ERF054 Antibody

What is ERF054 and what cellular functions does it perform?

ERF054 (Ethylene-responsive transcription factor 054) is a member of the AP2/ERF transcription factor family, specifically belonging to the ERF subfamily. It functions primarily as a transcriptional activator that binds to the GCC-box pathogenesis-related promoter element. The protein plays a significant role in regulating gene expression in response to various stress factors and components of stress signal transduction pathways. As a transcription factor, ERF054 is primarily localized in the nucleus where it can directly interact with DNA to influence gene expression.

Recent research on the Rosa multiflora homolog (RmERF54) has shown that this transcription factor is specifically involved in cold tolerance mechanisms, further supporting its role in stress response regulation . Understanding the basic function of ERF054 is essential for researchers designing experiments to investigate its role in plant stress physiology.

How does ERF054 relate to plant stress response mechanisms?

ERF054 has been demonstrated to play a crucial role in plant stress responses, particularly in cold tolerance. In Rosa multiflora, overexpression of RmERF54 significantly increases cold resistance in both transgenic tobacco and rose somatic embryos, while virus-induced gene silencing (VIGS) of RmERF54 increases cold susceptibility . This indicates that ERF054 is a positive regulator of cold tolerance mechanisms.

The transcription factor achieves this by extensive transcriptional reprogramming of stress response genes and antioxidant enzyme systems. Specifically, RmERF54 binds to the promoters of genes encoding the PODP7 peroxidase and the cold-related COR47 protein, activating their expression . Plants with increased ERF054 expression demonstrate higher antioxidant enzyme activities and considerably lower levels of reactive oxygen species (ROS) under cold stress conditions . Additionally, ERF054 functions within the DREB/COR signaling pathway, being a target of the Dehydration-Responsive-Element-Binding factor RmDREB1E .

What experimental model systems have been used to study ERF054 function?

Several experimental model systems have been successfully employed to investigate ERF054 function:

• Rosa multiflora: As a cold-tolerant species, it has been used to study the native function of RmERF54 in cold tolerance mechanisms .

• Transgenic tobacco: Heterologous expression of RmERF54 in tobacco has demonstrated the capability of this transcription factor to confer increased cold resistance even in a different plant species .

• Rose somatic embryos: These have been used as a system for overexpression studies to understand the impact of increased ERF054 levels on cold tolerance .

• Virus-induced gene silencing (VIGS) models: This approach has been used to reduce the expression of ERF054 in plants, allowing researchers to investigate the effects of reduced ERF054 function on stress tolerance .

• Arabidopsis thaliana: While not directly studying ERF054, related research on ERF4 in Arabidopsis has provided insights into how ERF family proteins function in plant development and stress responses .

How is ERF054 expression regulated by the circadian clock?

ERF054 appears to be subject to circadian regulation based on its promoter structure. The promoter regions of ERF53 and ERF54 contain Evening Elements (EEs), which are binding sites for circadian clock-related transcription factors . This suggests that ERF054 expression may oscillate throughout the day, potentially impacting its stress response functions at different times.

Research on related transcription factors has shown that the Arabidopsis RVE4 and RVE8 transcription factors interact with these EEs and may regulate ERF expression . Interestingly, these regulators function differently under normal versus stress conditions. While they regulate circadian clock-related target genes under normal temperature conditions, they switch to regulating stress-related target genes under cold and heat stress conditions . This mechanism of "target gene switching" provides an elegant system for plants to reprogram their transcriptional responses when environmental conditions change.

Researchers investigating ERF054 should consider these temporal aspects, as the timing of stress application relative to the circadian phase may significantly affect experimental outcomes. Time-course experiments that sample across the day/night cycle are recommended to fully capture the dynamics of ERF054 expression and function.

What molecular mechanisms underlie ERF054-mediated cold tolerance?

ERF054 employs several molecular mechanisms to confer cold tolerance in plants:

• Direct transcriptional activation: RmERF54 binds directly to the promoters of stress-response genes such as RmPODP7 (peroxidase) and RmCOR47 (cold-responsive protein) to activate their expression .

• Enhanced antioxidant capacity: Plants overexpressing RmERF54 demonstrate higher antioxidant enzyme activities, which helps protect cellular components from oxidative damage during cold stress .

• Reduced ROS accumulation: As a consequence of enhanced antioxidant enzyme activity, ERF054-overexpressing plants maintain lower levels of reactive oxygen species under cold stress conditions .

• Integration with DREB/COR pathway: ERF054 functions within the well-established DREB/COR cold response pathway, being identified as a target of RmDREB1E (a Dehydration-Responsive-Element-Binding factor) . This suggests that ERF054 may act downstream of DREB factors to execute specific aspects of the cold response program.

Unlike some transcription factors that respond to multiple stresses, ERF054 appears to have a more specialized role in cold tolerance. This specialization may allow for more precise regulation of cold-specific response mechanisms without triggering unnecessary responses to other stresses.

How does ERF054 interact with other transcription factors in stress response networks?

ERF054 does not function in isolation but rather as part of complex transcriptional networks that orchestrate plant stress responses. Several important interactions have been identified:

• DREB pathway integration: ERF054 has been identified as a target of RmDREB1E, placing it within the DREB/COR signaling pathway for cold stress responses . This suggests a hierarchical relationship where DREB factors may regulate ERF054 expression or activity.

• Co-expressed genes: In Arabidopsis, the related ERF4 is predicted to be co-expressed with multiple genes including other transcription factors such as STK, LUH/MUM1, MYB52, and GLABRA2 (GL2) . While this co-expression network was identified for ERF4 rather than ERF054 directly, it suggests that ERF family members function within complex regulatory networks involving multiple transcription factor families.

• Potential for protein-protein interactions: Many transcription factors form complexes with other proteins to regulate gene expression. Although specific interacting partners for ERF054 have not been directly identified in the provided search results, its role in complex stress response networks suggests that protein-protein interactions likely influence its activity.

Understanding these interactions is crucial for researchers aiming to place ERF054 within the broader context of plant stress response networks. Techniques such as yeast two-hybrid screens, co-immunoprecipitation followed by mass spectrometry, or split-luciferase complementation assays could be employed to identify direct protein interactors of ERF054.

What are the optimal approaches for detecting and studying ERF054 protein?

Several methodological approaches can be used to effectively detect and study ERF054 protein:

What techniques are most effective for studying ERF054 binding to target promoters?

To investigate how ERF054 interacts with target gene promoters, researchers can employ several complementary techniques:

• Chromatin Immunoprecipitation (ChIP): This is the gold standard for identifying in vivo binding sites of transcription factors. Using an ERF054-specific antibody, researchers can immunoprecipitate chromatin fragments bound by ERF054, followed by qPCR of specific promoter regions or sequencing (ChIP-seq) to identify genome-wide binding sites.

• Electrophoretic Mobility Shift Assay (EMSA): This in vitro technique can confirm direct binding of purified ERF054 protein to specific DNA sequences, such as the GCC-box elements it is known to recognize.

• DNA-affinity purification (DAP): Using immobilized DNA fragments containing putative ERF054 binding sites to capture the protein from nuclear extracts.

• Reporter gene assays: Constructing reporter plasmids with putative ERF054 target promoters driving expression of reporter genes (like luciferase or GFP) to assess transcriptional activation in response to ERF054 expression.

• Yeast one-hybrid assays: To test the interaction between ERF054 and specific DNA sequences in a heterologous system.

In Rosa multiflora, RmERF54 has been shown to bind to the promoters of RmPODP7 and RmCOR47 genes . Similar approaches could be used to identify and validate ERF054 binding sites in other plant species and experimental systems.

How can researchers effectively modify ERF054 expression to study its function?

Several genetic approaches can be employed to manipulate ERF054 expression levels and study the resulting phenotypes:

• Overexpression strategies:

  • Constitutive overexpression using strong promoters (e.g., 35S)

  • Tissue-specific overexpression

  • Inducible overexpression systems (e.g., estrogen-inducible or dexamethasone-inducible)

• Loss-of-function approaches:

  • T-DNA insertion mutants (in model species like Arabidopsis)

  • CRISPR/Cas9 gene editing to generate knockout or knockdown lines

  • RNA interference (RNAi)

  • Virus-induced gene silencing (VIGS), which has been successfully used for RmERF54 in Rosa multiflora

• Protein modification approaches:

  • Creating dominant-negative versions by removing activation domains

  • Fusion with repressor domains (e.g., SRDX repression domain)

  • Creation of constitutively active versions

• Complementation studies:

  • Expressing ERF054 in knockout/knockdown backgrounds to confirm function

  • Heterologous expression in different species to test conservation of function

When designing such experiments, it's important to include appropriate controls and to verify the expression levels achieved using techniques such as RT-qPCR for transcript analysis and western blotting with ERF054 antibody for protein levels.

What unresolved questions remain about ERF054 function in plant stress responses?

Despite recent advances, several important questions remain unanswered about ERF054 function:

• Stress specificity: While ERF054 has been implicated in cold tolerance , its potential roles in other abiotic or biotic stress responses remain largely unexplored. Does ERF054 specifically respond to cold, or does it function in a broader range of stress responses?

• Evolutionary conservation: How conserved is ERF054 function across different plant species? Do homologs in other species perform similar functions in stress responses?

• Post-translational regulation: Are there specific post-translational modifications that regulate ERF054 activity in response to stress signals? Phosphorylation, ubiquitination, or other modifications could potentially fine-tune its function.

• Temporal dynamics: How quickly does ERF054 respond to stress signals, and what is the duration of its activity? Understanding these temporal aspects could provide insights into its role in immediate versus long-term stress adaptation.

• Interaction with hormone signaling: How does ERF054 integrate with plant hormone signaling networks, particularly ethylene signaling given its classification as an ethylene-responsive factor?

Future research addressing these questions will provide a more comprehensive understanding of ERF054's role in plant stress responses.

How might ERF054 research contribute to improving crop stress resilience?

Research on ERF054 has significant potential applications for improving crop stress tolerance through several approaches:

• Marker-assisted selection: Identifying natural variants of ERF054 associated with enhanced cold tolerance could allow breeders to select for these alleles in breeding programs.

• Genetic engineering: The demonstrated ability of RmERF54 overexpression to enhance cold tolerance suggests that similar approaches could be used to improve cold tolerance in crop species. This could be achieved through:

  • Constitutive or stress-inducible overexpression

  • Precision breeding using CRISPR/Cas9 to modify endogenous ERF054 genes

  • Stacking of ERF054 with other stress tolerance genes

• Pathway engineering: Understanding how ERF054 integrates with broader stress response networks could allow for more sophisticated engineering of these pathways.

• Cross-species applications: The successful transfer of cold tolerance through RmERF54 expression in tobacco suggests that ERF054-based approaches might be transferable across diverse plant species.

• Climate change adaptation: As climate variability increases, tools for enhancing plant resilience to temperature extremes become increasingly valuable. ERF054 manipulation could help develop crops better adapted to cold snaps in otherwise warm regions.

When considering such applications, researchers should design experiments that evaluate not only stress tolerance but also potential trade-offs in terms of growth, yield, and other agronomically important traits.