Recombinant Oryza sativa subsp. japonica Auxin-responsive protein IAA7 (IAA7)

What is the functional role of Oryza sativa IAA7 protein in plant development?

IAA7 functions as a core component of the auxin signaling pathway that regulates numerous growth and developmental processes in plants. As an Aux/IAA family protein, it acts as a transcriptional repressor in auxin signal transduction, regulating auxin-responsive genes by interacting with Auxin Response Factors (ARFs). In its native context, IAA7 helps mediate responses to auxin, including stem elongation, cell expansion, and lateral root formation. Mutations in IAA7 (also known as AXR2) can result in developmental phenotypes including dwarfism, reduced hypocotyl elongation, and altered leaf morphology due to disruption of normal auxin signaling .

How does the structure of IAA7 relate to its function in auxin signaling?

IAA7 contains four conserved domains (I-IV) that are characteristic of Aux/IAA proteins. Domain I functions in transcriptional repression. Domain II contains the critical degron motif (GWPPV) that is recognized by the TIR1/AFB auxin receptors in the presence of auxin, leading to IAA7 ubiquitination and degradation. This degron motif is highly conserved across IAA proteins and is essential for auxin-mediated degradation. Domains III and IV mediate protein-protein interactions, particularly with ARF transcription factors and for IAA7 dimerization. Mutation of the conserved proline in domain II (as in the axr2-1 mutant, changing Pro-87 to Ser) stabilizes the protein against degradation, resulting in constitutive repression of auxin response genes .

What expression patterns are observed for IAA7 in different plant tissues and developmental stages?

Transcriptomic analyses reveal that IAA7 is expressed in multiple plant tissues with particularly high expression in stems. According to studies in Brassica napus (which shares homology with rice IAA7), expression is detected in seedling roots and leaves, stems during booting and flowering stages, flowers during flowering stage, and siliques during pod stage. The highest expression is observed in stem tissue, suggesting a crucial role in stem development and elongation. Expression patterns may vary between different plant species, but the predominant stem expression appears to be conserved across species, correlating with IAA7's role in regulating cell expansion during stem elongation .

How does the degron motif in IAA7 determine protein stability and auxin response?

The degron motif (GWPPV) in domain II of IAA7 serves as the molecular switch for auxin-dependent degradation. In the presence of auxin, the hormone acts as molecular glue, enhancing the interaction between IAA7 and the TIR1/AFB F-box proteins, components of SCF-type E3 ubiquitin ligase complexes. This interaction leads to IAA7 polyubiquitination and subsequent proteasomal degradation. Mutations in the degron motif, such as changing the conserved proline to serine (GWPPV to GWSPV) or glycine to glutamic acid (GWPPV to EWPPV), stabilize the protein against auxin-induced degradation. This stabilization prevents the de-repression of auxin-responsive genes, resulting in reduced auxin sensitivity and altered growth phenotypes. The highly conserved nature of this motif across Aux/IAA proteins underlies the fundamental mechanism of auxin signaling .

What interactions between IAA7 and ARF transcription factors have been characterized?

IAA7 interacts with specific ARF transcription factors to modulate auxin-responsive gene expression. Studies in Brassica napus have shown that BnaIAA7 interacts with BnaARF6 and BnaARF8, which regulate genes involved in cell expansion, such as BnaEXPA5 (encoding an expansin protein). This interaction occurs through domains III and IV of IAA7 and the C-terminal domains of ARFs. In the absence of auxin, IAA7 binds to these ARFs and recruits co-repressors, suppressing the transcription of target genes. When auxin levels rise, IAA7 degradation liberates the ARFs, allowing them to activate target gene expression. The specific ARF partners of IAA7 determine which downstream genes are regulated, creating specificity in auxin responses .

What is the significance of IAA7 mutations in understanding plant hormone signaling networks?

Mutations in IAA7 provide valuable insights into auxin signaling mechanisms and cross-talk with other hormonal pathways. The dominant gain-of-function mutations (such as axr2-1) that stabilize IAA7 result in pleiotropic phenotypes, including reduced hypocotyl elongation, decreased gravitropic response, and altered root development. These phenotypes have helped elucidate the role of auxin in regulating cell expansion, gravitropism, and lateral root formation. Additionally, IAA7 mutants often show altered sensitivity to other hormones, revealing integration points between auxin and other signaling pathways. For example, axr2-1 mutants show altered responses to ethylene and brassinosteroids, indicating that IAA7 functions at a node in the plant's hormonal signaling network. These mutations have been instrumental in mapping the molecular basis of hormone cross-talk in plants .

How can IAA7-based degron systems be implemented for conditional protein regulation?

