Recombinant Arabidopsis thaliana Probable inactive receptor kinase At5g16590 (At5g16590)

What is At5g16590 and what is its basic characterization in Arabidopsis thaliana?

At5g16590 (also known as LRR1) is a gene in Arabidopsis thaliana that encodes a probable inactive receptor kinase belonging to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family. This protein contains an extracellular domain with leucine-rich repeats, a transmembrane domain, and a cytoplasmic kinase domain. The full length of the protein is 625 amino acids, with the mature protein spanning positions 24-625 .

According to phosphoproteomic studies, a specific phosphopeptide from At5g16590 has been identified: "LIEEVSRSPAS(ph)PGPLSD" . Functionally, LRR1 has been implicated in abscisic acid (ABA) signaling pathways and has been observed as one of the most highly responding kinases during zinc resupply experiments at early time points (10 min, 30 min) and under zinc sufficiency conditions .

How are LRR-RLKs classified in Arabidopsis and where does At5g16590 fit in this classification?

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) represent the largest group of Arabidopsis RLKs with approximately 235 members. The LRR-RLK family is divided into 13 subfamilies (LRR I to XIII) classified according to the organization of LRRs in the extracellular domain .

The distribution of the number of LRRs per sequence in these receptors shows three peaks at 5, 20, or 21, suggesting these numbers may be optimal for the 3D conformation of these receptors and their interactions in homo- or heterocomplexes . This structural organization is important as it likely influences ligand binding and downstream signaling specificity.

To determine where At5g16590 fits within this classification, phylogenetic analysis comparing its sequence with other characterized LRR-RLKs is necessary. Researchers typically use the following methodology:

• Sequence alignment of the kinase domains or full-length proteins

• Construction of phylogenetic trees using methods like maximum likelihood or neighbor-joining

• Bootstrap analysis to assess the reliability of the tree branches

• Comparison with established LRR-RLK subfamily classifications

What are the available genetic resources for studying At5g16590 function?

Several genetic resources are available for studying At5g16590 function in Arabidopsis:

To utilize these resources effectively, researchers typically:

• Confirm the T-DNA insertion position via PCR-based genotyping

• Verify gene expression levels using RT-PCR or qRT-PCR to confirm knockout/knockdown

• For overexpression lines, quantify transcript levels relative to wild-type

• Perform complementation tests to confirm that phenotypes are specifically due to the disruption of At5g16590

What signaling pathways is At5g16590 involved in based on current research?

Current research indicates that At5g16590 (LRR1) is involved in several signaling pathways:

• Abscisic acid (ABA) signaling pathway: LRR1 has been implicated in the ABA signaling pathway that regulates plant responses to environmental stresses . A recent study utilized Arabidopsis thaliana as a model plant and induced stress by administering abscisic acid to investigate this phenomenon.

• Defense signaling: LRR1 has been characterized as "a leucine rich repeat receptor kinase involved in defense signaling" .

• Zinc homeostasis response: LRR1 was identified as one of the most highly responding kinases at early time points (10 min, 30 min) following zinc resupply and under zinc sufficiency conditions .

For studying these pathways, researchers typically employ:

• Transcriptional analysis (RNA-seq, qRT-PCR) to measure expression changes

• Phosphoproteomic studies to identify phosphorylation events

• Phenotypic analysis of mutants under various stress conditions

• Analysis of downstream signaling components using genetic and biochemical approaches

What experimental approaches can be used to study the phosphorylation status of At5g16590?

To study the phosphorylation status of At5g16590, several advanced experimental approaches can be employed:

A. Phosphoproteomic Analysis:

• Sample Preparation:

  • Isolate proteins from plant tissues under different conditions (e.g., control vs. stress)

  • Reduce and alkylate proteins with DTT and iodoacetamide

  • Precipitate proteins using methods like the 2D Clean-up Kit

  • Reconstitute in ammonium bicarbonate and digest with Trypsin overnight at 37°C

  • Perform a second Trypsin digestion in 80% Acetonitrile

• Phosphopeptide Enrichment:

  • Use titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC)

  • Enrich phosphopeptides prior to LC-MS/MS analysis

• LC-MS/MS Analysis:

  • Inject approximately 1.5μg protein into UPLC system

  • Separate peptides using a gradient increasing from 0-40% acetonitrile

  • Analyze using tandem mass spectrometry

  • Process data using software like MaxQuant for phosphosite identification

B. In Vitro Phosphorylation Assays:

• Express recombinant At5g16590 kinase domain

• Perform in vitro kinase assays with:

  • [γ-32P]ATP to detect autophosphorylation

  • Potential substrates to assess kinase activity

  • Known upstream kinases to identify regulatory phosphorylation sites

C. Phospho-specific Antibodies:

• Generate antibodies against predicted phosphorylation sites

• Use Western blotting to detect phosphorylation status in different conditions

• Employ immunoprecipitation followed by mass spectrometry for validation

One identified phosphopeptide from At5g16590 is "LIEEVSRSPAS(ph)PGPLSD" , which provides a starting point for targeted phosphorylation studies.

