CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) is a MYB-related transcription factor with a conserved SHAQKYF motif that serves as a core component of the plant circadian clock system. CCA1 functions as a master regulator that controls circadian rhythms through both transcriptional repression and activation depending on target gene context. It binds to specific cis-regulatory elements including evening elements (EE) and CCA1-binding sites (CBS) in target gene promoters such as APRR1/TOC1, TCP21/CHE, CAB2A, CAB2B, JMJ14, and BHLH80/FBH1. CCA1 works synergistically with LHY (LATE ELONGATED HYPOCOTYL) as morning-expressed clock genes and forms both homodimers and heterodimers with LHY, as well as larger protein complexes with CK2 subunits (CKB1, CKB2, CKB3) and co-regulators LNK1 and LNK2. The protein undergoes phosphorylation by casein kinase 2 (CK2) which modulates its DNA-binding affinity and is essential for temperature compensation of the circadian clock. Beyond circadian regulation, CCA1 modulates endoplasmic reticulum stress responses through the unfolded protein response (UPR), regulates reactive oxygen species homeostasis, and influences developmental processes including hypocotyl elongation, flowering time, and leaf movements. CCA1 is nuclear localized where it functions as part of multi-protein transcriptional complexes.
Definition: The process in which circadian clock components modulate the unfolded protein response and ER stress recovery in a time-dependent manner
Justification: Falcon research indicates CCA1 is involved in the regulation of endoplasmic reticulum (ER) stress responses. By modulating target genes involved in the unfolded protein response (UPR), CCA1 helps facilitate recovery from ER stress, thereby coupling the circadian clock to cellular proteostasis mechanisms
Parent term: response to endoplasmic reticulum stress
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: CCA1 is a MYB-related transcription factor with well-documented DNA binding activity. Multiple experimental studies demonstrate direct DNA binding to promoter sequences of clock-regulated genes. Reason: Experimental evidence from multiple publications demonstrates CCA1 binds DNA directly. This is a core molecular function essential for its role as a transcription factor. Supporting Evidence: PMID:9144958 A Myb-related transcription factor is involved in the phytochrome regulation file:ARATH/CCA1/CCA1-falcon-research.md CCA1 is classified as a MYB-related transcription factor, defined by its highly conserved Myb DNA-binding domain. Within this domain, the SHAQKYF motif serves as a signature element that is critical for binding specific DNA sequences in target gene promoters file:ARATH/CCA1/CCA1-deep-research-falcon.md binds to canonical promoter motifs—most prominently the **Evening Element (EE)** and the **CCA1-binding site (CBS)**—to regulate target gene expression. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: CCA1 nuclear localization is well-established experimentally. As a transcription factor, nuclear localization is essential for its function in regulating gene expression. Reason: Multiple experimental studies confirm CCA1 localizes to the nucleus. This is consistent with its role as a transcription factor and is supported by presence of a nuclear localization signal. Supporting Evidence: PMID:19218364 CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically PMID:9144958 Myb-related transcription factor is involved in the phytochrome regulation file:ARATH/CCA1/CCA1-deep-research-falcon.md Functional evidence places CCA1 in the **nucleus**, where it binds promoters and is assayed by ChIP/ChIP-seq. |
| GO:0010468 regulation of gene expression | IEA GO_REF:0000117 | ACCEPT | Summary: CCA1 regulates gene expression as a transcription factor, controlling multiple clock-regulated genes including TOC1, LHY, and CAB genes. This is a core function of CCA1. Reason: Abundant experimental evidence demonstrates CCA1 regulates expression of multiple target genes through direct binding to their promoters. This is a fundamental aspect of its function as a transcription factor. Supporting Evidence: PMID:11486091 Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock PMID:9144958 A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene file:ARATH/CCA1/CCA1-falcon-research.md CCA1 binds to promoter regions of key clock genes—including APRR1/TOC1, TCP21/CHE, CAB2A, and CAB2B—to either repress or promote transcription, as appropriate. CCA1 represses the expression of evening phase genes, including TOC1 and various PRR genes, while simultaneously activating morning-specific genes |
