Recombinant Oryza sativa subsp. japonica IAA-amino acid hydrolase ILR1-like 3 (ILL3), partial

What is the biochemical function of IAA-amino acid hydrolase ILR1-like 3 (ILL3) in rice?

IAA-amino acid hydrolase ILR1-like 3 (ILL3) belongs to a family of enzymes that hydrolyze amide-linked conjugates of indole-3-acetic acid (IAA). These enzymes release free IAA from various amino acid conjugates, thereby contributing to the active auxin pool in plants. ILL3 is part of a larger family that includes other hydrolases such as ILR1, IAR3, ILL2, ILL5, and ILL6, each with overlapping but distinct substrate specificities for different IAA-amino acid conjugates including IAA-Ala, IAA-Leu, and IAA-Phe . By hydrolyzing these conjugates, ILL3 plays a crucial role in auxin homeostasis, which is essential for virtually all aspects of plant growth and development.

How does ILL3 differ from other members of the IAA-amino acid hydrolase family?

ILL3 is one member of a multigene family of IAA-conjugate hydrolases that differ in their substrate preferences, expression patterns, and physiological roles. While comprehensive substrate specificity data specifically for ILL3 is limited in the provided search results, research on related family members provides valuable insights. For example, IAR3 shows preference for IAA-Ala, while ILR1 is more effective at hydrolyzing IAA-Leu and IAA-Phe .

Expression analysis of these genes has revealed overlapping but distinct patterns across different plant tissues and developmental stages. ILL3 and ILL5 are closely related genes, with IAR3 and ILL5 being closely linked on chromosome 1 in Arabidopsis . The differential expression and substrate specificities of these hydrolases suggest specialized roles in modulating auxin responses in specific tissues or developmental contexts.

What is the evolutionary relationship between rice ILL3 and homologous proteins in other plant species?

Rice (Oryza sativa) ILL3 belongs to a conserved family of IAA-amino acid hydrolases found across many plant species. This family has been extensively studied in Arabidopsis, where six IAA-conjugate hydrolases have been identified, including ILR1, IAR3, ILL1, ILL2, ILL3, and ILL5 . The conservation of these enzymes across different plant species suggests their fundamental importance in auxin homeostasis throughout plant evolution.

Comparative studies between rice and Arabidopsis hydrolases indicate functional conservation, as demonstrated by the ability of rice OsGH3-8 to function in Arabidopsis and the inhibition of both rice and Arabidopsis IAA conjugation pathways by the same chemical inhibitors . This evolutionary conservation makes rice ILL3 a valuable model for understanding auxin conjugate hydrolysis across plant species.

What are the recommended methods for expressing and purifying recombinant ILL3 protein for in vitro studies?

For expressing and purifying recombinant ILL3 protein, researchers typically use bacterial expression systems such as E. coli. Based on protocols used for similar IAA-amino acid hydrolases, the following methodology is recommended:

• Cloning: Amplify the full-length ILL3 coding sequence from rice cDNA and clone it into an appropriate expression vector (e.g., pET series) with a histidine tag for purification.

• Expression: Transform the construct into an E. coli expression strain (BL21(DE3) or similar) and induce protein expression with IPTG at optimal conditions (typically 16-20°C for 16-20 hours to improve solubility).

• Purification: Lyse cells under native conditions and purify the recombinant protein using nickel affinity chromatography, followed by size exclusion chromatography to obtain highly pure protein.

• Activity verification: Confirm enzyme activity using appropriate IAA-amino acid conjugate substrates and HPLC or LC-MS/MS analysis to detect the released free IAA .

The purified protein can then be used for biochemical characterization, substrate specificity studies, and structural analysis. When working with recombinant ILL3, it's essential to determine optimal buffer conditions (pH, salt concentration) that maintain enzyme stability and activity.

How can researchers measure the enzymatic activity of ILL3 in vitro?