How can protein-protein interactions involving At5g16590 be investigated using contemporary methods?

Investigating protein-protein interactions involving At5g16590 requires multiple complementary approaches:

A. Yeast-Based Methods:

• Yeast Two-Hybrid (Y2H):

  • Clone At5g16590 into bait vector and potential interactors into prey vectors

  • Transform into yeast and select for interaction-dependent reporter activation

  • Perform quantitative β-galactosidase assays to assess interaction strength

• Split-Ubiquitin System:

  • Particularly useful for membrane proteins like At5g16590

  • Fuse N-terminal and C-terminal ubiquitin fragments to potential interacting proteins

  • Interaction reconstitutes ubiquitin and releases a transcription factor

B. In Planta Methods:

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse potential interacting proteins with split YFP/GFP fragments

  • Transform into Arabidopsis protoplasts or tobacco leaves

  • Visualize fluorescence using confocal microscopy to confirm interaction

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of At5g16590 in plants

  • Immunoprecipitate protein complexes using antibodies against the tag

  • Identify interacting partners by Western blot or mass spectrometry

• Proximity-Dependent Labeling:

  • Fuse At5g16590 with BioID or TurboID

  • Identify nearby proteins through biotinylation and streptavidin pulldown

  • Analyze by mass spectrometry

C. In Vitro Methods:

• Pull-Down Assays:

  • Express recombinant At5g16590 with affinity tags

  • Incubate with plant extracts or purified proteins

  • Identify binding partners through Western blot or mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize purified At5g16590 on a sensor chip

  • Flow potential interacting proteins over the surface

  • Measure kinetics and affinity of interactions

What methodological approaches can determine why At5g16590 is classified as a probable inactive receptor kinase?

Several methodological approaches can help determine why At5g16590 is classified as a probable inactive receptor kinase:

A. Sequence Analysis of Catalytic Motifs:

• Examine critical catalytic residues in the kinase domain:

  • ATP-binding site (G-loop)

  • Catalytic loop (HRD motif)

  • Activation loop (DFG motif)

  • Metal-binding sites

• Compare with active kinases to identify deviations

B. Structural Biology Approaches:

• X-ray crystallography:

  • Express and purify the kinase domain

  • Crystallize under various conditions

  • Collect diffraction data and solve structure

  • Compare with active kinase structures

• Cryo-electron microscopy:

  • Analyze the 3D structure in near-native conditions

  • Identify structural features that would prevent catalysis

C. Biochemical Activity Assays:

• In vitro kinase assays:

  • Express recombinant kinase domain

  • Test activity using [γ-32P]ATP

  • Compare with active kinases and known pseudokinases

• ATP binding assays:

  • Thermal shift assays with ATP or ATP analogues

  • Isothermal titration calorimetry

D. Molecular Dynamics Simulations:

• Build computational models of the kinase domain

• Simulate protein dynamics to assess catalytic competence

• Compare with simulations of active kinases

Research on inactive TrkA kinase domain suggests that features related to the αC-helix positioning and dimer formation could be relevant to At5g16590's inactivity. The study states: "symmetrical dimers of the inactive TrkA TKD resembling those found in other RTKs, possibly reflecting an arrangement of kinase domains in a pre-formed TrkA dimer."

How does At5g16590 function in the abscisic acid signaling pathway?

