ARR10 Antibody

What is ARR10 and what is its role in cytokinin signaling?

ARR10 is a type-B Arabidopsis response regulator (ARR) protein that functions as a transcription factor in the cytokinin signaling pathway. It acts in concert with other type-B ARRs, particularly ARR1 and ARR12, to mediate cytokinin-regulated transcriptional responses . Type-B ARRs like ARR10 function downstream of histidine kinase receptors and phosphotransfer proteins in the cytokinin signaling pathway, where they bind to DNA and regulate the expression of cytokinin-responsive genes .

In the signaling cascade, ARR10 becomes activated through phosphorylation following cytokinin perception, which enables it to bind to specific DNA sequences and regulate gene expression. This regulation leads to various physiological responses to cytokinin, including cell division, shoot formation, and stress responses .

In which plant species is ARR10 conserved?

ARR10 is found in multiple plant species, with the antibody showing cross-reactivity across several important research and crop plants. According to product specifications, the ARR10 antibody recognizes the protein in the following species :

This cross-reactivity makes the antibody valuable for comparative studies of cytokinin signaling across different plant species, particularly within the Brassicaceae family and beyond to important crop and model plants .

What are the primary DNA binding motifs for ARR10?

ARR10 recognizes specific DNA sequence motifs to regulate gene expression. Protein-binding microarray studies have identified the primary DNA-binding motifs for ARR10, which include :

• Primary motif: AGATACGG

• Secondary motifs: AGATY (where Y represents C or T)

These binding sites are enriched near the transcription start sites of cytokinin-regulated genes, consistent with ARR10's role as a transcriptional regulator . The binding motifs are particularly prevalent in the promoter regions of type-A ARRs, which are primary cytokinin-responsive genes .

Researchers using ARR10 antibody for chromatin immunoprecipitation should consider these binding motifs when analyzing their results, as enrichment of these sequences provides validation for successful immunoprecipitation of ARR10-bound chromatin regions .

How can ARR10 antibody be effectively used in ChIP-seq experiments?

ARR10 antibody is a valuable tool for chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments to identify genome-wide binding sites of ARR10. For optimal results in ChIP-seq applications, researchers should consider the following methodological approach :

• Sample preparation: Use plants expressing tagged ARR10 (such as ARR10-GFP fusion) for enhanced antibody specificity and binding. Transgenic lines expressing ARR10-GFP under the 35S promoter in an arr1 arr10 arr12 mutant background have proven effective for ChIP-seq studies .

• Cytokinin treatment: Treat plant material with cytokinin (e.g., 5 μM 6-benzylaminopurine) for approximately 30 minutes before tissue collection. This timing captures the primary transcriptional response, as cytokinin-induced gene expression begins within 10 minutes and often peaks within 2 hours .

• Chromatin crosslinking and fragmentation: Use formaldehyde for crosslinking followed by sonication to generate appropriately sized chromatin fragments.

• Immunoprecipitation: Use anti-GFP antibodies for tagged ARR10 or direct ARR10 antibodies for native protein, depending on your experimental system.

• Data analysis: Use peak-calling algorithms like MACS (Model-based Analysis of ChIP-seq) to identify binding sites, with particular attention to peaks near transcription start sites, which represent the majority of ARR10 binding regions .

A comprehensive ChIP-seq analysis of ARR10 binding sites revealed 4,861 common binding sites across biological replicates, with binding sites predominantly found in intergenic regions (67%) and concentrated near transcription start sites .

What are the differences between ARR10 and other type-B ARRs in experimental systems?

ARR10 presents several unique characteristics compared to other type-B ARRs that influence experimental design considerations :

• Enhanced stability: ARR10 exhibits greater protein stability compared to other type-B ARRs like ARR1 and ARR12, making it particularly useful for overexpression studies and protein-based analyses .

• Hypersensitivity phenotype: When ARR10 is expressed from the ARR1 promoter or overexpressed under the 35S promoter, it confers a cytokinin hypersensitivity phenotype, despite having transcript levels comparable to other lines . This hypersensitivity is observed in:

  • Root growth inhibition at all cytokinin concentrations (0.001 to 1 μM)

  • Enhanced shoot induction responses

  • Amplified transcriptional responses to cytokinin

• Functional redundancy: While ARR10 functions redundantly with ARR1 and ARR12 in many cytokinin responses, its unique properties create differences in experimental outcomes when these proteins are expressed in the same genetic context .

