Recombinant Arabidopsis thaliana RING-H2 finger protein ATL54 (ATL54)

Basic Research Questions

• What is ATL54 and what are its structural characteristics?

ATL54 (At1g72220) is a RING-H2 finger protein in Arabidopsis thaliana that functions as an E3 ubiquitin ligase. It belongs to the ATL (Arabidopsis Tóxicos en Levadura) family, which is characterized by a RING-H2 domain, a hydrophobic region likely serving as a transmembrane domain, and a conserved GLD region .

The protein consists of 413 amino acids with the following key features:

• A RING-H2 domain that binds to E2 ubiquitin-conjugating enzymes

• A hydrophobic region at amino acid positions 82-104 predicted to be a transmembrane domain

• A characteristic cross-brace arrangement of cysteine and histidine residues that coordinate zinc ions in the RING-H2 domain

• Where is ATL54 localized within plant cells?

ATL54 is primarily localized to the plasma membrane. This localization has been experimentally verified through fluorescent protein fusion studies. When the C-terminus of ATL54 is tagged with YFP and expressed in Arabidopsis T87 protoplasts, the fluorescent signal is detected specifically on the plasma membrane .

In contrast, when only YFP is expressed or when the N-terminus of ATL54 is fused with YFP, the signal is distributed throughout the cytoplasm and nucleus. This suggests that the N-terminal region of ATL54 is crucial for proper membrane localization, likely due to the transmembrane domain (amino acids 82-104) predicted by the TMHMM 2.0 program .

• How is ATL54 expression regulated in Arabidopsis tissues?

According to the Arabidopsis eFP browser data cited by Noda et al., ATL54 is highly expressed at the base of inflorescence stems . Expression analysis using quantitative RT-PCR on different segments of 20-cm-high inflorescence stems showed distinct expression patterns along the stem axis.

The expression level varies by stem position, with differential patterns observed between wild-type plants and genetically modified lines. In ATL54-overexpressed plants (designated OX1, OX9, and OX10), transcripts were significantly increased compared to wild-type. In contrast, correct ATL54 transcripts were not detected in knock-out mutants, confirming the effectiveness of the T-DNA insertion in disrupting gene expression .

Advanced Research Questions

• How can the E3 ubiquitin ligase activity of ATL54 be experimentally demonstrated?

The E3 ubiquitin ligase activity of ATL54 can be demonstrated through an auto-ubiquitination assay, which is considered a reliable method for confirming E3 ligase functionality. The experimental approach involves:

• Protein preparation: Express recombinant ATL54 in E. coli as a fusion protein (MBP-ATL54-6myc) and purify it.

• Ubiquitination reaction: Mix the purified ATL54 protein with:

  • Ubiquitin

  • ATP

  • E1 ubiquitin-activating enzyme (UBE1)

  • E2 ubiquitin-conjugating enzyme (UbcH5b)

• Incubate at 30°C for 2 hours

• Analysis: Perform Western blotting using anti-ubiquitin antibodies to detect ubiquitinated proteins

Results from Noda et al. showed that ubiquitinated proteins were only detected when all components (ubiquitin, ATP, E1, E2, and MBP-ATL54-6myc) were present in the reaction. When any component was missing, no ubiquitination was observed, confirming that ATL54 functions as an E3 ubiquitin ligase .

• How does ATL54 function in the context of the ubiquitin-proteasome system?

ATL54 functions as an E3 ubiquitin ligase within the ubiquitin-proteasome system (UPS), which requires three enzyme types working in sequence:

• E1 (ubiquitin-activating enzyme): Activates ubiquitin in an ATP-dependent manner

• E2 (ubiquitin-conjugating enzyme): Carries the activated ubiquitin

• E3 (ubiquitin ligase, like ATL54): Recognizes specific substrates and facilitates ubiquitin transfer from E2 to the target protein

As a RING-type E3 ligase, ATL54 binds directly to both the E2 enzyme and the substrate, bringing them into proximity to facilitate ubiquitin transfer . ATL54 specifically works with members of the Ubc4/Ubc5 subfamily of E2 conjugases, showing E3 ligase activity when paired with UbcH5b in experimental settings .

