bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293779; this version posted September 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Diurnal Arabidopsis proteome dynamics 1 Diurnal Dynamics of the Arabidopsis Rosette Proteome and Phosphoproteome 2 R. Glen Uhrig1,4,*, Sira Echevarría-Zomeño1,*, Pascal Schlapfer1, Jonas Grossmann2, Bernd 3 Roschitzki2, Niklas Koerber3, Fabio Fiorani3 and Wilhelm Gruissem1,5# 4 #Corresponding Author: 5 Dr. Wilhelm Gruissem 6 Department of Biology 7 ETH Zurich 8 Universitätstrasse 2 9 8092 Zurich 10 Switzerland 11 Phone: +41 44 632 08 57 12 E-Mail: [email protected] 13 1 Institute of Molecular Plant Biology, Department of Biology, ETH Zurich, 8092 Zurich, 14 Switzerland 15 2 Functional Genomics Center Zurich, University of Zurich, 8057 Zurich, Switzerland 16 3 Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich GmbH, 17 52425 Jülich, Germany 18 4 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada 19 5 Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan 20 21 * denotes equal contribution and should be considered joint first authors. 22 23 24 Key Words: Arabidopsis thaliana, diurnal cycle, proteome, phosphoproteome, quantitative 25 proteomics 26 27 28 Running Head: Diurnal Arabidopsis proteome dynamics 29 30 Funding: This work was funded by TiMet - Linking the Clock to Metabolism (Grant Agreement 31 245143) supported by the European Commission (FP7-KBBE-2009-3). Experiments conducted at 32 Forschungszentrum Jülich were partly funded by the Helmholtz Association. 33 34 Summary Statement: The manuscript provides quantitative information of diurnal changes in the 35 accumulation and phosphorylation of proteins in Arabidopsis thaliana rosettes grown in a 12 h 36 photoperiod. The highly resolved time-scale of the datasets offer new proteome-level insights for 37 future targeted studies. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293779; this version posted September 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Diurnal Arabidopsis proteome dynamics 38 ABSTRACT 39 Plant growth depends on the diurnal regulation of cellular processes, but it is not well 40 understood if and how transcriptional regulation controls diurnal fluctuations at the protein-level. 41 Here we report a high-resolution Arabidopsis thaliana (Arabidopsis) leaf rosette proteome 42 acquired over a 12 h light : 12 h dark diurnal cycle and the phosphoproteome immediately before 43 and after the light-to-dark and dark-to-light transitions. We quantified nearly 5000 proteins and 44 800 phosphoproteins, of which 288 fluctuated in their abundance and 226 fluctuated in their 45 phosphorylation status. Of the phosphoproteins, 60% were quantified for changes in protein 46 abundance. This revealed six proteins involved in nitrogen and hormone metabolism that had 47 concurrent changes in both protein abundance and phosphorylation status. The diurnal proteome 48 and phosphoproteome changes involve proteins in key cellular processes, including protein 49 translation, light perception, photosynthesis, metabolism and transport. The phosphoproteome at 50 the light-dark transitions revealed the dynamics at phosphorylation sites in either anticipation of 51 or response to a change in light regime. Phosphorylation site motif analyses implicate casein kinase 52 II and calcium/calmodulin dependent kinases among the primary light-dark transition kinases. The 53 comparative analysis of the diurnal proteome and diurnal and circadian transcriptome established 54 how mRNA and protein accumulation intersect in leaves during the diurnal cycle of the plant. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293779; this version posted September 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Diurnal Arabidopsis proteome dynamics 55 INTRODUCTION 56 Plant growth and biomass production are direct functions of the diurnal cellular carbon 57 balance, which is regulated by a combination of light responses and the circadian clock. Light 58 responses are triggered by a change in regime (i.e., presence or absence of light), while the 59 circadian clock is comprised of transcriptional regulators that operate in anticipation of a change 60 (e.g. transition from light to dark) and whose activities span the 24 hour (h) photoperiod (Nohales 61 & Kay, 2016; Oakenfull & Davis, 2017; Seluzicki, Burko, & Chory, 2017). The core clock 62 transcriptional regulators include CCA1/LHY, PRR5, PRR7 and PRR9, which form the morning 63 loop, and