Recombinant Arabidopsis thaliana Putative RING-H2 finger protein ATL36 (ATL36)

How does ATL36 relate to other members of the ATL gene family?

ATL36 belongs to the ATL (Arabidopsis Tóxicos en Levadura) gene family, which comprises approximately 80 members in Arabidopsis thaliana and 121 in Oryza sativa (rice) . The family is characterized by:

• The presence of a conserved RING-H2 finger domain essential for ubiquitin ligase activity

• Predominantly intronless gene structure (90% of ATL genes), suggesting evolution as functional modules

• Involvement in various aspects of plant development and stress responses

Phylogenetic analysis indicates that about 60% of rice ATLs cluster with Arabidopsis ATLs, suggesting potential orthologous relationships between species . While ATL31 and ATL6 have been well-characterized in carbon/nitrogen nutrient responses and pathogen defense, the specific functional relationship between ATL36 and these better-studied family members remains to be fully elucidated .

What experimental evidence supports ATL36's function as a ubiquitin ligase?

Although direct experimental evidence for ATL36's ubiquitin ligase activity is not explicitly detailed in the search results, its classification is supported by:

• The presence of the RING-H2 finger domain, which is characteristic of a subclass of E3 ubiquitin ligases

• Its membership in the ATL family, which has been characterized as comprising RING-type E3 ubiquitin transferases

• Structural homology to other ATL proteins with demonstrated ubiquitin ligase activity

To definitively confirm ATL36's function, researchers should conduct:

• In vitro ubiquitination assays with purified recombinant ATL36

• E2 binding assays to identify compatible ubiquitin-conjugating enzymes

• Substrate identification and verification studies

• In vivo ubiquitination studies in Arabidopsis

What are the optimal conditions for working with recombinant ATL36 protein?

Based on commercial product information, researchers should observe these guidelines when working with recombinant ATL36:

Storage and Handling:

• Store lyophilized protein at -20°C/-80°C

• After reconstitution, store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

Reconstitution Protocol:

• Briefly centrifuge the vial before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration for long-term storage

Buffer Considerations:

• The protein is typically provided in a Tris/PBS-based buffer with 6% trehalose at pH 8.0

• For enzymatic assays, consider supplementing with:

  • Zinc or other relevant cofactors

  • Reducing agents (DTT or β-mercaptoethanol) to maintain RING domain integrity

  • Protease inhibitors to prevent degradation

How can researchers effectively generate and characterize ATL36 mutants?

To generate and thoroughly characterize ATL36 mutants, researchers should consider this comprehensive approach:

Mutant Generation Methods:

• T-DNA insertion lines:

  • Screen public repositories (TAIR, NASC, ABRC) for available insertion lines

  • Verify homozygosity through PCR genotyping

  • Confirm knockdown/knockout by RT-PCR or qRT-PCR

• CRISPR/Cas9 gene editing:

  • Design guide RNAs targeting ATL36 coding sequence, preferably the RING-H2 domain

  • Transform Arabidopsis using established Agrobacterium-mediated protocols

  • Confirm mutations by sequencing and isolate homozygous lines

Important consideration: Some ATL family genes appear to be essential for viability as homozygous T-DNA insertion plants couldn't be recovered in certain cases . If ATL36 proves essential, consider conditional or tissue-specific knockout strategies.

Characterization Protocol:

• Phenotypic analysis:

  • Growth parameters under normal conditions and various stresses

  • Morphological changes at different developmental stages

  • Response to light of varying intensities (given the light-dependent phenotype of some ATL mutants)

  • Chlorophyll content and photosynthetic efficiency measurements

• Molecular analysis:

  • Transcriptome profiling (RNA-seq) to identify affected pathways

  • Proteomics to detect changes in protein abundance and modification

  • Analysis of potential substrates (looking for proteins that accumulate in the mutant)

• Genetic analysis:

  • Complementation studies with wild-type ATL36

  • Generation of double mutants with related ATL genes to uncover redundant functions

  • Suppressor screens to identify interacting components

What assays are recommended to study ATL36 ubiquitin ligase activity?

To comprehensively characterize ATL36's ubiquitin ligase activity, researchers should implement the following assays:

In vitro ubiquitination assays:

• Purify recombinant ATL36 protein (His-tagged as described in commercial preparations)

• Combine with E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), tagged ubiquitin, ATP, and potential substrate proteins

• Incubate at 30°C for 1-2 hours

• Analyze by SDS-PAGE and western blotting with anti-ubiquitin antibodies

• Include appropriate controls (reactions lacking E1, E2, ATP, or using catalytically inactive ATL36)

E2 enzyme screening:

• Test interactions with different E2 enzymes using yeast two-hybrid or pull-down assays

• Assess activity with different E2s in the in vitro ubiquitination assay

• Verify physiologically relevant E2 partners through in vivo co-immunoprecipitation

Substrate identification approaches:

• Yeast two-hybrid screening with ATL36 as bait

• Co-immunoprecipitation coupled with mass spectrometry

• Comparative proteomics between wild-type and atl36 mutant plants

• In vitro protein arrays to detect direct interactions

In vivo ubiquitination verification:

• Express tagged versions of ATL36 and candidate substrates in Arabidopsis

• Immunoprecipitate the substrate under native conditions

• Detect ubiquitination by western blotting with anti-ubiquitin antibodies

• Use proteasome inhibitors (MG132) to stabilize ubiquitinated proteins

What is known about ATL36's role in plant development and stress responses?

While the specific function of ATL36 is not explicitly detailed in the provided search results, its role can be hypothesized based on related ATL family members:

Potential Developmental Roles:

• Some ATL genes are essential for viability, suggesting crucial developmental functions

• ATL8, for example, is expressed mainly in young siliques, indicating a potential role in embryogenesis

Stress Response Functions:

• Other characterized ATL proteins like ATL31 and ATL6 regulate carbon/nitrogen nutrient responses and pathogen defense mechanisms

• The atl31atl6 double mutant shows impaired 5-aminolevulinic acid biosynthesis, leading to a light intensity-dependent pale-green leaf phenotype

• ATL43 has been implicated in abscisic acid (ABA) response pathways

To determine ATL36's specific functions, researchers should:

• Analyze expression patterns across tissues, developmental stages, and stress conditions

• Characterize atl36 mutant phenotypes under various environmental challenges

• Identify ATL36 substrates and signaling partners

• Investigate potential redundancy with other ATL family members

How might ATL36 function in chloroplast development based on ATL family studies?

The atl31atl6 double mutant exhibits a light intensity-dependent pale-green leaf phenotype caused by disruption of 5-aminolevulinic acid biosynthesis, a rate-limiting step in chlorophyll production . This finding suggests potential roles for ATL family proteins in chloroplast development and function.

Insights from the atl31atl6 mutant:

• The pale-green phenotype is light-intensity dependent

• Plastid ultrastructure is abnormal with reduced thylakoid proteins

• HEMA1 expression (encoding a key enzyme for 5-aminolevulinic acid synthesis) is down-regulated

• GLK1 transcription factor, which directly promotes HEMA1 expression, is significantly decreased

• Application of 5-aminolevulinic acid restores the green phenotype

Potential roles for ATL36 in chloroplast functions:

• Regulation of chlorophyll biosynthesis pathway components

• Modulation of transcription factors controlling chloroplast development

• Involvement in light signaling pathways that impact chloroplast differentiation

• Targeted degradation of negative regulators of chloroplast function

Researchers should investigate whether ATL36 has similar or complementary functions to ATL31/ATL6 in chloroplast development through:

• Detailed phenotypic characterization of chloroplasts in atl36 mutants

• Analysis of chlorophyll biosynthesis gene expression

• Investigation of potential genetic interactions with atl31 and atl6

How does ATL36 compare to ATL31 and ATL6 in terms of function and regulation?

