Recombinant Arabidopsis thaliana Ubiquitin carboxyl-terminal hydrolase 18 (UBP18)

How should recombinant UBP18 be stored and handled for optimal activity?

For optimal activity maintenance, recombinant UBP18 should be stored according to these guidelines:

Storage recommendations:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (default recommendation is 50%)

• Aliquot for long-term storage at -20°C/-80°C

Storage buffer composition:

• Tris-based buffer

• 50% glycerol

• pH 8.0

What is the general function of UBP18 in Arabidopsis thaliana?

UBP18 functions as a deubiquitinating enzyme (DUB) that belongs to the ubiquitin-specific protease (UBP) family, which is the largest subfamily of DUBs in plants. It plays several critical roles in the ubiquitin/26S proteasome system:

• Ubiquitin recycling: UBP18 releases ubiquitin monomers from polyubiquitinated proteins through hydrolysis of peptide-ubiquitin bonds

• Protein stability regulation: By removing ubiquitin from target proteins, it can protect them from degradation by the 26S proteasome

• Processing of ubiquitin precursors: Participates in generating mature ubiquitin from precursor proteins

The UBP family in Arabidopsis consists of 27 members divided into 14 subfamilies, and research has shown that these proteins are involved in various plant biological processes including development and abiotic stress responses .

What are the recommended experimental parameters for using recombinant UBP18 in deubiquitination assays?

When designing deubiquitination assays with recombinant UBP18, consider the following parameters based on research protocols:

Recommended assay conditions:

• Buffer composition: 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 5 mM DTT, 0.01% Triton X-100

• Temperature: 30°C is optimal for enzymatic activity

• Incubation time: 1-2 hours for standard assays

• Enzyme concentration: 100-500 nM purified recombinant UBP18

• Substrate concentration: 0.5-2 μM of ubiquitinated proteins or 1-5 μM of ubiquitin-AMC for fluorescence-based assays

Controls to include:

• Heat-inactivated UBP18 (95°C for 5 minutes)

• Catalytically inactive mutant (e.g., cysteine-to-serine mutation at the active site)

• Broad-spectrum DUB inhibitor (e.g., N-ethylmaleimide at 5-10 mM)

• No-enzyme control

For analysis of deubiquitination, Western blotting with anti-ubiquitin antibodies is the standard approach for visualizing substrate deubiquitination .

How can I generate mutant versions of UBP18 to study its function in plants?

To generate UBP18 mutants for functional studies in Arabidopsis thaliana, several approaches have been successfully implemented:

T-DNA insertion mutations:

• Screen publicly available T-DNA insertion collections such as those from the University of Wisconsin or ABRC (Arabidopsis Biological Resource Center)

• Verify T-DNA insertions using PCR with gene-specific primers and T-DNA border primers

• Confirm loss-of-function by RT-PCR to ensure no full-length transcripts are present

Example screening primers:

• Gene-specific forward: 5'-TCCTTCGTTGCTTCGTCGCTTTGGACAGA-3'

• Gene-specific reverse: 5'-AACTGTACATGGCCTTTTTCAAAGTATAC-3'

• T-DNA left border primer: JL-202 (for Wisconsin collection) or L primer (for ABRC collection)

CRISPR/Cas9 mutagenesis:

• Design guide RNAs targeting conserved cysteine or histidine catalytic motifs

• Introduce through Agrobacterium-mediated transformation

• Screen and validate mutants by sequencing

Overexpression and complementation:
For comprehensive functional studies, generate both loss-of-function mutants and overexpression lines using the following approach:

• Clone the full-length UBP18 coding sequence into a plant expression vector under a constitutive promoter (e.g., 35S)

• Generate catalytically inactive versions by site-directed mutagenesis of active site residues

• Transform into wild type and ubp18 mutant backgrounds

• Select transformants on appropriate antibiotics and confirm expression levels by qRT-PCR

What methods are effective for detecting UBP18 expression in plant tissues?

