Recombinant Oryza sativa subsp. japonica IAA-amino acid hydrolase ILR1-like 5 (ILL5)

What is the biological function of IAA-amino acid hydrolase ILR1-like 5 (ILL5) in rice?

ILL5 is an enzyme belonging to the IAA-amino acid hydrolase family that plays a crucial role in auxin homeostasis in rice (Oryza sativa). It functions primarily to hydrolyze amino acid-type indole-3-acetic acid (IAA) conjugates, particularly IAA-Asp and IAA-Glu, thereby releasing free bioactive IAA. This enzyme is part of the GH3-ILR1-DAO pathway, which represents the main oxidative inactivation pathway for auxin in plants . Through this hydrolytic activity, ILL5 contributes to maintaining optimal endogenous auxin levels, which are essential for various developmental processes in rice plants. The regulation of auxin levels by ILL5 and related hydrolases is particularly important during germination, seedling establishment, and reproductive development .

How does ILL5 differ structurally and functionally from other members of the ILR1/ILL family?

ILL5 shares significant structural and functional similarities with other members of the ILR1/ILL family but possesses distinctive features that determine its substrate specificity and activity. Based on sequence analysis from recombinant protein data, rice ILL5 consists of 407 amino acids (residues 20-426) when expressed as a recombinant protein . While ILR1, ILL2, IAR3, and ILL3 collectively contribute to auxin homeostasis as demonstrated in Arabidopsis mutant studies, ILL5 has evolved specific substrate preferences and tissue-specific expression patterns in rice . The conserved functional domains include a hydrolase active site with metal-binding residues essential for catalytic activity. Unlike some family members that may have broader substrate specificity, ILL5 appears more specialized for hydrolyzing certain IAA-amino acid conjugates, particularly contributing to the conversion of storage forms of auxin to active forms during specific developmental windows or stress responses in rice.

What is the expression pattern of ILL5 in different rice tissues and developmental stages?

The expression of ILL5 in rice follows specific spatial and temporal patterns that correlate with its function in auxin homeostasis. Similar to its family members in Arabidopsis, rice ILL5 is primarily expressed in morphogenic auxin maxima—regions of the plant where auxin concentration peaks to drive developmental processes . High expression levels are observed in actively growing tissues such as root tips, shoot apical meristems, and developing reproductive organs. During seed germination and early seedling establishment, ILL5 expression is dynamically regulated in response to environmental conditions, particularly under submergence or hypoxic conditions where auxin metabolism is altered . Expression studies in rice varieties with differing anaerobic germination tolerance have shown that ILL5 and related genes involved in auxin metabolism are differentially regulated in response to submergence, suggesting their role in adaptive responses to environmental stresses. The precise tissue-specific expression patterns of ILL5 reflect its specialized function in modulating local auxin concentrations during critical developmental transitions.

What expression systems have been successfully used for producing recombinant Oryza sativa ILL5 protein?

Several expression systems have been employed for the production of recombinant Oryza sativa ILL5, each with distinct advantages for different research applications. The yeast expression system has proven particularly effective for producing functional ILL5 protein with proper post-translational modifications that maintain enzymatic activity . This system offers advantages in terms of cost-effectiveness while still providing eukaryotic protein processing capabilities. The recombinant protein expressed in yeast systems can undergo modifications such as glycosylation, acylation, and phosphorylation that ensure proper protein folding and native conformation .

Bacterial expression systems using E. coli have also been utilized for high-yield production, though they may lack some post-translational modifications. For studies requiring mammalian-type modifications, mammalian cell expression systems have been employed, albeit at higher cost and lower yield. The baculovirus-insect cell system represents an intermediate option that balances yield with eukaryotic processing. Each system produces recombinant ILL5 with characteristics that may be optimized for specific experimental purposes, from structural studies to enzymatic assays.

What purification strategies yield the highest activity for recombinant ILL5 protein?

The purification of recombinant ILL5 to maintain optimal enzymatic activity requires careful consideration of multiple factors. The most successful purification strategy involves affinity chromatography using His-tag fusion proteins, which allows for selective binding to nickel or cobalt resins . This approach typically yields protein with >90% purity suitable for most applications. To preserve enzymatic activity, it is essential to maintain appropriate buffer conditions throughout the purification process.

The following protocol has been established for optimal purification of active ILL5:

This purification approach typically yields ILL5 protein with specific activity comparable to commercial standards . Metal ions, particularly zinc or manganese, may need to be added during activity assays as they are cofactors for optimal hydrolase function.

