Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase UPL3 (UPL3), partial

What is UPL3 and what is its biological significance?

UPL3 is a HECT-domain E3 ubiquitin ligase in Arabidopsis thaliana that catalyzes the final step in protein ubiquitination by facilitating the transfer of ubiquitin to target proteins. UPL3 belongs to a small family of seven HECT-containing ubiquitin-protein ligases (UPL1-UPL7) in Arabidopsis that can be grouped into four subfamilies . As a member of the third subfamily along with UPL4, UPL3 is a large protein (~200 kDa) containing four Armadillo repeats similar to those found in the nuclear pore protein importin-α, suggesting that it identifies targets through binding to nuclear localization sequences .

UPL3 plays critical roles in multiple biological processes including plant immunity regulation, hormone signaling, and trichome development. Specifically, UPL3 is essential for controlling trichome morphology, as upl3 mutants exhibit aberrant trichome branching patterns and altered endoreplication .

How does UPL3 function at the molecular level?

UPL3 functions as an E3 ubiquitin ligase that associates with the 26S proteasome to provide ubiquitin ligase activity. The enzyme transfers ubiquitin from an E2 ubiquitin-conjugating enzyme to specific target proteins, marking them for degradation by the proteasome. Studies have shown that UPL3 is the primary active ligase associated with the proteasome in vitro, as proteasomes from immune-induced upl3 knockout mutants display substantial reduction in proteasome-associated ubiquitin ligase activity .

UPL3 performs critical "11th hour" polyubiquitination of target proteins at the proteasome, which is necessary for their processive degradation. This function distinguishes UPL3 from other E3 ligases that may initially recognize and ubiquitinate targets but require UPL3 for final processing at the proteasome .

What phenotypes are associated with UPL3 mutation in Arabidopsis?

UPL3 mutants exhibit several distinctive phenotypes:

• Trichome abnormalities: Instead of developing the typical three branches, many upl3 trichomes contain five or more branches. Additionally, these trichomes often undergo an additional round of endoreplication, resulting in enlarged nuclei with ploidy levels up to 64C .

• Hormone hypersensitivity: upl3 plants show hypersensitivity to gibberellic acid-3 (GA3), consistent with the known role of gibberellins in trichome development .

• Immune response defects: Lack of UPL3 activity is associated with failure to reprogram the transcriptome upon activation of immunity .

• Hormone signaling alterations: UPL3 mutants show disrupted salicylic acid and ethylene signaling pathways due to altered stability of transcriptional activators like NPR1 and EIN3 .

Notably, the trichome phenotype of upl3 mutants resembles that of previously described kaktus mutants, and genetic analyses have confirmed that upl3 and kaktus-2 are allelic, with kaktus-2 plants harboring a splice-site mutation in the UPL3 gene .

How does UPL3 interact with plant hormone signaling pathways?

UPL3 plays crucial roles in multiple hormone signaling pathways by regulating the stability of key transcriptional activators:

• Salicylic acid (SA) pathway: UPL3 regulates NPR1, a key transcriptional activator in SA signaling. UPL3 is the last in a relay of three ubiquitin ligases that polyubiquitinate NPR1, ensuring that transcriptionally inactive NPR1 is cleared from target gene promoters by the proteasome. In upl3 mutants, NPR1 accumulates at target promoters even in the absence of SA stimulation, disrupting proper SA responses .

• Ethylene pathway: UPL3, together with UPL4, regulates the stability of EIN3, a master transcription factor in ethylene signaling. SCF^EBF1/2 E3 ligases physically relay EIN3 to UPL3/4 at the proteasome. When UPL3/4 are absent, EIN3 accumulates at proteasomes but fails to be degraded efficiently, resulting in constitutive ethylene signaling .

The interaction of UPL3 with hormone signaling demonstrates a sophisticated mechanism where pathway-specific E3 ligases recognize hormone-responsive transcription factors and relay them to proteasome-associated UPL3/4 for final processing and degradation.

What is the role of UPL3 in plant immunity?

