Recombinant Arabidopsis thaliana Protein TOO MANY MOUTHS (TMM)

What is the TOO MANY MOUTHS (TMM) protein and its primary function in Arabidopsis thaliana?

TMM is a leucine-rich repeat receptor-like protein (LRR-RLP) that plays a critical role in regulating stomatal development and patterning in Arabidopsis thaliana. It functions as a specificity switch for the regulation of stomatal development by forming complexes with receptor kinases from the ERECTA family (ERfs) . The absence of TMM results in abnormal stomatal clustering, demonstrating its essential role in proper stomatal patterning . TMM lacks a cytoplasmic kinase domain and acts by modulating the signaling capabilities of its partner receptor kinases.

How does TMM interact with other proteins to regulate stomatal development?

TMM forms ligand-independent complexes with the leucine-rich repeat receptor kinases ER, ERL1, and ERL2 (collectively known as ERfs). These constitutive TMM-ERf complexes function as receptors for signaling peptides including EPF1, EPF2, and EPFL9 (Stomagen) . Multiple biochemical assays including gel filtration, pull-down assays, and isothermal titration calorimetry (ITC) have demonstrated that TMM complexes with ERfs to recognize these signaling peptides with much higher affinity than ERfs alone . This molecular partnership enables precise regulation of stomatal development in response to different signaling peptides.

What phenotypes are observed in TMM mutants?

TMM mutations produce tissue-specific stomatal development phenotypes:

Notably, tmm mutations also affect the subcellular trafficking of ERL1 receptors. In tmm mutants, there is a significant reduction in ERL1-YFP-positive endosomes in meristemoid cells, indicating that TMM is required for proper ERL1 endocytosis and internalization .

What are the best methods for expressing and purifying recombinant TMM protein?

For successful expression and purification of recombinant TMM protein:

• Expression System: Insect cells (typically Sf9 or High Five) provide an optimal eukaryotic expression system that enables proper folding and post-translational modifications of plant LRR proteins .

• Construct Design: Express the extracellular LRR domain of TMM (TMM LRR) with appropriate affinity tags (His-tag or GST-tag) for purification purposes .

• Purification Protocol:

  • Use affinity chromatography (Ni-NTA for His-tagged proteins)

  • Follow with size exclusion chromatography to obtain homogeneous protein

  • Confirm protein quality by SDS-PAGE and Western blotting

  • Verify proper folding using circular dichroism spectroscopy

For functional interaction studies, co-express TMM LRR with ERf LRR domains in the same insect cell system to obtain pre-formed complexes for biochemical and structural analyses .

How can researchers effectively assay TMM-ERf interactions and their binding to signaling peptides?

Several complementary approaches are recommended:

• Pull-down Assays: Co-express His-tagged and GST-tagged proteins in insect cells and perform reciprocal pull-down experiments to verify direct interactions between TMM LRR and ERf LRR domains .

• Gel Filtration: Use size exclusion chromatography to detect complex formation between purified TMM LRR, ERf LRR, and EPF/EPFL peptides. Complex formation is indicated by co-migration of proteins at higher molecular weights .

• Isothermal Titration Calorimetry (ITC): Quantitatively measure binding affinities between TMM-ERf complexes and peptide ligands. ITC studies have shown that:

  • EPF1 binds to ERL1 LRR-TMM LRR complex with a Kd of 1.3 μM

  • EPF2 binds to ERL1 LRR-TMM LRR complex with a Kd of 1.1 μM

  • EPFL9 binds to ERL1 LRR-TMM LRR complex with a Kd of 4 μM (approximately 10 times higher affinity than binding to ERL1 LRR alone)

• In vivo Verification: Generate transgenic Arabidopsis plants expressing mutant versions of TMM or ERfs to verify the functional relevance of specific protein-protein interactions identified in vitro .

How does TMM regulate the subcellular dynamics of ERf receptors?

TMM plays a critical role in controlling the subcellular trafficking of ERf receptors, particularly ERL1:

• Endocytosis/Internalization: The absence of TMM (tmm mutant) results in significantly reduced ERL1-YFP-positive endosomes per meristemoid cell (p=1.58e-14) and reduced fluorescent volume intensity ratio of ERL1-YFP-positive endosomes (p=0.0049) .

