The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana

• DOI: 10.1016/j.mcpro.2021.100172
• Publication Date: 2021-11-03T05:16:07Z
• Authors: Johanna Krahmer; Matthew Hindle; Laura K. Perby; Helle K. Mogensen; Tom H. Nielsen; Karen J. Halliday; Gerben van Ooijen; Thierry Le Bihan; Andrew J. Millar
• Keywords: Phosphoprotein; Molecular clock

•Circadian (phospho)proteomics time courses of plants with or without functional clock.•Most protein abundance/phosphorylation rhythms require a transcriptional oscillator.•The majority rhythmic phosphosites peak around subjective dawn ("phospho-dawn").•A phosphorylated serine the metabolic enzyme F2KP has relevance. Twenty-four-hour, circadian control many eukaryotic mRNA levels, whereas levels their more stable proteins are not expected to reflect RNA rhythms, emphasizing need test regulation abundance and modification. Here we present proteomic phosphoproteomic series from Arabidopsis thaliana under constant light conditions, estimating that just 0.4% quantified but much larger proportion phospho-sites were rhythmic. Approximately half most at dawn, pattern term "phospho-dawn." Members SnRK/CDPK family kinases candidate regulators. A CCA1-overexpressing line disables clock gene circuit lacked phosphorylation. However, few fluctuated despite CCA1-overexpression still tended in close suggesting canonical mechanism is necessary for perhaps all phosphorylation rhythms. To potential relevance our datasets, conducted phosphomimetic experiments using bifunctional fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The diverse targets controlled by circuit, implicating posttranslational mechanisms transmission timing information plants. Most observations well-characterized plant species can be explained genetic network mostly negatively interacting transcription factors (1Pokhilko A. Fernández A.P. Edwards K.D. Southern M.M. Halliday K.J. Millar A.J. includes repressilator additional feedback loops.Mol. Syst. Biol. 2012; 8: 574Crossref PubMed Scopus (299) Google Scholar, 2Fogelmark K. Troein C. Rethinking activation arabidopsis clock.PLoS Comput. 2014; 10e1003705Crossref (90) Scholar). 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(Col-0 accession) over-expressing ("CCA1-OX," Scholar)) study. Seeds germinated grown plates (2.15 g/l Murashige & Skoog medium Basal Salt Mixture (Duchefa Biochemie), pH 5.8 (adjusted KOH), 12 agar (Sigma)) 85 μmol m−2 s−1 white fluorescent lights 21 °C Percival incubators 11 days h light, dark cycles. Seedlings transferred soil intensity 110 light–dark cycle. After had total 22 days, ZT 0 day 23 remained switched continuously collection material started (dataset I) 24 II). dataset I, six points 4 intervals taken five replicates each point, harvesting eight rosettes II, intervals, WT, (16 each) was therefore sampled ZT12 ZT32, ZT24 48 (CCA1-OX) ZT52 (WT). rationale starting either rather than later point enhance detection reducing desynchronization after longer periods (28Yakir Hassidim Melamed-Book Hilman Kron I. R.M. autonomous cell-type specific Arabidopsis.Plant 68: 520-531Crossref typically relatively small amplitudes technical variability compared (29Graf Coman Uhrig Walsh Flis Stitt Parallel scales regulation.Open 7Crossref Statistical assessment rhythmicity. both variance (ANOVA) JTK_CYCLE statistical tools (see below details). Rosettes roots harvested flash-freezing liquid nitrogen. extraction precipitation carried out according method "IGEPAL-TCA" described (30Krahmer Hindle S.F. Le Bihan Sample preparation thaliana.Methods Enzymol. 