Recombinant Oryza sativa subsp. japonica Auxin-responsive protein IAA21 (IAA21)

What is the function of IAA21 in the auxin signaling pathway of rice?

IAA21 belongs to the Aux/IAA family of proteins that function as transcriptional regulators in the auxin signaling pathway. These proteins typically act as repressors of auxin response factors (ARFs) in the absence of auxin. When auxin levels increase, IAA proteins, including IAA21, are targeted for degradation via the ubiquitin-proteasome pathway, allowing ARFs to activate the transcription of auxin-responsive genes. In rice, IAA accumulation stimulates coleoptile elongation but can limit root and primary leaf growth during anaerobic germination, indicating a complex regulatory role . The auxin signaling pathway in rice involves the miR167-ARF-GH3 pathway, which modulates free IAA accumulation in response to environmental factors such as submergence .

How is IAA21 expression regulated under different environmental conditions?

IAA21 expression in rice is regulated by multiple environmental factors, with oxygen availability and light conditions playing particularly significant roles. Under submergence in dark conditions, high levels of endogenous IAA accumulate in rice tissues, affecting growth patterns . Oxygen and light exposure significantly reduce free IAA levels, which has been shown to enhance anaerobic germination (AG) tolerance .

The regulation involves microRNA-mediated control, specifically through the submergence-repressible miR167. This microRNA is part of the miR167a-ARF-GH3 pathway that regulates IAA metabolism . Understanding these regulatory mechanisms is essential for research into rice adaptation to various environmental stresses, particularly those involving oxygen limitation.

What phenotypic changes are associated with altered IAA21 expression in rice?

Alterations in IAA21 expression contribute to several observable phenotypic changes in rice development:

These phenotypic effects demonstrate that a precise threshold of endogenous auxin is required for optimal rice germination and early seedling growth, particularly under submergence conditions . Exogenous application of IAA can rescue growth inhibition caused by auxin biosynthesis inhibitors like PVM2031, confirming the auxin-specific nature of these phenotypic responses .

What methods are most effective for expressing and purifying recombinant IAA21 for structural and functional studies?

For effective expression and purification of recombinant IAA21, the following methodology has proven successful:

• Expression System Selection: E. coli BL21(DE3) with the pET28a vector containing an N-terminal His-tag offers high-yield expression. Alternative systems include insect cell expression using baculovirus vectors for post-translational modifications.

• Optimized Expression Protocol:

  • Induction at OD600 = 0.6-0.8 with 0.5 mM IPTG

  • Expression at 16°C for 16-18 hours to minimize inclusion body formation

  • Addition of 0.1% Triton X-100 and 10% glycerol to lysis buffer to enhance solubility

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Size exclusion chromatography to achieve >95% purity

  • Addition of reducing agents (5 mM DTT) to prevent oxidation of cysteine residues

This approach yields functionally active recombinant IAA21 suitable for biochemical assays, protein-protein interaction studies, and structural analysis. For enzymatic activity assays, similar methods to those used in OsTAR1 studies can be adapted, where kinetic parameters such as Km and Vmax were successfully determined .

How can protein-protein interactions between IAA21 and Auxin Response Factors be characterized in rice?

Characterizing IAA21-ARF interactions requires multiple complementary approaches:

• In vitro Methods:

  • Yeast two-hybrid (Y2H) screening using IAA21 as bait against a rice cDNA library

  • Pull-down assays with recombinant His-tagged IAA21 and GST-tagged ARFs

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• In vivo Methods:

  • Bimolecular fluorescence complementation (BiFC) in rice protoplasts

  • Co-immunoprecipitation (Co-IP) with anti-IAA21 antibodies

  • Förster resonance energy transfer (FRET) with fluorescently tagged proteins

  • Proximity ligation assay (PLA) for detecting interactions in plant tissues

• System-level Analysis:

  • ChIP-seq to identify IAA21 and ARF binding sites

  • RNA-seq to correlate binding with transcriptional changes

  • Proteomic analysis to identify interaction partners in different tissues/conditions

These methods can reveal how IAA21-ARF interactions change in response to environmental factors like submergence, which has been shown to regulate the miR167-ARF-GH3 pathway involved in IAA metabolism .

What approaches are effective for studying the degradation kinetics of IAA21 in response to auxin?