Several techniques have proven effective for detecting UBP18 expression in plant tissues:

Quantitative RT-PCR (qRT-PCR):

• Extract total RNA using standard methods (e.g., LiCl precipitation or commercial kits like RNeasy)

• Synthesize cDNA using reverse transcriptase

• Perform qRT-PCR with UBP18-specific primers

• Normalize to appropriate reference genes (e.g., ACTIN2/AT3G18780)

Recommended primers for qRT-PCR:

• Forward: 5'-AAGGTGAACGCTACTTTGGATCGAGAA-3'

• Reverse: 5'-TTCTGCTTTCTCAGCTTTCACTTGGTC-3'

Western blotting:

• Extract total protein from plant tissues

• Separate proteins by SDS-PAGE

• Transfer to membrane and probe with anti-UBP18 antibodies

• For recombinant His-tagged UBP18, anti-His antibodies can also be used

Protein extraction buffer:

• 50 mM Tris-HCl pH 7.5

• 150 mM NaCl

• 1 mM EDTA

• 10% glycerol

• 0.1% Triton X-100

• 1 mM PMSF

• Protease inhibitor cocktail

How does UBP18 contribute to abiotic stress responses in Arabidopsis thaliana?

UBP18 plays significant roles in multiple abiotic stress response pathways in Arabidopsis thaliana, as evidenced by extensive research:

Salt stress response:
Transcriptomic analysis reveals that UBP18 expression is induced by salt (NaCl) treatment, with approximately 2-fold upregulation after 12 hours of exposure to 140 mM NaCl . This induction suggests UBP18's involvement in salt stress adaptation mechanisms.

Osmotic stress response:
UBP18 is also responsive to osmotic stress induced by mannitol treatment (320 mM), with significant upregulation observed after 8 hours of treatment .

ABA signaling pathway:
UBP18 expression is induced by abscisic acid (ABA) treatment (10 μM), indicating its involvement in ABA-mediated stress responses. Research suggests that UBP18 may function in multiple aspects of ABA signaling, including:

• ABA synthesis

• Stomatal closure regulation

• Transcription factors in the ABA pathway

Experimental evidence from phenotypic analysis:
Studies comparing wild-type plants with ubp18 mutants and UBP18 overexpression lines reveal distinct phenotypic differences under stress conditions:

• UBP18 overexpression lines show enhanced salt and drought tolerance

• UBP18 overexpression plants maintain higher survival rates under continuous 100 mM NaCl irrigation

• UBP18 overexpression lines retain more vigorous appearance after water withholding for 20 days

• UBP18 overexpression leaves lose water content more slowly than wild-type

• UBP18 overexpression plants accumulate higher levels of proline under stress conditions

These findings collectively establish UBP18 as a positive regulator of abiotic stress tolerance in Arabidopsis.

What is known about UBP18's potential substrates and interaction networks?

The identification of UBP18's substrates and interaction partners is an active area of research. Current evidence suggests several potential targets and regulatory mechanisms:

Potential direct substrates:
While specific substrates of UBP18 have not been definitively identified, pathway analysis suggests several candidates:

• Ring domain ligase1 (RGLG1) - May be a direct substrate in drought stress response regulation

• Transcription factors involved in stress response pathways

Regulatory networks:
UBP18 appears to function in several regulatory circuits:

• DDF1 pathway: UBP18 may regulate salt-stress tolerance by modulating the dwarf and delayed flowering 1 (DDF1) pathway through a cascade reaction

• ERF53 pathway: UBP18 potentially operates in a feed-forward loop mechanism in drought-stress responses via ethylene response factor 53 (ERF53) and its ubiquitin ligase RGLG1

Methodological approaches for substrate identification:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express epitope-tagged UBP18 in plants

  • Purify UBP18 along with interacting proteins

  • Identify binding partners by mass spectrometry

• Ubiquitin remnant profiling:

  • Compare ubiquitination sites in wild-type and UBP18 mutant/overexpression plants

  • Sites with increased ubiquitination in UBP18 mutants suggest potential substrates

• Yeast two-hybrid screening:

  • Use UBP18 as bait to screen Arabidopsis cDNA libraries

  • Validate interactions biochemically and in planta

How does UBP18 function differ from other UBP family members in Arabidopsis?