UPL3 plays a critical role in plant immunity primarily through its regulation of NPR1, a master regulator of salicylic acid-mediated immune responses. UPL3 participates in the degradation of NPR1, which is essential for proper immune function.

In normal immune responses, NPR1 is recruited to promoters of defense genes (like PR1) in response to SA. UPL3 is constitutively associated with these promoters and facilitates the removal of NPR1 after it has activated transcription, allowing for precise control of immune gene expression .

In upl3 mutants, NPR1 accumulates to much higher levels at defense gene promoters both before and after SA treatment, disrupting the proper timing and magnitude of immune responses. Consequently, upl3 mutants are compromised in SA-induced expression of PR1 and fail to properly reprogram their transcriptome during immune activation .

This demonstrates how UPL3-mediated protein turnover at gene promoters helps maintain the balance between immune activation and homeostasis.

How does UPL3 function in the ubiquitin-proteasome system?

UPL3 functions at the intersection of target recognition and proteasomal degradation within the ubiquitin-proteasome system:

• Proteasome association: UPL3 physically associates with both 19S and 20S proteasome subcomplexes, suggesting interaction with the full 26S proteasome holoenzyme .

• Proteasome-associated ligase activity: UPL3 endows the proteasome with ubiquitin ligase activity. Purified proteasomes from wild-type plants can generate ubiquitin conjugates, while those from upl3 mutants show substantially reduced activity .

• Ubiquitin relay hub: UPL3 functions as a final relay point in ubiquitin ligase cascades. For example, in ethylene signaling, SCF^EBF1/2 ligases physically hand over EIN3 to UPL3 at the proteasome for final processing .

• Processive degradation facilitator: UPL3-mediated "11th hour" polyubiquitination is necessary for efficient processive degradation of target proteins. Without UPL3, targets like EIN3 can still reach the proteasome but accumulate there without efficient degradation .

This system creates a sophisticated mechanism where pathway-specific E3 ligases provide initial target recognition, while UPL3 at the proteasome provides the final processing needed for efficient degradation.

What approaches can be used to produce recombinant UPL3?

Producing functional recombinant UPL3 presents several challenges due to its large size (~200 kDa) and complex domain structure. Researchers can employ the following approaches:

• Domain-based expression: Rather than expressing the full protein, researchers can focus on functional domains. The C-terminal HECT domain of UPL1, a related protein, has been successfully expressed and shown to be necessary and sufficient for ubiquitin conjugation in vitro . A similar approach can be used for UPL3's HECT domain.

• Expression systems:

  • Bacterial expression: E. coli systems can be used for individual domains but may not be suitable for the full-length protein due to size limitations and lack of eukaryotic post-translational modifications.

  • Insect cell expression: Baculovirus-infected insect cells provide a eukaryotic environment better suited for large proteins like UPL3.

  • Plant-based expression: Transient expression in Nicotiana benthamiana or stable transformation of Arabidopsis cell cultures may provide the most native environment for proper folding and modifications.

• Fusion tags: Strategic use of tags can enhance solubility and facilitate purification:

  • N-terminal GST or MBP tags improve solubility

  • His-tags allow for metal affinity purification

  • FLAG or HA epitope tags enable immunoprecipitation

• Co-expression strategies: Co-expressing UPL3 with interacting partners or chaperones may improve stability and solubility of the recombinant protein.

How can the enzymatic activity of recombinant UPL3 be measured?

The enzymatic activity of recombinant UPL3 can be assessed using several complementary approaches:

• In vitro ubiquitination assays: This is the most direct method to measure UPL3 activity. The basic reaction requires:

  • Purified recombinant UPL3 (or its HECT domain)

  • E1 ubiquitin-activating enzyme

  • E2 ubiquitin-conjugating enzyme (preferably from the UBC8 family, which has been shown to work with HECT E3 ligases in Arabidopsis)

  • Ubiquitin (can be tagged with fluorescent or radioactive markers)

  • ATP and buffer components

  • Known substrate or general substrate like RPN10

  Activity is typically measured by detecting ubiquitin conjugates via western blotting or fluorescence/radioactive signal quantification.