TOC1, ELF3, ELF4 and LUX, which form the evening loop (Flis et al., 2015; Staiger, 64 Shin, Johansson, & Davis, 2013). More than 30% of all Arabidopsis genes are regulated by the 65 circadian clock at the transcript level (Blasing et al., 2005; Covington, Maloof, Straume, Kay, & 66 Harmer, 2008). However, less is known about how the resulting diurnal transcriptome relates to 67 protein abundance (Abraham et al., 2016; Choudhary, Nomura, Wang, Nakagami, & Somers, 68 2015; Graf et al., 2017) and post-translational protein modifications (Choudhary et al., 2015; 69 Uhrig, Schlapfer, Roschitzki, Hirsch-Hoffmann, & Gruissem, 2019), both of which may also affect 70 protein function at light-dark transitions and throughout the diurnal cycle. Transcript and protein 71 abundance changes are often disconnected because changes in transcript levels show no 72 corresponding change in protein abundance (Baerenfaller et al., 2012; Seaton et al., 2018). For 73 example, this disconnect was found in the circadian clock mutants CCA1/LHY, PRR7/PRR9, 74 TOC1 and GI (Graf et al., 2017) and for the variability in the timing of peak transcript and protein 75 levels (translational coincidence) as a function of the photoperiod-dependent coordination between 76 transcriptome and proteome changes (Seaton et al., 2018). Variable delays between peak transcript 77 and protein abundance have implicated post-transcriptional regulation (e.g. splicing), translational 78 regulation (e.g. translation rate) as well as post-translational regulation (e.g. protein 79 phosphorylation) as possible mechanisms to explain the temporal differences in RNA and protein 80 abundance. More recent studies of plant protein-level regulation have also found extensive 81 variability in protein turnover (Li et al., 2017; Seaton et al., 2018), which adds further regulatory 82 complexity because conventional quantitative proteome workflows cannot easily account for 83 protein turnover. Although transcript and protein synthesis, stability and turnover all contribute to 84 the coordination of transcript and protein abundance, how these mechanisms are integrated is 85 currently not well understood. Insights into this regulatory complexity requires protein-level time- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293779; this version posted September 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Diurnal Arabidopsis proteome dynamics 86 course experimentation and, in particular, an understanding of protein post-translational 87 modifications (PTMs). To address this, we undertook a large-scale quantitative proteomics 88 approach to determine the extent of diurnal protein abundance and/or phosphorylation changes in 89 Arabidopsis rosette proteins over a 24h photoperiod. 90 Reversible protein phosphorylation is the most abundant PTM in eukaryotes (Adam & 91 Hunter, 2018; Rao, Thelen, & Miernyk, 2014). In non-photosynthetic eukaryotes phosphorylation 92 is found to modulate more than 70% of all cellular processes (Olsen et al., 2006), including the 93 circadian clock itself (Robles, Humphrey, & Mann, 2017). The extent of regulatory protein 94 phosphorylation events is likely similar in land plants as they have significantly larger kinomes 95 (Lehti-Shiu & Shiu, 2012; Zulawski, Schulze, Braginets, Hartmann & Schulze, 2014) compared 96 to humans, which encode 518 protein kinases (Manning, Whyte, Martinez, Hunter, & Sudarsanam, 97 2002). Conversely, both plants and humans have an equally comparable number of protein 98 phosphatases (Kerk, Templeton, & Moorhead, 2008). However, most protein phosphatases require 99 association with regulatory subunits to achieve their specificity (Moorhead et al., 2008; Uhrig, 100 Labandera, & Moorhead, 2013), suggesting similar complexity in how plants manage protein 101 dephosphorylation through a likely expansion of protein phosphatase regulatory subunits. 102 In plants, diurnal protein phosphorylation is regulated either in response to light, by the 103 circadian clock (Choudhary et al., 2015), or both (Uhrig et al., 2019), while the clock itself is 104 regulated by phosphorylation (Kusakina & Dodd, 2012; Uehara et al., 2019). Recent studies of the 105 circadian phosphoproteome combining the analysis of a free-running cycle and the circadian clock 106 mutants elf4 (Choudhary et al., 2015) or CCA1-OX over-expression (Krahmer et al., 2019) have 107 revealed temporally modified phosphorylation sites related to casein kinase II (CKII) and sucrose 108 non-fermenting