Recombinant Arabidopsis thaliana Two-component response regulator-like APRR5 (APRR5), partial

What is APRR5 and what functional pathways does it participate in?

APRR5 (Arabidopsis Pseudo-Response Regulator 5) is a member of the APRR1/TOC1 family of proteins that function within the plant circadian clock system. These proteins share structural similarities with bacterial two-component signal transduction systems, though they typically lack conserved phosphoacceptor residues found in authentic response regulators. APRR5 participates in both circadian rhythm regulation and abscisic acid (ABA) signaling pathways .

The two-component signaling system in Arabidopsis consists of histidine protein kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs), and response regulators (ARRs), with APRR5 being part of this extended family. This ancient signaling mechanism has been evolutionary conserved across prokaryotes and eukaryotes and plays important roles in plant signal transduction related to hormones, stress responses, and light signaling .

How does APRR5 expression change throughout the day-night cycle?

APRR5 exhibits a distinct rhythmic expression pattern under continuous light conditions. The APRR1/TOC1 family genes accumulate in a sequential manner after dawn in the precise order of APRR9→APRR7→APRR5→APRR3→APRR1/TOC1 . This sequential expression is a defining characteristic of the circadian clock mechanism in Arabidopsis and allows for the temporal coordination of various clock-regulated processes.

Importantly, this sequential expression doesn't require the PRR genes to be connected as a series of activators, as demonstrated by mathematical modeling of the Arabidopsis circadian clock . Instead, the system relies heavily on repressive interactions with specific activation points to maintain the rhythmic expression patterns.

What phenotypes are observed in APRR5 overexpression and knockout plants?

Plants overexpressing APRR5 (APRR5-ox) display several distinctive phenotypes:

• Altered circadian rhythms: Free-running rhythms are considerably changed for several clock-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 family members, GI, and CAB2, though the rhythms remain sustained even under continuous light .

• Early flowering: APRR5-ox plants flower significantly earlier than wild-type plants, particularly under short-day conditions, suggesting APRR5's involvement in photoperiodic flowering control pathways .

• Hypersensitivity to red light: APRR5-ox plants exhibit the SRL (short-hypocotyls under red light) phenotype, indicating enhanced sensitivity to red light during early photomorphogenesis. This phenotype is also observed in APRR1-ox and APRR9-ox plants .

• Enhanced ABA responses: Overexpression of PRR5 enhances ABA signaling, resulting in inhibited seed germination .

In contrast, disrupting both PRR5 and PRR7 simultaneously renders germinating seeds hyposensitive to ABA, demonstrating their redundant positive role in ABA signaling during seed germination .

How does APRR5 interact with plant hormone signaling pathways?

APRR5 plays a significant role in abscisic acid (ABA) signaling pathways, particularly during seed germination. Recent research has demonstrated that APRR5 physically associates with ABSCISIC ACID-INSENSITIVE5 (ABI5), a crucial transcription factor in the ABA signaling cascade .

This interaction has functional consequences:

• APRR5 and PRR7 positively modulate ABA signaling during seed germination in a redundant manner.

• APRR5 stimulates the transcriptional function of ABI5 without affecting its stability.

• The expression of several ABA-responsive genes is upregulated by PRR proteins.

• Genetic analysis confirms that APRR5 promotes ABA signaling mainly dependent on ABI5 .

This connection provides a direct molecular link between the circadian clock and hormone signaling, demonstrating how these regulatory systems are integrated through transcriptional complexes involving both circadian components and hormone response factors.

What experimental approaches are most effective for studying APRR5 protein-protein interactions?

