CYCC1-1 Antibody

What is CycC1;1 and why are antibodies against it important in plant research?

CycC1;1 (C-type Cyclin1;1) is a nuclear protein in Arabidopsis thaliana that functions as a negative regulator in two critical plant processes: salt stress tolerance and ABA signaling . CycC1;1 antibodies are crucial for studying these regulatory mechanisms because they allow researchers to detect and isolate the native protein in plant tissues. The antibodies enable chromatin immunoprecipitation (ChIP) experiments that reveal CycC1;1's association with target gene promoters, such as SOS1 and ABI5 . Without specific antibodies, it would be impossible to determine where and when CycC1;1 binds to DNA in vivo, a critical aspect of understanding its transcriptional regulatory function.

Where is CycC1;1 expressed in plants, and how does this inform antibody-based detection strategies?

CycC1;1 is highly expressed in Arabidopsis roots and during early vegetative growth stages, with particularly high expression in germinating seeds and seedlings . This tissue-specific and developmental expression pattern means that researchers should focus their antibody-based detection methods on these tissues for optimal results. GUS reporter assays have confirmed that CycC1;1 expression is lower in mature leaves, cauline leaves, flowers, and siliques . Subcellular localization studies using GFP-tagged CycC1;1 have demonstrated that it is primarily a nuclear protein, as evidenced by co-localization with DAPI nuclear staining . This nuclear localization should be considered when designing extraction protocols for immunodetection, as nuclear extraction buffers would be more effective than total protein extractions.

What role does CycC1;1 play in salt stress response, and how are antibodies used to study this function?

CycC1;1 negatively regulates salt tolerance in Arabidopsis by interfering with WRKY75-mediated transcriptional activation of SOS1, a key component in plant salt tolerance . Disruption of CycC1;1 promotes SOS1 expression and enhances salt tolerance. Anti-CycC1;1 antibodies are used in ChIP-qPCR experiments to demonstrate that CycC1;1 associates with the SOS1 promoter at the -500 bp upstream region and TATA box, but not the coding region or terminator . These antibody-based techniques have revealed that CycC1;1 occupies the SOS1 promoter and interferes with RNA polymerase II recruitment, thereby suppressing SOS1 expression under low salinity conditions . This understanding of CycC1;1's molecular mechanism would not be possible without specific antibodies that enable precise protein localization on chromatin.

How does CycC1;1 affect ABA signaling, and what insights have antibody-based methods provided?

CycC1;1 negatively modulates ABA signaling during seed germination by interacting with and inhibiting ABSCISIC ACID INSENSITIVE5 (ABI5) . The loss-of-function cycc1;1 mutant shows hypersensitivity to ABA during germination and increased expression of ABI5 and its downstream targets . Antibody-based techniques have demonstrated that CycC1;1 physically interacts with ABI5 to occupy the promoters of ABI5-targeted genes and ABI5 itself, thereby interfering with ABI5's transcriptional activation activity . ABA treatment reduces the interaction between CycC1;1 and ABI5, relieving this inhibition . These molecular interactions were elucidated through immunoprecipitation techniques using CycC1;1 antibodies, providing crucial insights into the mechanism of ABA signaling regulation.

How does the CycC1;1-WRKY75 complex regulate SOS1 transcription under different salinity conditions?

The CycC1;1-WRKY75 complex exhibits dynamic regulation of SOS1 transcription that varies with salinity conditions. Under low salinity, CycC1;1 forms a complex with the transcription factor WRKY75, which binds to the W-box motif in the SOS1 promoter . This interaction prevents WRKY75 from activating SOS1 transcription, effectively repressing salt tolerance mechanisms. ChIP-qPCR experiments using anti-CycC1;1 antibodies have demonstrated that CycC1;1 association with the SOS1 promoter depends on WRKY75, as this association is significantly reduced in the wrky75-25 mutant .

Under high salinity conditions, the regulatory dynamics change: WRKY75 expression increases while CycC1;1 expression decreases, shifting the balance toward WRKY75-mediated activation of SOS1 transcription . This results in enhanced SOS1 expression and salt tolerance. Interestingly, high salinity treatment increases the enrichment of CycC1;1-associated SOS1 promoter fragments in wild-type plants, but this enrichment is greatly repressed in the wrky75-25 mutant . This suggests that the salt stress response involves complex changes in the CycC1;1-WRKY75 interaction that go beyond simple expression changes, potentially involving post-translational modifications that could be detected using phospho-specific antibodies in future research.