The enzymatic activity of ILL3 can be measured using several complementary approaches:

• HPLC-based assay: Incubate purified recombinant ILL3 with IAA-amino acid conjugates (e.g., IAA-Ala, IAA-Leu, IAA-Phe) in appropriate buffer conditions. At timed intervals, stop the reaction and analyze the samples by HPLC to detect and quantify the released free IAA.

• LC-MS/MS analysis: For more sensitive detection, use liquid chromatography coupled with tandem mass spectrometry to measure both the disappearance of IAA-conjugates and the appearance of free IAA .

• Colorimetric assays: Coupled enzyme assays that produce a colorimetric readout when IAA is released can be developed for high-throughput screening.

• Substrate specificity profiling: Test multiple IAA-amino acid conjugates to determine the substrate preference of ILL3, calculating kinetic parameters (Km, Vmax) for each substrate.

When conducting these assays, it's important to include appropriate controls such as heat-inactivated enzyme, no-substrate controls, and positive controls using related hydrolases with known activity profiles (e.g., ILR1 or IAR3) .

What approaches can be used to study the in vivo function of ILL3 in rice plants?

Several approaches can be employed to investigate the in vivo function of ILL3 in rice:

• Gene knockout/knockdown: Use CRISPR/Cas9 genome editing or RNAi approaches to generate ILL3 loss-of-function mutants in rice.

• Overexpression studies: Create transgenic rice lines overexpressing ILL3 under constitutive or inducible promoters to assess gain-of-function phenotypes.

• Expression analysis: Employ quantitative RT-PCR, RNA-seq, or promoter-reporter fusions (e.g., ILL3 promoter::GUS) to characterize the spatial and temporal expression patterns of ILL3.

• Chemical genetics approach: Utilize specific inhibitors like KKI that target IAA metabolism pathways to acutely disrupt auxin homeostasis and observe the resulting phenotypes .

• Multiple gene knockout: Create double or triple mutants with other IAA-amino acid hydrolases to reveal functional redundancy, as demonstrated in Arabidopsis with ilr1 iar3 ill2 triple mutants .

• Auxin response assays: Assess sensitivity to exogenous IAA and IAA-amino acid conjugates in wild-type versus ILL3-modified plants by measuring root/hypocotyl elongation, lateral root formation, and other auxin-responsive growth parameters .

• Metabolite analysis: Use LC-MS/MS to quantify endogenous levels of free IAA and IAA-conjugates in wild-type versus ILL3-modified plants to determine the impact on auxin homeostasis .

How does ILL3 contribute to auxin homeostasis during rice development?

ILL3 contributes to auxin homeostasis by hydrolyzing IAA-amino acid conjugates to release free IAA, thus increasing the active auxin pool available for signaling. Based on research with related hydrolases, this function is particularly important during specific developmental stages and in certain tissues.

In Arabidopsis, studies with the triple hydrolase mutant ilr1 iar3 ill2 have shown that these enzymes significantly contribute to the free IAA pool during seed germination and early seedling development . The triple mutant exhibited reduced IAA levels in imbibed seeds and seedlings (by 33% and 47%, respectively) along with increased levels of IAA-Ala and IAA-Leu conjugates .

For rice ILL3 specifically, its contribution likely depends on:

• Temporal expression patterns: When the gene is expressed during development.

• Spatial expression patterns: Which tissues express the gene.

• Substrate availability: The presence of specific IAA-amino acid conjugates in different tissues.

• Functional redundancy: The presence and activity of other IAA-amino acid hydrolases.

Growth assays with IAA and IAA conjugates in rice have shown that modulating auxin conjugation pathways affects primary root elongation, mesocotyl elongation, and other developmental processes, suggesting ILL3 may influence these aspects of rice development through its role in auxin homeostasis .

What phenotypes are associated with altered ILL3 expression or function in rice?