• Proteasome-associated activity assays: Since UPL3 endows proteasomes with ubiquitin ligase activity, researchers can purify proteasomes and assess their ability to generate ubiquitin conjugates with and without recombinant UPL3 .

• Substrate degradation assays: Using known UPL3 substrates like NPR1 or EIN3, researchers can monitor degradation rates in cell-free degradation systems supplemented with recombinant UPL3.

• Binding assays: While not directly measuring enzymatic activity, assessing the ability of recombinant UPL3 to bind substrates, E2 enzymes, or proteasome components provides insight into its functional state.

What are the known protein-protein interactions of UPL3?

UPL3 engages in multiple protein-protein interactions that are critical for its function:

• Proteasome components: UPL3 interacts with components of both the 19S and 20S proteasome subcomplexes, including the regulatory subunit RPN1 (S2) located at the base of the 19S particle .

• Substrate interactions:

  • NPR1: UPL3 interacts with this salicylic acid-responsive transcriptional activator

  • EIN3: UPL3 receives this ethylene-responsive transcription factor through a physical handover from SCF^EBF1/2

• E2 enzyme interactions: While specific E2 partners for UPL3 are not directly mentioned in the provided search results, related HECT E3 ligases in Arabidopsis, such as UPL1, interact with E2 enzymes from the UBC8 family .

• Other E3 ligases: UPL3 functions in relay with other E3 ligases:

  • Pathway-specific ubiquitin ligases that initially recognize and ubiquitinate targets

  • For NPR1 processing, UPL3 is the last in a relay of three ubiquitin ligases

  • For EIN3 processing, SCF^EBF1/2 physically hands over EIN3 to UPL3

These interactions position UPL3 as a hub in ubiquitin relay mechanisms and highlight its role as a terminal processor for proteins targeted for proteasomal degradation.

How can recombinant UPL3 be used to identify novel substrates?

Identification of novel UPL3 substrates can be approached using several complementary strategies:

• Protein stability profiling: Compare proteome-wide protein stability in wild-type versus upl3 mutant plants. Proteins showing increased stability in mutants are potential UPL3 substrates. This can be accomplished through:

  • Quantitative proteomics with stable isotope labeling

  • Pulse-chase experiments with protein synthesis inhibitors like cycloheximide

  • Global protein stability profiling using tagged or reporter-fused proteins

• In vitro ubiquitination screens: Use purified recombinant UPL3 in combination with protein arrays or cell extracts to identify proteins that become ubiquitinated in a UPL3-dependent manner.

• Affinity purification approaches:

  • Express epitope-tagged UPL3 with catalytically inactive HECT domain (mutation of the active site cysteine) to trap substrates

  • Co-immunoprecipitation followed by mass spectrometry to identify interacting proteins

  • Proximity-based labeling methods (BioID or TurboID fused to UPL3) to identify proteins in close proximity to UPL3 in vivo

• Transcriptome and phenotype analysis: Since UPL3 regulates transcription factors like NPR1 and EIN3, comparing transcriptome changes between wild-type and upl3 mutants can identify pathways potentially regulated by UPL3 substrates. The table below summarizes phenotypes observed in upl3 mutants that may guide substrate identification:

What is the role of UPL3 in ubiquitin relay mechanisms?

UPL3 plays a critical role in ubiquitin relay mechanisms, serving as the terminal processor in multi-step ubiquitination cascades:

• Sequential ubiquitination: UPL3 often acts as the final E3 ligase in a sequence of ubiquitination events. For example, in salicylic acid signaling, UPL3 is the last in a relay of three ubiquitin ligases that polyubiquitinate NPR1 .

• Physical handover: In ethylene signaling, SCF^EBF1/2 ligases physically hand over the partially ubiquitinated EIN3 substrate to UPL3/4 at the proteasome. This physical relay is required for EIN3's recruitment to UPL3 .

