">PTM</jats:styled-content>s simultaneously to elucidate when, where and how they are involved in plant cell regulation.</jats:p>