Arabidopsis thaliana contains 27 UBP proteins divided into 14 subfamilies, each with specific functions in plant biology. Research has revealed both overlapping and distinct functions among these family members:

Comparative analysis of UBP family members:

Unique features of UBP18:

• UBP18 is specifically induced by multiple abiotic stresses (salt, osmotic) and ABA treatment

• Appears to function in several distinct stress response pathways simultaneously

• Potentially acts through both direct substrate deubiquitination and broader transcriptional regulation

Functional redundancy:
Some UBP family members show functional redundancy (e.g., UBP3/UBP4), while others like UBP18 appear to have more specialized functions. This is evidenced by the different phenotypes observed in single and double mutants of various UBP genes .

What technical challenges exist in purifying active recombinant UBP18?

Producing and purifying active recombinant UBP18 presents several technical challenges that researchers should consider:

Expression system selection:
Various expression systems have been used for UBP18 production with different success rates:

• E. coli: Most commonly used system; protein is typically expressed with N-terminal His-tag for purification purposes

• Yeast or baculovirus: Alternative systems that may provide better folding for plant proteins

• Cell-free expression systems: Another option for difficult-to-express proteins

Solubility challenges:
Deubiquitinating enzymes often face solubility issues when expressed in heterologous systems. To improve solubility:

• Optimize expression conditions (temperature, IPTG concentration)

• Use solubility-enhancing fusion tags (MBP, SUMO, GST)

• Co-express with chaperones

• Consider truncated constructs focusing on the catalytic domain

Maintaining enzymatic activity:
Preserving the catalytic activity of UBP18 during purification requires:

• Addition of reducing agents (DTT or β-mercaptoethanol) in all buffers

• Inclusion of protease inhibitors lacking thiol-reactive components

• Purification at 4°C to minimize degradation

• Careful pH control (typically pH 7.5-8.0)

• Avoidance of metal chelators that might affect the catalytic site

Quality control methods:

• Purity assessment: SDS-PAGE with Coomassie staining (should be >90% pure)

• Activity assays: Using model substrates like ubiquitin-AMC or di-ubiquitin chains

• Thermal stability testing: Differential scanning fluorimetry to assess protein folding

For long-term storage of purified UBP18, research indicates that addition of 50% glycerol and storage at -20°C/-80°C, with avoidance of repeated freeze-thaw cycles, is optimal for maintaining activity .

How can UBP18 be utilized to enhance stress tolerance in crop plants?

Based on UBP18's demonstrated role in stress tolerance, several strategies can be implemented for crop improvement:

Transgenic approaches:

• Overexpression strategy: Constitutive or stress-inducible expression of Arabidopsis UBP18 in crop plants could enhance their tolerance to multiple abiotic stresses

• Genome editing: CRISPR/Cas9 modification of endogenous UBP18 homologs in crops to enhance their activity or stress responsiveness

• Promoter engineering: Modification of UBP18 promoters to optimize expression patterns under stress conditions

Supporting research evidence:
Research with UBP18 overexpression in Arabidopsis demonstrated:

• Enhanced salt and drought tolerance

• Higher survival rates under continuous salt irrigation

• Improved recovery after drought stress (>95% survival in OE plants vs. lower in wild-type)

• Reduced water loss rate

• Increased proline accumulation under stress conditions

Potential crop applications:

• Cereals (rice, wheat, maize) grown in regions affected by drought or soil salinity

• Vegetable crops sensitive to osmotic and salt stress

• Integration with breeding programs targeting marginal agricultural lands

Risk assessment considerations:

• Potential growth-yield trade-offs under non-stress conditions

• Effects on disease resistance pathways

• Possible unintended consequences on plant development or reproduction

What is the evolutionary relationship between UBP18 and related deubiquitinating enzymes across plant species?