Several complementary techniques have proven effective for investigating APRR5 protein-protein interactions:

• Bimolecular Fluorescence Complementation (BiFC): This technique has successfully demonstrated the in vivo interaction between APRR5 and ABI5 in plant cells. The procedure involves:

  • Fusing the full-length coding sequence of APRR5 with the N-terminal YFP fragment (APRR5-nYFP)

  • Fusing the interaction partner (e.g., ABI5) with the C-terminal YFP fragment (ABI5-cYFP)

  • Co-expressing these constructs transiently in leaf cells (typically Nicotiana benthamiana)

  • Visualizing YFP fluorescence in the nucleus of transformed cells (confirmed by DAPI staining)

• Co-immunoprecipitation (CoIP): This approach provides robust biochemical evidence of protein associations in vivo:

  • Generate transgenic Arabidopsis plants simultaneously overexpressing tagged versions of APRR5 and its putative interactor (e.g., 35S:2FLAG-PRR5 and 35S:ABI5-4MYC)

  • Extract proteins under non-denaturing conditions

  • Immunoprecipitate one protein using tag-specific antibodies

  • Detect the co-precipitated partner protein by western blotting

• Yeast Two-Hybrid Assays: While not specifically mentioned in the search results for APRR5, this system remains valuable for initial screening of potential interaction partners.

• In vitro pull-down assays: These can be used to confirm direct physical interactions using purified recombinant proteins.

When designing experiments to study APRR5 interactions, researchers should consider the circadian nature of APRR5 expression and potentially conduct experiments at different time points throughout the day-night cycle to capture time-dependent interactions.

What methods are optimal for expressing and purifying recombinant APRR5 protein?

Based on current research approaches for plant circadian clock proteins, the following methods are recommended for expressing and purifying recombinant APRR5:

• Expression system selection:

  • E. coli systems (BL21 derivatives) work well for partial domains of APRR5

  • Full-length APRR5 may require eukaryotic expression systems like insect cells (Sf9) to ensure proper folding and post-translational modifications

  • Consider codon optimization for the expression system

• Tags and fusion strategies:

  • N-terminal 6xHis or GST tags facilitate purification while minimizing interference with function

  • TEV or PreScission protease cleavage sites allow tag removal after purification

  • For difficult-to-express proteins, MBP (maltose-binding protein) fusions can enhance solubility

• Purification protocol:

  • Two-step purification combining affinity chromatography and size exclusion is recommended

  • Consider timing of expression and purification, as circadian proteins may exhibit time-dependent properties

  • Buffer optimization is critical (typically 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol)

  • Presence of phosphatase inhibitors may be necessary to maintain native phosphorylation states

• Functional validation:

  • Assess protein activity through DNA-binding assays if applicable

  • Verify proper folding through circular dichroism or limited proteolysis

  • Confirm the ability to interact with known partners through in vitro binding assays

Due to potential challenges with expressing plant transcription factors in heterologous systems, it may be beneficial to express functional domains separately rather than the complete protein.

How can researchers effectively study the role of APRR5 in circadian rhythm regulation?

To effectively study APRR5's role in circadian rhythm regulation, researchers should implement multi-faceted approaches:

When analyzing data from these experiments, it's important to remember that "the many negative feedbacks in the system should discourage intuitive interpretations of mutant phenotypes. The dynamics of the clock are difficult to predict without mathematical modelling, and the clock is better viewed as a tangled web than as a series of loops" .

What experimental approaches best elucidate the interaction between APRR5 and ABI5 in ABA signaling?

To thoroughly investigate the APRR5-ABI5 interaction in ABA signaling, researchers should employ these approaches:

• Biochemical characterization of the interaction:

  • Map interaction domains through truncation and mutation analysis

  • Determine binding affinity and kinetics using techniques like surface plasmon resonance

  • Assess whether the interaction is direct or requires additional factors

  • Investigate if the interaction is regulated by ABA or circadian timing

• Functional analysis of the transcriptional complex:

  • Perform ChIP-seq with both APRR5 and ABI5 to identify co-regulated target genes

  • Use sequential ChIP (re-ChIP) to confirm co-occupancy at specific loci

  • Employ reporter gene assays to quantify how APRR5 affects ABI5's transcriptional activity

  • Investigate whether APRR5 affects ABI5 DNA binding, cofactor recruitment, or activation potential

• Physiological relevance testing:

  • Create a series of genetic combinations: single and double mutants, as well as complementation lines