The IAA7 degron has been successfully employed in auxin-inducible degron (AID) systems for conditional protein regulation. To implement this system, researchers should:

• Clone the minimal IAA7 degron sequence (mini-IAA7 or mIAA7) as a fusion tag with their protein of interest

• Express the fusion protein along with the OsTIR1 F-box protein in the target organism

• Induce degradation by adding auxin (IAA) or synthetic auxin analogs

For improved performance, the AID2 system utilizing OsTIR1(F74G) and 5-Ph-IAA offers several advantages:

The mini-IAA7 degron shows less basal degradation compared to other degrons but may work less efficiently for induced degradation due to its shorter length and fewer lysine residues for ubiquitination. This system has been successfully applied in yeast, mammalian cells, and even in mice .

What experimental factors influence the efficiency of IAA7-based degron systems?

Several factors significantly impact the efficiency of IAA7-based degron systems:

• TIR1 variant selection: OsTIR1(F74G) shows superior performance with reduced basal degradation and increased sensitivity to synthetic auxin analogs compared to wild-type OsTIR1.

• Degron tag design: The length and composition of the IAA7-derived degron affect both basal stability and induced degradation efficiency. Mini-IAA7 shows less basal degradation but may have reduced degradation efficiency.

• Expression levels: The relative expression levels of TIR1 and the target protein affect system performance. OsTIR1(F74G) works efficiently at lower expression levels compared to wild-type OsTIR1.

• Ligand selection and concentration: 5-Ph-IAA works at ~670-fold lower concentrations than IAA with OsTIR1(F74G), minimizing potential off-target effects.

• Target protein accessibility: The degron tag must be accessible to the TIR1-auxin complex for efficient degradation. The tag position (N- or C-terminal) should be optimized for each target protein.

• Cell type and organism context: The efficiency of the system varies between organisms and cell types due to differences in protein expression, proteasome activity, and cellular environment .

How can IAA7-based systems be optimized for studying essential genes?

When studying essential genes using IAA7-based degron systems, researchers should consider:

• Degron system selection: The AID2 system with OsTIR1(F74G) and 5-Ph-IAA shows minimal basal degradation, critical for studying essential genes where even slight reduction in protein levels can be lethal.

• Inducible TIR1 expression: Placing OsTIR1(F74G) under an inducible promoter (such as copper-inducible MT2 promoter) allows tight control over the degradation system.

• Degron tag optimization: Using mini-IAA7 reduces basal degradation but may affect degradation kinetics. The tag position should be optimized to minimize interference with protein function before induction.

• Titration of degradation: Using varying concentrations of 5-Ph-IAA allows for fine-tuning the degree of protein depletion to determine the minimal functional threshold.

• Genetic backup: Implementing the degron system in a heterozygous background or with a rescue construct that can be conditionally inactivated provides experimental flexibility.

• Rapid reversibility: The system's reversibility allows for pulse-chase experiments to determine the acute versus sustained effects of protein depletion.

These optimizations have enabled successful studies of essential genes like MCM10 and SLD3 in yeast and DHC1 in human cells that were previously challenging to investigate due to lethality issues .

How can researchers address the challenge of basal degradation in IAA7-based systems?

Basal degradation (degradation without auxin addition) has been a significant challenge with traditional AID systems. Researchers can minimize this issue through:

• Using the AID2 system with OsTIR1(F74G): This mutant shows virtually undetectable basal degradation compared to wild-type OsTIR1.

• Alternative degron designs: Mini-IAA7 (mIAA7) shows reduced basal degradation compared to full-length or other minimal IAA degrons.

• Controlled TIR1 expression: Using inducible or tissue-specific promoters for TIR1 expression limits unwanted degradation.

• Alternative TIR1 homologs: AtAFB2 (from Arabidopsis thaliana) has shown lower basal degradation than OsTIR1 in some contexts.

• Protein expression levels: Increasing the expression of the target protein can compensate for basal degradation.

• System optimization for specific cell types: Different cell types may require different combinations of TIR1 variants and degron designs.

Quantitative comparisons show that when combined with OsTIR1(F74G), both mAID and mIAA7 tags allow successful generation of cell lines for essential genes, with mIAA7 showing less basal degradation but lower efficiency upon induction .

What are the kinetic properties of IAA7 degradation and how can they be measured accurately?

The kinetics of IAA7-based degradation systems can be characterized by several parameters:

• Basal degradation rate: Measured by comparing protein levels in uninduced conditions to a non-degron control.

• Induced degradation half-life (T1/2): The AID2 system achieves a T1/2 of approximately 62 minutes for highly expressed reporter proteins, compared to 147 minutes for the original AID system.

• DC50 value: The ligand concentration required for 50% degradation (0.45 nM for OsTIR1(F74G) with 5-Ph-IAA versus 300 nM for wild-type OsTIR1 with IAA).

• Recovery kinetics: After ligand removal, protein levels typically recover within 3 hours, depending on protein synthesis rates.

For accurate measurement:

• Use time-course sampling with quantitative western blotting

• Employ fluorescent reporter systems for real-time monitoring

• Account for protein synthesis by using translation inhibitors when necessary

• Normalize to internal controls to account for loading variations

• Consider using mathematical modeling to extract rate constants

For essential proteins, degradation kinetics should be monitored relative to the appearance of phenotypic consequences to establish the critical threshold of protein depletion .