To investigate At5g16590's role in abscisic acid (ABA) signaling, the following research approaches are recommended:

A. Genetic Analysis:

• Characterize phenotypes of mutant lines under ABA treatment:

  • Compare lrr1-1 and lrr1-2 T-DNA insertion lines with wild-type plants

  • Analyze overexpression lines (LRR1ox2, LRR1ox10)

  • Measure germination rates, root growth, and stomatal responses

• Genetic interaction studies:

  • Create double mutants with known ABA signaling components

  • Analyze epistatic relationships to place At5g16590 in the pathway

B. Molecular Response Analysis:

• Transcriptional profiling:

  • Perform RNA-seq or microarray analysis of wild-type vs. mutants

  • Compare ABA-responsive gene expression patterns

  • Identify differentially regulated pathways

• Phosphoproteomics:

  • Analyze phosphorylation changes after ABA treatment

  • Compare wild-type vs. mutant phosphorylation patterns

C. Biochemical Interaction Studies:

• Identify direct interactions with ABA signaling components:

  • Pull-down assays with known ABA receptors (PYR/PYL/RCAR)

  • Co-IP with SnRK2 kinases or PP2C phosphatases

  • Y2H screens for novel interactors

• Reconstitution assays:

  • Express components in heterologous systems

  • Test if At5g16590 modulates ABA-dependent interactions

D. Cell Biology Approaches:

• Subcellular localization studies:

  • GFP fusion proteins to track localization changes after ABA treatment

  • Co-localization with known ABA signaling components

• FRET/FLIM analysis:

  • Measure protein-protein interactions in vivo

  • Assess dynamic changes in response to ABA

The involvement of At5g16590 in the ABA signaling pathway was investigated using Arabidopsis thaliana as a model plant, with stress induced by administering abscisic acid .

What are the optimal methodologies for recombinant expression and purification of At5g16590?

For optimal recombinant expression and purification of At5g16590, several approaches can be employed:

A. Bacterial Expression Systems:

• E. coli expression:

  • Clone the coding sequence into pET vectors with appropriate tags (His, GST, MBP)

  • Transform into expression strains like BL21(DE3), Rosetta, or Arctic Express

  • Optimize expression conditions (temperature, IPTG concentration, induction time)

  • For membrane proteins, consider specific E. coli strains designed for membrane protein expression

• Purification strategy:

  • Lysis using French press or sonication in appropriate buffers

  • Affinity chromatography using the protein tag

  • Size exclusion chromatography for further purification

  • Ion exchange chromatography if needed

B. Gateway Cloning System for Flexibility:

• Create Gateway entry clones:

  • Clone the sequence into pDONR/zeo vector

  • Generate different constructs (full-length, kinase domain only, extracellular domain only)

• Transfer to expression vectors:

  • Use LR Clonase reaction to transfer into destination vectors

  • Choose vectors based on expression system and application

C. Eukaryotic Expression Systems:

• Insect cell expression:

  • Use baculovirus expression systems for better post-translational modifications

  • Clone into vectors like pFastBac with secretion signals

  • Express in Sf9 or High Five cells

• Plant-based expression:

  • Use Agrobacterium-mediated transient expression in Nicotiana benthamiana

  • For stable expression, transform Arabidopsis or tobacco

  • Extract using plant protein extraction buffers with appropriate detergents

D. Cell-Free Expression Systems:

• Wheat germ or rabbit reticulocyte lysate systems:

  • Useful for rapid screening of expression constructs

  • Good for proteins toxic to host cells

According to search results, a recombinant full-length Arabidopsis thaliana probable inactive receptor kinase At5g16590 protein, His-tagged, expressed in E. coli is commercially available , indicating successful expression in bacterial systems.

What advanced techniques can be used to determine the 3D structure of At5g16590?

Determining the 3D structure of At5g16590 requires several advanced techniques:

A. X-ray Crystallography:

• Protein production and purification:

  • Express domains separately (extracellular domain, kinase domain)

  • Ensure high purity (>95%) and homogeneity

  • Remove flexible regions that may hinder crystallization

• Crystallization screening:

  • Set up crystallization trials with commercial screens

  • Optimize promising conditions (pH, salt, precipitant)

  • Consider co-crystallization with ligands or interacting proteins

• Data collection and structure determination:

  • Collect diffraction data at synchrotron radiation facilities

  • Process data and solve structure using molecular replacement or experimental phasing

  • Build and refine the model

B. Cryo-Electron Microscopy:

• Sample preparation:

  • Prepare protein in detergent micelles or nanodiscs for membrane proteins

  • Optimize buffer conditions and protein concentration

  • Vitrify samples on grids

• Data acquisition:

  • Collect images using high-end cryo-EM equipment

  • Process data using motion correction and CTF estimation

• Image processing and 3D reconstruction:

  • Perform particle picking and 2D classification

  • Generate initial 3D models and refine

  • Build atomic models into the density map

C. Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Isotope labeling:

  • Express protein with 15N, 13C, and/or 2H labels

  • Purify labeled protein to high homogeneity

• Spectrum acquisition:

