TOC1 clock protein phosphorylation controls complex formation with NF‐YB/C to repress hypocotyl growth
2021; Springer Nature; Volume: 40; Issue: 24 Linguagem: Inglês
10.15252/embj.2021108684
ISSN1460-2075
AutoresJiapei Yan, Shibai Li, Yeon Jeong Kim, Qingning Zeng, Amandine Radziejwoski, Lei Wang, Yuko Nomura, Hirofumi Nakagami, David E. Somers,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle2 November 2021Open Access Source DataTransparent process TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth Jiapei Yan Jiapei Yan orcid.org/0000-0003-2573-2702 Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Shibai Li Shibai Li orcid.org/0000-0001-9293-6006 Molecular Genetics, Ohio State University, Columbus, OH, USA Memorial Sloan Kettering Cancer Center, Molecular Biology Program, New York, NY, USA Search for more papers by this author Yeon Jeong Kim Yeon Jeong Kim Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Qingning Zeng Qingning Zeng Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Amandine Radziejwoski Amandine Radziejwoski POSTECH, Division of Integrative Biosciences and Biotechnology, Pohang, South Korea Search for more papers by this author Lei Wang Lei Wang orcid.org/0000-0002-4912-5387 Molecular Genetics, Ohio State University, Columbus, OH, USA The Chinese Academy of Sciences, Institute of Botany, Beijing, China Search for more papers by this author Yuko Nomura Yuko Nomura RIKEN Center for Sustainable Resource Science (CSRS), Plant Proteomics Research Unit, Yokohama, Japan Search for more papers by this author Hirofumi Nakagami Hirofumi Nakagami orcid.org/0000-0003-2569-7062 RIKEN Center for Sustainable Resource Science (CSRS), Plant Proteomics Research Unit, Yokohama, Japan Max Planck Institute for Plant Breeding Research, Protein Mass Spectrometry, Cologne, Germany Search for more papers by this author David E Somers Corresponding Author David E Somers [email protected] orcid.org/0000-0001-5170-2430 Molecular Genetics, Ohio State University, Columbus, OH, USA POSTECH, Division of Integrative Biosciences and Biotechnology, Pohang, South Korea Search for more papers by this author Jiapei Yan Jiapei Yan orcid.org/0000-0003-2573-2702 Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Shibai Li Shibai Li orcid.org/0000-0001-9293-6006 Molecular Genetics, Ohio State University, Columbus, OH, USA Memorial Sloan Kettering Cancer Center, Molecular Biology Program, New York, NY, USA Search for more papers by this author Yeon Jeong Kim Yeon Jeong Kim Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Qingning Zeng Qingning Zeng Molecular Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Amandine Radziejwoski Amandine Radziejwoski POSTECH, Division of Integrative Biosciences and Biotechnology, Pohang, South Korea Search for more papers by this author Lei Wang Lei Wang orcid.org/0000-0002-4912-5387 Molecular Genetics, Ohio State University, Columbus, OH, USA The Chinese Academy of Sciences, Institute of Botany, Beijing, China Search for more papers by this author Yuko Nomura Yuko Nomura RIKEN Center for Sustainable Resource Science (CSRS), Plant Proteomics Research Unit, Yokohama, Japan Search for more papers by this author Hirofumi Nakagami Hirofumi Nakagami orcid.org/0000-0003-2569-7062 RIKEN Center for Sustainable Resource Science (CSRS), Plant Proteomics Research Unit, Yokohama, Japan Max Planck Institute for Plant Breeding Research, Protein Mass Spectrometry, Cologne, Germany Search for more papers by this author David E Somers Corresponding Author David E Somers [email protected] orcid.org/0000-0001-5170-2430 Molecular Genetics, Ohio State University, Columbus, OH, USA POSTECH, Division of Integrative Biosciences and Biotechnology, Pohang, South Korea Search for more papers by this author Author Information Jiapei Yan1, Shibai Li1,2, Yeon Jeong Kim1, Qingning Zeng1, Amandine Radziejwoski3, Lei Wang1,4, Yuko Nomura5, Hirofumi Nakagami5,6 and David E Somers *,1,3 1Molecular Genetics, Ohio State