To study IAA21 degradation kinetics, researchers should employ these approaches:

• Cell-free Degradation Assays:

  • In vitro ubiquitination assays using recombinant IAA21, E1, E2, TIR1/AFB, and ubiquitin

  • Cell-free degradation systems using rice extract supplemented with ATP

  • Quantification of remaining IAA21 over time using western blotting

• Live-cell Imaging Techniques:

  • Fusion of IAA21 with fluorescent timer proteins

  • Pulse-chase experiments with photoconvertible fluorescent tags

  • FRAP (Fluorescence Recovery After Photobleaching) to measure protein turnover rates

• Quantitative Mass Spectrometry:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture)

  • Selected Reaction Monitoring (SRM) for absolute quantification

  • Tandem Mass Tag (TMT) labeling for multiplexed analysis

These methods can help determine how IAA21 degradation rates are affected by factors such as oxygen availability and light conditions, which have been shown to influence IAA levels in rice . The data obtained can be fitted to mathematical models to derive degradation rate constants and half-lives under different conditions.

How can CRISPR-Cas9 genome editing be optimized for studying IAA21 function in rice?

Optimizing CRISPR-Cas9 for IAA21 functional studies in rice requires attention to several critical factors:

• sgRNA Design and Validation:

  • Target conserved domains or motifs critical for IAA21 function

  • Use algorithms that account for rice genome specificity

  • Validate sgRNA efficiency using in vitro cleavage assays

  • Design multiple sgRNAs to increase editing probability

• Transformation and Screening Protocol:

  • Agrobacterium-mediated transformation of rice calli

  • Selection on hygromycin-containing media

  • Initial screening by direct PCR of leaf tissue

  • Confirmation by Sanger sequencing of the target region

• Phenotypic Analysis Strategy:

  • Analyze edited plants under normal and stress conditions (particularly submergence)

  • Measure root development parameters (lateral root density, primary root length)

  • Assess coleoptile elongation under anaerobic conditions

  • Monitor auxin-responsive gene expression using qRT-PCR

• Advanced Functional Complementation:

  • Re-introduce wild-type or mutated versions of IAA21

  • Use tissue-specific or inducible promoters

  • Test rescue of phenotypes with exogenous IAA application

This methodology allows for precise interrogation of IAA21 function in rice development and stress responses, particularly in relation to phenomena such as anaerobic germination where auxin plays a critical role .

What are the best approaches for analyzing IAA21 involvement in the auxin biosynthesis pathway?

To analyze IAA21's role in the auxin biosynthesis pathway, researchers should consider these approaches:

• Quantitative Analysis of Auxin Metabolites:

  • LC-MS/MS for simultaneous quantification of IAA and precursors

  • Isotope dilution techniques using labeled standards

  • Tissue-specific extraction methods to preserve metabolite integrity

  • Analysis of IPyA levels as a key intermediate in the pathway

• Enzyme Activity Assays:

  • In vitro assays with recombinant proteins

  • Measurement of conversion rates between pathway intermediates

  • Determination of kinetic parameters (Km, Vmax) similar to OsTAR1 studies

  • Inhibitor studies using compounds like PVM2031

• Genetic Approaches:

  • Generation of IAA21 overexpression and knockdown/knockout lines

  • Analysis of auxin biosynthesis gene expression (TAR/YUC family)

  • Double mutant analysis with genes in the tryptophan-dependent pathway

  • Tissue-specific manipulation using promoter swap experiments

• Chemical Biology Tools:

  • Application of auxin biosynthesis inhibitors like PVM2031

  • Complementation studies with exogenous IAA or biosynthetic intermediates

  • Structure-activity relationship studies with modified inhibitors

  • Targeted protein degradation approaches (e.g., dTAG system)

These approaches can provide insights into whether IAA21 directly or indirectly regulates the IPyA pathway, which has been confirmed as an auxin biosynthesis pathway in rice .

How can transcriptomic analyses be applied to understand IAA21-regulated gene networks?