UBP18 belongs to an evolutionarily conserved family of deubiquitinating enzymes with homologs across multiple plant species. Understanding these relationships provides insights into functional conservation and specialization:

Evolutionary conservation:
The UBP family appears to be conserved across land plants, with varying numbers of members in different species. Phylogenetic analysis reveals that:

• UBP18 in Arabidopsis has homologs in other plant species including:

  • Maize (ZmUBP15, ZmUBP16, and ZmUBP19)

  • Rice (OsUBP homologs)

  • Other crop species

• Functional conservation extends across species, with homologs often playing similar roles:

  • Maize ZmUBP15, ZmUBP16, and ZmUBP19 are involved in responses to cadmium and salt stress

  • Pepper CaUBP12 positively modulates dehydration resistance

Structural conservation:

• The catalytic domains containing signature cysteine and histidine motifs are highly conserved

• N-terminal and C-terminal regions show more divergence, potentially accounting for substrate specificity differences

• Key structural elements for ubiquitin recognition are maintained across species

Methodological approaches for evolutionary analysis:

• Multiple sequence alignment of UBP18 homologs across species

• Phylogenetic tree construction using maximum likelihood or Bayesian methods

• Domain architecture analysis to identify conserved and species-specific features

• Synteny analysis to understand genomic context conservation

Understanding these evolutionary relationships can guide the identification of functionally important UBP18 residues and domains, as well as inform cross-species applications for crop improvement.

How does UBP18 interact with the broader ubiquitin-proteasome system during plant stress responses?

UBP18 functions within the complex network of the ubiquitin-proteasome system (UPS), which plays crucial roles in plant stress responses. Understanding these interactions requires examination of multiple regulatory layers:

Integration with the ubiquitin-proteasome system:

• Ubiquitin recycling: UBP18, like other UBPs, contributes to maintaining the pool of free ubiquitin by disassembling polyubiquitin chains and releasing ubiquitin monomers

• Counteracting E3 ligases: UBP18 may specifically counteract the activity of E3 ubiquitin ligases involved in stress responses, such as RGLG1

• Substrate stabilization: By removing ubiquitin from specific target proteins, UBP18 can prevent their degradation by the 26S proteasome, thus stabilizing positive regulators of stress responses

Regulatory networks during stress responses:
Research has identified several potential regulatory mechanisms:

• ABA signaling pathway: UBP18 appears to function in multiple aspects of ABA signaling:

  • ABA synthesis regulation

  • Stomatal closure modulation

  • Regulation of ABA-responsive transcription factors

• Salt stress pathway: UBP18 may regulate salt-stress tolerance by modulating the DDF1 pathway through a cascade reaction

• Drought stress response: UBP18 potentially functions in a feed-forward loop mechanism involving ERF53 and its ubiquitin ligase RGLG1

Transcriptomic evidence:
Enrichment analysis of differentially expressed genes in ubp18 mutants revealed alterations in multiple stress-related and metabolic pathways, suggesting that UBP18 influences the expression of numerous genes involved in stress responses .

These findings collectively indicate that UBP18 serves as a key integrator within the broader UPS-mediated stress response network, with both direct deubiquitination functions and indirect effects on transcriptional regulation.

What emerging technologies can advance our understanding of UBP18 function in plant biology?