How can researchers verify the enzymatic activity of recombinant ILL5 in vitro?

Verification of ILL5 enzymatic activity requires specific assays that measure the hydrolysis of IAA-amino acid conjugates. The most direct method involves quantitative measurement of free IAA released from IAA-amino acid conjugates using liquid chromatography-mass spectrometry (LC-MS/MS). A standard in vitro activity assay protocol involves:

• Incubation of purified recombinant ILL5 (50-100 ng) with substrate (IAA-Asp or IAA-Glu, 50-100 μM) in reaction buffer (50 mM Tris-HCl pH 7.5, 1 mM MnCl₂) at 28°C for 30-60 minutes.

• Termination of the reaction with equal volume of methanol containing internal standards (typically deuterated IAA).

• Quantification of free IAA using LC-MS/MS with multiple reaction monitoring.

Alternative methods include:

• Colorimetric assays using synthetic substrates that release chromogenic groups upon hydrolysis

• Radiolabeled substrate assays using ³H-IAA conjugates

• Fluorescence-based assays for high-throughput screening

Control experiments should include heat-inactivated enzyme and substrate-only controls. Kinetic parameters (Km, Vmax) can be determined by varying substrate concentrations and analyzing data using Michaelis-Menten or Lineweaver-Burk plots. The specific activity of different recombinant ILL5 preparations can be compared to ensure consistency between batches and expression systems .

How does ILL5 function contribute to rice adaptation to anaerobic germination conditions?

ILL5's role in rice adaptation to anaerobic germination (AG) conditions is connected to the complex interplay between auxin homeostasis and hypoxic stress responses. Under submergence or stagnant water conditions, rice seedlings experience altered auxin metabolism that significantly impacts germination and early seedling establishment . Research has shown that oxygen and light can reduce IAA levels, which promotes seedling establishment and enhances rice AG tolerance.

The function of ILL5 and related hydrolases in this context involves the regulation of free IAA levels through hydrolyzing IAA-amino acid conjugates. Under hypoxic conditions, rice seedlings accumulate higher levels of free IAA compared to aerobic counterparts, with the highest levels detected at 4 days after imbibition . This excessive auxin accumulation can inhibit root and primary leaf growth in rice seedlings under stagnant water. The metabolism of IAA shifts to promote degradation during AG, and ILL5 likely contributes to this adaptive response by modulating the balance between active and storage forms of auxin.

Comparative studies across rice varieties with differential AG tolerance (such as Nipponbare, IR64, and IR42) have revealed that auxin metabolism patterns correlate with submergence tolerance. AG-tolerant varieties show more effective regulation of IAA levels, suggesting that ILL5 and related enzymes play a key role in determining adaptation to submergence during germination. This understanding opens possibilities for enhancing rice germination under flooded conditions through targeted modifications of auxin metabolism pathways .

What role does ILL5 play in rice disease resistance and stress responses?

While ILL5 primarily functions in auxin homeostasis, emerging evidence suggests connections between auxin signaling pathways and rice disease resistance mechanisms. Auxin homeostasis modulation has been implicated in responses to various biotic and abiotic stresses. Though not directly studied in the context of rice yellow mottle virus (RYMV) resistance, IAA metabolism enzymes like ILL5 may indirectly influence defense responses through their effects on plant development and stress physiology .

The nucleoporin gene OsCPR5.1 (RYMV2) has been identified as a recessive resistance locus against RYMV in African rice (Oryza glaberrima) . While this resistance mechanism operates primarily through altered nuclear pore complex function rather than auxin metabolism, the broader signaling networks involving hormonal balance during pathogen response likely include auxin homeostasis components.

Research on plant-pathogen interactions has revealed that pathogens often manipulate auxin signaling to promote susceptibility, while plants may alter auxin metabolism as part of defense responses. ILL5 and other IAA hydrolases could therefore contribute to reestablishing hormonal homeostasis during infection. Further research is needed to elucidate the specific roles of ILL5 in rice immunity, but its function in maintaining appropriate auxin levels suggests potential involvement in coordinating growth and defense responses during pathogen challenges.

How does ILL5 activity influence rice lodging resistance and yield components?

The influence of ILL5 on rice lodging resistance and yield components is linked to its role in auxin homeostasis, which affects plant architecture, stem strength, and carbohydrate partitioning. Research on the prl5 locus, which improves lodging resistance in rice, has shown that delayed leaf senescence results in carbohydrate reaccumulation in the stem after grain filling, enhancing mechanical strength . While ILL5 has not been directly implicated in this specific pathway, its function in auxin metabolism is relevant to these developmental processes.