• Proteasome-specific processing: UPL3-mediated "11th hour" polyubiquitination at the proteasome is necessary for the processive degradation of target proteins. Without this final processing step, targets can still be recruited to the proteasome but stall during degradation .

• Pathway integration: By serving as a common endpoint for multiple signaling pathways, UPL3 may integrate various cellular signals at the level of protein degradation. This is evidenced by its involvement in both salicylic acid and ethylene signaling pathways .

This system appears to ensure precision in protein degradation, with pathway-specific E3 ligases providing initial target recognition and UPL3 at the proteasome providing the final processing needed for efficient degradation.

How does UPL3 coordinate with other HECT E3 ligases in Arabidopsis?

UPL3 is one of seven HECT-domain E3 ubiquitin ligases (UPL1-UPL7) in Arabidopsis, which can be grouped into four subfamilies based on their domain structure and evolutionary relationships . UPL3 and UPL4 form one subfamily characterized by the presence of four Armadillo repeats similar to those in nuclear pore protein importin-α .

The coordination between UPL3 and other HECT E3 ligases involves:

• Functional redundancy: UPL3 shows partial functional overlap with its closest homolog UPL4. Both are required for optimal control of EIN3 stability and ethylene signaling, as demonstrated by the enhanced phenotypes in upl3 upl4 double mutants compared to single mutants .

• Distinct substrate specificity: Despite some overlap, each UPL appears to have unique substrate preferences:

  • UPL3 has a demonstrated role in trichome development not shared by other UPLs

  • Different UPLs contain distinct N-terminal domains that likely determine substrate specificity

• Differential expression and localization: Different UPLs may function in distinct tissues, developmental stages, or subcellular compartments, allowing for tissue-specific or context-dependent regulation of substrate proteins.

• Evolutionary conservation: The fact that different UPL subfamilies in Arabidopsis often appear more similar to HECT E3s from other species than to each other suggests that these subfamilies arose before the split of animal, fungal and plant kingdoms, and have catalytic activities that cannot be replaced by other E3 types .

What are the technical challenges in expressing and purifying functional recombinant UPL3?

Expressing and purifying functional recombinant UPL3 presents several technical challenges:

• Size limitations: At approximately 200 kDa, UPL3 is a large protein that can be difficult to express in full-length form. This size can cause:

  • Poor expression efficiency

  • Incomplete translation

  • Protein misfolding

  • Aggregation during purification

• Domain complexity: UPL3 contains multiple domains including four Armadillo repeats and the C-terminal HECT domain. Each domain may have different requirements for proper folding and stability.

• Post-translational modifications: As a eukaryotic protein, UPL3 may require specific post-translational modifications for full activity that might not be properly added in bacterial expression systems.

• Protein stability: HECT E3 ligases often have regions of intrinsic disorder that can contribute to instability during purification.

• Catalytic activity preservation: The catalytic cysteine in the HECT domain is susceptible to oxidation, which can inactivate the enzyme during purification.

• Co-factor requirements: Proper folding and activity may require co-factors or interacting proteins that are not present in heterologous expression systems.

• Auto-ubiquitination: E3 ligases can undergo auto-ubiquitination, potentially leading to self-degradation during expression.

To address these challenges, researchers often take domain-based approaches (expressing functional domains separately) or use eukaryotic expression systems like insect cells or plant-based systems that can better handle large proteins with complex folding requirements.

What recent advances have been made in understanding UPL3 function?

Recent research has revealed several important aspects of UPL3 function:

• Ubiquitin ligase relays: A major advance is the discovery that UPL3 functions as a terminal processor in ubiquitin ligase relay systems. In both salicylic acid and ethylene signaling pathways, transcriptional activators (NPR1 and EIN3) are relayed from pathway-specific ubiquitin ligases to proteasome-associated UPL3/4 for final processing .

• Proteasome-associated activity: UPL3 has been found to endow proteasomes with ubiquitin ligase activity. Proteasomes from upl3 knockout mutants display substantially reduced ability to generate ubiquitin conjugates compared to wild-type proteasomes .