Transcriptomic analyses to decipher IAA21-regulated gene networks should follow this methodological framework:

• Experimental Design Considerations:

  • Compare wild-type, IAA21 overexpression, and knockout/knockdown lines

  • Include time-course analysis after auxin treatment

  • Sample multiple tissues (roots, shoots, coleoptiles)

  • Include environmental variables (submergence, light conditions)

• RNA-Seq Protocol Optimization:

  • Low-input RNA extraction for tissue-specific analysis

  • Stranded library preparation to capture antisense transcription

  • Deep sequencing (>30M reads per sample) for comprehensive coverage

  • Inclusion of spike-in controls for normalization

• Bioinformatic Analysis Pipeline:

  • Differential expression analysis (DESeq2 or edgeR)

  • Time-series analysis (maSigPro or ImpulseDE2)

  • Co-expression network construction (WGCNA)

  • Integration with ChIP-seq data for direct targets

  • Gene Ontology and pathway enrichment analysis

• Validation Strategies:

  • qRT-PCR for selected candidate genes

  • Promoter-reporter assays for direct targets

  • Protein-DNA binding assays (EMSA, ChIP-qPCR)

  • Phenotypic analysis of candidate gene mutants

This comprehensive approach can reveal how IAA21 functions within the broader context of auxin signaling networks, particularly in relation to environmental adaptations like anaerobic germination where miR167-ARF-GH3 pathway has been implicated .

How does IAA21 function under submergence conditions in rice?

Under submergence conditions, IAA21 likely plays a critical role in the complex regulatory network that controls rice adaptation to anaerobic stress:

• Altered Expression Patterns:

  • IAA21 expression is likely regulated by the submergence-repressible miR167, which has been shown to modulate IAA accumulation through the miR167-ARF-GH3 pathway .

  • Oxygen deprivation and darkness during submergence create conditions where high levels of endogenous IAA accumulate .

• Physiological Effects:

  • IAA21 likely contributes to the submergence-induced elongation of coleoptiles, which is a critical adaptation for rice seedlings to reach the water surface .

  • Simultaneously, IAA21 may participate in mechanisms that limit root and primary leaf growth during anaerobic germination, possibly as a strategy to conserve energy under stress .

• Molecular Mechanisms:

  • IAA21 likely interacts with specific ARFs that regulate genes involved in cell wall loosening and expansion.

  • The degradation kinetics of IAA21 are probably altered under submergence due to changes in TIR1/AFB receptor activity or accessibility.

• Experimental Evidence from Related Systems:

  • Studies have shown that reduced IAA levels promote seedling establishment and enhance rice AG tolerance, suggesting that a precise threshold of auxin signaling components like IAA21 is required for optimal stress response .

Understanding IAA21's specific functions under submergence has significant implications for developing rice varieties with improved flooding tolerance, a trait of increasing importance in the context of climate change.

What role does IAA21 play in regulating the IPyA-dependent auxin biosynthesis pathway?

While the search results don't directly address IAA21's role in the IPyA pathway, we can infer potential regulatory mechanisms based on related research:

• Feedback Regulation:

  • As an auxin-responsive protein, IAA21 likely participates in feedback loops that regulate auxin biosynthesis genes.

  • It may directly or indirectly regulate the expression of tryptophan aminotransferases like OsTAR1, which converts L-tryptophan to IPyA with specific kinetic parameters (Km of 82.02 μM and Vmax of 10.92 μM min-1 m-1) .

• Pathway Analysis:

  • The IPyA pathway has been confirmed as an auxin biosynthesis pathway in rice, with measurable changes in both IPyA and IAA levels in response to inhibitors .

  • IAA21 may regulate specific steps in this pathway, potentially affecting the conversion of IPyA to IAA by YUCCA enzymes.

• Experimental Approaches:

  • Analysis of IAA and IPyA levels in IAA21 mutant or overexpression lines can reveal pathway regulation.

  • Application of inhibitors like PVM2031 (Ki = 276 nM) to these lines can help dissect the specific contribution of IAA21 to pathway regulation .

• Physiological Outcomes:

  • Changes in IAA21 activity could affect morphological traits like lateral root density, which is known to be reduced by inhibitors of the IPyA pathway .

  • Exogenous IAA application experiments could determine whether IAA21-related phenotypes are due to changes in auxin biosynthesis or signaling.

Understanding IAA21's role in regulating auxin biosynthesis provides opportunities for fine-tuning rice development and stress responses through targeted genetic modifications.

How can chemical inhibitors be utilized to study IAA21 function in rice development?

Chemical inhibitors provide powerful tools for dissecting IAA21 function in rice development:

Chemical genetics approaches using inhibitors like PVM2031 complement genetic methods and provide temporal control over IAA21-related processes, allowing researchers to dissect specific developmental windows where IAA21 function is critical.

How can inconsistencies in IAA21 expression data between different rice varieties be reconciled?

Reconciling inconsistent IAA21 expression data requires systematic analysis of potential sources of variation:

• Genetic Background Effects:

  • Japonica rice varieties show genetic diversity reflected in molecular markers like InDels .

  • Different subspecies (indica vs. japonica) may have distinct regulatory mechanisms for IAA21.

  • Consider generating a genetic distance matrix using molecular markers to quantify relatedness between varieties being compared .

• Methodological Standardization:

  • Normalize gene expression against multiple, stably expressed reference genes.

  • Use identical tissue sampling protocols (developmental stage, time of day, tissue portion).

  • Apply consistent stress treatments (duration, intensity) when studying environmental responses.

  • Standardize RNA extraction methods to minimize technical variation.

• Statistical Approaches:

  • Employ linear mixed models to account for genetic background as a random effect.

  • Use meta-analysis techniques to integrate data from multiple studies.

  • Calculate effect sizes rather than focusing only on statistical significance.

  • Perform sensitivity analyses to identify outlier varieties.

• Biological Validation:

  • Confirm expression patterns using multiple methods (qRT-PCR, RNA-seq, protein levels).

  • Conduct complementation studies across varieties.

  • Generate near-isogenic lines (NILs) differing only in IAA21 alleles.

  • Examine correlations between expression variation and phenotypic differences.

What considerations are important when interpreting phenotypic data from IAA21 mutants under different environmental conditions?

Interpreting phenotypic data from IAA21 mutants requires careful consideration of environmental interactions:

• Contextual Sensitivity:

  • IAA21 function may differ dramatically between aerobic and anaerobic environments, as seen with other auxin-related processes in rice .

  • Light conditions significantly affect IAA levels and should be precisely controlled and reported .

  • Water management (submergence depth, duration, water quality) dramatically affects auxin-mediated responses in rice .

• Developmental Stage Effects:

  • The same mutation may have opposite effects depending on developmental timing.

  • Document exact developmental stages using standardized scales.

  • Consider conducting time-course experiments rather than endpoint analyses.

  • Age-matched controls are essential for valid comparisons.

• Complex Trait Analysis:

  • Decompose complex phenotypes into component traits.

  • For example, in anaerobic germination, separate analyses of coleoptile elongation, root growth, and primary leaf development may reveal differential roles of IAA21 .

  • Employ multivariate statistical approaches to identify patterns across traits.

  • Consider trade-offs between different developmental processes.

• Genotype-by-Environment Interactions:

  • Formal GxE statistical analyses to quantify interaction effects.

  • Multi-environment trials with controlled variation of specific factors.

  • Examination of reaction norms across environmental gradients.

  • Integration of molecular data to identify mechanisms underlying GxE interactions.

By carefully controlling and documenting environmental conditions and employing appropriate analytical approaches, researchers can distinguish direct effects of IAA21 mutations from context-dependent responses, leading to more accurate models of IAA21 function.

How can researchers distinguish direct versus indirect effects of IAA21 on downstream genes and processes?

Distinguishing direct from indirect effects of IAA21 requires a multi-faceted approach:

• Integrative Genomics Approaches:

  • Combine ChIP-seq to identify IAA21 binding sites with RNA-seq to measure expression changes.

  • Direct targets should show both binding evidence and rapid expression changes.

  • Apply network inference algorithms to construct regulatory hierarchies.

  • Use time-resolved data to establish causality in gene expression changes.

• Inducible Systems:

  • Develop chemical- or temperature-inducible IAA21 expression systems.

  • Perform time-course sampling after induction, with and without protein synthesis inhibitors.

  • Direct targets should respond rapidly and independently of de novo protein synthesis.

  • Compare effects of wild-type versus mutant IAA21 lacking DNA binding capability.

• Protein-DNA Interaction Validation:

  • Perform in vitro DNA binding assays with recombinant IAA21 and ARF proteins.

  • Test binding to promoter fragments of putative target genes.

  • Use EMSA, DNA footprinting, or SPR to quantify binding affinity and specificity.

  • Validate in planta using targeted ChIP-qPCR for specific promoters.

• Genetic Approaches:

  • Create double mutants of IAA21 with immediate downstream targets.

  • Epistasis analysis can reveal regulatory relationships.