Auxin homeostasis regulates numerous aspects of plant development that contribute to lodging resistance, including:

• Stem cell division and elongation

• Vascular development and lignification

• Secondary cell wall formation

• Senescence timing and progression

Near-isogenic rice lines carrying prl5 showed higher carbohydrate content in stems at 2 weeks after heading and higher carbohydrate reaccumulation at 6 weeks after heading compared to control plants . This pattern correlates with delayed chlorophyll degradation in leaf blades, suggesting that senescence timing is a key factor in lodging resistance.

ILL5, through its role in regulating active auxin levels, potentially influences these developmental processes by modulating the timing of senescence and source-sink relationships during grain filling and maturation. Precise manipulation of ILL5 expression or activity could therefore be explored as a strategy to enhance lodging resistance without yield penalties in rice breeding programs. Future research targeting the temporal and spatial regulation of ILL5 activity during reproductive development could reveal specific mechanisms connecting auxin metabolism to lodging resistance and yield stability.

What are the most effective genetic approaches for studying ILL5 function in rice?

Multiple genetic approaches have proven effective for studying ILL5 function in rice, each with specific advantages for different research questions. Based on current research methodologies, the following approaches are recommended:

• CRISPR/Cas9 genome editing: This has emerged as the most precise method for generating loss-of-function mutations in ILL5. Unlike the challenges faced with traditional breeding approaches for transferring traits between rice subspecies , CRISPR/Cas9 allows direct modification of the target gene in elite varieties. When designing guide RNAs, targeting conserved catalytic domains yields complete loss-of-function, while modifications to the N-terminal region may produce altered activity phenotypes.

• RNAi and artificial microRNA approaches: These methods offer advantages for studying genes where complete knockout may be lethal or for achieving tissue-specific or inducible knockdown. For ILL5, construct design should target unique regions to avoid off-target effects on other ILR1/ILL family members.

• Overexpression studies: Constitutive or inducible overexpression of ILL5 using vectors like pUbi or pOsActin provides insights into gain-of-function phenotypes. These studies have revealed that increased expression of IAA-amino acid hydrolases can significantly alter auxin homeostasis and enhance tolerance to submergence .

• Reporter gene fusions: Promoter:GUS or protein:GFP fusions enable detailed analysis of spatial and temporal expression patterns, revealing that ILL5 expression localizes to morphogenic auxin maxima .

• TILLING (Targeting Induced Local Lesions IN Genomes): For subtle modifications or in varieties recalcitrant to transformation, TILLING populations offer an alternative approach to identify missense mutations affecting specific protein domains.

Each approach should be selected based on the specific research question, with consideration of genetic background effects, transformation efficiency of the target variety, and whether complete or partial loss of function is desired.

What analytical methods provide the most accurate quantification of ILL5 substrates and products in plant tissues?

Accurate quantification of ILL5 substrates (IAA-amino acid conjugates) and products (free IAA) in plant tissues requires sophisticated analytical methods with high sensitivity and specificity. Based on current research standards, the following approaches yield the most reliable results:

• Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): This represents the gold standard for auxin metabolite analysis, offering unparalleled sensitivity (detection limits in the picogram range) and specificity. The recommended protocol involves:

  • Sample extraction with methanol/water/formic acid (15:4:1, v/v/v)

  • Solid-phase extraction cleanup

  • Analysis using multiple reaction monitoring (MRM) with deuterium-labeled internal standards

• Ultra-Performance Liquid Chromatography (UPLC): When coupled with high-resolution mass spectrometry, UPLC provides enhanced chromatographic separation of structurally similar auxin metabolites.

• Immunoassay techniques: While less specific than MS-based methods, immunoassays offer advantages for high-throughput screening when antibodies with appropriate specificity are available.

The accurate measurement of IAA and its conjugates requires careful attention to sample handling to prevent degradation or interconversion of metabolites. Plants grown under different conditions (aerobic vs. hypoxic) show distinctive profiles of IAA conjugates, with hypoxic seedlings generally containing lower IAA-Ala but higher IAA-Glu, IAA-Asp, and oxIAA compared to aerobic counterparts . This metabolic shift illustrates the importance of standardized sampling and analysis protocols when comparing experimental treatments.

For developmental studies, analysis of IAA metabolites at multiple timepoints is essential, as demonstrated by the dynamic changes observed in rice seedlings, where IAA levels typically peak at 4 days after imbibition and decline afterward under both aerobic and hypoxic conditions .