These differences should be considered when designing experiments using ARR10 antibodies, particularly when comparing results with other type-B ARRs or when using ARR10 as a model for type-B ARR function .

How does ARR10 binding correlate with cytokinin-regulated gene expression?

ChIP-seq studies using ARR10 antibodies have revealed important insights about the relationship between ARR10 binding and cytokinin-regulated gene expression :

• Binding site distribution: ARR10 binding peaks are significantly enriched near transcription start sites of both cytokinin-induced and cytokinin-repressed genes, suggesting direct regulation of both activation and repression responses .

• Target gene overlap: Of 2,848 non-redundant differentially expressed genes identified through multiple cytokinin expression studies, 804 corresponded to direct ARR10 targets based on ChIP-seq data .

• Enrichment in cytokinin-responsive genes: ARR10 candidate targets were substantially enriched for cytokinin-regulated genes:

  • 48% (76 of 158) of cytokinin up-regulated genes from the "golden list" (meta-analysis of 13 cytokinin transcriptome studies) corresponded to ARR10 targets

  • 25% (17 of 68) of cytokinin down-regulated genes from the golden list corresponded to ARR10 targets

• Correlation with robustness of response: Genes supported by a higher number of microarray experiments (indicating more robust cytokinin responses) were more likely to be associated with ARR10 binding peaks .

This correlation provides strong evidence for direct transcriptional regulation by ARR10 and demonstrates the value of ARR10 antibodies in linking cytokinin signaling to downstream transcriptional networks .

What are the optimal storage and handling conditions for ARR10 antibody?

For maximum effectiveness in research applications, ARR10 antibody requires specific storage and handling protocols :

• Storage temperature: Store lyophilized antibody at the recommended temperature immediately upon receipt. Avoid repeated freeze-thaw cycles by using a manual defrost freezer .

• Shipping conditions: The product is typically shipped at 4°C and should be properly stored upon arrival .

• Working solution preparation: When preparing working solutions, consider using BSA or other stabilizers to maintain antibody activity, particularly for diluted solutions that will be stored.

• Cross-reactivity considerations: When working with species other than Arabidopsis thaliana, pilot experiments to confirm cross-reactivity and specificity are recommended, as antibody performance may vary across the recognized species (Brassica rapa, Brassica napus, Nicotiana tabacum, and Medicago truncatula) .

How can ARR10 antibody be used to study the mechanistic details of cytokinin response?

ARR10 antibody can be employed in multiple experimental approaches to elucidate the mechanistic details of cytokinin response :

• Mapping the transcriptional cascade: ChIP-seq with ARR10 antibody has revealed a transcriptional cascade operating downstream of type-B ARRs. This technique can identify primary, secondary, and tertiary response genes in the cytokinin signaling pathway .

• Temporal dynamics analysis: By performing ChIP at different time points following cytokinin treatment, researchers can track the temporal dynamics of ARR10 binding to different target genes, revealing the sequence of regulatory events in the cytokinin response pathway .

• Combinatorial studies with other transcription factors: Co-immunoprecipitation and sequential ChIP approaches using ARR10 antibody alongside antibodies for other transcription factors can reveal combinatorial regulation patterns in cytokinin-mediated transcriptional networks .

• Structure-function analysis: ARR10 antibody can be used to assess how mutations in the ARR10 protein affect its DNA binding capacity and regulatory function, providing insights into the structural determinants of ARR10 activity .

What controls should be included when using ARR10 antibody in ChIP experiments?

To ensure robust and reliable results when using ARR10 antibody in ChIP experiments, several controls should be included :

• Negative genetic controls: Include arr10 knockout mutants or arr1 arr10 arr12 triple mutants as negative controls to establish background signal levels .

• Input controls: Always process a portion of the chromatin before immunoprecipitation as an input control to normalize for chromatin abundance.

• Mock IP controls: Perform parallel immunoprecipitations with non-specific IgG or pre-immune serum to identify non-specific binding.

• Known target validation: Include analysis of known ARR10 target genes, such as type-A ARRs (particularly ARR5, ARR7, and ARR15), which have well-documented ARR10 binding sites in their promoter regions .

• Motif enrichment analysis: Validate ChIP-seq results by confirming enrichment of the known ARR10 binding motifs (AGATACGG and AGATY) in the immunoprecipitated DNA fragments .

• Cytokinin treatment controls: Include both cytokinin-treated and untreated samples to distinguish constitutive from cytokinin-induced ARR10 binding events .