While the search results don't provide specific information about ILL3 mutant phenotypes in rice, insights can be drawn from studies of related hydrolases in Arabidopsis and experiments manipulating auxin conjugation pathways in rice:

• Root development: Altered hydrolase function in Arabidopsis affects root sensitivity to IAA-amino acid conjugates and can lead to shorter primary roots and fewer lateral roots on unsupplemented medium . In rice, both IAA and inhibitors of IAA conjugation (like KKI) suppress primary root elongation .

• Shoot development: In rice, IAA and KKI (which inhibits IAA conjugation) promote mesocotyl elongation , suggesting that changes in ILL3 activity could affect shoot development.

• Auxin responsiveness: Plants with altered IAA-conjugate hydrolase function show changed sensitivity to both IAA and IAA conjugates. For example, the Arabidopsis ilr1 iar3 ill2 triple mutant roots are slightly less responsive to exogenous IAA .

• Gene expression changes: Modulation of auxin homeostasis through altered hydrolase activity affects the expression of auxin-responsive genes. In rice, inhibition of IAA conjugation induces expression of auxin-responsive genes such as OsIAA1 and OsGH3-8 .

• IAA and IAA-conjugate levels: Plants with altered hydrolase function show changes in endogenous levels of free IAA and IAA-conjugates, as demonstrated in the Arabidopsis triple mutant which had reduced IAA levels and elevated IAA-Ala and IAA-Leu conjugate levels .

How does ILL3 interact with other components of the auxin signaling and metabolism network?

ILL3 functions within a complex network of auxin metabolism and signaling components:

• Coordination with GH3 enzymes: ILL3 functionally counteracts GH3 family proteins, which conjugate IAA to amino acids. While GH3 enzymes inactivate auxin by conjugation, ILL3 and related hydrolases reactivate auxin by cleaving these conjugates .

• Feedback regulation: Auxin homeostasis involves feedback regulation between biosynthesis, inactivation, and transport. When IAA-amino acid hydrolases are inhibited, IAA levels increase rapidly (within 10 minutes), suggesting these enzymes play a critical role in IAA turnover under steady-state conditions .

• Transcriptional regulation: Auxin signaling typically induces expression of genes involved in auxin metabolism, including GH3 genes. This creates a feedback loop where increased auxin levels stimulate both its conjugation and, subsequently, the potential for conjugate hydrolysis .

• Spatial and temporal coordination: The expression patterns of ILL3 likely complement those of other auxin homeostasis proteins, creating tissue-specific regulation of active auxin levels.

• Integration with development: The IAA conjugate hydrolase system represents a reservoir of inactive auxin that can be rapidly mobilized during specific developmental events or in response to environmental stimuli.

Research indicates that most endogenous IAA in plants would be turned over within 10 minutes, highlighting the dynamic nature of auxin homeostasis and the importance of inactivation pathways (including the balance between conjugation and hydrolysis) in maintaining appropriate auxin levels .

What structural features of ILL3 determine its substrate specificity?

The substrate specificity of IAA-amino acid hydrolases like ILL3 is determined by specific structural features of their active sites. Although detailed structural information specifically for rice ILL3 is not provided in the search results, insights from related hydrolases suggest several key structural determinants:

• Active site architecture: The binding pocket that accommodates IAA-amino acid conjugates likely contains specific residues that interact with both the IAA moiety and the amino acid portion of the substrate.

• Catalytic residues: As metalloenzymes, these hydrolases typically contain zinc-binding motifs essential for catalytic activity.

• Substrate entry channel: The size and chemical properties of the channel leading to the active site influence which IAA-conjugates can access the catalytic center.

• Recognition residues: Specific amino acids in the binding pocket likely form hydrogen bonds or hydrophobic interactions with different amino acid moieties of the IAA-conjugates, explaining the differential activity toward IAA-Ala, IAA-Leu, or IAA-Phe.