551: 405-431Crossref (5) Briefly, extracted precipitated TCA phase separation, then washed methanol acetone. total, 500 μg resuspended digested standard in-solution protocol peptides desalted. Before drying, eluted separated into two parts: 490 digest phosphopeptide enrichment, 10 saved analysis. Phosphopeptides enriched Titansphere spin tip kit (GL Sciences Inc) desalted BondElut Omix tips (Agilent) manufacturers' instructions. LC-MS/MS measurement subsequent previously Scholar): Dried dissolved μl (phosphoproteomics) 20 (global proteomics) 0.05% TFA passed through Millex-LH 0.45 μm (Millipore) filters. 8 run on-line capillary- HPLC-MSMS consisting micropump (1200 binary HPLC Agilent) coupled hybrid LTQ-Orbitrap XL instrument (Thermo-Fisher). Reverse-phase buffer LC-MS separation (2.5% acetonitrile, 0.1% FA H2O) B (10% H2O, 90% formic acid, 0.025% TFA). LC peptide initial 80 min linear gradient 0% 35% B, steeper up 98% over period min, remaining 15 until quick drop before end 120 min. Peak lists Progenesis software (version 4.1.4924.40586). tair Arabidopsis_1rep 20110103, 27,416 entries) database data-dependent detection, Mascot search engine 2.4), including sequences rank smaller 5. Search parameters follows: charges 2+, 3+ 4+, trypsin enzyme, allowing missed cleavages, carbamidomethyl (C) fixed modification, Oxidation (M), Phospho (ST) (Y), Acetyl(Protein N-term) variable modifications, tolerance 7 ppm, MS/MS 0.4 Da, 3+, ESI-trap instrument, decoy cutoff 20. cases, resulted false-discovery rate (FDR), less 5% exception (phosphoproteomics I: 3.5%, II: 3.2, 6.8%, 4.5%, calculated formula 2∗d/(n+d) (31Elias J.E. Gygi S.P. Target-decoy strategy spectrometry-based proteomics.Methods Mol. 2010; 604: 55-71Crossref (384) n d being number hits normal databases, respectively, score 20). Peptides area Progenesis, sum quantitation unique peptides. Where matched very multiple accession numbers shown exported results (supplemental Data S1 S2; Table 2).Table 2List CCA1-OX datasetsA. Phosphopeptides, WTAccessionDescriptionPeptide ID IPeptide IIPeptide sequencePhospho residue(s)p-value Ip-value IIq-value Iq-value IIPeak IPeak IIAT4G32340TPR-like superfam. pr.1566812823SASSLDLNLR39.5E-051.9E-095.7E-032.1E-061232AT2G33830DRM262462709TVAAVAGSPGTPTTPGSAR13/112.0E-022.0E-086.5E-028.9E-061240AT4G31700RPS6A86241785SRLSSAAAKPSVTA1,4,52.0E-062.8E-074.9E-043.9E-052424778SRLSSAAAKPSVTA1,4,5/1,4,112.0E-062.4E-084.9E-048.9E-06242421SRLSSAAAKPSVTA1,4,5/1,42.0E-069.5E-074.9E-041.1E-04242424893985LSSAAAKPSVTA91.5E-033.6E-032.1E-021.2E-013248AT5G10360RPS6B41, 173628SRLSSAPAKPVAA4/1, 47.4E-034.8E-074.0E-025.9E-053224AT3G47470CAB4715636DLSFTSIGSSAK31.3E-061.6E-074.9E-043.0E-052832AT3G07650COL9818211184AGEAYDYDPLTPTRSY153.0E-032.4E-072.7E-023.8E-052440AT5G48250BBX81103316261SGEAYDYDPMSPTRSY15/167.4E-031.6E-064.0E-021.6E-042440AT1G69870NRT1.771428476ISSPGSILDAEK31.7E-043.0E-066.8E-032.8E-042448AT5G40890CLC-A17831852HRTLSSTPLALVGAK3, 55.1E-051.1E-044.0E-037.3E-032424AT5G53420CCT101.19501997LGAGLVQSPLDR86.0E-031.1E-043.6E-027.3E-032848AT5G20670DUF167742025127TSSSGALPGIDGVESR3/41.9E-031.6E-042.3E-021.0E-022848AT1G73980TTM1100018639LSLDDDTVSSPK101.7E-021.9E-045.9E-021.1E-022824AT1G78020FLZ610297, 17003249, 1093LLSMVTPR31.3E-052.1E-041.5E-031.2E-0224242081, 5542, 55422113, 737, 2081RHSGDFSDAGHFLR32.4E-021.5E-037.4E-026.2E-022424AT1G11310MLO214963909SVENYPSSPSPR7/8, 102.3E-041.9E-037.7E-036.9E-023224AT2G32240PICC58837383DIDLSFSSPTKR81.1E-024.0E-034.9E-021.3E-012448AT3G13290VCR41406826TLSYPTPPLNPQSPR133.0E-035.4E-032.7E-021.6E-012848AT4G35100PIP3A140214ALGSFRSNATN4, 71.1E-028.0E-034.9E-021.9E-012824AT5G38640NagB/RpiA/CoA transferase-like pr.46831690DFPDGSTTASPGR106.0E-038.8E-033.6E-022.0E-013224AT1G37130NIA243822045, 2045VHDDDEDVSSEDENETHNSNAVYYK9, 