How does ARR10 contribute to drought response regulation?

ARR10 works together with other type-B ARRs to regulate drought responses in plants, though with some functional specialization :

• Redundant function: ARR1, ARR10, and ARR12 function redundantly as regulators of drought response, with experiments showing overlapping but distinct roles .

• Relative contribution: Among these three ARRs, ARR1 appears to be the most critical for drought response, suggesting a hierarchical organization within this functional redundancy .

• Methodological approach to study: To investigate ARR10's specific contribution to drought stress responses, researchers can:

  • Use ARR10 antibody in ChIP-seq experiments under both normal and drought conditions to identify stress-specific binding patterns

  • Compare binding patterns and transcriptional responses between single, double, and triple mutants of arr1, arr10, and arr12

  • Analyze the effect of ARR10 overexpression on drought tolerance phenotypes

Such studies may reveal how cytokinin signaling integrates with abscisic acid (ABA) pathways during drought stress, and how ARR10 specifically contributes to this regulatory network .

What are the genomic features of ARR10 binding sites?

ChIP-seq studies using ARR10 antibody have revealed several key genomic features of ARR10 binding sites :

• Genomic distribution: ARR10 binding peaks are distributed across different genomic regions, with 67% in intergenic regions, 19% in introns, and 19% in exons (when normalized per kilobase of region type) .

• Proximity to transcription start sites: The highest frequency of ARR10 binding occurs near transcription start sites (TSSs), consistent with its role as a transcriptional regulator .

• Type-A ARR regulation: Nine out of ten type-A ARR genes have ARR10 binding peaks in their promoter regions, confirming direct regulation of these primary cytokinin response genes .

• Motif enrichment: ARR10 binding sites show significant enrichment for the DNA-binding motifs identified through protein-binding microarrays, confirming the specificity and physiological relevance of these binding sites .

• Relationship to chromatin features: ARR10 binding sites may correlate with specific chromatin modifications and accessibility states, though this relationship requires further investigation using combinations of ChIP-seq for ARR10 and various histone marks.

Understanding these genomic features provides insight into the mechanisms by which ARR10 regulates gene expression and mediates cytokinin responses throughout the plant genome .

How does ARR10 function compare across different plant developmental stages?

ARR10 shows developmental stage-specific functions that can be investigated using ARR10 antibody in stage-specific experiments :

• Root development: ARR10 plays a role in cytokinin-mediated inhibition of root growth, with ARR10 overexpression lines showing hypersensitivity to cytokinin in root growth inhibition assays .

• Shoot regeneration: ARR10 contributes to shoot induction and regeneration from callus tissue in response to cytokinin, with ARR10 overexpression enhancing this response beyond wild-type levels .

• Germination and seedling development: ARR10, along with other type-B ARRs, regulates various aspects of early development, though with partial functional redundancy with ARR1 and ARR12 .

• Temporal dynamics of binding: ChIP-seq analysis at different developmental stages could reveal how ARR10 binding patterns change throughout plant development, potentially explaining stage-specific cytokinin responses.

Researchers can use ARR10 antibody in ChIP experiments across different developmental stages to track changes in ARR10 binding patterns and correlate these with stage-specific gene expression changes and phenotypic outcomes .

How can ARR10 antibody be used to study cross-talk between cytokinin and other hormone signaling pathways?

ARR10 antibody is a valuable tool for investigating interactions between cytokinin and other plant hormone signaling pathways :

• Combined hormone treatments: Researchers can treat plants with cytokinin plus other hormones (auxin, ethylene, abscisic acid, etc.) before performing ChIP with ARR10 antibody to identify how these hormones affect ARR10 binding patterns.

• Genome-wide binding analysis: ChIP-seq with ARR10 antibody can reveal binding to promoters of genes involved in other hormone signaling pathways, identifying direct cross-talk points.

• Hormone crosstalk study design:

  • Compare ARR10 binding patterns in wild-type vs. mutants defective in other hormone signaling pathways

  • Analyze how ARR10 binding changes in response to combination treatments with multiple hormones

  • Identify hormone-specific and shared ARR10 target genes through comparative ChIP-seq and RNA-seq analyses

• Gene ontology enrichment: Analysis of ARR10 targets identified by ChIP-seq has revealed enrichment for various biological processes, likely including genes involved in multiple hormone signaling pathways .

Such studies can provide molecular mechanisms underlying the well-documented interactions between cytokinin and other phytohormones in regulating plant growth and development .