How can researchers design experiments to distinguish ILL5 functions from those of other IAA-amino acid hydrolases?

Designing experiments to distinguish the specific functions of ILL5 from other IAA-amino acid hydrolases requires multifaceted approaches that address the challenge of functional redundancy. The following experimental strategies have proven effective:

• Genetic approaches with multiple mutants: Since single mutants may show limited phenotypes due to redundancy, higher-order mutants combining mutations in multiple hydrolase genes provide clearer functional insights. Studies in Arabidopsis using ilr1 ill2 iar3 ill3 quadruple mutants revealed specific roles of these hydrolases that were masked in single mutants . Similar approaches in rice would help delineate ILL5's unique functions.

• Substrate specificity analysis: In vitro enzymatic assays using purified recombinant proteins can determine the substrate preferences of ILL5 compared to other family members. A comprehensive panel of different IAA-amino acid conjugates (IAA-Ala, IAA-Leu, IAA-Phe, IAA-Asp, IAA-Glu) should be tested to establish substrate specificity profiles.

• Tissue-specific expression analysis: High-resolution expression mapping using promoter-reporter constructs or cell-type-specific transcriptomics can identify spatial and temporal domains where ILL5 is uniquely expressed compared to other family members.

• Complementation experiments: Selective complementation of multiple hydrolase mutants with individual genes can determine which functions can be rescued by ILL5 versus other family members.

• Inducible expression systems: Temporal control of gene expression using inducible promoters allows examination of immediate versus long-term consequences of altering ILL5 activity, helping distinguish direct from indirect effects.

• Enzyme inhibitor studies: Development of specific inhibitors targeting the unique structural features of ILL5 could provide pharmacological tools to complement genetic approaches.

In rice, comparative studies across different varieties and growth conditions have shown that IAA metabolism responds distinctively to environmental challenges such as submergence . These natural variations provide valuable experimental systems for dissecting the specific contributions of ILL5 to adaptive responses.

How does ILL5 coordinate with other auxin metabolism enzymes in the GH3-ILR1-DAO pathway during rice development?

The coordination between ILL5 and other enzymes in the GH3-ILR1-DAO pathway represents a sophisticated regulatory network controlling auxin homeostasis throughout rice development. This pathway encompasses three major enzymatic activities: conjugation (GH3), hydrolysis (ILL5/ILR1), and oxidation (DAO) . Current research indicates that these enzymes operate in a balanced system where perturbation of one component leads to compensatory changes in others.

In the GH3-ILR1-DAO pathway, GH3 proteins conjugate IAA to amino acids, forming storage or inactive forms. ILL5 and related hydrolases can release free IAA from these conjugates, while DAO enzymes catalyze oxidative inactivation of IAA. The functional integration of these enzymes is evidenced by studies showing that dao1-1 mutants accumulate IAA-Asp and IAA-Glu, which can be hydrolyzed by ILR1/ILL enzymes, resulting in high-auxin phenotypes .

During normal development, these enzymes are differentially expressed in response to developmental cues and environmental signals. Under submergence, for example, rice exhibits altered IAA metabolism with reduced levels of IAA-Ala conjugates and increased levels of IAA-Glu, IAA-Asp, and oxIAA compared to aerobic conditions . This metabolic shift suggests coordinated regulation of the entire pathway to adapt auxin signaling to hypoxic stress.

The miR167a-ARF-GH3 pathway has been identified as a regulatory module that controls IAA metabolism under submergence, with reduced miR167 levels promoting auxin metabolism, reducing endogenous free IAA levels, and enhancing rice anaerobic germination tolerance . ILL5, as part of this broader regulatory network, likely responds to these upstream signals to fine-tune local auxin concentrations during stress adaptation and normal developmental transitions.

What structural features of ILL5 determine its substrate specificity and catalytic efficiency?

The substrate specificity and catalytic efficiency of ILL5 are determined by specific structural features that have been partly elucidated through sequence analysis, protein modeling, and functional studies. Based on analysis of the recombinant protein sequence , several key structural elements appear to influence its catalytic properties:

• Catalytic domain architecture: ILL5 contains a conserved M20 peptidase domain characteristic of the IAA-amino acid hydrolase family. Within this domain, specific residues coordinating metal ions (likely zinc or manganese) form the catalytic center essential for the hydrolysis reaction.