• BFA Body Formation: Treatment with Brefeldin A (BFA), which inhibits protein trafficking, shows significantly reduced ERL1-YFP-positive BFA bodies in tmm mutants compared to wild type (p=4.64e-11) .

• MVB/LE Trafficking: Wortmannin (Wm) treatment, which affects multivesicular bodies/late endosomes (MVB/LE), reveals that TMM mediates the trafficking of ERL1 to these compartments without affecting general endocytic trafficking .

• ER Retention: In the absence of TMM, ERL1-YFP shows enhanced accumulation in ring-like structures that co-localize with endoplasmic reticulum markers, suggesting that TMM also plays a role in facilitating the exit of ERL1 from the ER .

These findings indicate that TMM regulates both the plasma membrane localization and endocytic trafficking of ERf receptors, which is likely essential for proper signal transduction during stomatal development.

What are the structural determinants of TMM-ERf-ligand interactions?

Critical structural elements defining TMM-ERf-ligand interactions include:

• TMM-ERf Interface: Specific amino acid residues are essential for TMM-ERf complex formation:

  • TMM L281 and E379 are critical residues for interaction with ERL1

  • ERL1 E114 is important for interaction with TMM

  • Mutation of these residues disrupts complex formation and affects stomatal development in vivo

• Ligand Recognition:

  • EPF1 and EPF2 recognition requires TMM-ERf complex formation, as ERfs alone show little to no binding to these peptides

  • The "GS" motif in EPF1, particularly S3, is crucial for binding to the TMM-ERL1 complex

  • EPF1 G13 and P50 are important residues that pack against TMM LRR

  • Mutations in these EPF1 residues (S3R, G13R, P50R) significantly reduce peptide activity in vivo

• Differential Ligand Recognition:

  • EPF1 and EPF2 require TMM for high-affinity binding to ERfs

  • EPFL9 (Stomagen) can weakly bind to ERL1 alone but shows ~10-fold higher affinity when binding to the TMM-ERL1 complex

  • This differential recognition pattern explains how TMM functions as a specificity switch for different EPF/EPFL ligands

How can researchers effectively employ genetic approaches to study TMM function?

To effectively study TMM function through genetic approaches:

• Complementation Analysis:

  • Transform tmm mutants with wild-type or mutated TMM constructs under native promoters to assess functional relevance of specific amino acids

  • Quantify stomatal development phenotypes in different plant tissues (cotyledons vs. stems) to assess tissue-specific effects

• Domain Swap Experiments:

  • Create chimeric proteins by swapping domains between TMM and other RLPs to identify functional domains

  • Express these chimeras in tmm backgrounds to assess their ability to rescue mutant phenotypes

• CRISPR/Cas9 Gene Editing:

  • Generate precise mutations in TMM to create an allelic series that affects specific protein functions

  • Target interaction interfaces with ERfs or ligands based on structural data

• Conditional Expression Systems:

  • Use inducible promoters to control TMM expression temporally

  • Create tissue-specific promoter constructs to express TMM in different cell types to understand its context-dependent functions

• Reporter Systems:

  • Fuse TMM to fluorescent proteins to track subcellular localization and dynamics

  • Use FRET/BiFC systems to study protein-protein interactions in vivo

Why might recombinant TMM protein show reduced functionality in biochemical assays?