Several cutting-edge technologies hold promise for deepening our understanding of UBP18's functions:

Advanced imaging techniques:

• Super-resolution microscopy: Tracking UBP18 subcellular localization and dynamics during stress responses with nanometer resolution

• FRET-FLIM (Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging): Visualizing UBP18 interactions with putative substrates in living cells

• Proximity labeling (BioID, TurboID): Identifying proteins in close proximity to UBP18 in different cellular compartments and under various conditions

Proteomics approaches:

• Ubiquitinome analysis: Comparing ubiquitination patterns between wild-type and ubp18 mutant plants to identify potential substrates

• Targeted proteomics: Using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) to quantify specific UBP18 substrate candidates

• Protein turnover analysis: Pulse-chase proteomics to measure protein stability changes dependent on UBP18 activity

Structural biology:

• Cryo-EM: Determining the structure of UBP18 alone and in complex with substrates

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Mapping conformational changes upon substrate binding

• AlphaFold2 and related AI tools: Prediction of protein-protein interactions and complex structures

Systems biology approaches:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to build comprehensive models of UBP18 function

• Network analysis: Using protein-protein interaction networks to position UBP18 within stress response pathways

• Mathematical modeling: Developing computational models to simulate and predict the effects of UBP18 perturbation on stress response dynamics

Genome editing technologies:

• CRISPR base editors and prime editors: Introduction of precise mutations in UBP18 to understand structure-function relationships

• Conditional knockouts: Using inducible CRISPR systems to study UBP18 function in specific tissues or developmental stages

• CRISPR activation/interference: Modulating UBP18 expression without permanent genetic changes

These technologies, especially when used in combination, have the potential to provide unprecedented insights into UBP18's role in plant stress responses and development.

What are common challenges in UBP18 activity assays and how can they be overcome?

Researchers frequently encounter several challenges when conducting UBP18 activity assays. Here are the most common issues and recommended solutions:

Enzyme activity loss during storage:

• Problem: Recombinant UBP18 may lose activity during storage due to oxidation of catalytic cysteine residues

• Solution:

  • Add reducing agents (1-5 mM DTT) to all buffers

  • Store in small aliquots with 50% glycerol at -80°C

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, lyophilize the protein in the presence of trehalose (6%) as a stabilizer

Background DUB activity in protein extracts:

• Problem: Plant extracts contain multiple DUBs that can interfere with UBP18-specific activity measurements

• Solution:

  • Use selective inhibitors to block activity of other DUB families

  • Perform immunoprecipitation to isolate UBP18 before activity assays

  • Include appropriate controls with broad-spectrum DUB inhibitors

Substrate specificity issues:

• Problem: Generic DUB substrates may not reflect UBP18's natural specificity

• Solution:

  • Test multiple ubiquitin chain types (K48, K63, linear) to determine preference

  • Use physiologically relevant substrates when possible

  • Compare activity with other UBP family members as reference points

Inefficient detection methods:

• Problem: Traditional Western blotting has limited sensitivity for detecting deubiquitination

• Solution:

  • Use fluorescent ubiquitin substrates (Ub-AMC) for real-time activity monitoring

  • Implement FRET-based di-ubiquitin substrates for enhanced sensitivity

  • Consider mass spectrometry-based approaches for comprehensive analysis

Data interpretation challenges:

• Problem: Distinguishing UBP18-specific effects from other deubiquitinating activities

• Solution:

  • Always include catalytically inactive UBP18 mutant controls

  • Perform parallel assays in extracts from ubp18 knockout plants

  • Use ubiquitin chain type-specific antibodies for precise analysis

When studying UBP18 in stress responses, what experimental design considerations are critical?