ATL31 and ATL6 are well-characterized ATL family members that control carbon/nitrogen nutrient and pathogen responses in Arabidopsis . A comparative analysis reveals:

Functional Similarities and Differences:

• All contain the characteristic RING-H2 finger domain essential for ubiquitin ligase activity

• ATL31 and ATL6 show functional redundancy in carbon/nitrogen response and chloroplast development

• The double mutant atl31atl6 exhibits a light intensity-dependent pale-green phenotype not observed in the single mutants

• ATL36's specific function requires further characterization, but may involve similar or distinct pathways

Regulatory Mechanisms:

• The atl31atl6 mutant shows down-regulation of HEMA1 and GLK1, suggesting these ATLs regulate chlorophyll biosynthesis gene expression

• Application of 5-aminolevulinic acid rescues the pale-green phenotype in atl31atl6, indicating a specific block in this biosynthetic step

Experimental approaches to compare ATL36 with ATL31/6:

• Generate single, double, and triple mutant combinations

• Perform cross-complementation experiments

• Compare substrate specificity through proteomics and interaction studies

• Analyze expression patterns to identify overlapping or distinct regulation

• Investigate potential heterodimerization or other interactions between ATL family members

What are the potential substrates of ATL36 and how can they be identified?

As a putative E3 ubiquitin ligase, ATL36 likely targets specific proteins for ubiquitination and subsequent degradation. Identifying these substrates is crucial for understanding ATL36 function.

Potential substrate classes based on ATL family studies:

• Transcription factors controlling chloroplast development (such as GLK1)

• Components of stress signaling pathways

• Metabolic enzymes involved in carbon/nitrogen metabolism

• Regulatory proteins in pathogen response pathways

Comprehensive substrate identification strategy:

• Interactome mapping:

  • Yeast two-hybrid screening with ATL36 as bait

  • Affinity purification-mass spectrometry (AP-MS) using tagged ATL36

  • Proximity labeling approaches (BioID or TurboID fused to ATL36)

• Proteomics-based approaches:

  • Quantitative proteomics comparing wild-type and atl36 mutants

  • Di-Gly remnant profiling to identify changes in the ubiquitinated proteome

  • Protein stability profiling to identify proteins with altered half-lives

• Candidate-based testing:

  • Based on known substrates of related ATL proteins

  • Proteins functioning in pathways affected in atl36 mutants

  • In vitro ubiquitination assays with recombinant candidates

• Validation protocol for identified candidates:

  • Confirm direct interaction with ATL36

  • Demonstrate ubiquitination in vitro and in vivo

  • Show altered stability/abundance in atl36 mutants

  • Map ubiquitination sites and generate non-ubiquitinatable mutants

  • Determine functional consequences of ubiquitination

How is ATL36 gene expression regulated under various developmental and stress conditions?

Understanding the transcriptional and post-transcriptional regulation of ATL36 can provide insights into its biological functions. While specific information about ATL36 regulation is not provided in the search results, a comprehensive investigation would include:

Transcriptional regulation analysis:

• Promoter isolation and characterization to identify regulatory elements

• Reporter gene assays to monitor activity under different conditions

• ChIP-seq to identify transcription factors binding to the ATL36 promoter

• Analysis of ATL36 expression across public transcriptome datasets

Environmental and developmental regulation:

• qRT-PCR analysis of ATL36 expression across:

  • Different tissues and developmental stages

  • Responses to abiotic stresses (light, temperature, drought, nutrients)

  • Exposure to pathogens and elicitors

  • Hormone treatments

Post-transcriptional regulation:

• Analysis of mRNA stability and alternative splicing

• Investigation of potential miRNA-mediated regulation

• Polysome profiling to assess translational efficiency

Integration with ATL family regulation:

• Comparative expression analysis with other ATL genes

• Investigation of potential feedback regulation within the ATL family

• Correlation analysis with known ATL-regulated pathways

What emerging technologies could advance our understanding of ATL36 function?