• Substrate binding pocket: The specificity for different IAA-amino acid conjugates is determined by the amino acid residues lining the substrate binding pocket. The pocket must accommodate both the indole ring of IAA and the variable amino acid moiety of the conjugate.

• N-terminal region: The N-terminal portion (approximately residues 20-100) may play a regulatory role, as analogous regions in related proteins have been implicated in protein-protein interactions or subcellular localization.

• Metal-binding sites: The sequence contains conserved histidine and aspartate residues that likely coordinate metal ions essential for catalysis. The specific arrangement of these residues influences catalytic efficiency.

Structural comparison with related enzymes suggests that subtle differences in the substrate binding pocket architecture explain the preferential hydrolysis of certain IAA-amino acid conjugates over others. While IAA-Ala and IAA-Leu appear to be minor metabolites in rice , IAA-Asp and IAA-Glu serve as significant storage forms that can be hydrolyzed by ILL5 and related enzymes.

Advanced structural biology approaches, including X-ray crystallography or cryo-electron microscopy of ILL5 in complex with various substrates, would provide definitive insights into the structural basis of substrate recognition and catalytic mechanism. Such structural information would guide rational enzyme engineering for enhanced specificity or activity.

How does post-translational regulation of ILL5 respond to environmental stresses in rice?

Post-translational regulation of ILL5 in response to environmental stresses represents an important but understudied aspect of auxin homeostasis in rice. While transcriptional regulation has been more extensively characterized, emerging evidence suggests that post-translational modifications and protein-protein interactions significantly influence ILL5 activity under stress conditions.

Several potential mechanisms of post-translational regulation warrant investigation:

• Redox regulation: The catalytic mechanism of ILL5 likely involves metal coordination that may be sensitive to cellular redox status. Under hypoxic conditions during submergence, altered redox environments could directly affect enzyme activity independent of expression levels.

• Phosphorylation: Proteomic studies in related plant systems have identified phosphorylation sites on auxin metabolism enzymes that respond to stress signaling cascades. These modifications can alter enzyme activity, stability, or subcellular localization.

• Protein-protein interactions: ILL5 may interact with other components of auxin metabolism pathways or stress response proteins to form functional complexes with altered catalytic properties or substrate accessibility.

• Subcellular compartmentalization: Dynamic changes in ILL5 localization between cellular compartments could regulate access to substrates, as IAA conjugates may not be uniformly distributed within cells.

Under submergence stress, rice plants show significant changes in IAA metabolism, with altered patterns of conjugation and oxidation . These changes likely involve coordinated regulation of multiple enzymes including ILL5. The observation that oxygen and light can reduce IAA levels and enhance anaerobic germination tolerance suggests that environmental factors directly influence the auxin homeostasis machinery.

The miR167a-ARF-GH3 pathway has been identified as responsive to submergence, with reduced miR167 levels enhancing auxin metabolism and promoting tolerance . How this transcriptional regulatory module interfaces with post-translational control of ILL5 remains an important question for future research, particularly for understanding the rapid adaptive responses required under changing environmental conditions.

What biotechnological applications might exploit engineered variants of ILL5 for improving rice crop resilience?

Engineered variants of ILL5 offer promising biotechnological applications for enhancing rice crop resilience through precise modulation of auxin homeostasis. Based on current understanding of ILL5 function, several strategic approaches could be developed:

• Stress-inducible expression systems: Engineering ILL5 variants under stress-responsive promoters could enable dynamic auxin regulation specifically during challenging conditions. For submergence tolerance, variants with enhanced catalytic efficiency could be expressed under hypoxia-inducible promoters to prevent excessive IAA accumulation that inhibits seedling establishment .

• Substrate specificity engineering: Structure-guided modifications to the substrate binding pocket could create ILL5 variants with altered preferences for specific IAA-amino acid conjugates, allowing more precise control over which auxin storage forms are mobilized under different conditions.

• Tissue-specific expression optimization: Targeted expression in specific tissues, such as developing stems or reproductive organs, could enhance lodging resistance by modulating auxin-regulated processes affecting stem strength and architecture .

• Integration with disease resistance pathways: While direct connections between ILL5 and pathogen resistance remain to be fully established, engineered coordination between auxin homeostasis and defense response pathways could potentially enhance broad-spectrum disease resistance without yield penalties.

• Climate adaptation applications: As climate change increases the frequency of flooding events, ILL5 variants optimized for function under anaerobic conditions could improve germination and establishment in flood-prone regions.