Several factors can affect TMM protein functionality in biochemical assays:

• Improper Folding: LRR domains are notoriously difficult to express correctly. Ensure proper disulfide bond formation by:

  • Using eukaryotic expression systems (insect cells preferred over bacterial systems)

  • Adding protein disulfide isomerase co-expression constructs

  • Including low concentrations of reducing agents in buffers

• Glycosylation Issues: TMM is likely glycosylated in planta, and improper glycosylation may affect functionality:

  • Check for N-linked glycosylation sites using prediction tools

  • Consider expressing in glycosylation-competent systems

  • Evaluate the effects of glycosidase treatments on protein activity

• Buffer Optimization:

  • Test various pH conditions (typically pH 6.5-8.0)

  • Optimize salt concentration (150-300 mM NaCl)

  • Include stabilizers like glycerol (5-10%)

  • Consider adding divalent cations (Ca²⁺, Mg²⁺) that might be required for activity

• Protein Aggregation: TMM may form aggregates that reduce functional activity:

  • Add non-ionic detergents at low concentrations (0.01-0.05% Triton X-100)

  • Include arginine and glutamic acid (50-100 mM) to reduce aggregation

  • Perform dynamic light scattering to assess protein homogeneity

How should researchers address conflicting results between in vitro biochemical data and in vivo phenotypic observations?

When faced with discrepancies between biochemical and in vivo data:

• Validate Protein Functionality:

  • Ensure recombinant proteins retain native conformation using circular dichroism or limited proteolysis

  • Verify that tagged versions used for biochemical studies can complement genetic mutants in vivo

• Consider Complex Formation Requirements:

  • TMM functions as part of multiprotein complexes; absence of partner proteins in biochemical assays may explain functional differences

  • Include all relevant complex components in biochemical assays

• Examine Tissue-Specific Effects:

  • TMM function varies between tissues (cotyledons vs. stems)

  • Ensure in vivo studies examine multiple tissues to capture tissue-specific effects

• Evaluate Protein Levels:

  • Check if protein expression levels in biochemical assays match physiological concentrations

  • Use quantitative Western blots to compare recombinant protein levels to endogenous protein levels

• Consider Post-Translational Modifications:

  • Phosphorylation, glycosylation, or other modifications may be required for function

  • Use mass spectrometry to identify post-translational modifications present in native but not recombinant protein

What emerging technologies could advance our understanding of TMM function?

Several cutting-edge approaches show promise for deepening our understanding of TMM:

• Cryo-Electron Microscopy:

  • Determine high-resolution structures of TMM-ERf-ligand complexes

  • Visualize conformational changes upon ligand binding

  • Map the complete interaction interface between complex components

• Proximity Labeling Proteomics:

  • Fuse TMM to BioID or TurboID enzymes to identify proximal proteins in vivo

  • Map the dynamic TMM interactome under different developmental conditions or in response to environmental stimuli

• Single-Molecule Techniques:

  • Use single-molecule FRET to study conformational dynamics of TMM-ERf complexes

  • Apply total internal reflection fluorescence (TIRF) microscopy to track TMM-containing complexes at the plasma membrane

• Advanced Microscopy:

  • Implement super-resolution microscopy to visualize nanoscale organization of TMM complexes

  • Use fluorescence correlation spectroscopy to determine complex stoichiometry and assembly kinetics

• Phosphoproteomics:

  • Analyze phosphorylation events downstream of TMM-ERf activation

  • Identify temporal sequence of signaling events following ligand perception

How might research on TMM inform broader understanding of plant receptor function and evolution?

TMM research has significant implications for plant receptor biology:

• Receptor Complex Evolution:

  • TMM represents an important model for understanding the evolution of receptor-coreceptor systems in plants

  • Comparative genomics across plant species can reveal evolutionary patterns of RLP-RLK partnerships

• Signaling Specificity Mechanisms:

  • TMM acts as a specificity switch, enhancing ERf binding to some ligands while potentially inhibiting others

  • This mechanism may represent a common strategy for generating signaling specificity in plant receptor systems

• Cell-Type Specific Signaling:

  • TMM functions differently in different tissues

  • Understanding this context-dependency could reveal mechanisms for tissue-specific signal interpretation

• Receptor Trafficking Regulation:

  • TMM's role in controlling ERf endocytosis and subcellular trafficking provides insights into how receptor-like proteins can modulate signaling through trafficking control

  • This mechanism may be conserved across other RLP-RLK pairs

• Translational Applications:

  • Knowledge of how TMM regulates stomatal development could inform strategies for engineering crops with improved water-use efficiency

  • Understanding fundamental receptor complex formation principles may guide protein engineering for synthetic plant signaling systems