| GO:0048511 rhythmic process | IEA GO_REF:0000043 | MODIFY | Summary: This term is too general for CCA1. CCA1 specifically functions in circadian rhythms, not just any rhythmic process. The more specific circadian rhythm term (GO:0007623) better captures its function. Reason: While CCA1 is involved in rhythmic processes, this term is overly broad. CCA1 specifically functions in circadian rhythms, and the more specific term GO:0007623 (circadian rhythm) better represents its core function. Proposed replacements: circadian rhythm Supporting Evidence: PMID:9657153 Expression of the CCA1 gene is transiently induced by phytochrome and oscillates with a circadian rhythm |
| GO:0005515 protein binding | IPI PMID:10535927 The protein kinase CK2 is involved in regulation of circadia... | REMOVE | Summary: This annotation represents another CCA1-CK2 interaction study. While the interaction is functionally relevant for clock regulation, the generic protein binding term should be consolidated with the specific protein kinase binding annotation. Reason: This represents a duplicate annotation for CCA1-CK2 interaction already captured more specifically in the protein kinase binding annotation. Removing to avoid redundancy and improve annotation specificity. Supporting Evidence: PMID:10535927 The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis |
| GO:0005515 protein binding | IPI PMID:14978263 CK2 phosphorylation of CCA1 is necessary for its circadian o... | MARK AS OVER ANNOTATED | Summary: This annotation is based on CCA1 interaction with CK2 components. While the interaction is well-established, protein binding is too generic. The specific kinase binding or enzyme binding terms would be more informative. Reason: CCA1 interacts specifically with CK2 kinase subunits. More specific binding terms would provide better functional annotation than generic protein binding. Supporting Evidence: PMID:14978263 CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function |
| GO:0005515 protein binding | IPI PMID:9724822 Protein kinase CK2 interacts with and phosphorylates the Ara... | MODIFY | Summary: This annotation represents CCA1 interaction with CK2 kinase subunits. The interaction is functionally important for CCA1 phosphorylation and circadian function. However, the generic protein binding term should be replaced with the more specific protein kinase binding term. Reason: While the CCA1-CK2 interaction is well-documented and functionally important, generic protein binding provides limited information. The more specific protein kinase binding term better captures this functionally relevant interaction. Proposed replacements: protein kinase binding Supporting Evidence: PMID:9724822 Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein PMID:14978263 CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function |
| GO:0000987 cis-regulatory region sequence-specific DNA binding | IDA PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis c... | ACCEPT | Summary: CCA1 binds to specific cis-regulatory elements in promoters of clock-regulated genes. This term accurately captures its sequence-specific DNA binding activity to regulatory regions. Reason: Experimental evidence demonstrates CCA1 binds to specific regulatory sequences in target gene promoters. This term precisely describes this core molecular function. Supporting Evidence: PMID:25246594 CCA1 binds in vivo to the FBH1 promoter and regulates its expression PMID:11486091 Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock |
| GO:0007623 circadian rhythm | IMP PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis c... | ACCEPT | Summary: CCA1 is a core component of the circadian clock and its function is central to maintaining circadian rhythms. This annotation captures its primary biological function. Reason: Abundant evidence demonstrates CCA1 is essential for proper circadian rhythm function. Loss of CCA1 results in altered circadian rhythms, confirming its core role in this process. Supporting Evidence: PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis circadian clock PMID:9657153 Constitutive expression of CCA1 protein in transgenic plants abolished the circadian rhythm of several genes |