109.0E-048.8E-031.7E-022.0E-012828AT3G13530MAPKKK735156312SKLPLVGVSSFR103.0E-038.8E-032.7E-022.0E-012840AT1G35580CINV19031066SVLDTPLSSAR5, 81.7E-029.7E-035.9E-022.1E-012424AT1G74780Major Facilitator Superfam. pr.62794702, 5728TVPHDYSPLISSPK123.0E-039.7E-032.7E-022.1E-012848AT3G27700zinc finger fam. pr.95639073LDTASDSGAAIASPK134.8E-031.1E-023.4E-022.2E-012848AT5G65010ASN249754842AGSDLVDPLPK36.9E-041.3E-021.6E-022.4E-012824AT4G31160DCAF1, DDB1-CUL4 187547154, 8583VHEGAPDTEVLLASPR143.5E-021.3E-029.3E-022.4E-012848AT4G13510AMT1;1214110SPSPSGANTTPTPV13.5E-021.4E-029.3E-022.5E-013248AT5G23660SWEET121480617187LGTLTSPEPVAITVVR69.1E-031.8E-024.3E-023.0E-012440AT3G26730RING/U-box pr.57946017NQTQSLSPPDVSR71.3E-042.2E-026.2E-033.5E-012424AT4G20910HEN11564813859, 13859SSSPNVFAAPPILQK3/2, 39.5E-052.6E-025.7E-033.7E-012824AT2G07360TASH32769, 50859464, 8286YQSTYEGYGSPIREEPPPPYSYSEPQSR101.1E-022.6E-024.9E-023.7E-012436AT4G26130unknown pr.5416, 7652, 15187456TTSIGDGGEEGVDDKASNFINK(FK)31.4E-022.6E-025.3E-023.7E-012440AT1G44800nodulin MtN21737734568SQELPITNVVK14.4E-062.6E-026.5E-043.7E-012852AT2G46920POL784811608SNFSAPLSFR81.9E-033.1E-022.3E-024.1E-012840AT2G42600, AT3G14940PPC2/PPC31672, 5762, 40, 4070MASIDAQLR37.4E-034.0E-024.0E-024.7E-013224AT3G60240EIF4G15851579QVLQGPSATVNSPR123.8E-034.3E-023.0E-024.9E-012848AT1G70770DUF2359514169, 800MTAIDSDDDGVVR67.4E-034.3E-024.0E-024.9E-012448AT5G40890, AT3G27170CLC-A/CLC-B749406TLSSTPLALVGAK31.3E-044.3E-026.2E-034.9E-012848AT4G12770, AT4G12780AUXILIN-LIKE 1/2.238, 238318FENVFSSISSSPTK116.0E-034.7E-023.6E-025.2E-012848B. CCA1-OXAccessionDescriptionPeptide I (h)Peak (h)AT2G33830DRM21348, 432812709TVAAVAGSPGTPTTPGSAR110.0230.0069111224AT1G51805SIF326172514, 1136VEGTLPSYMQASDGRSPR160.0190.021112424C. CCA1-OX, 12–20 hAccessionDescriptionPeptide (h)AT1G77760NIA147761968SVSSPFMNTASK30.0130.000670.420.252448AT2G45820REMORIN 1.31234, 5319698, 2343ALAVVEKPIEEHTPK130.0190.00450.420.682424D. Global proteomics, WTAccessionDescriptionp-value (h)AT4G39800MI-1-P SYNTHASE6.22E-068.7E-120.00595.78E-092824AT4G17090BAM30.00564.6E-050.230.0152044AT5G13630GUN50.0300.000100.430.0283252AT1G78570RHM10.000450.000590.0710.0872848AT1G15820LHCB60.0440.0230.520.931236AT5G54190 AT4G27440PORA PORB0.000250.0300.06511236AT3G08940 AT2G40100LHCB4.2 LHCB4.30.00130.0210.160.881236AT2G05070 AT2G05100LHCB2.2 LHCB2.10.0130.000320.310.0621232E. CCA1-OXAccessionDescriptionp-value (h)AT3G47070Unknown0.0340.0120.630.912040AT3G49190O-acyltransferase pr.0.00160.0220.240.923244AT4G39800MI-1-P SYNTHASE0.000370.034060.180.992424AT1G08200 AT2G27860AXS2 AXS10.00560.00260.350.562044Abbreviations: fam., family; pr, protein.All p-values analysis, q-values BH-corrected p-values.In phosphopeptides, location residues differs slightly between datasets (shifted 1–2 phosphates missing), case, noted I/dataset II).Bold denotes difference h. Open table new tab Abbreviations: protein. All p-values. Bold order remove duplicates due alternative modifications other qpMerge (32Hindle Krahmer Noordally Z.B. Simpson T.I. qpMerge: Merging isoforms motif centric strategy.bioRxiv. ([preprint])https://doi.org/10.1101/047100Crossref publicly available pep2pro (33Baerenfaller Svozil Hull Russenberger Bischof Lu Baginsky pep2pro: tool comprehensive reveal about organ-specific thaliana.Integr. (Camb). 3: 225-237Crossref (61) http://fgcz-pep2pro.uzh.ch (Assembly names "ed.ac.uk I," II," II") deposited ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via PRIDE partner repository (34Vizcaíno Deutsch provides globally coordinated submission dissemination.Nat. Biotechnol. 32: 223-226Crossref (1998) identifier PXD009230. Exported.csv files quantifications online suppl