Recombinant Arabidopsis thaliana Protein IDA-LIKE 3 (IDL3)

What is IDL3 and what is its role in Arabidopsis thaliana?

IDL3 (IDA-LIKE 3) is a member of the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) family of proteins in Arabidopsis thaliana. It functions as a putative peptide ligand that likely acts through receptor-like kinases (RLKs) to regulate developmental processes, particularly floral abscission . IDL3 contains a secretion signal peptide and a conserved C-terminal motif known as the extended PIP (EPIP) domain, which is essential for its biological function . The gene is identified as At5g09805 and is also referenced as F17I14 and MYH9 in genomic databases.

How is IDL3 expressed in different tissues and developmental stages?

The expression pattern of IDL3 differs from other IDL family members, suggesting specialized functions. Based on comprehensive studies using in silico data, qRT-PCR, and GUS promoter lines:

While IDA is specifically expressed in floral abscission zones, IDL3 shows a broader expression pattern across different tissues and developmental stages . Detailed expression analysis indicates that IDL genes are differentially regulated, reflecting their potentially diverse functions in plant development .

How is IDL3 expression regulated under stress conditions?

IDL3 expression is significantly affected by both biotic and abiotic stresses. Research has demonstrated that certain IDL family members, including IDL3, are strongly and rapidly induced under stress conditions . This stress-responsive expression pattern suggests that IDL3 may function beyond developmental processes, potentially playing a role in plant stress responses:

• Biotic stress: IDL3 may be induced during pathogen interactions

• Abiotic stress: Environmental factors such as drought, salt, or temperature stress can trigger IDL3 expression

This dual function in both development and stress response is characteristic of many plant signaling peptides, suggesting IDL3 participates in crosstalk between growth and stress pathways.

What expression systems are suitable for recombinant IDL3 production?

Multiple expression systems have been successfully used for recombinant IDL3 production, each with specific advantages and limitations:

For structural studies where post-translational modifications are less critical, E. coli expression systems can provide sufficient quantities of protein. For functional assays requiring properly processed IDL3, eukaryotic expression systems may be preferable .

What challenges exist in purifying functional IDL3 protein and how can they be addressed?

Purification of functional IDL3 presents several challenges:

• Small size: As a small peptide (<100 amino acids), IDL3 can be difficult to isolate using standard chromatography techniques.

• Protein processing: The biologically active form of IDL3 likely requires processing similar to IDA, where the EPIP-C domain is cleaved from the precursor protein . Evidence suggests IDA can be processed by an activity from cauliflower meristems, similar to CLV3 processing .

• Solubility issues: Recombinant expression often leads to inclusion body formation, especially in E. coli systems.

Recommended solutions:

• Fusion tags: Use solubility-enhancing tags such as GST, MBP, or SUMO

• Refolding protocols: For proteins expressed as inclusion bodies, consider refolding methods similar to those developed for other Arabidopsis proteins like RGL-3, which achieved 87% recovery of renatured protein after solubilization in 8M urea followed by 20-fold dilution

• In vitro processing: To obtain the active peptide form, consider in vitro processing using cauliflower extracts as demonstrated for IDA

How can I determine if IDL3 shares functional redundancy with other IDL family members?

Research indicates potential functional redundancy among IDL family members, as overexpression of all IDL genes resulted in phenotypes similar to IDA overexpression, although with varying severity . To study functional redundancy:

• Genetic approaches:

  • Create and characterize idl3 single mutants

  • Generate multiple idl mutant combinations

  • Perform complementation studies with different IDL genes

• Domain swap experiments:

  • Generate constructs where the EPIP-C domain of IDL3 is replaced with that of other IDL proteins

  • Create chimeric proteins with different variable regions but the same EPIP domains

  • Test these constructs for functional complementation in ida mutants

Experimental evidence shows that the EPIP-C domain of some IDL proteins could partially substitute for IDA function, suggesting overlapping but distinct activities . The variable region of IDA appears to positively support IDL EPIP-C functionality, indicating complex structure-function relationships within this family .

What receptor-like kinases (RLKs) interact with IDL3, and how can these interactions be studied?

Based on studies with IDA, the likely receptors for IDL3 include members of the HAESA (HAE) and HAESA-LIKE (HSL) receptor-like kinase family:

To study IDL3-RLK interactions:

• In vitro binding assays:

  • Surface plasmon resonance (SPR)

  • Pull-down assays with tagged IDL3 peptides

  • Yeast two-hybrid assays with receptor ectodomains

• In vivo approaches:

  • Co-immunoprecipitation

  • Bimolecular fluorescence complementation (BiFC)

  • FRET-based interaction assays

• Genetic approaches:

  • Generation of receptor mutants and testing IDL3 responsiveness

  • Overexpression of IDL3 in receptor mutant backgrounds

Current evidence suggests that the HAE-HSL2 receptor system may be shared among multiple IDL proteins, but with differing affinities or downstream effects .

How can synthetic IDL3 peptides be used to study signaling pathways?