| GO:0009408 response to heat | IEP PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis c... | KEEP AS NON CORE | Summary: CCA1 expression is modulated in response to temperature changes as part of temperature compensation of the circadian clock. However, this is a secondary function related to its core clock role. Reason: While CCA1 does respond to heat/temperature changes, this is primarily in the context of temperature compensation of the circadian clock rather than a general heat response. This represents a non-core function. Supporting Evidence: PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis circadian clock file:ARATH/CCA1/CCA1-falcon-research.md Environmental cues such as light and temperature have been shown to significantly impact the expression dynamics of CCA1. The involvement of CK2-mediated phosphorylation in modifying CCA1's activity serves as a key regulatory mechanism that adjusts its response to temperature fluctuations |
| GO:0010378 temperature compensation of the circadian clock | IMP PMID:25246594 FBH1 affects warm temperature responses in the Arabidopsis c... | ACCEPT | Summary: Temperature compensation is an important property of circadian clocks, allowing them to maintain consistent periods across temperature ranges. CCA1 contributes to this function. Reason: Experimental evidence shows CCA1 is involved in temperature compensation of the circadian clock, maintaining relatively constant period lengths across temperature ranges. This is a core aspect of circadian clock function. Supporting Evidence: PMID:25246594 We found that upon temperature changes, overexpression of FBH1 alters the pace of CCA1 expression by causing a period shortening and thus preventing the clock from buffering against this change in temperature file:ARATH/CCA1/CCA1-falcon-research.md CK2-mediated phosphorylation in modifying CCA1's activity serves as a key regulatory mechanism that adjusts its response to temperature fluctuations, ensuring that the circadian clock remains stable across a range of thermal conditions |
| GO:0005515 protein binding | IPI PMID:31948398 Receptor kinase FERONIA regulates flowering time in Arabidop... | MARK AS OVER ANNOTATED | Summary: Another generic protein binding annotation that lacks specificity. Without more specific characterization of the interaction, this provides limited functional information. Reason: Generic protein binding terms are over-annotations that provide little functional insight. More specific binding terms would be preferable if the specific interaction type was characterized. Supporting Evidence: PMID:31948398 Receptor kinase FERONIA regulates flowering time in Arabidopsis |
| GO:0043254 regulation of protein-containing complex assembly | IMP PMID:20007447 The role of casein kinase II in flowering time regulation ha... | ACCEPT | Summary: CCA1 can form homodimers and heterodimers with LHY, and is part of larger protein complexes. This annotation may reflect its role in clock protein complex formation. Reason: CCA1 forms homodimers, heterodimers with LHY, and is part of larger protein complexes essential for clock function. This represents a legitimate aspect of its molecular function. Supporting Evidence: PMID:19218364 CCA1 and LHY are present in the same large complex in plants file:ARATH/CCA1/CCA1-falcon-research.md CCA1 forms both homodimers and heterodimers (particularly with LHY), and these interactions are critical for its efficient binding to target promoters. CCA1 interacts with CKB1, CKB2 and CKB3. Interacts with LNK1 and LNK2 PMID:20007447 Dec 9. The role of casein kinase II in flowering time regulation has diversified during evolution. |
| GO:0005515 protein binding | IPI PMID:28790150 Phosphorylation of Histone H2A at Serine 95: A Plant-Specifi... | MARK AS OVER ANNOTATED | Summary: Yet another generic protein binding annotation. Like other protein binding terms, this lacks specificity and should be replaced with more informative molecular function terms. Reason: Generic protein binding annotations provide minimal functional information and represent over-annotation. More specific interaction terms would be more useful. Supporting Evidence: PMID:28790150 Phosphorylation of Histone H2A at Serine 95: A Plant-Specific Mark Involved in Flowering Time Regulation and H2A.Z Deposition. |
| GO:0043565 sequence-specific DNA binding | IDA PMID:28790150 Phosphorylation of Histone H2A at Serine 95: A Plant-Specifi... | ACCEPT | Summary: CCA1 exhibits sequence-specific DNA binding activity to regulatory elements in target gene promoters. This is a core molecular function of this transcription factor. Reason: Multiple experimental studies demonstrate CCA1 binds to specific DNA sequences in target gene promoters. This is fundamental to its function as a transcription factor. Supporting Evidence: PMID:11486091 both proteins bind to a region in the TOC1 promoter that is critical for its clock regulation PMID:28790150 Phosphorylation of Histone H2A at Serine 95: A Plant-Specific Mark Involved in Flowering Time Regulation and H2A.Z Deposition. |