The differential substrate preferences observed among family members (e.g., IAR3 preferring IAA-Ala while ILR1 is more active with IAA-Leu and IAA-Phe) suggest that even subtle differences in the active site architecture can significantly impact substrate specificity . Detailed structural studies using X-ray crystallography or cryo-EM, combined with site-directed mutagenesis of key residues, would be necessary to fully characterize the structural determinants of ILL3 substrate specificity.

How do environmental factors influence ILL3 expression and activity in rice?

While the search results don't specifically address environmental regulation of rice ILL3, research on auxin metabolism suggests several potential mechanisms by which environmental factors might influence ILL3 expression and activity:

• Light conditions: Light quality and photoperiod often regulate auxin-related genes, potentially affecting ILL3 expression patterns.

• Temperature stress: Heat or cold stress may alter the balance between auxin conjugation and hydrolysis as part of stress adaptation.

• Nutrient availability: Nutrient status, particularly nitrogen availability, can affect auxin metabolism and potentially ILL3 expression.

• Water stress: Drought or flooding conditions might trigger changes in auxin homeostasis as part of stress responses.

• Pathogen attack: Biotic stress often involves auxin signaling modulation, potentially affecting ILL3 regulation.

Research methodologies to investigate these environmental influences would include:

• Quantitative RT-PCR or RNA-seq analysis of ILL3 expression under varied environmental conditions

• Promoter-reporter studies (ILL3 promoter::GUS) to visualize spatial expression changes in response to environmental cues

• Biochemical assays to determine if ILL3 enzymatic activity is directly affected by pH, temperature, or other environmental parameters

• Phenotypic analysis of ILL3 mutants or overexpression lines under different environmental conditions to assess functional significance

What is the relationship between ILL3 activity and the dynamics of auxin distribution in rice tissues?

The relationship between ILL3 activity and auxin distribution dynamics in rice involves complex spatial and temporal regulation:

• Localized auxin release: ILL3 activity in specific tissues can create localized increases in free IAA from stored conjugates, potentially establishing or maintaining auxin gradients essential for development.

• Temporal control: The rapid turnover of IAA (within approximately 10 minutes under steady-state conditions) suggests that ILL3 and related hydrolases provide a mechanism for quick adjustments to auxin levels.

• Integration with transport: ILL3-mediated hydrolysis likely works in concert with auxin transporters to fine-tune auxin distribution. Changes in IAA conjugate hydrolase activity can potentially compensate for alterations in auxin transport or biosynthesis.

• Developmental context: The significance of ILL3 for auxin distribution may vary by developmental stage or tissue type, with potentially greater importance during seed germination and early seedling development, as observed with related hydrolases in Arabidopsis .

• Mathematical modeling: The search results mention that "many computational models of the auxin system assume parameters for synthesis, transport, and inactivation" and that "the inactivation rates of IAA in specific tissues and cells need to be considered in IAA distribution models together with IAA biosynthesis and polar transport" .

Methodological approaches to study this relationship would include:

• Use of auxin reporters such as DR5::GFP or the newer AuxSen FRET-based biosensor in wild-type versus ILL3-modified plants

• Tissue-specific or inducible expression/suppression of ILL3 to observe localized effects on auxin distribution

• Combining chemical inhibition approaches (e.g., using KKI to inhibit conjugation) with auxin transport inhibitors to dissect the relative contributions of different pathways

How does rice ILL3 differ from its homologs in Arabidopsis and other model plants?

Rice ILL3 belongs to a conserved family of IAA-amino acid hydrolases found across plant species, but with potential species-specific adaptations:

• Sequence conservation: While the core catalytic domains are likely conserved, rice ILL3 may have sequence variations that reflect adaptation to rice-specific developmental processes or environmental conditions.

• Expression patterns: Expression analysis of IAA-amino acid hydrolases in Arabidopsis has shown tissue-specific patterns, with IAR3 expressed most strongly in roots, stems, and flowers . Rice ILL3 may show distinct expression patterns reflecting the different developmental programs of monocots compared to dicots.