• "11th hour" polyubiquitination: UPL3 performs critical polyubiquitination of substrates at the proteasome, which is necessary for their processive degradation. Without this step, substrates like EIN3 can still be recruited to proteasomes but stall during degradation .

• Physical handover mechanisms: For EIN3 processing, researchers have discovered that SCF^EBF1/2 physically relays EIN3 to UPL3 at the proteasome. This physical handover is required for EIN3's recruitment to UPL3, suggesting a new mechanism for substrate transfer between E3 ligases .

• Chromatin association: UPL3 has been found to constitutively associate with target gene promoters independent of hormone treatment, suggesting a direct role in regulating transcription factor dynamics at the chromatin level .

How can genome editing approaches be used to study UPL3 function?

Genome editing technologies, particularly CRISPR/Cas9, offer powerful approaches to study UPL3 function:

• Domain-specific mutations: Rather than complete knockout, researchers can introduce precise mutations to specific domains or residues:

  • Mutation of the catalytic cysteine in the HECT domain to eliminate ubiquitin ligase activity

  • Targeted modifications to Armadillo repeats to alter substrate recognition

  • Introduction of point mutations that mimic or prevent phosphorylation or other post-translational modifications

• Reporter fusions: Genome editing can be used to create endogenous fusions of UPL3 with reporters:

  • Fluorescent protein tags for live-cell imaging of UPL3 localization and dynamics

  • Proximity labeling tags (BioID/TurboID) for in vivo substrate identification

  • Degron tags for controlled degradation of UPL3 to study acute loss of function

• Substrate modification: CRISPR can be used to modify putative UPL3 substrates:

  • Mutation of ubiquitination sites to confirm direct targeting

  • Creation of stabilized variants resistant to UPL3-mediated degradation

  • Introduction of tags for monitoring substrate levels and localization

• Promoter engineering: Modifying the UPL3 promoter can provide temporal or tissue-specific control of expression to study context-dependent functions.

• Paralog replacement: Replace UPL3 coding sequence with that of other UPL family members to test functional redundancy and domain specificity.

These approaches can provide more nuanced insights than traditional knockout studies and allow for the separation of different aspects of UPL3 function.

What are the implications of UPL3 research for crop improvement?

Research on UPL3 has several potential implications for crop improvement:

• Enhanced stress resistance: UPL3's role in plant immunity and hormone signaling suggests that modulating its activity could enhance crop resistance to pathogens. Since UPL3 regulates NPR1, a master regulator of salicylic acid-mediated defense responses, careful engineering of UPL3 or its targets could potentially enhance broad-spectrum disease resistance .

• Growth-defense balance optimization: UPL3 participates in both defense (SA) and growth (ethylene) hormone pathways, positioning it at the nexus of growth-defense tradeoffs. Engineering crops with optimized UPL3 activity could potentially break the negative correlation between growth and defense that limits crop productivity under stress conditions .

• Trichome engineering: UPL3's role in trichome development opens possibilities for engineering trichome density, branching, and size in crops. Trichomes contribute to pest resistance, reduce water loss, and affect leaf temperature regulation, making them important targets for crop improvement .

• Hormone sensitivity modulation: upl3 mutants show altered sensitivity to plant hormones including gibberellins and ethylene. Engineering UPL3 activity could potentially allow fine-tuning of hormone responses to optimize growth under specific environmental conditions .

• Protein degradation engineering: Understanding UPL3's role in the ubiquitin-proteasome system provides knowledge for engineering protein degradation pathways. This could be used to control levels of specific proteins that limit crop productivity or quality traits.

The conservation of HECT E3 ligases across plant species suggests that findings from Arabidopsis UPL3 research could be transferable to crop species, though validation in specific crops would be necessary.

What controls are essential when studying UPL3 activity in vitro?

When studying UPL3 activity in vitro, several essential controls should be included:

• Enzyme activity controls:

  • Catalytic cysteine mutant: A mutant version of UPL3 with the HECT domain active site cysteine mutated to serine or alanine should be included as a negative control to confirm that observed ubiquitination is due to UPL3's catalytic activity.