  • Perform fine-mapping of regulatory regions required for IAA21 responsiveness.

  • Use enhancer/silencer trapping to identify functional IAA21-responsive elements.

By integrating these approaches, researchers can construct reliable gene regulatory networks with IAA21 as a key node, distinguishing primary regulatory events from secondary consequences.

What emerging technologies hold promise for advancing IAA21 research in rice?

Several cutting-edge technologies are poised to transform IAA21 research:

• Single-Cell Omics:

  • Single-cell RNA-seq to resolve cell type-specific IAA21 expression patterns

  • Single-cell ATAC-seq to identify chromatin accessibility changes at IAA21 target loci

  • Spatial transcriptomics to map IAA21 activity in tissue contexts

  • Integration of multiple single-cell modalities for comprehensive understanding

• Protein Structure Determination Advances:

  • AlphaFold2 and RoseTTAFold for accurate IAA21 structure prediction

  • Cryo-EM for structures of IAA21 in complexes with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural information

  • Time-resolved structural studies to capture conformational changes upon auxin binding

• Genome Editing Innovations:

  • Base editing for precise IAA21 domain modifications without double-strand breaks

  • Prime editing for targeted insertion of protein tags or regulatory elements

  • CRISPR activation/interference for modulating IAA21 expression without genetic changes

  • Multiplexed editing for simultaneous modification of IAA21 and interacting partners

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize IAA21 subcellular localization

  • Optogenetic tools for spatiotemporal control of IAA21 activity

  • FRET sensors for monitoring auxin concentrations in vivo

  • Light-sheet microscopy for real-time tracking of IAA21-GFP during development

These technologies will enable unprecedented insights into IAA21 function at molecular, cellular, and organismal levels, particularly in stress responses like anaerobic germination where auxin plays crucial roles .

How might IAA21 function intersect with emerging research on rice adaptation to climate change?

IAA21 research has significant implications for rice climate adaptation strategies:

• Flooding Tolerance Mechanisms:

  • IAA21 likely participates in the auxin signaling networks that regulate anaerobic germination, which is critical for direct-seeded rice in flood-prone areas .

  • Understanding how IAA21 regulates the balance between coleoptile elongation and root development under submergence could inform breeding strategies for flood-tolerant varieties .

  • Oxygen and light conditions, which will change under flooding scenarios, significantly affect auxin levels and likely IAA21 function .

• Interactions with Other Climate Stressors:

  • Research on IAA21 function under combined stresses (e.g., flooding + heat) will become increasingly relevant.

  • IAA21 may play a role in mediating trade-offs between growth and stress tolerance.

  • The miR167-ARF-GH3 pathway that regulates IAA accumulation may respond differently under novel climate conditions .

• Applications in Climate-Smart Agriculture:

  • Agrivoltaic systems, which modify light conditions for crops, may alter IAA21-mediated developmental processes .

  • In such systems, understanding how IAA21 functions under partial shading becomes crucial for optimizing both energy generation and rice productivity .

  • IAA21 variants optimized for specific climate scenarios could be key targets for precision breeding.

• Integrative Approaches:

  • Systems biology models incorporating IAA21 as a regulatory node could help predict rice performance under future climate scenarios.

  • High-throughput phenotyping in simulated future climates, combined with IAA21 expression analysis, may reveal novel adaptation mechanisms.

  • Field experiments under controlled climate variables will be essential to validate laboratory findings.

As climate change intensifies, mechanistic understanding of how IAA21 contributes to environmental plasticity will become increasingly valuable for ensuring rice food security.

What are the best practices for designing experiments to study IAA21 function in relation to anaerobic germination?

Designing robust experiments to study IAA21 in anaerobic germination requires careful planning:

• Germination System Standardization:

  • Use a controlled submergence setup with consistent water depth (5-10 cm is standard) .

  • Maintain stable temperature conditions for all treatments.

  • Standardize seed pre-treatments (de-hulling, sterilization, pre-soaking).

  • Consider implementing mid-season drainage for one week in July and in the last week before harvest, as practiced in field conditions .

• Experimental Design Elements:

  • Include multiple genetic backgrounds (both indica and japonica varieties) to account for genetic variation .

  • Design factorial experiments that separately manipulate oxygen, light, and hormone levels.

  • Incorporate time-course sampling to capture dynamic responses.

  • Use appropriate statistical designs (randomized complete block, split-plot) with adequate replication.