What biochemical evidence confirms the direct interaction between CycC1;1 and its partner proteins, and how can antibodies improve interaction studies?

Multiple complementary techniques have confirmed direct interactions between CycC1;1 and its partner proteins. For the CycC1;1-WRKY75 interaction, researchers employed:

• Yeast two-hybrid (Y2H) screening that initially identified WRKY75 as a CycC1;1-interacting partner

• Bimolecular fluorescence complementation (BiFC) assays showing reconstituted YFP fluorescence in nuclei when CycC1;1 and WRKY75 fusion proteins were co-expressed

• Co-immunoprecipitation (Co-IP) experiments that detected HA-tagged CycC1;1 in protein precipitants immunoprecipitated by anti-GFP antibodies from leaves expressing GFP-tagged WRKY75

• GST pull-down assays demonstrating that purified His-WRKY75 was specifically pulled down by GST-CycC1;1 but not by GST alone

For the CycC1;1-ABI5 interaction, similar methods confirmed direct binding . Antibodies played crucial roles in these experiments, particularly in Co-IP assays where they were essential for selectively precipitating protein complexes. Future interaction studies could be enhanced by developing monoclonal antibodies against specific domains of CycC1;1, which would allow researchers to determine which regions of the protein are involved in different protein-protein interactions.

How can phosphorylation-specific antibodies advance our understanding of CycC1;1 regulation during stress responses?

Phosphorylation-specific antibodies for CycC1;1 would significantly advance our understanding of its regulation during stress responses. Research has shown that ABA-induced phosphorylation of ABI5 at Ser-42 disrupts its interaction with CycC1;1, relieving CycC1;1's inhibitory effect . This suggests that phosphorylation events may similarly regulate CycC1;1's interactions with partner proteins.

Developing phospho-specific antibodies that recognize specific phosphorylated residues in CycC1;1 would enable researchers to:

• Track dynamic changes in CycC1;1 phosphorylation status during salt stress and ABA responses

• Correlate phosphorylation with changes in protein-protein interactions and transcriptional regulatory activity

• Identify the kinases responsible for CycC1;1 phosphorylation by combining phospho-antibody detection with kinase inhibitor treatments

• Determine whether phosphorylation affects CycC1;1's subcellular localization or stability

Such antibodies would provide temporal resolution to our understanding of CycC1;1 regulation that current genetic approaches cannot achieve. Additionally, they would permit the simultaneous analysis of phosphorylation and protein-protein interactions through sequential immunoprecipitation techniques.

What are the technical challenges in generating specific antibodies against CycC1;1 and its post-translationally modified forms?

Generating specific antibodies against CycC1;1 presents several technical challenges. First, C-type cyclins share conserved domains with other cyclin family members, potentially causing cross-reactivity . Researchers must carefully select unique epitopes within CycC1;1 that differ from CycC1;2 and other cyclins to ensure specificity.

For post-translationally modified forms, the challenges include:

• Identifying the specific modification sites through mass spectrometry analysis

• Synthesizing phosphopeptides that accurately represent the modified regions

• Ensuring that the modifications are stable during immunization

• Developing screening methods that can distinguish between modified and unmodified forms

The search results indicate that researchers have successfully generated anti-CycC1;1 antibodies for ChIP-qPCR experiments , suggesting these challenges can be overcome with careful epitope selection and validation protocols. Future antibody development could focus on the regions of CycC1;1 that mediate interactions with WRKY75 and ABI5, as these are likely key regulatory domains subject to post-translational modification.

What are the optimal conditions for ChIP-qPCR when using CycC1;1 antibodies to study promoter binding?

For optimal ChIP-qPCR experiments using CycC1;1 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Use Arabidopsis seedlings subjected to relevant stress conditions (salt stress or ABA treatment) alongside untreated controls. The research indicates high CycC1;1 expression in roots and young seedlings, making these ideal tissues for ChIP experiments .

• Crosslinking conditions: Based on successful experiments in the literature, formaldehyde crosslinking (typically 1%) for 10-15 minutes under vacuum is effective for capturing CycC1;1-DNA interactions .

• Sonication parameters: Adjust sonication to produce chromatin fragments averaging 200-500 bp, which is optimal for resolving binding at specific promoter regions.

• Antibody selection: Anti-CycC1;1 antibodies should be validated for ChIP applications specifically. The research successfully used such antibodies to detect binding at the SOS1 promoter .

