Recombinant Oryza sativa subsp. japonica Auxin-responsive protein IAA15 (IAA15)

What is the functional role of IAA15 in Oryza sativa root development?

IAA15 is an auxin-responsive protein that functions as a transcriptional repressor in the auxin signaling pathway. Research indicates IAA15 plays a key negative regulatory role in lateral root formation by interacting with ARF7 and ARF19 transcription factors. This interaction inhibits the transcription of auxin-responsive genes, particularly LBD16 and LBD29, which are positive regulators of lateral root formation. Studies using IAA15 P75S OX plants (containing a mutation in domain II) showed auxin-deficient phenotypes, including shortened primary roots and decreased lateral roots compared to wild-type plants . Expression analysis using IAA15pro::GUS reporter constructs revealed that IAA15 transcripts are primarily detected in lateral root primordia and mature lateral root tips .

How is IAA15 regulated at the post-translational level in rice?

IAA15 undergoes several key post-translational modifications that regulate its stability and function:

• Ubiquitination: Wild-type IAA15 is subject to auxin-induced polyubiquitination, which targets it for degradation by the 26S proteasome. This process can be inhibited by proteasome inhibitors like MG132 .

• Phosphorylation: IAA15 is a direct substrate of mitogen-activated protein kinases (MPKs), specifically MPK3 and MPK6, which phosphorylate IAA15 at Ser-2 and Thr-28 residues. This phosphorylation significantly enhances protein stability by inhibiting polyubiquitination .

• Stabilization mechanisms: The IAA15 P75S mutation in domain II prevents normal protein degradation, resulting in stabilized IAA15 that more effectively represses auxin-responsive gene expression .

These regulatory mechanisms are critical for modulating IAA15 activity in response to environmental stimuli, particularly during stress responses.

What experimental evidence supports IAA15's role in drought response?

Studies have established a direct link between IAA15 function and drought response in plants:

• IAA15 was identified as a novel substrate of MPK3 and MPK6, which are activated during drought stress .

• Transgenic plants overexpressing a phospho-mimicking mutant of IAA15 (IAA15 DD) showed reduced lateral root development, similar to the drought response phenotype .

• Phosphorylation by MPKs induces the stabilization of IAA15 protein by inhibiting polyubiquitination, enhancing its repressive activity on auxin-responsive genes .

• The reduced lateral root development in IAA15 phospho-mimetic plants is caused by the downregulation of LBD genes, leading to enhanced tolerance to drought .

This evidence suggests IAA15 functions as a key molecular switch that suppresses lateral root development as an adaptive response to water limitation.

What are the optimal methods for expression and purification of recombinant rice IAA15?

Based on published protocols, the following approaches are recommended for recombinant IAA15 expression and purification:

Expression systems:

• Bacterial expression: Full-length IAA15 can be amplified by PCR from a cDNA library of rice plants using gene-specific primers and cloned into appropriate expression vectors .

• Vector selection: For E. coli expression, pET-series vectors with His-tags or GST-fusion systems have been successfully used.

• Induction conditions: Typically, expression is induced with IPTG (0.5-1.0 mM) at lower temperatures (16-20°C) to improve protein solubility.

Purification strategy:

• Initial purification: Affinity chromatography using Ni-NTA columns for His-tagged proteins or glutathione sepharose for GST-fusion proteins.

• Additional purification: Size exclusion chromatography or ion exchange chromatography to achieve higher purity.

• Quality assessment: SDS-PAGE and Western blotting with anti-Flag antibodies or specific anti-IAA15 antibodies .

For functional studies, researchers should consider including a protease inhibitor cocktail during extraction to prevent degradation, as IAA15 is subject to rapid turnover.

What are the recommended methods for studying IAA15 protein-protein interactions?

Several complementary approaches have been successfully employed:

• Yeast two-hybrid (Y2H) analysis:

  • Full-length IAA15 can be cloned into pGAD424 (AD) vector containing the Leu2 selection marker

  • Potential interaction partners (such as MPK3, MPK6, TIR1, ARF7, and ARF19) should be cloned into pAS2-1 (BD) vector containing the Trp1 selection marker

  • Cotransformation into yeast strain pJ69-4A allows testing of interactions through HIS3 or LacZ reporter gene activation

• In vitro pull-down assays:

  • Using recombinant GST-IAA15 or His-IAA15 proteins

  • Incubation with plant extracts or other recombinant proteins

  • Analysis by SDS-PAGE and Western blotting

• Co-immunoprecipitation (Co-IP):

  • Expression of tagged IAA15 (Flag-IAA15) in plant systems

  • Immunoprecipitation using anti-Flag antibody coupled to agarose beads

  • Detection of interacting proteins by Western blotting or mass spectrometry

• BiFC (Bimolecular Fluorescence Complementation):

  • For visualizing interactions in vivo

  • Fusion of IAA15 and potential partners with complementary fragments of fluorescent proteins

What are the key considerations for designing IAA15 mutant studies?