| GO:0010468 regulation of gene expression | IMP PMID:23638299 Accurate timekeeping is controlled by a cycling activator in... | ACCEPT | Summary: Duplicate of earlier regulation of gene expression annotation. CCA1 regulates gene expression as its primary function as a transcription factor. Reason: This annotation is supported by experimental evidence and represents a core function of CCA1. While it duplicates an earlier annotation, both have different evidence codes and references. Supporting Evidence: PMID:23638299 Accurate timekeeping is controlled by a cycling activator in Arabidopsis |
| GO:0048574 long-day photoperiodism, flowering | IGI PMID:19011118 Circadian clock proteins LHY and CCA1 regulate SVP protein a... | KEEP AS NON CORE | Summary: CCA1 contributes to photoperiodic flowering control through its role in the circadian clock. However, flowering regulation is a downstream consequence of its core clock function rather than a primary function. Reason: While CCA1 affects flowering time through its circadian clock function, flowering regulation is a downstream developmental process rather than its core molecular function. This represents a non-core function. Falcon deep research frames flowering as one of many pleiotropic clock outputs (up to ~30-90% of the transcriptome is clock-controlled), consistent with this being non-core. Supporting Evidence: PMID:19011118 Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis file:ARATH/CCA1/CCA1-deep-research-falcon.md A 2024 review notes that under constant conditions ~**30%** of the transcriptome can be clock-controlled, and with light/temperature cues this can reach **up to 90%**, illustrating the potentially broad downstream impact of core clock TFs such as CCA1. |
| GO:0045892 negative regulation of DNA-templated transcription | IMP PMID:11486091 Reciprocal regulation between TOC1 and LHY/CCA1 within the A... | ACCEPT | Summary: CCA1 functions as a transcriptional repressor for several target genes including TOC1 and its own expression. This represents a core aspect of its transcriptional regulatory function. Reason: Well-documented experimental evidence shows CCA1 negatively regulates transcription of target genes like TOC1 and LHY. This is a fundamental aspect of its function in circadian feedback loops. Supporting Evidence: PMID:11486091 Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock file:ARATH/CCA1/CCA1-deep-research-falcon.md It represses evening-phased genes such as **TOC1** and evening-complex components; TOC1/CHE-mediated pathways reciprocally repress CCA1, generating interlocked feedback. |
| GO:0045893 positive regulation of DNA-templated transcription | IMP PMID:9144958 A Myb-related transcription factor is involved in the phytoc... | ACCEPT | Summary: CCA1 can also function as a transcriptional activator for some target genes like CAB genes. This demonstrates its dual regulatory capacity as both activator and repressor. Reason: CCA1 positively regulates transcription of some target genes such as CAB genes, demonstrating its role as both transcriptional activator and repressor depending on target and context. Supporting Evidence: PMID:9144958 A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene |
| GO:0007623 circadian rhythm | IEP PMID:21499259 Coordinated transcriptional regulation underlying the circad... | ACCEPT | Summary: Another circadian rhythm annotation with different evidence. CCA1 is central to circadian rhythms and this annotation is well-supported. Reason: Multiple lines of evidence support CCA1 role in circadian rhythms. This annotation with IEP evidence complements other circadian rhythm annotations. Supporting Evidence: PMID:21499259 Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis |
| GO:0042754 negative regulation of circadian rhythm | IMP PMID:21499259 Coordinated transcriptional regulation underlying the circad... | KEEP AS NON CORE | Summary: CCA1 is a transcriptional repressor of evening-phased clock genes (TOC1, ELF4, etc.), and this repressive activity within the interlocked feedback loop constitutes negative regulation of the circadian oscillator. The IMP source (PMID:21499259) directly shows CCA1/LHY suppress ELF4 expression at dawn, and overexpression (PMID:9657153) abolishes rhythms, both consistent with a negative regulatory role. This is a genuine but secondary characterization relative to CCA1's core "circadian rhythm" and DNA-binding transcription factor functions. Reason: The original REMOVE rationale conflated "CCA1 is essential for the clock" with "CCA1 cannot negatively regulate the clock." These are not mutually exclusive. CCA1's mechanism is repression of evening clock genes, which is a form of negative regulation of the