Synthetic peptides corresponding to the functional domain of IDL3 can be powerful tools for dissecting signaling pathways:

• Design considerations:

  • Focus on the EPIP or EPIP-C domain based on evidence from IDA studies

  • Include potential post-translational modifications

  • Consider synthetic peptide variants to test structure-function relationships

• Application strategies:

  • Exogenous application: Apply synthetic peptides to wild-type or mutant plants to analyze phenotypic effects

  • Competitive inhibition: Use IDL3 peptides to potentially disrupt signaling of related peptides

  • Transcriptomic analysis: Perform RNA-seq after peptide treatment to identify downstream targets, similar to studies with PIPL3 peptide that revealed roles in biotic stress responses and cell wall modification

• Controls and validation:

  • Use scrambled peptide sequences as negative controls

  • Compare effects with other IDL family peptides

  • Verify receptor specificity using receptor mutants

What role does IDL3 play in the crosstalk between developmental and stress response pathways?

Evidence suggests IDL3 may function at the intersection of developmental regulation and stress responses:

• Developmental functions:

  • May regulate floral abscission similar to IDA

  • Potentially involved in other developmental processes based on expression patterns

• Stress response functions:

  • Rapidly induced by both biotic and abiotic stresses

  • Shares structural features with PIP/PIPL peptides involved in immune responses

Research approaches to study this crosstalk:

• Temporal expression analysis: Monitor IDL3 expression under different stresses and developmental stages

• Mutant phenotyping: Analyze idl3 mutants under both normal and stress conditions

• Hormone interaction studies: Examine interactions with stress hormones (JA, SA, ABA) and developmental hormones (auxin, cytokinin)

• Downstream target analysis: Compare transcriptomic responses to IDL3 in different contexts

Preliminary data suggests that, like the PIP/PIPL peptides, IDL3 may help regulate biotic stress responses and cell wall modification processes that are important in both development and stress adaptation .

How has the IDL family evolved in plants, and what does this tell us about IDL3 function?

The IDA/IDL family appears to have evolved alongside the HAESA receptor-like kinase family to regulate various developmental processes in plants. Comparative analysis reveals:

• Family expansion: The IDL family in Arabidopsis consists of at least 8 members (IDA and IDL1-8), with three recently identified members (IDL6-8)

• Functional divergence: While all IDL proteins maintain the core PIP motif, differences in expression patterns and subtle sequence variations suggest functional specialization

• Structural relationships: The IDL family shows similarity to other peptide families:

  • Shares the SGPS motif with PIP/PIPL peptides

  • The PIP motif has similarity to the active CLV3 peptide

This evolutionary relationship suggests that IDL3 may have evolved from an ancestral signaling peptide that diversified to regulate various developmental and stress response pathways in modern plants.

What translational applications might IDL3 research have for crop improvement?

Understanding IDL3 function could have several translational applications:

• Abscission control: Manipulating IDL3 or related genes could help control fruit drop, flower shedding, or leaf abscission in crops

• Stress tolerance: Given its induction by stress, IDL3 pathway manipulation might enhance plant resilience to environmental challenges

• Developmental regulation: Engineering IDL3 expression could potentially modulate specific developmental processes in crops

• Translational research platform: Arabidopsis IDL3 studies can serve as a model for identifying candidate genes for crop improvement, similar to how Corteva Agriscience used Arabidopsis for pre-screening genes that improved yield and drought tolerance in maize

The pathway from basic research to application would involve:

• Identifying crop homologs of IDL3

• Characterizing their function in crop species

• Developing targeted breeding or engineering approaches based on pathway knowledge

• Field testing under relevant environmental conditions

What emerging technologies might advance our understanding of IDL3 function?

Several cutting-edge approaches could significantly enhance IDL3 research:

• CRISPR-Cas9 genome editing:

  • Generate precise IDL3 mutants

  • Create reporter knock-ins at the endogenous locus

  • Modify receptor binding sites to alter specificity

• Proximity labeling proteomics:

  • Identify proteins that interact with IDL3 in vivo

  • Map the complete signaling complex around receptors

  • Track temporal changes in the interactome during development or stress

• Single-cell transcriptomics:

  • Resolve cell-type specific responses to IDL3

  • Identify rare cell populations that express or respond to IDL3

  • Track developmental trajectories influenced by IDL3 signaling

• Structural biology approaches:

  • Cryo-EM structures of receptor-ligand complexes

  • NMR studies of IDL3 peptide conformation

  • Computational modeling of ligand-receptor interactions

How can Integrative Data Analysis (IDA) approaches be applied to IDL3 research?

Integrative Data Analysis (IDA) offers powerful frameworks for combining multiple datasets to gain deeper insights into IDL3 function:

• Benefits of IDA for IDL3 research:

  • Larger sample sizes enable detection of subtle phenotypes

  • Increased sample heterogeneity improves external validity

  • Complex models can be fitted that wouldn't be possible with individual studies

• Implementation strategies:

  • Pool raw data from multiple IDL3 expression studies

  • Combine transcriptomic responses across different conditions

  • Integrate phenotypic data from various genetic backgrounds

• Challenges and solutions:

  • Between-study heterogeneity requires careful normalization

  • Different experimental designs need compatible analytical frameworks

  • Data sharing and standardization requires community coordination

By applying IDA approaches to IDL3 research, scientists can leverage the collective power of multiple studies to build more comprehensive models of IDL3 function in plant development and stress responses.