When designing mutation studies for IAA15, researchers should focus on:

• Domain-specific mutations:

  • Domain II mutations: The P75S mutation in the conserved domain II stabilizes IAA15 by preventing auxin-induced degradation

  • Phosphorylation site mutations: Creating phospho-null (S2A, T28A) or phospho-mimetic (S2D, T28D) variants to study the effects of MPK-mediated phosphorylation

• Expression systems:

  • Dexamethasone (DEX)-inducible systems have been effective for controlled expression of IAA15 variants

  • Tissue-specific promoters can target expression to relevant root tissues

• Phenotypic assays:

  • Root architecture analysis, including lateral root number and primary root length

  • Drought tolerance assays

  • Auxin response assays using reporter constructs like DR5::GUS

• Molecular characterization:

  • Protein stability assays (treatment with cycloheximide and auxin)

  • Ubiquitination assays (MG132 treatment followed by immunoprecipitation)

  • Transcriptional analysis of target genes such as LBD16 and LBD29

How can researchers effectively study the phosphorylation dynamics of IAA15?

To investigate IAA15 phosphorylation, consider these methodological approaches:

• In vitro kinase assays:

  • Incubate recombinant IAA15 with active MPKs (MPK3/MPK6)

  • Use [γ-32P]ATP to detect phosphorylation

  • Analyze by autoradiography or phospho-imaging

• Phosphorylation site identification:

  • Mass spectrometry (LC-MS/MS) analysis of phosphorylated IAA15

  • Targeted analysis focusing on Ser-2 and Thr-28 residues

  • Generation of phospho-specific antibodies

• In vivo phosphorylation detection:

  • Utilize phospho-mimetic (S2D/T28D) and phospho-null (S2A/T28A) variants

  • Employ Phos-tag SDS-PAGE to detect mobility shifts in phosphorylated proteins

  • Immunoprecipitation followed by Western blotting with phospho-specific antibodies

• Functional analysis of phosphorylation:

  • Compare wild-type IAA15 with phospho-variant proteins in:

    • Protein stability assays

    • Interaction studies with ARFs

    • Transcriptional repression assays

• Genetic approaches:

  • Analyze IAA15 phosphorylation in mpk3/mpk6 mutant backgrounds

  • Use MPK inhibitors to block phosphorylation events

  • Create transgenic lines expressing phospho-variant IAA15 proteins

What methodologies are most appropriate for analyzing the transcriptional repression activity of IAA15?

Several complementary approaches can be employed:

• Reporter gene assays:

  • DR5::GUS reporter lines to visualize auxin-responsive gene expression

  • LBD16pro::GUS and LBD29pro::GUS constructs to directly monitor IAA15 target gene expression

  • Transient expression systems using luciferase reporters

• Chromatin immunoprecipitation (ChIP):

  • Use tagged IAA15 (Flag-IAA15) to identify direct binding to target promoters

  • Analysis of ARF7/ARF19 binding in the presence of wild-type versus mutant IAA15

  • Sequential ChIP to identify IAA15-ARF complexes on target promoters

• Transcriptome analysis:

  • RNA-seq comparison between wild-type and IAA15 variant lines

  • Identification of genes differentially expressed in response to IAA15 overexpression

  • Time-course analysis following auxin treatment or stress exposure

• Protein-DNA binding studies:

  • Electrophoretic mobility shift assays (EMSA) to analyze IAA15-ARF binding to auxin response elements

  • DNA-protein pull-down assays using promoter fragments of target genes

  • In vitro transcription assays to directly measure repression activity

• Genetic interaction studies:

  • Combining IAA15 variants with arf7/arf19 mutants

  • Analysis of lateral root phenotypes in various genetic backgrounds

  • Suppressors/enhancers screens to identify additional components

How does IAA15 function differ from other Aux/IAA proteins in rice root development?

While research specifically comparing rice IAA15 with other Aux/IAA proteins is limited, several distinguishing features can be identified:

How does cadmium stress affect IAA15 expression and function in rice?

Based on transcriptome analysis of rice under cadmium stress:

• Genes encoding auxin-responsive proteins including IAA family members are significantly up-regulated in rice shoots exposed to cadmium (75 μmol/L CdCl2) .

• This up-regulation occurs alongside changes in other signaling pathways, including down-regulation of TIFY family, ERF, and bZIP transcription factors .

• The cadmium-induced changes in IAA gene expression correlate with enhanced oxidative stress, as indicated by the differential expression of genes involved in oxidoreductase activity, catalytic activity, and oxidation-reduction processes .

• The altered expression of IAA genes, potentially including IAA15, may contribute to the growth inhibition observed under cadmium toxicity, particularly through interference with normal auxin signaling in roots and shoots .

• The contrasting regulation patterns between IAA family genes (up-regulated) and other signaling components (down-regulated) suggests a complex transcriptional response to cadmium stress that may involve IAA15-mediated repression of auxin-responsive growth processes.

What experimental approaches can effectively assess IAA15's role in modifying root architecture under stress conditions?