• Primer design: Design primers to target specific regions of interest in promoters, including:

  • W-box elements (for detecting WRKY75-mediated binding)

  • TATA box regions (shown to bind CycC1;1)

  • Coding regions and terminators (as negative controls)

• Controls: Include the following essential controls:

  • Input chromatin (non-immunoprecipitated)

  • No-antibody controls to assess non-specific binding

  • Immunoprecipitation with IgG as a negative control

  • ChIP in relevant mutant backgrounds (e.g., wrky75 mutant)

The research demonstrates that CycC1;1 specifically associates with the -500 bp upstream region and TATA box of the SOS1 promoter but not the coding region or terminator . This provides valuable positive and negative control regions for validating new ChIP experiments.

How can I validate the specificity of CycC1;1 antibodies for immunoprecipitation experiments?

To validate the specificity of CycC1;1 antibodies for immunoprecipitation experiments, implement the following comprehensive validation steps:

• Western blot analysis:

  • Test the antibody against recombinant CycC1;1 protein

  • Compare signal from wild-type plant extracts versus cycc1;1 mutant extracts

  • Evaluate potential cross-reactivity with CycC1;2 (closely related)

• Immunoprecipitation controls:

  • Perform IP using the cycc1;1 mutant as a negative control

  • Include the cycc1;1/1;2 double mutant to assess cross-reactivity with CycC1;2

  • Use competing peptides to demonstrate binding specificity

• Epitope-tagged validation:

  • Compare IP results between native antibodies and antibodies against epitope-tagged CycC1;1 (e.g., HA-CycC1;1 or CycC1;1-GFP) in complementation lines

  • Verify co-precipitation of known interacting partners like WRKY75 or ABI5

• Mass spectrometry confirmation:

  • Analyze immunoprecipitated proteins by mass spectrometry to confirm CycC1;1 identity

  • Check for co-precipitation of known complex components

The research successfully used anti-CycC1;1 antibodies in ChIP-qPCR experiments, indicating that specific and functional antibodies can be developed . The specificity was confirmed in part by the differential enrichment of specific promoter regions (SOS1 promoter regions versus coding or terminator regions) and by the dependence of this enrichment on WRKY75 .

What complementary techniques should be used alongside CycC1;1 antibody-based approaches to verify protein-protein interactions?

To robustly verify CycC1;1 protein-protein interactions, researchers should employ multiple complementary techniques alongside antibody-based approaches:

The research on CycC1;1 exemplifies this multi-technique approach, using Y2H to identify potential interactors, BiFC to visualize interactions in planta, Co-IP to confirm interactions in vivo, and GST pull-down to demonstrate direct physical interaction in vitro . For the WRKY75-DNA interaction, EMSA was used to confirm binding to the W-box element in the SOS1 promoter . This comprehensive approach provides convincing evidence for the biological relevance of these interactions.

How can dual-luciferase reporter assays be optimized to study CycC1;1-mediated transcriptional regulation?

Dual-luciferase reporter assays have been successfully employed to study CycC1;1-mediated transcriptional regulation . To optimize these assays for investigating CycC1;1 function, researchers should:

• Promoter selection: Use the native promoters of CycC1;1 target genes, such as SOS1 or ABI5-responsive promoters. The research successfully used the SOS1 promoter driving LUC expression .

• Control selection: Use 35S pro:Renilla luciferase (REN) as an internal control to normalize for transformation efficiency and expression levels .

• Expression system: Nicotiana benthamiana leaves have been successfully used for transient expression of these reporter constructs .

• Experimental design:

  • Test CycC1;1 as an effector by co-expressing 35S pro:CycC1;1-GFP with the reporter constructs

  • Include relevant transcription factors (e.g., WRKY75) to test their interactions with CycC1;1

  • Apply relevant treatments (salt, ABA) to assess condition-dependent regulation

  • Include dose-response experiments by varying the ratios of CycC1;1 to transcription factor constructs

• Controls and variations:

  • Empty vector controls

  • Mutated promoter elements (e.g., W-box mutations) to confirm binding specificity

  • Truncated or mutated CycC1;1 constructs to identify regulatory domains

The research demonstrated that LUC activity driven by the SOS1 promoter was significantly repressed in the presence of CycC1;1, confirming its negative regulatory role . Similar approaches could be used to study other CycC1;1 target genes and to investigate how different stress conditions affect this regulation.

How can CycC1;1 antibodies be used to study the temporal dynamics of transcriptional regulation during stress responses?

CycC1;1 antibodies can provide unique insights into the temporal dynamics of transcriptional regulation during stress responses through time-course experiments. Research has shown that under high salinity conditions, the balance between CycC1;1 and WRKY75 changes, with increased WRKY75 expression and decreased CycC1;1 expression leading to enhanced SOS1 transcription . Similarly, ABA treatment reduces the interaction between CycC1;1 and ABI5 .