  • E1/E2 dependency controls: Reactions lacking E1, E2, or ATP should be included to confirm the ubiquitination cascade dependency.

  • Positive control substrate: Include a known UPL3 substrate like NPR1 or EIN3 as a positive control when testing potential new substrates .

• Specificity controls:

  • E2 enzyme panel: Test multiple E2 enzymes to confirm specificity, with UBC8 family members expected to work with UPL3 based on studies of related HECT E3 ligases .

  • Other HECT E3 ligases: Include other UPL family members (particularly UPL4) to assess substrate specificity differences.

  • Auto-ubiquitination assessment: Distinguish between auto-ubiquitination of UPL3 and true substrate ubiquitination.

• Technical controls:

  • Ubiquitin variants: Include lysine-mutant ubiquitin or methylated ubiquitin to assess chain type specificity.

  • Proteasome activity: When studying proteasome-associated activity, include proteasome inhibitors to confirm degradation-dependency.

  • Storage stability control: Test enzyme activity after various storage conditions to ensure stability during experimental timeframes.

• Validation approaches:

  • In vitro to in vivo correlation: Confirm that in vitro results correlate with in vivo observations using genetic approaches.

  • Domain requirement analysis: Test individual domains and combinations to map functional requirements.

How can researchers distinguish between different ubiquitin chain types generated by UPL3?

Distinguishing between different ubiquitin chain types generated by UPL3 is critical for understanding the functional outcomes of UPL3-mediated ubiquitination:

• Ubiquitin mutant approaches:

  • Lysine-to-arginine mutants: Use ubiquitin variants with specific lysine residues mutated to arginine (K48R, K63R, etc.) to determine which lysine residues are used for chain formation.

  • Lysine-only ubiquitin: Use ubiquitin variants where all lysines except one are mutated to arginine to force chain formation through a specific lysine.

  • Methylated ubiquitin: Use methylated ubiquitin to restrict chain formation and analyze mono-ubiquitination.

• Chain-specific antibodies:

  • Commercial antibodies that specifically recognize K48-linked, K63-linked, or other ubiquitin chain types can be used in western blotting after in vitro reactions.

• Mass spectrometry approaches:

  • Ubiquitin remnant profiling: After trypsin digestion, ubiquitinated lysines retain a di-glycine remnant that can be identified by mass spectrometry.

  • Top-down proteomics: Analyze intact ubiquitin chains to determine linkage types and chain length.

  • Selected reaction monitoring: Use targeted mass spectrometry to quantify specific ubiquitin linkage types.

• Ubiquitin chain restriction analysis:

  • Deubiquitinating enzymes (DUBs): Use linkage-specific DUBs to cleave specific chain types and observe changes in ubiquitination patterns.

  • Ubiquitin binding domains: Use specific ubiquitin-binding domains that preferentially bind certain chain types as detection reagents.

• Functional validation:

  • Proteasomal degradation assays: K48-linked chains typically signal proteasomal degradation, so coupling ubiquitination assays with degradation assays can provide indirect evidence for K48 linkages.

  • Signaling pathway analysis: K63 and other atypical chains often mediate signaling rather than degradation, so assessing functional outcomes can provide clues about chain types.

What are common pitfalls in the analysis of UPL3-mediated ubiquitination?

Researchers studying UPL3-mediated ubiquitination should be aware of several common pitfalls:

• Misinterpretation of auto-ubiquitination:

  • E3 ligases like UPL3 commonly undergo auto-ubiquitination in vitro

  • This self-modification can be mistaken for substrate ubiquitination

  • Control reactions without substrate and careful size analysis of ubiquitinated products are essential

• E2 enzyme selection issues:

  • Using inappropriate E2 enzymes can lead to false negatives

  • HECT E3 ligases in Arabidopsis have been shown to work with E2s from the UBC8 family

  • Testing multiple E2s or using E2 mixtures can prevent missing activity

• Buffer and reaction condition problems:

  • UPL3 activity may be sensitive to buffer conditions (pH, salt, reducing agents)

  • The catalytic cysteine in the HECT domain is susceptible to oxidation

  • Include reducing agents (DTT or β-mercaptoethanol) to maintain cysteine reactivity

• Substrate state considerations:

  • Some substrates may require specific modifications (phosphorylation, etc.) for recognition

  • Substrate conformation may affect recognition

  • Recombinant substrates may lack necessary co-factors or interacting partners

• Overlooking relay mechanisms:

  • UPL3 functions in ubiquitin relay systems with other E3 ligases

  • Direct in vitro assays with UPL3 alone might miss substrates that require prior modification by another E3

  • Consider including other E3 ligases in reconstitution experiments

• Technical detection limitations:

  • Western blotting may lack sensitivity for detecting low levels of ubiquitination

  • Background bands can complicate interpretation

  • Consider using tagged ubiquitin (His, biotin, fluorescent) for improved detection

• Physiological relevance assessment:

  • In vitro ubiquitination doesn't guarantee in vivo relevance

  • Validation through genetic approaches and in vivo substrate stability analysis is essential

  • Consider proteasome association of UPL3 when designing experiments, as this is critical for its in vivo function

What Arabidopsis mutant lines and transgenic resources are available for UPL3 research?

Several genetic resources are available for researchers studying UPL3 in Arabidopsis:

• T-DNA insertion mutants:

  • T-DNA disruptions of the UPL3 locus have been characterized

  • The upl3 mutants show distinctive trichome phenotypes with supernumerary branches

  • These mutants can be obtained from Arabidopsis stock centers like ABRC or NASC

• Point mutation alleles:

  • kaktus-2 has been identified as an allele of upl3 with a splice-site mutation

  • This allele may be useful for studying partial loss of function

• Double mutants:

  • upl3 upl4 double mutants have been generated and show enhanced phenotypes in ethylene signaling

  • upl3 ein3 double mutants and upl3 upl4 ein3 triple mutants are available for studying the relationship between UPL3 and ethylene signaling

• Tagged lines:

  • Transgenic lines expressing tagged versions of UPL3 have been used for studies:

    • UPL3-GFP for localization and chromatin immunoprecipitation studies

    • Epitope-tagged UPL3 for protein interaction studies

• Inducible/tissue-specific expression lines:

  • Lines with UPL3 under control of inducible or tissue-specific promoters may be available or can be generated

• Substrate reporter lines:

  • NPR1-GFP expressed in npr1-1 and upl3 npr1-1 backgrounds for studying UPL3's effect on NPR1 localization and stability

  • Similar reporter lines for EIN3 and other substrates

When requesting these resources, researchers should consider the specific genetic background and confirm the mutation or transgene by genotyping, as background mutations can sometimes confound phenotypic analysis.

What bioinformatic tools can help analyze UPL3 structure and function?

Several bioinformatic tools can assist researchers in analyzing UPL3 structure and function:

• Protein domain prediction tools:

  • InterPro, Pfam, SMART: Identify functional domains like the HECT domain and Armadillo repeats

  • COILS, MARCOIL: Predict coiled-coil regions that may be involved in protein-protein interactions

  • DisEMBL, PONDR: Identify intrinsically disordered regions common in E3 ligases

• Structural prediction resources:

  • AlphaFold or RoseTTAFold: Generate structural predictions for UPL3 domains

  • SWISS-MODEL: Homology modeling based on known structures of HECT domains

  • Molecular dynamics simulation tools for studying domain flexibility and interactions

• Comparative genomics tools:

  • BLAST, HMMER: Identify UPL3 homologs in other plant species

  • Clustal Omega, MUSCLE: Multiple sequence alignment of UPL3 homologs

  • MEGA, PhyML: Phylogenetic analysis of the UPL family

• Post-translational modification prediction:

  • NetPhos: Predict potential phosphorylation sites

  • UbPred, UbiSite: Predict potential ubiquitination sites in UPL3 and its substrates