University, Columbus, OH, USA 2Memorial Sloan Kettering Cancer Center, Molecular Biology Program, New York, NY, USA 3POSTECH, Division of Integrative Biosciences and Biotechnology, Pohang, South Korea 4The Chinese Academy of Sciences, Institute of Botany, Beijing, China 5RIKEN Center for Sustainable Resource Science (CSRS), Plant Proteomics Research Unit, Yokohama, Japan 6Max Planck Institute for Plant Breeding Research, Protein Mass Spectrometry, Cologne, Germany *Corresponding author. Tel: +1 614 292 2551; E-mail: [email protected] The EMBO Journal (2021)40:e108684https://doi.org/10.15252/embj.2021108684 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Plant photoperiodic growth is coordinated by interactions between circadian clock and light signaling networks. How post-translational modifications of clock proteins affect these interactions to mediate rhythmic growth remains unclear. Here, we identify five phosphorylation sites in the Arabidopsis core clock protein TIMING OF CAB EXPRESSION 1 (TOC1) which when mutated to alanine eliminate detectable phosphorylation. The TOC1 phospho-mutant fails to fully rescue the clock, growth, and flowering phenotypes of the toc1 mutant. Further, the TOC1 phospho-mutant shows advanced phase, a faster degradation rate, reduced interactions with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) and HISTONE DEACETYLASE 15 (HDA15), and poor binding at pre-dawn hypocotyl growth-related genes (PHGs), leading to a net de-repression of hypocotyl growth. NUCLEAR FACTOR Y subunits B and C (NF-YB/C) stabilize TOC1 at target promoters, and this novel trimeric complex (NF-TOC1) acts as a transcriptional co-repressor with HDA15 to inhibit PIF-mediated hypocotyl elongation. Collectively, we identify a molecular mechanism suggesting how phosphorylation of TOC1 alters its phase, stability, and physical interactions with co-regulators to precisely phase PHG expression to control photoperiodic hypocotyl growth. SYNOPSIS Photoperiodic growth of plants is coordinated by an interplay between light signaling and circadian clock. This study reports that phosphorylation of a key Arabidopsis clock protein, TOC1, is essential for its function in repressing hypocotyl growth in the dark. Alanine substitution of five newly discovered phosphosites in the N-terminus of TOC1 phenocopies clock and developmental defects of the toc1 mutant. TOC1 phospho-mutations alter the protein expression profile, diminishing TOC1 stability, chromatin binding and interaction with PIF3. TOC1 forms a trimeric complex with NF-YB/C and recruits HDA15 to repress pre-dawn-phased hypocotyl growth-related genes at night. TOC1 phospho-mutations diminish interactions with NF-YB/C, decreasing chromatin residence of TOC1. Introduction The rotation of the earth on its axis subjects plants to diurnal photocycles which drive a cyclic pattern of many biological processes with a period of approximately 24 h. These rhythms are regulated by an internal circadian clock that enables plants to anticipate periodic environmental changes which allow optimal phasing of molecular, physiological, and behavioral responses to specific times of day (Dunlap, 1999; Harmer et al, 2001; Yakir et al, 2007). Light is the most important environmental cue in the entrainment of the clock, facilitating a stable phase relationship with external photoperiods (Roenneberg & Foster, 1997). Hypocotyl elongation is such a physiological response that under diurnal growth conditions, displays rhythmic pattern with maximal elongation rate at the end of night (Nozue et al, 2007; de Montaigu et al, 2010). Rhythmic hypocotyl growth under photoperiodic conditions is controlled by coordination between the circadian clock and light signaling networks (Nozue et al, 2007; Niwa et al, 2009; de Montaigu et al, 2010; Nomoto et al, 2012). Upon light exposure, phytochromes interact with a subset of basic helix–loop–helix (bHLH) phytochrome-interacting factors (PIFs) which trigger phosphorylation