Researchers investigating IAA15's function in stress-responsive root architecture should consider:

• Advanced root phenotyping methods:

  • X-ray microCT imaging for non-destructive visualization of root architecture in soil

  • Transparent growth systems for real-time monitoring of root development

  • Automated root phenotyping platforms for high-throughput analysis

• Genetic resources:

  • IAA15 overexpression and knockdown/knockout lines

  • Phospho-variant lines (IAA15 DD for phospho-mimetic; IAA15 AA for phospho-null)

  • Lines with mutations in the IAA15 interaction domain (domain II)

• Stress application protocols:

  • Controlled drought stress using precisely regulated soil moisture content

  • Polyethylene glycol (PEG)-mediated osmotic stress in hydroponic systems

  • Combined stress treatments (e.g., drought + heat, cadmium + drought)

• Analytical techniques:

  • Time-course analysis of root development under stress conditions

  • Quantification of lateral root number, length, and angles

  • Measurement of root hair development and elongation

  • Analysis of water use efficiency and stress tolerance metrics

• Molecular analyses:

  • Expression and phosphorylation status of IAA15 during stress progression

  • ChIP-seq to identify stress-specific binding sites of IAA15-ARF complexes

  • Hormone measurements in root tissues under stress conditions

How can multi-omics approaches be integrated to comprehensively understand IAA15 function?

A systems biology approach to IAA15 research should incorporate:

• Genomics:

  • Analysis of IAA15 gene structure and regulatory elements

  • Identification of natural variation in IAA15 across rice varieties

  • CRISPR/Cas9 editing to create precise mutations

• Transcriptomics:

  • RNA-seq comparison of wild-type vs. IAA15 variant lines

  • Tissue-specific and cell-type-specific transcriptome analysis

  • Time-course expression studies during stress responses

  • Single-cell RNA-seq to capture cellular heterogeneity in IAA15 responses

• Proteomics:

  • Identification of the IAA15 interactome under different conditions

  • Quantitative analysis of IAA15 protein abundance and stability

  • Phosphoproteomics to identify IAA15 phosphorylation sites and dynamics

  • Analysis of IAA15 ubiquitination patterns

• Metabolomics:

  • Profiling of auxin and other hormone levels in IAA15 variant lines

  • Analysis of metabolic changes associated with altered root development

  • Identification of metabolic signatures of stress responses

• Phenomics:

  • High-throughput phenotyping of root architecture traits

  • Field-based phenotyping under various environmental conditions

  • Correlation of molecular and phenotypic datasets

• Computational integration:

  • Network analysis to place IAA15 in broader signaling contexts

  • Predictive modeling of IAA15 function under various conditions

  • Machine learning approaches to identify patterns across multi-omics datasets

What computational tools are most effective for predicting IAA15 interactions and functional domains?

Researchers studying IAA15 structure and interactions should consider:

• Protein structure prediction tools:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • I-TASSER for modeling domains and full-length protein

  • SWISS-MODEL for homology-based structural modeling

• Interaction prediction software:

  • STRING database for known and predicted protein-protein interactions

  • PRISM for protein-protein interaction site mapping

  • PredictProtein for functional analysis from sequence

• Phosphorylation site prediction:

  • NetPhos or PhosphoSitePlus for identifying potential phosphorylation sites

  • GPS (Group-based Prediction System) for kinase-specific phosphorylation site prediction

  • KinasePhos for prediction of kinase-specific phosphorylation sites

• Domain analysis tools:

  • SMART or Pfam for identification of functional domains

  • MEME Suite for motif discovery

  • Conservation analysis using ConSurf or similar tools

• Molecular dynamics simulations:

  • GROMACS or AMBER for analyzing protein dynamics

  • Specifically useful for understanding how phosphorylation affects IAA15 structure

  • Analysis of protein-protein interaction dynamics

These computational approaches can guide experimental design and help interpret experimental results, providing insights into IAA15 function that may not be immediately apparent from experimental data alone.

What novel technologies are emerging for manipulating IAA15 function in crop improvement?

Several cutting-edge approaches show promise for IAA15-focused crop improvement:

• Precision genome editing:

  • Base editing for creating specific mutations in IAA15 phosphorylation sites

  • Prime editing for precise modifications without double-strand breaks

  • Multiplex editing to modify IAA15 and related genes simultaneously

• Synthetic biology approaches:

  • Engineering synthetic IAA15 variants with altered stability or specificity

  • Development of orthogonal auxin signaling systems

  • Design of synthetic promoters for precise control of IAA15 expression

• Optogenetic and chemogenetic tools:

  • Light-controlled degradation or activation of IAA15

  • Chemical-inducible systems for temporal control of IAA15 function

  • Spatially restricted manipulation of IAA15 activity

• Advanced phenotyping technologies:

  • Digital twin modeling of IAA15-mediated root development

  • AI-assisted image analysis for root architecture phenotyping

  • Field-deployable sensors for monitoring root responses in real-time

• Speed breeding integration:

  • Rapid cycling of IAA15 variants through multiple generations

  • High-throughput phenotypic screening platforms

  • Integration with genomic selection approaches

These technologies offer unprecedented precision in manipulating IAA15 function, potentially leading to rice varieties with enhanced stress tolerance and resource use efficiency through optimized root architecture.