  • Analyze ABA sensitivity in these genetic backgrounds using germination assays with different ABA concentrations

  • Test responses across different developmental stages and environmental conditions

  • Measure ABA-responsive gene expression using qRT-PCR or RNA-seq approaches

• Structural biology approaches:

  • Determine the crystal or cryo-EM structure of the APRR5-ABI5 complex

  • Use this information to design specific mutations that disrupt the interaction

  • Test the effects of these mutations in vivo to confirm the structural basis of functional interaction

These approaches should be complemented with careful temporal analysis, as both circadian regulation and ABA signaling are highly dependent on timing and environmental conditions.

How do APRR5 and other PRR family members function redundantly in the circadian clock?

The redundancy between APRR5 and other PRR family members requires specialized experimental approaches to unravel:

• Higher-order mutant analysis:

  • Generate double, triple, and higher-order mutants of PRR family members

  • Compare single prr5 mutants with various mutant combinations (e.g., prr5 prr7 double mutants)

  • Analyze circadian phenotypes in these genetic backgrounds under various conditions

  • Look for enhanced or synergistic effects in multiple mutants that aren't present in single mutants

• Domain swapping experiments:

  • Create chimeric proteins between APRR5 and other PRR proteins

  • Express these under the control of either native promoter

  • Assess the ability of different domains to complement specific mutant phenotypes

  • This approach can reveal which domains confer functional specificity versus redundancy

• Tissue-specific and temporal expression analysis:

  • Compare expression patterns of PRR family members at high temporal resolution

  • Use cell-type specific promoters for expression analysis

  • Determine if redundancy occurs due to co-expression in the same cells or compensatory expression in different tissues

  • Employ techniques like INTACT or FACS to isolate specific cell types for analysis

• Integration of data through systems biology:

  • Build mathematical models incorporating all PRR family members

  • Test how different connection architectures affect clock function

  • Predict outcomes of perturbations (mutations, environmental changes)

  • Compare model predictions with experimental data to refine understanding

Research has shown that PRR5 and PRR7 function redundantly in ABA signaling during seed germination, as disrupting both simultaneously renders germinating seeds hyposensitive to ABA . The sequential expression of PRR genes (APRR9→APRR7→APRR5→APRR3→APRR1/TOC1) does not necessarily imply a series of activations, as mathematical modeling suggests the clock relies more heavily on repressive interactions .

What are the best practices for designing experiments to study APRR5 phosphorylation status?

Phosphorylation likely plays a crucial role in APRR5 function, given its similarity to response regulators in two-component signaling systems. To effectively study APRR5 phosphorylation:

• Detection methods:

  • Phos-tag SDS-PAGE for mobility shift detection of phosphorylated species

  • Mass spectrometry (LC-MS/MS) for identification of specific phosphorylation sites

  • Phospho-specific antibodies for western blot analysis (once sites are identified)

  • 2D gel electrophoresis for separating differentially phosphorylated isoforms

• Temporal considerations:

  • Sample collection at multiple time points throughout the day/night cycle

  • Rapid harvest and extraction in the presence of phosphatase inhibitors

  • Consider the effects of light conditions on phosphorylation status

• Functional analysis of phosphorylation:

  • Site-directed mutagenesis of predicted phosphorylation sites (Ser/Thr to Ala or Asp)

  • Create phospho-mimetic (S/T to D/E) and phospho-null (S/T to A) variants

  • Test these variants in complementation assays in aprr5 mutant backgrounds

  • Assess how phosphorylation affects protein-protein interactions, particularly with ABI5

• Identification of kinases/phosphatases:

  • Candidate approach testing known clock-associated kinases

  • Kinase inhibitor screens to identify classes of kinases involved

  • Affinity purification followed by mass spectrometry to identify interacting kinases/phosphatases

  • In vitro kinase assays to confirm direct phosphorylation

Given the involvement of APRR5 in both circadian and ABA signaling pathways, it's important to examine how phosphorylation status might change in response to both timing cues and hormone treatment .