Several cutting-edge technologies and approaches could significantly enhance ATL36 research:

CRISPR/Cas-based technologies:

• Base editing or prime editing for precise modification of ATL36 regulatory or functional domains

• CRISPRi/CRISPRa for conditional regulation of expression

• CRISPR screens to identify genetic interactions with ATL36

Structural biology approaches:

• Cryo-EM to determine the structure of ATL36 in complex with E2 enzymes and substrates

• Hydrogen-deuterium exchange mass spectrometry to map protein interaction surfaces

• AlphaFold2 or RoseTTAFold structure prediction and validation

Single-cell and spatial analysis:

• Single-cell transcriptomics to capture cell-type specific expression patterns

• Spatial transcriptomics to map ATL36 expression within plant tissues

• Super-resolution microscopy to track ATL36 localization at nanoscale resolution

Systems biology integration:

• Multi-omics approaches (transcriptomics, proteomics, metabolomics) in atl36 mutants

• Network analysis to position ATL36 within cellular signaling pathways

• Mathematical modeling of ubiquitin-mediated protein degradation dynamics

Synthetic biology approaches:

• Engineered ATL36 variants with altered substrate specificity

• Optogenetic or chemically-inducible versions for temporal control

• Biosensors to monitor ATL36 activity in vivo

Plant space biology applications:

• Study of ATL36 function under spaceflight conditions

• Analysis of radiation effects on ATL36-mediated pathways

• Exploration of ATL36's role in DNA damage response signaling networks

How conserved is ATL36 across plant species and what does this reveal about its function?

The ATL gene family shows significant conservation across plant species, with approximately 80 members in Arabidopsis thaliana and 121 in Oryza sativa (rice) . About 60% of rice ATLs cluster with Arabidopsis ATLs, suggesting many potential orthologous relationships .

Evolutionary conservation analysis framework:

• Sequence conservation assessment:

  • Identify ATL36 orthologs in other plant species through reciprocal BLAST searches

  • Analyze sequence conservation, particularly in the RING-H2 domain

  • Determine whether conservation extends beyond the RING domain to substrate-binding regions

• Structural conservation:

  • Compare domain architecture across species

  • Analyze conservation of key functional residues

  • Identify species-specific variations that might reflect functional adaptations

• Functional conservation testing:

  • Cross-species complementation studies (expression of orthologs in Arabidopsis atl36 mutants)

  • Comparative biochemical analysis of orthologous proteins

  • Testing for conserved interaction partners and substrates

• Evolutionary rate analysis:

  • Calculate substitution rates to determine selective pressures

  • Compare evolutionary rates with other ATL family members

  • Identify potential signatures of adaptive evolution

The high degree of intronless structure (90%) across ATL genes suggests they evolved as functional modules , which may indicate conservation of core functions across species.

What role might ATL36 play in DNA damage response based on ATL family functions?

Recent research has investigated the role of Arabidopsis genes in DNA damage response during spaceflight and other stressful conditions . While ATL36 is not specifically mentioned in this context, the ATL family's involvement in stress responses suggests potential functions in DNA damage response pathways:

Potential mechanisms for ATL36 in DNA damage response:

• Targeted degradation of DNA repair proteins:

  • E3 ubiquitin ligases often regulate the abundance and activity of DNA repair factors

  • ATL36 might target specific DNA repair proteins for degradation or activation

  • This could be involved in switching between different repair pathways

• Signaling cascade regulation:

  • ATL36 could mediate the degradation of signaling components in the ATR/ATM pathways

  • This might affect checkpoint activation and cell cycle progression after DNA damage

  • Ubiquitination could serve as a non-degradative signal in the DNA damage response

• Stress response integration:

  • ATL36 might link DNA damage responses to other stress response pathways

  • This could coordinate cellular responses to multiple simultaneous stresses

  • The protein could contribute to maintaining genome stability under stress conditions

Experimental approaches to investigate this role:

• Analyze atl36 mutant sensitivity to DNA damaging agents (UV, radiomimetic chemicals)

• Measure DNA repair efficiency in atl36 mutants

• Identify potential interactions between ATL36 and DNA repair proteins

• Study ATL36 expression and localization changes following DNA damage

• Investigate potential substrates involved in DNA damage response pathways