The RING-H2 domain of ATL54 is critical for E2 binding, with specific amino acid residues mediating this interaction. Structural studies on related ATL proteins have shown that mutations in key residues of the RING domain affect both E2 binding and E3 activity, demonstrating a direct correlation between the two functions .

• What is the relationship between ATL54 and secondary cell wall formation?

ATL54 plays a regulatory role in secondary cell wall formation, particularly during xylogenesis. This relationship was established through several experimental approaches:

• Gene co-expression network analysis: ATL54 was found to be highly co-expressed with genes involved in secondary wall formation, suggesting functional association .

• Genetic manipulation studies: Analysis of ATL54 knock-out and overexpression mutants revealed:

  • In apical stem portions of knock-out plants, several secondary wall biosynthetic genes were up-regulated, including:

    • Cellulose synthase genes (CesA8, CesA4, and CesA7)

    • Xylan biosynthesis genes (IRX9, IRX14, GUX1, and GUX2)

    • Lignin biosynthesis gene CCoAOMT1

• Effect on programmed cell death: In middle stem portions, both ATL54 knock-out and overexpression mutants showed down-regulation of XCP1 (Xylem Cysteine Peptidase1), a gene associated with programmed cell death in xylem tracheary elements .

These findings suggest that ATL54 functions as a regulatory component in secondary wall biosynthesis and programmed cell death during xylogenesis, likely by mediating the ubiquitination and subsequent degradation of proteins involved in these processes .

• How can researchers generate and validate ATL54 mutant lines?

To generate and validate ATL54 mutant lines, researchers can follow these methodological approaches:

For ATL54 knock-out mutants:

• Obtain T-DNA insertion lines (e.g., SALK_072859) from repositories like the Arabidopsis Biological Resource Center.

• Extract genomic DNA from seedling leaves using a simple protocol:

  • Immerse leaves in PrepMan Ultra Sample Preparation Reagent

  • Heat at 100°C for 10 minutes to extract DNA

• Validate homozygous insertion lines by PCR using:

  • Primers specific to the native ATL54 gene

  • Primers that span the T-DNA insertion site

• Confirm absence of ATL54 expression by RT-PCR or qRT-PCR

For ATL54 overexpression lines:

• Clone the ATL54 coding sequence from a full-length cDNA clone (e.g., RIKEN RAFL clone)

• Subclone into an entry vector (e.g., pENTR/D-TOPO)

• Transfer to an overexpression vector (e.g., pH35GS) using recombination (LR Clonase II)

• Transform Arabidopsis using Agrobacterium-mediated transformation

• Select transformants and verify increased expression by qRT-PCR

Expression analysis should be performed on specific tissues where ATL54 is normally expressed, such as inflorescence stems. Researchers should examine multiple independent transgenic lines to account for position effects of transgene insertion .

• What methodologies can be used to determine the impact of ATL54 on cell wall composition?

To determine the impact of ATL54 on cell wall composition, researchers can employ the following methodologies:

• Chemical analysis of cell wall components:

  • Analyze lignin content using the acetyl bromide method

  • Determine lignin composition through thioacidolysis followed by GC-MS analysis

  • Quantify cell wall neutral sugars using alditol acetate derivatives and GC analysis

  • Measure uronic acids using colorimetric methods

• Histochemical staining:

  • Use phloroglucinol-HCl staining for lignin visualization

  • Apply toluidine blue to detect polysaccharides

  • Perform immunohistochemistry with antibodies against specific cell wall components

• Gene expression analysis:

  • Conduct qRT-PCR to measure expression of cell wall biosynthetic genes

  • Sample different portions of stems to capture developmental differences

  • Compare expression patterns between wild-type, knock-out, and overexpression plants

In the studies by Noda et al., chemical analysis of mature stems showed no substantial changes in lignin content or composition between wild-type and ATL54 mutant plants, despite the altered expression of biosynthetic genes in specific stem regions. This suggests that ATL54 may have tissue-specific or developmentally regulated effects that might be compensated for in whole mature stems .