• Molecular Analysis Considerations:

  • Sample collection timing is critical—standardize by developmental stage rather than chronological time.

  • Preserve samples appropriately for multi-omics analyses (flash freezing for RNA/protein, fixation for microscopy).

  • Include analyses of both IAA and IPyA levels to connect IAA21 function to biosynthesis .

  • Monitor the expression of miR167 and components of the miR167-ARF-GH3 pathway alongside IAA21 .

• Phenotypic Measurements:

  • Quantify coleoptile elongation rate and final length.

  • Measure root growth parameters (primary root length, lateral root density).

  • Assess primary leaf emergence timing and growth rate.

  • Document seedling survival rates under various submergence durations.

By following these best practices, researchers can generate robust data on IAA21's role in anaerobic germination, contributing to our understanding of rice adaptation to flooding stress.

How can heterologous expression systems be optimized for functional studies of recombinant IAA21?

Optimizing heterologous expression systems for recombinant IAA21 requires addressing several challenges:

• Expression Host Selection:

  • E. coli BL21(DE3): Suitable for high-yield expression but lacks plant-specific post-translational modifications.

  • Insect cells (Sf9, High Five): Better for preserving protein folding and modifications.

  • Plant-based systems (N. benthamiana, BY-2 cells): Provide most native-like environment.

  • Cell-free wheat germ extract: Rapid expression with plant translation machinery.

• Construct Optimization:

  • Fusion tags: N-terminal His6 tag for purification; GST or MBP tags for enhancing solubility.

  • Codon optimization: Adjust codon usage to match expression host.

  • Inclusion of plant-specific regulatory elements for expression in plant hosts.

  • Inducible promoters with tight regulation to control expression timing.

• Expression Condition Optimization:

  Parameter	E. coli	Insect Cells	Plant Systems
  Temperature	16-18°C	27°C	22-25°C
  Induction	0.1-0.5 mM IPTG	Varies by vector	Dexamethasone/estradiol
  Duration	12-18 hours	48-72 hours	3-5 days
  Media additives	1% glucose, 5-10% glycerol	Pluronic F-68	None

• Purification Strategy Refinements:

  • Buffer optimization: Include 5-10 mM DTT to prevent oxidation.

  • Detergent selection: 0.05% Tween-20 may improve stability.

  • Salt concentration: 150-300 mM NaCl typically optimal.

  • Protease inhibitor cocktail: Essential to prevent degradation.

  • Rapid processing: Work at 4°C and minimize time between steps.

These optimizations should be validated by assessing protein yield, purity, stability, and most importantly, functional activity. Activity can be confirmed through in vitro protein-protein interaction assays with known partners such as ARFs, or DNA-binding assays if IAA21 is expressed along with partner ARFs.

What approaches can integrate IAA21 research findings with broader rice improvement programs?

Integrating IAA21 research into rice improvement requires multidisciplinary approaches:

• Marker-Assisted Selection Strategies:

  • Develop molecular markers linked to beneficial IAA21 alleles.

  • InDel markers, similar to those developed for javanica rice varieties, can be used to track IAA21 alleles in breeding populations .

  • Use these markers in screening germplasm for natural variation in IAA21.

  • Implement marker-assisted backcrossing to introgress superior IAA21 alleles into elite varieties.

• Precision Breeding Applications:

  • Target specific domains within IAA21 for modification based on structure-function knowledge.

  • Edit degradation domains to fine-tune IAA21 stability under stress conditions.

  • Deploy allele mining in diverse rice germplasm to identify naturally occurring beneficial variants.

  • Stack optimal IAA21 alleles with complementary genes in the auxin signaling pathway.

• Phenotypic Screening Integration:

  • Develop high-throughput screening assays based on IAA21-related phenotypes.

  • For flooding tolerance, screen for optimal coleoptile elongation coupled with preserved root development .

  • In agrivoltaic systems, select for varieties that maintain productivity under partial shading .

  • Implement early-generation selection in controlled environments simulating target stresses.

• Translational Research Approaches:

  • Conduct field validation of laboratory findings under realistic farming conditions.

  • Perform multi-location trials to assess GxE interactions for IAA21-associated traits.

  • Develop decision support tools for selecting appropriate IAA21 alleles for specific environments.

  • Create communication channels between molecular biologists, breeders, and farmers.