circadian rhythm. The IMP evidence (PMID:21499259) demonstrates CCA1/LHY directly suppress ELF4 at dawn, and constitutive CCA1 overexpression abolishes rhythms (PMID:9657153). The annotation is therefore correct and should be retained, but marked non-core because CCA1's core characterization is as a sequence-specific DNA-binding transcription factor in the core oscillator (GO:0007623) rather than the directional regulation term. Supporting Evidence: PMID:21499259 the circadian-controlled CCA1 and LHY proteins directly suppress ELF4 expression periodically at dawn through physical interactions with these transcription-promoting factors PMID:9657153 Constitutive expression of CCA1 protein in transgenic plants abolished the circadian rhythm of several genes |
| GO:0007623 circadian rhythm | IGI PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl re... | ACCEPT | Summary: Third circadian rhythm annotation with IGI evidence. Consistent with CCA1 core role in circadian clock function. Reason: CCA1 role in circadian rhythms is well-established through multiple types of evidence. This IGI evidence supports the core function annotation. Supporting Evidence: PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl regulate expression of the C-repeat binding factor (CBF) pathway in Arabidopsis |
| GO:0009409 response to cold | IGI PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl re... | KEEP AS NON CORE | Summary: CCA1 involvement in cold response is likely related to temperature compensation of the circadian clock rather than a general cold response. This is a secondary function. Reason: While CCA1 may respond to cold temperatures, this is primarily in the context of circadian clock temperature compensation rather than general cold stress response. This represents a non-core function. Falcon deep research places cold/CBF among the many downstream stress-output pathways enriched in the CCA1/LHY targetome, consistent with non-core. Supporting Evidence: PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl regulate expression of the C-repeat binding factor (CBF) pathway in Arabidopsis file:ARATH/CCA1/CCA1-deep-research-falcon.md Intersection with cca1 lhy mutant DEGs yielded **556** candidate direct regulated targets enriched for abiotic stress-related processes (cold, water deprivation, salt, wounding) as well as light and circadian terms. |
| GO:0043565 sequence-specific DNA binding | IDA PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl re... | ACCEPT | Summary: Duplicate sequence-specific DNA binding annotation with different reference. CCA1 sequence-specific DNA binding is well-established and represents a core molecular function. Reason: Multiple experimental studies demonstrate CCA1 sequence-specific DNA binding. This represents a fundamental molecular function essential for its transcriptional regulatory role. Supporting Evidence: PMID:21471455 Circadian clock-associated 1 and late elongated hypocotyl regulate expression of the C-repeat binding factor (CBF) pathway in Arabidopsis |
| GO:0005634 nucleus | IDA PMID:19218364 CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL fun... | ACCEPT | Summary: Duplicate nucleus annotation with experimental evidence. Nuclear localization is essential for CCA1 function as a transcription factor. Reason: Nuclear localization is experimentally demonstrated and essential for CCA1 transcriptional regulatory function. This IDA evidence supports the earlier IEA annotation. Supporting Evidence: PMID:19218364 CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically |
| GO:0007623 circadian rhythm | IMP PMID:19218364 CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL fun... | ACCEPT | Summary: Fourth circadian rhythm annotation, this one with IMP evidence from a key study on CCA1-LHY synergistic function in circadian clock. Reason: This IMP evidence from a detailed study of CCA1 and LHY function strongly supports CCA1 role in circadian rhythms. Multiple independent evidence types confirm this core function. Supporting Evidence: PMID:19218364 CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically in the circadian clock of Arabidopsis |
| GO:0003677 DNA binding | IDA PMID:18344319 Systems approach identifies an organic nitrogen-responsive g... | ACCEPT | Summary: Duplicate DNA binding annotation with experimental IDA evidence. DNA binding is a fundamental molecular function of CCA1 as a transcription factor. Reason: This experimental evidence supports the earlier IEA DNA binding annotation. DNA binding is essential for CCA1 function as a transcription factor. Supporting Evidence: PMID:18344319 Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1 |