and degradation of PIF proteins resulting in a cascade of transcriptional changes to alter growth (Castillon et al, 2007; Jiao et al, 2007; Bae & Choi, 2008; Leivar & Quail, 2011; Pham et al, 2018). The circadian system integrates with PIF-dependent light signaling and gates maximal light responsiveness of hypocotyl elongation to specific times of day mainly through two mechanisms. The first is through the direct transcriptional regulation of PIF4 and PIF5 genes by the circadian clock which coincides with control of PIF protein accumulation by light (Nozue et al, 2007; Niwa et al, 2009; Nusinow et al, 2011; Nomoto et al, 2013; Li et al, 2020). The second mechanism relies on the temporal inhibition of the transcriptional activation activity of PIFs by clock proteins, such as TIMING OF CAB EXPRESSION 1 (TOC1) (Soy et al, 2016; Zhu et al, 2016), PSEUDO-RESPONSE REGULATORs (PRRs) (Martin et al, 2018; Zhang et al, 2020), and GIGANTEA (GI) (Nohales et al, 2019), which co-occupy the same genomic targets with PIF proteins. Post-translational regulation of circadian systems has been widely studied in mammals, Drosophila, and Neurospora (Gallego & Virshup, 2007), and the role of phosphorylation in modulating transcriptional activity, subcellular localization, stability, and protein–protein interaction of clock proteins is well-documented (Saini et al, 2019; Brenna & Albrecht, 2020). Many plant clock proteins are also phosphorylated, and this modification is essential for their full biological function (Seo & Mas, 2014; Yan et al, 2021). CLOCK-ASSOCIATED 1 (CCA1), which comprises the morning loop with LATE ELONGATED HYPOCOTYL (LHY) in the autoregulatory feedback network of circadian clock, is phosphorylated by Casein kinase 2 (CK2), loss of which interferes with CCA1's transcriptional activity (Sugano et al, 1998; Daniel et al, 2004). Phosphorylation of PRR5 and TOC1 enhances interaction with the F-box protein ZEITLUPE (ZTL), facilitating their subsequent degradation (Fujiwara et al, 2008). TOC1/PRR3 phosphorylation-dependent interaction protects TOC1 from ZTL-dependent degradation, suggesting a complex interplay between phosphorylation and stability for these clock proteins. PRR5 enhances TOC1 phosphorylation and nuclear transport, implicating a phosphorylation-mediated mechanism of TOC1 nuclear import or subcellular re-distribution (Wang et al, 2010). However, direct evidence illustrating how TOC1's function is modulated by phosphorylation, which residues are phosphorylated and essential for TOC1 in sustaining circadian period and how it relates to gating of hypocotyl growth to photoperiods, is still lacking. In this study, we identified 5 phosphorylation sites near the N-terminus of TOC1. Substitution of all 5 residues by alanine (5X) results in clock and developmental defects, similar to the toc1-101 (hereafter toc1) mutant. Our genetic and biochemical evidence strongly suggests that TOC1 phosphorylation controls hypocotyl elongation through effects on circadian phase, protein stability, and chromatin residence of TOC1. TOC1 phosphorylation alters interactions with NUCLEAR FACTOR Y subunit C (NF-YC) and associations with HDA15 and PIF3, which together with NF-YB regulate downstream pre-dawn hypocotyl growth-related genes (PHGs) to precisely phase their expression to pre-dawn hours. PRR5 similarly interacts with NF-YB/C subunits and HDA15 and likely co-represses a subset of target genes together with TOC1. Collectively, our findings identify a molecular mechanism of how TOC1 phosphorylation controls photoperiodic hypocotyl elongation and highlight the essential role of post-translational regulation of TOC1 in synchronizing plant growth with cyclic environmental changes. Results Identification of TOC1 N-terminal phosphosites In an effort to determine the role of phosphorylation in the function of TOC1, we performed mass spectrometry of the N-terminus (aa 1–242) when transiently co-expressed in N. benthamiana with PRR5, which visibly