• Substrate preferences: The substrate specificity of rice ILL3 may differ from its Arabidopsis counterparts, potentially reflecting differences in the predominant IAA-amino acid conjugates present in each species.

• Functional importance: The relative importance of ILL3 versus other family members may differ between rice and Arabidopsis, reflecting differences in their developmental programs and environmental adaptations.

• Genetic redundancy: The degree of functional overlap between ILL3 and other hydrolase family members may differ between rice and Arabidopsis.

What methodological approaches can be used to study ILL3 function in crop improvement?

Several methodological approaches can be employed to study ILL3 function with applications to crop improvement:

• CRISPR/Cas9 gene editing: Create precise modifications in the ILL3 gene to alter its expression or activity, potentially enhancing desirable agronomic traits.

• Overexpression/ectopic expression: Generate transgenic rice with enhanced or tissue-specific ILL3 expression to modify auxin distribution patterns and potentially improve traits like root architecture, tillering, or grain development.

• Field trials: Evaluate ILL3-modified plants under various field conditions to assess performance related to yield, stress tolerance, and other agronomic traits.

• Association studies: Analyze natural variation in ILL3 sequences across rice cultivars to identify potential correlations with beneficial traits.

• Chemical genetics: Use compounds like KKI that target auxin homeostasis pathways to rapidly modulate auxin levels and phenocopy genetic modifications, providing proof-of-concept for genetic engineering strategies.

• Marker-assisted selection: Develop molecular markers based on beneficial ILL3 alleles for use in conventional breeding programs.

• Tissue-specific modulation: Use tissue-specific or inducible promoters to modify ILL3 expression only in target tissues or developmental stages to minimize unintended consequences.

These approaches could potentially lead to rice varieties with enhanced root systems for drought tolerance, modified architecture for increased yield, or altered developmental timing for adaptation to different growing seasons.

What are the current knowledge gaps in understanding ILL3 function compared to other IAA-amino acid hydrolases?

Despite advances in understanding IAA-amino acid hydrolases, several knowledge gaps remain regarding ILL3 function:

• Substrate specificity profile: Detailed biochemical characterization of rice ILL3 substrate preferences compared to other family members is lacking.

• Structural information: Crystal structures of ILL3 that would reveal the molecular basis of its substrate specificity have not been reported.

• Tissue-specific roles: The specific tissues and developmental stages where ILL3 function is most critical in rice remain to be fully characterized.

• Regulation mechanisms: Detailed understanding of how ILL3 expression and activity are regulated at transcriptional, post-transcriptional, and post-translational levels is incomplete.

• Interaction partners: Potential protein-protein interactions that might modulate ILL3 function in vivo are largely unknown.

• Subcellular localization: The precise subcellular compartmentalization of ILL3 and its significance for function requires further investigation.

• Environmental responsiveness: How environmental factors specifically modulate ILL3 expression and activity needs more detailed characterization.

• Evolutionary significance: The selective pressures that have shaped ILL3 evolution in rice compared to other plant species remain to be fully explored.

Addressing these knowledge gaps will require integrative approaches combining structural biology, biochemistry, molecular genetics, cell biology, and systems biology. The rapid development of new technologies for studying protein function and plant development provides opportunities to fill these gaps in our understanding of ILL3 and related hydrolases.

What are common challenges in working with recombinant ILL3 protein and how can they be addressed?

Researchers working with recombinant ILL3 protein may encounter several challenges:

• Protein solubility issues:

  • Challenge: IAA-amino acid hydrolases may form inclusion bodies when overexpressed.

  • Solution: Express at lower temperatures (16-20°C), use solubility-enhancing fusion tags (MBP, SUMO), or optimize induction conditions (lower IPTG concentration, shorter induction time).

• Maintaining enzyme activity:

  • Challenge: Loss of activity during purification or storage.