Designing experiments to study UBP18's role in stress responses requires careful consideration of multiple factors:

Timing and developmental stage:

• Challenge: UBP18's effect on stress responses may vary with developmental stage

• Recommendation:

  • Clearly define plant age and developmental stage for all experiments

  • Compare multiple developmental stages (seedling, vegetative, reproductive)

  • Consider time-course experiments to capture dynamic responses

Stress application methods:

• Challenge: Different stress application methods can yield conflicting results

• Recommendation:

  • For salt stress: Use both plate-based (acute) and soil irrigation (chronic) methods

  • For drought: Control soil water content precisely; consider both gradual and rapid drought imposition

  • For ABA treatment: Use both foliar application and media supplementation

  • Clearly document stress intensity, duration, and application method

Appropriate controls:

• Challenge: Distinguishing UBP18-specific effects from general stress responses

• Recommendation:

  • Include multiple genotypes: wild-type, ubp18 knockout, UBP18 overexpression, and catalytically inactive UBP18 complementation lines

  • Use other UBP family mutants (e.g., ubp15) as specificity controls

  • Include non-stressed controls for all genotypes and time points

Phenotypic assessment:

• Challenge: Selecting relevant and quantifiable stress response parameters

• Recommendation:

  • Combine morphological (survival rate, rosette size), physiological (water loss rate, electrolyte leakage), and biochemical (proline content, ROS levels) measurements

  • Quantify parameters using standardized methods and appropriate statistical analysis

  • Document experimental conditions thoroughly, including light intensity, humidity, and temperature

Gene expression analysis:

• Challenge: Identifying UBP18-regulated genes relevant to stress responses

• Recommendation:

  • Perform both targeted (qRT-PCR of known stress genes) and global (RNA-seq) expression analyses

  • Include multiple time points after stress imposition

  • Consider tissue-specific analyses (roots vs. shoots)

  • Validate key findings using multiple biological replicates

Research has shown that UBP18 expression is induced by salt (NaCl), osmotic stress (mannitol), and ABA treatments, with peak expression occurring at different time points for each stress (NaCl: 12h, mannitol: 8h, ABA: 4-6h) . These timing considerations should be incorporated into experimental designs.

What are the limitations of current models explaining UBP18's role in plant stress responses?

Current models explaining UBP18's role in plant stress responses face several limitations that should be acknowledged:

Substrate identification gaps:

• Limitation: While UBP18 is implicated in stress responses, specific substrates directly deubiquitinated by UBP18 remain largely unidentified

• Implication: Without confirmed substrates, mechanistic understanding remains incomplete

• Future direction: Implement global ubiquitinome analysis comparing wild-type and ubp18 mutants under stress conditions

Functional redundancy uncertainties:

• Limitation: Potential functional overlap with other UBP family members complicates interpretation of single mutant phenotypes

• Implication: Single ubp18 mutant phenotypes may underestimate UBP18's full importance

• Future direction: Generate and characterize higher-order mutants (e.g., ubp15 ubp18 double mutants) to address redundancy

Tissue-specific function gaps:

• Limitation: Current studies focus on whole-plant or seedling responses, potentially missing tissue-specific roles

• Implication: UBP18 may have different functions in different tissues that aren't captured in whole-plant analyses

• Future direction: Use tissue-specific promoters to express or silence UBP18 in specific cell types

Stress signaling integration:

• Limitation: How UBP18 integrates with hormone signaling beyond ABA remains unclear

• Implication: The cross-talk between UBP18 and other stress hormones (ethylene, jasmonate, salicylic acid) is poorly understood

• Future direction: Analyze UBP18 function in the context of multiple hormone signaling pathways

Molecular mechanism uncertainties:

• Limitation: Direct evidence for how UBP18 affects transcriptional regulation is lacking

• Implication: Current models rely heavily on correlative gene expression data rather than direct mechanistic insights

• Future direction: Implement chromatin immunoprecipitation (ChIP) studies to determine if UBP18 affects chromatin-associated proteins

Translation to crop species:

• Limitation: Most UBP18 research is conducted in the model plant Arabidopsis

• Implication: Function in economically important crops may differ

• Future direction: Characterize UBP18 homologs in major crop species under field-relevant stress conditions

Addressing these limitations will require integrative approaches combining biochemistry, genetics, cell biology, and systems biology to develop more comprehensive models of UBP18 function in plant stress responses.