enhances TOC1 phosphorylation-dependent gel mobility (Wang et al, 2010). We identified 5 phosphorylated residues within and near the N-terminus pseudo-response receiver domain (PRR; Fig 1A). To determine whether mutations of the 5 phosphosites affect this enhancement, progressive alanine substitutions of the 5 residues were introduced and each was co-expressed with PRR5 in N. benthamiana. Figure 1. Phosphorylation-dependent protein mobility shift is eliminated or strongly reduced in 5X and S175A Schematic diagrams of full-length (FL), N-terminus (NT), and C-terminus CCT domain of TOC1 used in this manuscript. The 5 phosphorylated residues identified from our mass spectrometry were progressively substituted by alanine, and their positions are displayed on top of TOC1FL. Mutations in S175 and 5X attenuate PRR5's enhancement on TOC1 phosphorylation. Wild-type and phosphosite mutants of TOC1NT were transiently expressed with or without PRR5 in N. benthamiana leaves, and total proteins were extracted 3 days after infiltration. Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12-h/12-h light/dark cycles were subject to heat (30°C 20 min), pyrophosphatase (PP), and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states was separated by 8% SDS–PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP. Source data are available online for this figure. Source Data for Figure 1 [embj2021108684-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint In most lines, there was an increased relative abundance of upper (phosphorylated) vs. lower (unphosphorylated) forms of TOC1 in TOC1 wild type (TOC1WT), T135A, S194A/S201A/T204A, and T135A/S194A/S201A/S204A (Fig 1B). In contrast, an enhancement of the upper form of TOC1 was not observed in S175A and the quintuple mutant T135A/S175A/S194A/S201A/S204A (subsequently termed 5X) (Fig 1B), indicating residue S175 is essential for TOC1 phosphorylation, as detected by mobility shift. We next generated stable transgenic lines expressing GFP-tagged TOC1 WT, T135A, S175A, and 5X driven by the TOC1 native promoter, and TOC1 WT and 5X driven by the 35S promoter in the toc1-101 background. TOC1 protein expression patterns in a time series (Appendix Fig S1A) and TOC1 protein expression levels at ZT1 and ZT13 (Appendix Fig S1B) were examined by Western blotting, and two independent lines for each transgene with similar expression level were selected for further characterization. Mobility shift assays revealed that 5X migrates faster than TOC1WT, indicating unphosphorylated 5X due to the phosphosite mutations (Fig 1C). Similar to 5X, the majority of S175A was in the unphosphorylated state, with a faint upper band. Pyrophosphatase treatment of TOC1 and T135A, but not 5X and S175A, was reverted by phosphatase inhibitors NaF/Na3VO4 (Fig 1C). These results suggest that mutation of all 5 phosphosites eliminates detectable TOC1 phosphorylation and S175 is the most essential residue for maintaining the phosphorylation state of TOC1. S175A and 5X fail to fully rescue clock and developmental defects of toc1 To determine the in vivo significance of the 5 phosphosites, we next examined hypocotyl elongation in native promoter lines expressing WT, 5X, T135A, and S175A under constant red light. Wild-type TOC1 and T135A fully rescued the long hypocotyl in toc1-101 at all fluence rates tested, whereas 5X and S175A failed to fully complement, especially at low fluence rates (Fig 2A–D). Overexpression of TOC1 (35S:TOC1) strongly shortens hypocotyl length relative to WT, whereas 35S:5X was much less effective (Fig 2E and F). Similar results were observed for both independent transgenic lines of each phospho-variant (Appendix Fig S2). Figure 2. 