To study these temporal dynamics:

• Time-course ChIP-seq experiments: Use CycC1;1 antibodies for ChIP followed by high-throughput sequencing at multiple time points during stress treatment to map genome-wide binding dynamics.

• Sequential ChIP (re-ChIP): Perform sequential immunoprecipitations with CycC1;1 antibodies followed by WRKY75 or ABI5 antibodies to track temporal changes in co-occupancy of target promoters.

• Quantitative Co-IP: Measure changes in protein-protein interactions over time following stress treatment using quantitative Western blotting of co-immunoprecipitated proteins.

• Chromatin conformation capture: Combine CycC1;1 ChIP with 3C/4C/Hi-C techniques to understand how CycC1;1 influences higher-order chromatin structure during stress responses.

These approaches would provide a comprehensive understanding of how CycC1;1-mediated transcriptional regulation changes dynamically during stress responses, potentially revealing novel regulatory mechanisms not evident from static analyses.

What insights could tissue-specific and cell-type-specific antibody-based detection of CycC1;1 provide for plant stress biology?

Tissue-specific and cell-type-specific antibody-based detection of CycC1;1 could significantly advance our understanding of plant stress biology. Research has shown that CycC1;1 is highly expressed in roots and during early vegetative growth stages , but its cell-type-specific expression patterns remain unexplored.

Implementing techniques such as:

• Immunohistochemistry: Using CycC1;1 antibodies on tissue sections to visualize expression patterns across different cell types during stress responses.

• Cell-type-specific ChIP: Combining INTACT (isolation of nuclei tagged in specific cell types) or FANS (fluorescence-activated nuclei sorting) with CycC1;1 ChIP to determine cell-type-specific binding patterns.

• Single-cell protein analysis: Adapting antibody-based detection for single-cell proteomics to understand cell-to-cell variation in CycC1;1 abundance and modification state.

Such approaches could reveal that:

• CycC1;1-mediated regulation differs between root cell types (e.g., epidermis vs. stele)

• Certain tissues may show unique CycC1;1 interactome profiles

• Stress-induced changes in CycC1;1 localization or modification may occur in specific cell types first

This information would be valuable for developing targeted stress tolerance strategies that modify CycC1;1 function in specific tissues or cell types without affecting its roles in other developmental processes.

How might cross-species reactive CycC1;1 antibodies contribute to comparative studies of stress tolerance mechanisms?

Cross-species reactive CycC1;1 antibodies would enable comparative studies of stress tolerance mechanisms across different plant species, particularly crop plants. The research focused on Arabidopsis thaliana , but understanding how CycC1;1 functions in crops could lead to practical applications in agriculture.

Such antibodies would allow researchers to:

• Compare CycC1;1 expression patterns: Determine whether tissue-specific expression patterns observed in Arabidopsis are conserved in crops like rice, wheat, or maize.

• Identify conserved protein-protein interactions: Test whether CycC1;1 interacts with WRKY75 and ABI5 orthologs in crop species through Co-IP experiments.

• Map chromatin binding profiles: Compare CycC1;1 binding patterns at stress-responsive gene promoters across species to identify conserved and divergent regulatory mechanisms.

• Assess post-translational modifications: Determine whether stress-induced modifications of CycC1;1 are conserved across species.

The data suggests that CycC1;1's role in stress responses may be broadly conserved, as experiments in both Arabidopsis and Nicotiana benthamiana demonstrated similar functions . Developing antibodies that recognize conserved epitopes in CycC1;1 across different plant species would facilitate transfer of knowledge from model systems to crops, potentially accelerating the development of stress-tolerant varieties.

What are the implications of CycC1;1's dual roles in salt stress and ABA signaling for agricultural applications?

The dual roles of CycC1;1 in negatively regulating both salt tolerance and ABA signaling have significant implications for agricultural applications. The research shows that disruption of CycC1;1 promotes SOS1 expression and salt tolerance , while also increasing sensitivity to ABA and enhancing expression of ABA-responsive genes .

This dual regulatory role presents both opportunities and challenges:

Antibody-based research tools will be essential for understanding these complex regulatory networks and identifying ideal intervention points. For example, antibodies against specific phosphorylated forms of CycC1;1 could help identify conditions that selectively modify its interaction with either WRKY75 or ABI5, potentially allowing for targeted modulation of either salt tolerance or ABA signaling without affecting the other pathway.