• How does ATL54 compare to other members of the ATL family?

ATL54 is one of approximately 80 members of the ATL family in Arabidopsis, characterized by their RING-H2 domain structure. When comparing ATL54 to other family members:

Structural similarities:

• All ATLs contain a RING-H2 domain with a specific arrangement of cysteine and histidine residues

• Most ATLs contain a hydrophobic region (likely transmembrane domain)

• Many contain a conserved GLD region with unknown function

Sequence homology:

• ATL54 shares limited sequence homology with other ATLs outside the conserved domains

• For example, ATL54 and ATL55/RING1 share only 23% identity and 43% similarity

Functional diversity:

• While ATL54 is associated with secondary cell wall formation and programmed cell death

• Other ATLs serve diverse functions:

  • AthATL2 is involved in early response to pathogen-associated molecular patterns (PAMPs)

  • ACRE-132 (a tobacco ATL) is induced in response to pathogen effectors

  • Other ATLs function in hormone signaling, stress responses, and development

E2 enzyme specificity:

• Most characterized ATLs, including ATL54, interact with members of the Ubc4/Ubc5 subfamily of E2 conjugases

• The structural basis for this E2-E3 recognition has been elucidated for some ATLs through NMR spectroscopy

The wide distribution and functional diversity of ATL proteins suggest they evolved to regulate various plant-specific processes, with ATL54 specialized for secondary cell wall formation .

Experimental Design and Methodology

• What experimental design would be optimal for studying ATL54's role in programmed cell death during xylogenesis?

An optimal experimental design for studying ATL54's role in programmed cell death (PCD) during xylogenesis would incorporate multiple approaches:

• In vitro xylem differentiation system:

  • Establish Arabidopsis cell suspensions that can be induced to undergo xylogenesis

  • Compare wild-type, ATL54 knock-out, and ATL54 overexpression lines

  • Monitor PCD progression using fluorescent markers or vital stains

  • Quantify timing and extent of PCD in different genetic backgrounds

• Spatial and temporal expression analysis:

  • Create ATL54 promoter-reporter fusions (e.g., GUS or fluorescent proteins)

  • Analyze expression patterns during different stages of xylem development

  • Perform laser capture microdissection of xylem cells at different developmental stages

  • Conduct single-cell transcriptomics to identify co-expressed genes

• Identification of ATL54 substrates:

  • Perform co-immunoprecipitation using tagged ATL54

  • Conduct yeast two-hybrid screening to identify interacting proteins

  • Use proximity labeling methods (BioID or TurboID) to identify proteins in close proximity to ATL54

  • Validate potential substrates with in vitro ubiquitination assays

• Analysis of XCP1 regulation:

  • Since XCP1 expression is affected in ATL54 mutants, investigate the mechanism by:

    • Analyzing XCP1 promoter for potential regulatory elements

    • Performing chromatin immunoprecipitation to identify transcription factors binding to XCP1 promoter

    • Determining if ATL54 ubiquitinates transcription factors that regulate XCP1

• Comparative analysis across vascular plants:

  • Identify ATL54 orthologs in other plant species

  • Determine if the function in PCD is conserved across different plant lineages

  • Analyze expression patterns in species with different wood formation strategies

This multi-faceted approach would provide comprehensive insights into ATL54's mechanistic role in regulating programmed cell death during wood formation, potentially revealing novel regulatory pathways in xylogenesis .

The research should focus particularly on the middle portions of inflorescence stems, where both ATL54 knock-out and overexpression were shown to affect XCP1 expression in previous studies .

I hope these FAQs provide valuable guidance for your research on ATL54. For further information or specific experimental protocols, please consult the cited primary literature.