  • Solution: Include zinc ions in purification buffers (as these are likely metalloenzymes), add reducing agents to prevent oxidation of catalytic cysteines, determine optimal pH and salt conditions for stability, and use glycerol in storage buffers.

• Substrate availability:

  • Challenge: IAA-amino acid conjugates for activity assays may not be commercially available.

  • Solution: Synthesize conjugates enzymatically using recombinant GH3 proteins and verify by mass spectrometry, or use chemical synthesis approaches followed by HPLC purification.

• Assay sensitivity:

  • Challenge: Detecting low levels of IAA released in activity assays.

  • Solution: Use sensitive LC-MS/MS methods, develop fluorescent or radioactive substrates, or employ coupled enzyme assays that amplify the detection signal.

• Determining substrate specificity:

  • Challenge: Testing multiple substrates efficiently.

  • Solution: Develop a high-throughput assay format using multi-well plates and automated sampling/analysis.

• Protein crystallization:

  • Challenge: Obtaining protein crystals for structural studies.

  • Solution: Screen multiple crystallization conditions, use surface entropy reduction mutations, try co-crystallization with substrates or inhibitors, consider nanobody-assisted crystallization.

How can researchers interpret conflicting results when studying ILL3 function in different experimental systems?

When encountering conflicting results about ILL3 function across different experimental systems, researchers should consider:

• Genetic background differences:

  • Different rice varieties may have different baseline expression of other IAA-amino acid hydrolases.

  • Solution: Include appropriate genetic controls and validate findings in multiple genetic backgrounds.

• Functional redundancy:

  • Single gene studies may show minimal phenotypes due to compensation by other family members.

  • Solution: Create and analyze multiple mutant combinations, as demonstrated with the ilr1 iar3 ill2 triple mutant in Arabidopsis .

• Experimental conditions:

  • Growth conditions can significantly impact auxin-related phenotypes.

  • Solution: Standardize growth conditions across experiments and explicitly report all parameters.

• Developmental timing:

  • Effects of altered ILL3 function may be developmental stage-specific.

  • Solution: Perform time-course analyses rather than single time-point observations.

• Tissue-specific effects:

  • ILL3 may have different roles in different tissues.

  • Solution: Analyze multiple tissues and consider tissue-specific gene manipulation approaches.

• Methodology differences:

  • Different methods for measuring IAA or assessing phenotypes can yield different results.

  • Solution: Validate findings using complementary methodological approaches.

• Data interpretation frameworks:

  • Conceptual models used to interpret data may differ between studies.

  • Solution: Critically evaluate underlying assumptions and develop integrative models that accommodate seemingly conflicting observations.

A systematic approach that considers these factors can help reconcile conflicting results and develop a more complete understanding of ILL3 function.

What are the most effective experimental designs for investigating ILL3 function in relation to other auxin metabolism enzymes?

To effectively investigate ILL3 function in relation to other auxin metabolism enzymes, researchers should consider these experimental designs:

• Higher-order mutant analysis:

  • Create and phenotype various combinations of mutants affecting ILL3 and related pathways (other hydrolases, GH3 enzymes, auxin biosynthesis enzymes).

  • This approach revealed significant findings in Arabidopsis, where the ilr1 iar3 ill2 triple mutant showed reduced endogenous IAA levels and accumulation of IAA conjugates .

• Conditional gene expression systems:

  • Use inducible promoters to control the timing of ILL3 expression.

  • This allows observation of immediate versus long-term consequences of altered ILL3 activity and avoids compensation effects from constitutive modifications.

• Chemical genetics approach:

  • Combine specific inhibitors targeting different aspects of auxin metabolism.

  • For example, combining KKI (which inhibits GH3 enzymes) with a hypothetical ILL3 inhibitor would reveal the dynamic interplay between conjugation and hydrolysis pathways.

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics analyses of ILL3-modified plants.

  • This comprehensive approach can reveal adaptive responses to altered ILL3 function across the entire auxin regulatory network.