5X and S175A fail to fully rescue the hypocotyl phenotype displayed in toc1-101 A–F. Shown are hypocotyl lengths of cRL-grown 4-day-old seedlings of toc1 complemented with wild-type TOC1 (A), 5X (B), T135A (C), and S175A (D) driven by TOC1 native promoter and wild-type TOC1 (E), 5X (F) driven by 35S promoter at sequential fluence rates. Different letters indicate statistically significant differences between genotypes (P < 0.01, one-way ANOVA followed by Tukey–Kramer HSD test), and error bars indicate SEM. Data are representative of three biological trials with similar results. Source data are available online for this figure. Source Data for Figure 2 [embj2021108684-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint We further examined the hypocotyl length of the phosphosite mutants under short days (SD). Consistent with previous work (Soy et al, 2016), toc1-101 hypocotyl is significantly longer than wild type, while TOC1 and T135A fully rescued the toc1-101 phenotype. Similar to tests under red light, 5X and S175A also failed to complement under SD (Fig 3A and Appendix Fig S3A). 35S:TOC1 strikingly shortened hypocotyl length, while 35S:5X displayed significant longer hypocotyl length than wild type, similar to TOC1:5X (Fig 3A and Appendix Fig S3A). Taken together, results under both growth conditions suggest the importance of these phosphorylations, especially at S175, in hypocotyl growth control by TOC1. Figure 3. 5X and S175A incompletely rescue toc1-101 defects in period length, expression phase, and hypocotyl growth A, B. Hypocotyl lengths under SD (A) and free-running period (B) of Col-0, toc1-101 and independent native and 35S promoter lines expressing wild-type TOC1 and each phosphosite mutant. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. C–E. Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: For A, seedlings were grown under white light and SD (8-h/16-h L/D) for 3 days. 100 seedlings from 4 independent trials were averaged. For B–E, 18–27 seedlings per line were entrained in 12-h/12-h light/dark cycles for 7 days then transferred to constant red light at ZT2 for image acquisition at 2-h intervals for 1 week. White and gray regions indicate subjective light and dark periods. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in hypocotyl length and period between genotypes (P < 0.01, one-way ANOVA followed by Tukey–Kramer HSD test). Source data are available online for this figure. Source Data for Figure 3 [embj2021108684-sup-0005-SDataFig3.xlsx] Download figure Download PowerPoint To examine the effects on circadian period, the CCA1:LUC reporter was crossed into the phosphosite mutant lines, and free-running period was measured. As expected, toc1-101 showed a 19-h period and advanced phase compared to Col-0 with an approximately 24-h period (Fig 3B and C). TOC1:TOC1 and TOC1:T135A more than rescued the short period and advanced phase, with both showing a slightly longer period, likely due to slightly higher than endogenous levels of TOC1 expression coming from the transgenes. In contrast, the period of TOC1:5X and TOC1:S175A was intermediate between toc1-101 and TOC1:TOC1; 2 h shorter than Col-0 and with an earlier phase (Fig 3B–D and Appendix Fig S3B). Overexpression of TOC1(35S:TOC1) significantly lengthened period to more than 29 h and delayed the phase of CCA1:LUC. 35S:5X displayed a much shorter period and more advanced phase than Col-0, statistically similar to TOC1:5X and TOC1:S175A (Fig 3B and E, and Appendix Fig S3B). The much longer period of 35S:TOC1 line #2 than #1 likely results from the higher TOC1 protein levels in this line (Appendix Fig S1B). Flowering time was also examined in these same native promoter lines grown under SD. TOC1 and T135A fully rescued the early flowering phenotype of toc1-101 whereas 5X and S175A did not (Appendix Fig S3C). Taken together, these results suggest that phosphorylation of the five residues in TOC1, particularly S175, is essential for its full function in controlling circadian period and development. Phospho-mutants de-repress PHGs TOC1 down-regulates PHGs, such as PIL1, AT5G02580, and CDF5, by repressing the transcriptional activity of PIF3 (Soy et al, 2016; Martin et al, 2018). The longer hypocotyl in the 5X and S175A lines