How do mutations in the IAA7 degron motif affect protein function and auxin responsiveness?

Mutations in the IAA7 degron motif have profound effects on protein stability and auxin responses:

• Domain II mutations (degron motif):

  • P87S mutation (as in axr2-1): Prevents auxin-induced degradation, resulting in constitutive repression of auxin response genes and dominant auxin-resistant phenotypes.

  • G-to-E substitution (GWPPV to EWPPV): Similarly stabilizes the protein against degradation.

• Domain I mutations (as second-site suppressors):

  • L15F mutation: Partially suppresses the dominant effect of the P87S mutation, suggesting it reduces the repression activity of domain I.

• Domain III mutations:

  • R138K mutation: Acts as an intragenic suppressor of P87S, likely by affecting interactions with ARF proteins or IAA7 dimerization.

These mutations reveal distinct functional roles of different IAA7 domains:

• Domain II primarily affects auxin-induced degradation

• Domain I is critical for transcriptional repression activity

• Domains III and IV mediate protein-protein interactions

The analysis of these mutations through phenotypic characterization, protein stability assays, and protein interaction studies provides valuable information about structure-function relationships in IAA7 and the molecular mechanisms of auxin signaling .

How is IAA7 being utilized in synthetic biology applications?

IAA7-derived degrons have become valuable tools in synthetic biology, with recent developments including:

• Conditional protein degradation systems: The AID2 system utilizing OsTIR1(F74G) and the synthetic auxin analog 5-Ph-IAA has demonstrated superior performance with no detectable leaky degradation and 670-times lower ligand concentration requirements compared to conventional AID systems.

• Metabolic engineering applications: In Yarrowia lipolytica, an industrially important oleaginous yeast, mini-IAA7 (mIAA7) degron coupled with OsTIR1 has been used to achieve conditional protein degradation. This system has successfully regulated canthaxanthin production, reducing it by approximately 50% when the degron system was activated, demonstrating its utility for controlling metabolic pathways.

• Orthogonal control systems: By using plant-derived components in non-plant organisms, IAA7-based systems provide orthogonal control mechanisms that don't interfere with endogenous signaling pathways in the host.

• Multi-level control circuits: IAA7 degrons can be combined with other synthetic biology tools to create sophisticated genetic circuits with multiple control points.

• In vivo applications: The AID2 system has been successfully applied in mice, opening avenues for sophisticated in vivo studies of protein function in mammals.

These applications highlight how fundamental understanding of plant hormone signaling can lead to innovative tools for synthetic biology across diverse organisms .

What advances have been made in understanding the IAA7 interactome?

Recent research has provided insights into the IAA7 protein interaction network:

• ARF interactions: Studies in Brassica napus have identified BnaARF6 and BnaARF8 as direct interactors of BnaIAA7. These interactions regulate the expression of BnaEXPA5, which encodes an expansin protein involved in cell wall loosening and cell expansion.

• Transcriptional targets: Transcriptome analyses between wild-type and IAA7 mutant plants have revealed downstream gene networks regulated by IAA7. These include cell wall-modifying enzymes, particularly expansins, which mediate cell expansion.

• Co-repressor recruitment: IAA7 functions by recruiting co-repressors to auxin-responsive promoters, with the specific co-repressors varying by plant species and cellular context.

• TIR1/AFB interactions: Structural studies of the TIR1-auxin-IAA7 complex have provided detailed molecular insights into how auxin promotes this interaction, functioning as a "molecular glue."

• Hormonal crosstalk nodes: IAA7 serves as an integration point for multiple hormone signaling pathways, with identified interactions between auxin signaling and other hormone response factors.

This expanding understanding of the IAA7 interactome helps elucidate how a single protein can mediate diverse developmental outcomes in response to the same hormonal signal .

What are the evolutionary implications of IAA7 conservation across plant species?

The high conservation of IAA7 across diverse plant species has significant evolutionary implications:

• Functional conservation: The four conserved domains of IAA7 are maintained across flowering plants, indicating strong selective pressure to preserve auxin-mediated degradation and transcriptional repression functions.

• Species-specific adaptations: Despite core conservation, species-specific variations in IAA7 sequences likely contribute to differences in growth patterns and environmental responses between plant species.

• Differential expansion: The Aux/IAA gene family has undergone differential expansion in various plant lineages, with IAA7 orthologs forming distinct clades. This suggests subfunctionalization or neofunctionalization following gene duplication events.

• Degron sequence conservation: The GWPPV degron motif in domain II is highly conserved, underscoring its fundamental importance in auxin perception mechanisms. Even conservative substitutions in this region can significantly alter auxin responsiveness.

• Cross-species utility: The high conservation of the degron motif allows IAA7-derived degrons from one species (Arabidopsis or rice) to function effectively in diverse organisms, from yeast to mammals, demonstrating the robustness of this molecular mechanism.