The potential for such applications is supported by observations that altered auxin metabolism significantly affects rice adaptation to submergence. Research has shown that reduced IAA levels promote seedling establishment and enhance anaerobic germination tolerance, suggesting that a "certain threshold level of auxin is essential for rice AG tolerance" . Biotechnological approaches targeting ILL5 could provide more precise control over this threshold than conventional breeding approaches.

How might comparative analysis across rice varieties with different stress tolerance inform ILL5 function?

Comparative analysis across rice varieties with different stress tolerance phenotypes offers a powerful approach to illuminate ILL5 function and its contribution to adaptive traits. Natural variation in auxin metabolism components correlates with differential stress responses, providing valuable genetic resources for dissecting ILL5's role in resilience mechanisms.

Research on anaerobic germination has already revealed significant differences in auxin metabolism patterns between rice varieties with contrasting submergence tolerance. All tested varieties (IR64, IR42, and Nipponbare) showed higher free IAA levels in hypoxic seedlings than in aerobic counterparts, but with variety-specific patterns in the accumulation of IAA conjugates . The highest IAA level was detected in IR64, with distinctive patterns of IAA-Ala, IAA-Glu, IAA-Asp, and oxIAA across varieties under stress conditions.

For future research, the following comparative approaches would be particularly informative:

• Allelic diversity analysis: Sequencing ILL5 and related genes across diverse rice germplasm could identify natural variants with altered enzymatic properties or expression patterns associated with enhanced stress tolerance.

• Transcriptome and metabolome comparisons: Integrated omics approaches comparing auxin-related gene expression and metabolite profiles between tolerant and susceptible varieties under various stresses would reveal how ILL5 functions within broader regulatory networks.

• Reciprocal introgression studies: Transferring ILL5 alleles between varieties with contrasting phenotypes could verify the contribution of specific genetic variants to stress tolerance traits.

• Evolutionary analysis across Oryza species: Comparing ILL5 orthologs from wild rice species adapted to diverse environments could reveal selection signatures associated with stress adaptation.

This comparative approach has already yielded insights with the identification of the recessive resistance locus rymv2 (OsCPR5.1) from African rice (Oryza glaberrima), which confers resistance to Rice yellow mottle virus . Similar strategies focusing specifically on ILL5 and auxin metabolism genes could identify valuable genetic variants for crop improvement programs targeting multiple stress resilience traits.

What emerging technologies could advance our understanding of ILL5's role in cellular auxin homeostasis?

Emerging technologies across multiple fields offer exciting opportunities to advance understanding of ILL5's role in cellular auxin homeostasis at unprecedented resolution. These innovative approaches could address current knowledge gaps regarding the spatial, temporal, and molecular details of ILL5 function:

• Single-cell transcriptomics and proteomics: These technologies allow examination of cell type-specific expression and activity of ILL5 and other auxin metabolism enzymes, revealing how auxin homeostasis varies across different cell populations within tissues. This approach could identify specialized cell types where ILL5 plays particularly crucial roles.

• CRISPR-based transcriptional modulators: Systems like CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) enable precise temporal and spatial control of ILL5 expression without permanent genetic modification, allowing detailed dissection of its function at specific developmental stages.

• Auxin biosensors with subcellular resolution: Genetically encoded fluorescent auxin sensors can visualize auxin dynamics in living cells with subcellular resolution, potentially revealing how ILL5 activity influences auxin gradients and signaling microdomains within cells.

• Proximity labeling proteomics: Techniques like BioID or TurboID fused to ILL5 could identify protein interaction partners under different conditions, revealing how ILL5 functions within larger protein complexes or signaling hubs.

• Cryo-electron microscopy: Advanced structural biology approaches could provide atomic-resolution structures of ILL5 in complex with substrates or regulatory partners, illuminating the molecular basis of its catalytic mechanism and regulation.

• Synthetic biology approaches: Engineered auxin metabolic circuits incorporating ILL5 variants with defined properties could test hypotheses about system-level properties of auxin homeostasis networks.

• Nanoscale metabolite imaging: Emerging mass spectrometry imaging technologies with cellular or subcellular resolution could visualize the spatial distribution of ILL5 substrates and products, revealing where and when hydrolysis occurs in planta.

These technologies, particularly when applied in combination, promise to transform our understanding of how ILL5 contributes to the complex and dynamic regulation of auxin homeostasis in rice cells. Such advanced insights would not only illuminate fundamental aspects of plant hormone biology but also inform more sophisticated strategies for crop improvement through precise manipulation of auxin metabolism.