could result from increased expression of these genes. We further investigated the expression level of PIL1, AT5G02580, and CDF5 during the pre-dawn phase in Col-0, toc1-101, TOC1:TOC1, and TOC1:5X. Consistent with previous results, the expression of the tested PHGs was strongly increased from ZT15 to ZT23 with the peak expression near the end of night. Loss of TOC1 function in toc1-101 resulted in an even stronger increased expression of these genes (Fig 4A–C). TOC1:TOC1 rescued the high expression of all three genes in toc1-101 to WT levels whereas TOC1:5X failed to fully repress their expression (Fig 4D–F). T135A rescued PIL1, AT5G02580, and CDF5 gene expression to WT levels, whereas S175A was unable to fully complement CDF5 expression at ZT18 (Fig EV1). These results indicate that the longer hypocotyl length of TOC1:5X and TOC1:S175A is at least partially due to de-repression of PHGs, and TOC1:S175A may affect a smaller set of genes than TOC1:5X. Figure 4. Mutations in 5X de-repress the pre-dawn hypocotyl growth-related genes (PHGs) A–C. PIL1, AT5G02580, and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data are representative of 3 biological trials. D–F. Expression of PIL1, AT5G02580, and CDF5 in 3-d-old SD-grown seedlings at ZT18. 5 biological trials were averaged, and error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (P < 0.05). Source data are available online for this figure. Source Data for Figure 4 [embj2021108684-sup-0006-SDataFig4.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Expression of PIL1, AT5G02580, and CDF5 in Col-0, toc1-101, and native promoter TOC1/5X/T135A/S175A lines at ZT18 Four biological trials were averaged, and error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (P < 0.05). Source data are available online for this figure. Download figure Download PowerPoint TOC1 phospho-mutations weaken PIF3 interaction and chromatin residence De-repression of the PHGs in the 5X line may result from a reduced physical interaction between TOC1 5X and PIF3. We then examined the effect of TOC1 phosphorylation on its interaction with PIF3 by transiently co-expressing PIF3-TAP with either TOC1-GFP or 5X-GFP in N. benthamiana. Wild-type TOC1 co-immunoprecipitated (Co-IPed) with PIF3, but interaction between PIF3 and 5X was only 60% of WT (Fig 5A and B and Appendix Fig S4A), suggesting TOC1 phosphorylation is important for PIF3 binding. However, further tests with TOC1NT (aa 1–242), which contains the five phosphorylation sites, found that neither WT nor 5X NT Co-IPed with PIF3 (Appendix Fig S4A). This suggests that despite the binding enhancement by N-terminal phosphorylation, additional domains of TOC1 are needed for PIF3 interaction. Figure 5. 5X interacts more poorly with PIF3 and is present to a lesser extent at PIF3 target promoters Co-IP of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively. Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, and asterisks indicate significant difference (*P < 0.05, Student's t-test). ChIP-qPCR of TOC1 binding to promoters of pre-dawn-phased PIF3 target genes at ZT14 in Col-0, native promoter TOC1 and 5X lines. ChIP-qPCR of the chromatin residence of TOC1-GFP at target promoters in toc1 and pif3/4/5 toc1 (pif345t1) mutant backgrounds. Data information: For C and D, 3-d-old SD-grown seedlings were harvested at ZT14, and TOC1-GFP was immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged, and error bars indicate SEM. Asterisks indicate significant differences (**P < 0.001, ***P < 0.0001, Student's t-test), n.s. = not significant. Source data are available online for this figure. Source Data for Figure 5 [embj2021108684-sup-0007-SDataFig5.zip] Download figure Download PowerPoint We next tested whether the diminished association between 5X and PIF3 altered enrichment of TOC1 at three PIF3 target promoters, PIL1, AT5G02580, and CDF5. ChIP-qPCR showed both TOC1 and 5X were significantly enriched at the promoter of all three genes. However, 5X