  • GPS-SUMO: Predict SUMOylation sites that might regulate UPL3 activity

• Protein-protein interaction prediction:

  • STRING, BioGRID: Explore known and predicted protein interactions

  • PRISM, HADDOCK: Protein-protein docking to model UPL3 interactions with substrates

  • MoRFpred: Identify molecular recognition features in disordered regions

• Expression analysis tools:

  • BAR eFP Browser: Visualize UPL3 expression patterns across tissues and conditions

  • GENEVESTIGATOR: Compare expression profiles of UPL3 and potential substrates

  • Co-expression databases to identify genes with similar expression patterns

• Molecular visualization tools:

  • PyMOL, UCSF Chimera: Visualize predicted structures and design mutations

  • VMD: Molecular dynamics visualization and analysis

These tools can provide valuable insights into UPL3 function and guide experimental design, particularly when structural information is limited.

What are the most significant open questions in UPL3 research?

Despite significant advances in understanding UPL3 function, several important questions remain:

• Substrate recognition mechanisms: How does UPL3 specifically recognize its diverse substrates? The role of the Armadillo repeats in UPL3 suggests interaction with nuclear localization sequences, but the exact recognition mechanisms remain unclear .

• Regulatory mechanisms: How is UPL3 activity regulated? Potential mechanisms including phosphorylation, changes in subcellular localization, or interactions with regulatory proteins have not been fully explored.

• Comprehensive substrate identification: While NPR1 and EIN3 have been identified as UPL3 substrates , a comprehensive catalog of UPL3 targets across different tissues and conditions remains to be established.

• Chain-type specificity: What types of ubiquitin chains does UPL3 preferentially build, and how does this affect substrate fate? The relationship between UPL3 activity and different ubiquitin chain topologies needs further investigation.

• Evolutionary conservation: How conserved is the UPL3 function across plant species, and has it been recruited for species-specific processes in different plants?

• Proteasome association mechanisms: What molecular interactions mediate UPL3's association with the proteasome, and how is this association regulated?

• Ubiquitin relay mechanisms: How widespread are the ubiquitin ligase relay mechanisms identified for NPR1 and EIN3 , and what determines the need for sequential action of multiple E3 ligases?

• Therapeutic potential: Can modulation of UPL3 activity be harnessed for agricultural applications to enhance crop immunity or developmental traits?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology.

How might UPL3 research impact our broader understanding of plant biology?

UPL3 research has the potential to significantly impact our understanding of plant biology in several areas:

• Protein quality control systems: UPL3's role as a proteasome-associated E3 ligase provides insights into how plants maintain protein homeostasis. As one of only seven HECT E3 ligases in Arabidopsis (compared to about 50 in mammals) , understanding UPL3 helps explain how plants accomplish sophisticated protein regulation with a relatively streamlined ubiquitin system.

• Transcriptional regulation: UPL3's role in regulating key transcription factors like NPR1 and EIN3 reveals mechanisms by which protein degradation directly influences transcriptional outputs, connecting protein turnover to gene expression networks.

• Hormone signaling integration: UPL3's involvement in both salicylic acid and ethylene signaling pathways provides a molecular framework for understanding how plants integrate responses to multiple hormonal inputs, potentially revealing coordination points between growth and defense pathways.

• Evolutionary adaptations: The conservation of HECT E3 ligases across kingdoms suggests fundamental roles, while their specific adaptations in plants may reveal unique aspects of plant biology that diverged during evolution .

• Cell differentiation mechanisms: UPL3's role in trichome development offers insights into how post-translational modifications contribute to cell-fate decisions and specialized cell development in plants.

• Ubiquitin relay systems: The discovery of ubiquitin ligase relays involving UPL3 introduces a new paradigm in protein degradation that may be widespread in plants and potentially in other organisms.

• Stress response mechanisms: UPL3's function in immunity and hormone signaling helps explain how plants modulate protein stability during stress responses, potentially revealing targets for enhancing crop resilience.