| GO:0003700 DNA-binding transcription factor activity | ISS PMID:11118137 Arabidopsis transcription factors: genome-wide comparative a... | ACCEPT | Summary: This annotation captures the essential molecular function of CCA1 as a DNA-binding transcription factor. This is the most accurate and informative molecular function term for CCA1. Reason: This term precisely describes CCA1 primary molecular function as a DNA-binding transcription factor. This is more specific and informative than general DNA binding terms. Supporting Evidence: PMID:11118137 Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes PMID:9144958 A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene file:ARATH/CCA1/CCA1-deep-research-falcon.md **Sequence-specific DNA-binding transcription factor** that can act mainly as a repressor in the core clock, but also as an activator for selected outputs depending on promoter context. |
| GO:0007623 circadian rhythm | TAS PMID:9657153 Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 ... | ACCEPT | Summary: Fifth circadian rhythm annotation with TAS evidence from the foundational paper establishing CCA1 role in circadian clock. This is the strongest evidence for CCA1 core function. Reason: This TAS evidence comes from the seminal paper that established CCA1 as a core component of the circadian clock. This represents the strongest evidence for its primary biological function. Supporting Evidence: PMID:9657153 Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression file:ARATH/CCA1/CCA1-deep-research-falcon.md CCA1/LHY are essential for robust circadian rhythms: single mutants often shorten period, while cca1 lhy double mutants show severe disruption/low amplitude. |
| GO:0046983 protein dimerization activity | NAS | NEW | Summary: Added to align core_functions with existing annotations. CCA1 forms homodimers and LHY heterodimers required for efficient promoter binding. Reason: Core function term not present in existing_annotations. Supporting Evidence: file:ARATH/CCA1/CCA1-deep-research-falcon.md Strongest functional partner is **LHY**; CCA1 and LHY can act redundantly and also form heterodimers in vivo. |
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Download this section (compressed HTML)Q: How does CCA1 switch between repressor and activator activity at different Evening Element / CCA1-binding-site target promoters, and what corepressor versus coactivator complexes mediate each mode in vivo?
Q: What are the in vivo functional consequences of CK2-mediated phosphorylation of CCA1 - does phosphorylation modulate DNA-binding affinity, dimerization, nuclear retention, or protein turnover, and does each role contribute independently to clock period determination?
Q: How do CCA1 and LHY partition their target binding sites genome-wide under matched circadian time points, and which CCA1-specific (non-LHY) targets account for the residual short-period phenotype of cca1 single mutants?
Experiment: Time-resolved CCA1 ChIP-seq paired with nascent transcript profiling (e.g. PRO-seq or GRO-seq) under matched dawn time points in wild-type and cca1 lhy double mutants, with parallel rve8 mutant samples to map the EE-binding repressor (CCA1)/activator (RVE) balance genome-wide.
Hypothesis: CCA1 occupancy is largely shared with LHY at EE/CBS motifs, but a defined subset of CCA1-specific or CCA1-preferred targets accounts for the residual short-period phenotype of cca1 single mutants.
Type: ChIP-seq with nascent transcription profiling
Experiment: CRISPR-engineered phosphosite mutants of CCA1 (CK2-target serines/threonines to alanine or aspartate) introduced at the endogenous locus, with parallel free-running clock phenotyping, leaf-movement assays, hypocotyl length, and CCA1 ChIP-seq to dissect which CK2 phosphorylations control which activities.
Hypothesis: Distinct CK2 phosphorylation events on CCA1 separately tune DNA-binding affinity, dimerization with LHY, and turnover; combinatorial mutation phenocopies different CK2 perturbations.
Type: CRISPR phosphosite editing with circadian phenotyping
Experiment: Inducible (estradiol-driven) CCA1 expression in cca1 lhy double-mutant plants combined with single-cell RNA-seq at the shoot apex across the day, to define which CCA1-controlled gene modules recover with which CCA1 dose and timing.
Hypothesis: Transient pulses of CCA1 at subjective dawn are sufficient to rescue most clock-output gene rhythms in cca1 lhy plants, with dose-dependent thresholds for separable output modules (cold response, nitrogen metabolism, flowering).
Type: Inducible complementation with single-cell RNA-seq
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