had 40–50% lower abundance at these promoter regions than TOC1 WT (Fig 5C), suggesting that TOC1 phosphorylation promotes chromatin residence at these gene promoters. As PIF proteins have been reported to anchor PRR association to target DNA (Zhang et al, 2020), we next crossed TOC1:TOC1-GFP into pif3/4/5 toc1 quadruple mutant background and assessed whether PIFs facilitate TOC1 binding to the promoter of PIL1, AT5G02580, and CDF5 by ChIP-qPCR. This could explain the reduced chromatin presence of 5X through the weaker interaction with PIF3. We ensured similar protein abundance between different mutant backgrounds at tissue harvest time (Appendix Fig S4B). Results showed significant enrichment of TOC1 at PIL1, AT5G02580, and CDF5 promoters, largely independent of PIF3, PIF4, and PIF5 (Fig 5D), similar to previous reports (Soy et al, 2016). However, we cannot exclude the possibility that other PIF proteins may recruit TOC1 to the target promoters (Li et al, 2020; Zhang et al, 2020). Taken together, these data suggest two functional defects of the unphosphorylated 5X are a diminished interaction with PIF3, and independently, a reduction in chromatin presence at key PHGs. Early phasing of S175A and 5X reduces TOC1 pre-dawn accumulation The phasing of TOC1 expression is important to its role in both circadian and diurnal gene regulation, so we further tested TOC1 protein accumulation in native promoter TOC1/5X/T135A/S175A lines under SD. Quantitation of TOC1 abundance at sequential time points showed similar TOC1 protein accumulation patterns for wild-type TOC1 and T135A. In both cases, TOC1 levels increased during the photoperiod, attaining maximum expression post-dusk (ZT8-14), with a gradual decrease to the end of night (Figs 6A and EV2A). Figure 6. TOC1 phospho-mutants show early-phased protein expression and reduced stability TOC1 protein expression pattern in 3-d-old seedlings in native promoter TOC1/5X/T135A/S175A lines grown in SD. White and black regions indicate light and dark periods. Data from 3 independent trials were averaged; error bars indicate SEM. Statistical analyses were performed by Student's t-test. Asterisks indicate significant differences between TOC1 wild-type and its phosphosite mutants (*P < 0.05). Measurement of TOC1-GFP protein turnover in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12-h/12-h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with Student's t-test, and different letters indicate significant differences (P < 0.05). Data from 3 independent trials were averaged. Data information: For A and B, TOC1 protein was detected by α-GFP, and ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ. Source data are available online for this figure. Source Data for Figure 6 [embj2021108684-sup-0008-SDataFig6.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Loss of phosphorylation in TOC1 results in early-phased protein expression but has no effect on nucleocytoplasmic distribution of TOC1 protein 5X and S175A display advanced protein expression phase compared with wild-type TOC1 and T135A. 3-d-old seedlings from the second TOC1/5X/T135A/S175A native promoter lines grown in SD condition were harvested at indicated time points. White and black regions indicate light and dark periods. Protein abundance was quantified by ImageJ, results from 3 biological trials were averaged, and error bars indicate SEM. Statistical analysis was performed by Student's t-test. Asterisks indicate significant differences between TOC1 wild-type and its phosphosite mutants (*P < 0.05; **P < 0.001). TOC1 protein nucleocytoplasmic distribution in wild-type TOC1 (WT) and 5X. 10-d-old seedlings of native promoter wild-type TOC1 and 5X grown in 12-h/12-h light/dark cycles were processed for nuclear, cytosolic, and total protein fractions at indicated time points. TOC1 was detected by α-GFP, and ADK and Histone 3 (H3) were detected by their specific antibody and used as
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