• Tissue-specific analysis:

  • Use tissue-specific promoters to modify ILL3 expression only in specific tissues.

  • Combine with cell-type specific transcriptomics to identify downstream effects in a spatially resolved manner.

• Quantitative time-course studies:

  • Monitor changes in IAA, IAA conjugates, and related metabolites over time after perturbing ILL3 function.

  • Research has shown that IAA turnover can occur within 10 minutes , highlighting the importance of temporal resolution in experimental design.

• Biosensor applications:

  • Utilize auxin biosensors like AuxSen to visualize real-time changes in auxin distribution in ILL3-modified backgrounds.

  • This approach provides spatial and temporal resolution of auxin dynamics that traditional analytical methods cannot achieve.

These experimental designs, especially when used in combination, can provide comprehensive insights into the functional role of ILL3 within the broader auxin homeostasis network.

What are the key findings to date regarding ILL3 function in rice and other plant systems?

The current research on IAA-amino acid hydrolases, including ILL3, has established several key findings:

• IAA-amino acid hydrolases contribute significantly to the free IAA pool by hydrolyzing IAA-amino acid conjugates, as demonstrated by reduced IAA levels in Arabidopsis triple hydrolase mutants .

• Different hydrolases show distinct but overlapping substrate preferences, with enzymes like IAR3 preferring IAA-Ala while ILR1 is more active with IAA-Leu and IAA-Phe .

• These enzymes play important roles during germination and early seedling development, affecting root and hypocotyl growth .

• Auxin homeostasis involves a rapid turnover of IAA, with studies suggesting most endogenous IAA in plants is turned over within 10 minutes .

• The IAA conjugate hydrolase system represents a reservoir of inactive auxin that can be rapidly mobilized during specific developmental events.

• Expression patterns of these hydrolases vary across tissues, with IAR3 (related to ILL3) expressed most strongly in roots, stems, and flowers in Arabidopsis .

• The hydrolase system functionally counteracts GH3 enzymes, which conjugate IAA to amino acids, creating a dynamic balance in auxin homeostasis .

While many of these findings come from Arabidopsis research, they provide a framework for understanding ILL3 function in rice and other plants, given the conservation of these enzyme families across plant species.

What emerging technologies and methodologies will advance our understanding of ILL3 function?

Several emerging technologies and methodologies hold promise for advancing our understanding of ILL3 function:

• CRISPR/Cas technologies: New developments in CRISPR systems allow for more precise gene editing, multiplexed targeting of several genes simultaneously, and conditional gene regulation, enabling more sophisticated genetic analyses of ILL3 and related genes.

• Single-cell 'omics: Single-cell transcriptomics and metabolomics can reveal cell-type specific roles of ILL3 that might be masked in whole-tissue analyses.

• Advanced auxin biosensors: New-generation biosensors like AuxSen, a fluorescence resonance energy transfer-based biosensor for IAA visualization , offer improved spatial and temporal resolution for studying auxin dynamics.

• Structural biology advances: Cryo-EM and integrated structural biology approaches can reveal detailed structural information about ILL3 and its interactions with substrates.

• Protein engineering: Directed evolution and rational design approaches can generate ILL3 variants with altered substrate specificities or enhanced activities for both research and potential biotechnological applications.

• Computational modeling: Advanced computational models incorporating auxin synthesis, transport, and inactivation parameters can help understand the system-level consequences of altered ILL3 function .

• Synthetic biology approaches: Reconstituting auxin homeostasis pathways with defined components in heterologous systems can reveal fundamental principles of pathway regulation.

• Chemical biology tools: Development of specific inhibitors or activity-based probes for ILL3 would enable acute perturbation of its function and in situ activity profiling.

These technologies, especially when used in combination, will provide unprecedented insights into ILL3 function and its role in auxin homeostasis.

What potential applications exist for manipulating ILL3 activity in agriculture and biotechnology?

Understanding and manipulating ILL3 activity offers several potential applications: