Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase ORTHRUS-LIKE 1 (ORTHL)

What is the domain structure of ORTHL1 and how does it differ from other ORTH family proteins?

ORTHL1 (also known as ORL1/VIM6) possesses a distinct domain architecture compared to other members of the ORTHRUS family in Arabidopsis thaliana. While ORTH1-5 proteins contain an N-terminal PHD domain, two RING domains flanking an SRA domain, ORTHL1 has a simpler structure with only one RING domain and one SRA domain. Specifically, ORTHL1 lacks the N-terminal PHD domain and the C-terminal RING domain that are present in other ORTH proteins .

This structural difference suggests a potentially specialized function for ORTHL1 within the broader ORTH family. The presence of both SRA and RING domains classifies ORTHL1 as an SRA-RING protein, a structural motif important for its function in ubiquitylation and DNA methylation processes .

What experimental evidence confirms the expression of ORTHL1 in Arabidopsis thaliana?

The expression of ORTHL1 has been experimentally verified through RT-PCR techniques that successfully isolated its cDNA. In contrast to ORTH3 and ORTH4, for which expression remains uncertain due to failed cDNA isolation attempts, ORTHL1 expression has been clearly demonstrated alongside ORTH1, ORTH2, and ORTH5 .

This confirmation of expression is significant as it validates ORTHL1 as an actively transcribed gene rather than a pseudogene, supporting its biological relevance in Arabidopsis development and regulation.

What evidence demonstrates that ORTHL1 functions as an E3 ubiquitin ligase?

ORTHL1 has been experimentally proven to function as an E3 ubiquitin ligase through in vitro ubiquitylation assays. When expressed as a GST fusion protein, recombinant ORTHL1 successfully catalyzed ubiquitylation reactions in the presence of the E2 conjugating enzyme AtUBC11 .

This activity confirms that despite its reduced domain architecture compared to other ORTH proteins, ORTHL1 retains functional E3 ligase capacity. The demonstration of catalytic activity indicates that the single RING domain in ORTHL1 is sufficient for mediating ubiquitin transfer from E2 enzymes to substrates .

How selective is ORTHL1 in its interactions with E2 ubiquitin-conjugating enzymes?

While specific E2 selectivity testing for ORTHL1 is not directly reported in the provided search results, insights can be drawn from studies of ORTH1, which showed differential activity with various E2 enzymes of the UBC8 family. ORTH1 exhibited robust activity with UBC8, UBC10, and UBC11, while showing diminished activity with more divergent UBC8 family members like UBC28 and UBC29 .

By extension, ORTHL1 likely demonstrates similar E2 selectivity patterns, with highest activity when paired with members of the UBC8 subfamily. This selectivity is significant for understanding the biological pathways in which ORTHL1 participates, as different E2-E3 combinations can target different substrate proteins for ubiquitylation.

What are the recommended approaches for expressing and purifying recombinant ORTHL1 for in vitro studies?

For successful expression and purification of functional recombinant ORTHL1:

• Expression System: GST fusion protein expression in E. coli has been successfully employed for ORTHL1 and other ORTH family members. This system provides both a purification tag and often enhances protein solubility .

• Purification Protocol:

  • Affinity chromatography using glutathione-agarose matrices

  • Buffer conditions maintaining the structural integrity of RING domains (typically including reducing agents such as DTT or β-mercaptoethanol)

  • Metal ion supplementation (zinc) may be necessary to maintain RING domain structure

• Functional Testing: Following purification, verify E3 ligase activity through in vitro ubiquitylation assays containing:

  • Purified recombinant ORTHL1

  • E1 activating enzyme

  • Selected E2 conjugating enzyme (preferably AtUBC11)

  • Ubiquitin

  • ATP

  • Reaction buffer with appropriate pH and salt concentration

This approach has proven effective for obtaining functionally active ORTHL1 protein suitable for biochemical characterization studies .

What methods can be employed to study ORTHL1-mediated DNA methylation effects in vivo?

To investigate ORTHL1's role in DNA methylation regulation, researchers can employ these methodologies:

• Transgenic Approaches:

  • Create ORTHL1 overexpression lines using appropriate plant promoters

  • Generate ORTHL1 knock-out or knock-down lines using CRISPR/Cas9 or RNAi

  • Develop complementation lines expressing ORTHL1 with specific domain mutations

• Methylation Analysis Techniques:

  • Bisulfite sequencing to quantify methylation at specific genomic regions

  • Methylation-sensitive PCR for targeted analysis of key loci (such as FWA or Cen180 repeats)

  • Whole-genome bisulfite sequencing for global methylation patterns

• Phenotypic Assays:

  • Flowering time measurements (as ORTH family overexpression affects flowering time)

  • Evaluation of epigenetic inheritance by tracking phenotypes across generations

  • Analysis of reporter gene expression in methylation-sensitive contexts

These methodologies can be combined to establish causal relationships between ORTHL1 activity and specific DNA methylation patterns, advancing understanding of its epigenetic regulatory function.

How does ORTHL1 influence DNA methylation patterns and what genomic regions are most affected?

ORTHL1, like other members of the ORTH family, plays a significant role in regulating DNA methylation patterns in Arabidopsis. Based on studies of ORTH proteins:

• Methylation Impact: Overexpression of ORTH proteins leads to hypomethylation at specific genomic regions, suggesting that ORTHL1 may function in maintaining proper methylation levels at these loci .

• Target Regions: The FWA gene and centromeric Cen180 repeats have been identified as regions where ORTH proteins influence methylation patterns. These regions are particularly important for developmental processes such as flowering time .

• Mechanism of Action: The SRA domain in ORTHL1 likely mediates interaction with methylated DNA, while its E3 ligase activity presumably regulates the stability or activity of proteins involved in the DNA methylation machinery.

• Epigenetic Memory: Once established, methylation changes induced by altered ORTH protein expression can persist even in the absence of the original trigger, demonstrating the capacity of ORTHL1 to influence epigenetic memory mechanisms .

The influence of ORTHL1 on these methylation patterns suggests it functions as part of a broader regulatory network controlling epigenetic states in the Arabidopsis genome.

What are the functional consequences of ORTHL1-mediated changes in DNA methylation?

The alteration of DNA methylation patterns by ORTHL1 and other ORTH family proteins results in several functional consequences:

• Altered Flowering Time: Overexpression of ORTH proteins leads to changes in flowering time, one of the most well-documented phenotypic consequences. This is consistent with the hypomethylation observed at the FWA locus, a known regulator of flowering time .

• Epigenetic Inheritance: The late-flowering phenotype induced by ORTH overexpression persists even after the transgene is no longer present. This demonstrates that ORTHL1-mediated methylation changes can establish stable, heritable epigenetic states .

• Genomic Stability: Changes in methylation at centromeric regions (Cen180 repeats) may impact chromosome stability and proper segregation during cell division.

• Gene Expression Alterations: Beyond flowering time, methylation changes likely affect the expression of multiple genes involved in various developmental and physiological processes.

These consequences highlight the significant role of ORTHL1 in epigenetic regulation and suggest its potential involvement in adapting plant development to environmental conditions through methylation-dependent mechanisms.

How do the structural differences between ORTHL1 and other ORTH family members relate to their functional specialization?

The structural differences between ORTHL1 and other ORTH family members likely contribute to functional specialization in the following ways:

These structural differences suggest that ORTHL1 may have evolved specialized functions:

• Substrate Targeting: The absence of the PHD domain likely alters ORTHL1's ability to interact with chromatin substrates compared to other ORTH proteins.

• Catalytic Activity: While still functional as an E3 ligase, ORTHL1 may exhibit different catalytic properties due to having only one RING domain. Studies with ORTH1 demonstrated that either RING domain alone is capable of promoting ubiquitylation, suggesting that ORTHL1's single RING domain is sufficient for activity .

• Regulatory Mechanisms: The simplified domain architecture of ORTHL1 may result in different regulatory mechanisms controlling its activity compared to other ORTH family members.

This structural and functional specialization may allow ORTHL1 to complement the activities of other ORTH proteins by targeting different substrates or operating in distinct cellular contexts .

What is known about the interaction between ORTHL1 and components of the DNA methylation machinery?

While direct interactions between ORTHL1 and specific components of the DNA methylation machinery are not explicitly detailed in the provided search results, several important inferences can be drawn:

• SRA Domain Interactions: The SRA domain in ORTHL1 likely mediates interactions with methylated DNA, potentially recognizing specific methylation patterns or contexts (CG, CHG, or CHH).

• E3 Ligase Targets: As an E3 ubiquitin ligase, ORTHL1 presumably targets specific proteins for ubiquitylation. These targets might include:

  • DNA methyltransferases or their regulators

  • Histone modifying enzymes that influence DNA methylation

  • Demethylases or proteins that protect against DNA methylation

• Regulatory Network: ORTHL1 operates within a complex regulatory network controlling DNA methylation. The hypomethylation at FWA and Cen180 repeats when ORTH proteins are overexpressed suggests that these proteins normally function in maintaining proper methylation levels .

• Functional Redundancy: The existence of multiple ORTH family members with similar domain architectures suggests potential functional redundancy or cooperative activities in regulating DNA methylation.

Further research utilizing techniques such as protein-protein interaction assays, ChIP-seq, and genetic epistasis analysis would be necessary to fully elucidate the specific components of the DNA methylation machinery that interact with ORTHL1.

How conserved is ORTHL1 across plant species and what does this reveal about its evolutionary importance?

The evolutionary conservation of ORTHL1 across plant species provides valuable insights into its biological significance:

This evolutionary conservation underscores the fundamental importance of ORTHL1 and related proteins in plant epigenetic regulation and suggests that insights gained from studying ORTHL1 in Arabidopsis may be broadly applicable across plant species.

What methods are most effective for analyzing the functional differences between ORTHL1 and other ORTH family members?

To effectively analyze functional differences between ORTHL1 and other ORTH family members, researchers should consider employing the following complementary approaches:

• Structural and Biochemical Analysis:

  • Domain-swapping experiments to determine the contribution of individual domains to protein function

  • Quantitative enzyme kinetics to compare E3 ligase activities

  • Protein-protein and protein-DNA interaction assays to identify differential binding partners and DNA targets

  • Structural studies (X-ray crystallography or cryo-EM) to visualize molecular interactions

• Genetic Approaches:

  • Generate single and combinatorial knockout/knockdown lines for different ORTH family members

  • Create transgenic lines expressing chimeric proteins with domains from different ORTH proteins

  • Perform genetic complementation assays to determine functional interchangeability

  • Analyze genetic interactions through epistasis studies

• Genomic and Epigenomic Profiling:

  • ChIP-seq to identify differential genomic binding sites

  • Whole-genome bisulfite sequencing to compare methylation patterns in various ORTH mutants

  • RNA-seq to identify differentially regulated genes

  • Proteomics to identify unique ubiquitylation targets

• Spatial and Temporal Expression Analysis:

  • Tissue-specific and developmental stage-specific expression profiling

  • Subcellular localization studies to identify potential compartmentalization differences

  • Inducible expression systems to examine acute versus chronic effects

By integrating these approaches, researchers can develop a comprehensive understanding of how the structural differences between ORTHL1 and other ORTH family members translate into functional specialization within the broader context of DNA methylation regulation and plant development.