  • Collect multidimensional NMR data

  • Assign backbone and side-chain resonances

• Structure calculation:

  • Derive distance and angular constraints

  • Calculate ensemble of structures

D. Integrative Structural Biology Approaches:

• Combine multiple techniques:

  • Small-angle X-ray scattering (SAXS)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Cross-linking mass spectrometry (XL-MS)

  • Computational modeling

• Homology modeling:

  • Use structures of related LRR-RLKs as templates

  • Validate models using experimental data

Research on related proteins suggests the LRR domains likely form a horseshoe-shaped solenoid structure, while the kinase domain would adopt a typical protein kinase fold with N-lobe (mainly β-sheets) and C-lobe (mainly α-helices) .

How can researchers investigate At5g16590's role in zinc homeostasis?

To investigate At5g16590's role in zinc homeostasis, researchers can implement the following methodological approaches:

A. Transcriptional Response Analysis:

• Gene expression studies:

  • Conduct time-course experiments with varying zinc concentrations

  • Perform qRT-PCR to measure At5g16590 expression

  • Compare with known zinc homeostasis genes

• Global transcriptome analysis:

  • Use RNA-seq to identify co-regulated genes

  • Compare wild-type vs. At5g16590 mutants under zinc deficiency/sufficiency

B. Proteome and Phosphoproteome Analysis:

• Dynamic protein response:

  • Design time-course experiments with zinc resupply (10min, 30min, etc.)

  • Extract proteins from different cellular fractions

  • Analyze protein abundance changes

• Phosphorylation dynamics:

  • Investigate phosphorylation changes in response to zinc

  • Compare wild-type vs. mutant phosphorylation patterns

C. Physiological Characterization:

• Zinc content analysis:

  • Measure zinc levels in different tissues using ICP-MS

  • Compare wild-type vs. mutant plants under various zinc conditions

• Growth phenotypes:

  • Analyze root and shoot growth under zinc deficiency/excess

  • Examine zinc deficiency symptoms in mutants

D. Protein Interaction Network:

• Identify interactions with zinc transporters:

  • Perform Y2H or Co-IP with ZIP and CDF transporters

  • Investigate the effect of zinc on these interactions

• Signaling pathway analysis:

  • Map the signaling cascade from zinc perception to response

  • Identify downstream targets using phosphoproteomics

Research has shown that LRR1 is "repeatedly one of the most highly responding kinases at 10, 30min and Zn sufficiency" , suggesting its importance in early zinc sensing or signaling. The study used a dynamic approach to zinc resupply, analyzing protein responses at multiple time points (10min, 30min, 120min, 480min) after reintroducing zinc to zinc-deficient plants.

How can genetic and phenotypic approaches be combined to elucidate At5g16590 function?

Combining genetic and phenotypic approaches provides powerful insights into At5g16590 function:

A. Comprehensive Mutant Analysis:

• Characterize multiple alleles:

  • Compare T-DNA insertion lines (lrr1-1, lrr1-2)

  • Generate CRISPR/Cas9 knockouts for precise gene editing

  • Create domain-specific mutations (e.g., kinase-dead versions)

• Develop expression lines:

  • Overexpression under constitutive promoters

  • Tissue-specific expression using appropriate promoters

  • Inducible expression systems for temporal control

B. Multilevel Phenotyping:

• High-throughput phenotyping:

  • Growth rate analysis under various conditions

  • Root architecture phenotyping (primary root length, lateral root number)

  • Automated imaging systems for continuous monitoring

• Stress response characterization:

  • Test responses to hormones (ABA, auxin, cytokinin)

  • Examine abiotic stress tolerance (drought, salt, oxidative stress)

  • Assess biotic stress responses (pathogen resistance)

• Cellular and subcellular phenotyping:

  • Cell-type specific markers to examine developmental effects

  • Live-cell imaging to track dynamic processes

C. Integration with -Omics Data:

• Connect genotype to molecular phenotypes:

  • Transcriptome analysis of mutants

  • Proteome and metabolome profiling

  • Epigenetic modifications

• Network analysis:

  • Construct gene regulatory networks

  • Identify key hubs and modules affected by At5g16590

D. Advanced Genetic Approaches:

• Suppressor screens:

  • Mutagenize At5g16590 mutants and screen for phenotypic reversion

  • Identify genetic interactors

• Synthetic lethality/enhancement screens:

  • Generate double mutants with genes in related pathways

  • Identify genetic interactions

• QTL analysis:

  • Use natural variation and AI-RIL populations

  • Map QTLs related to At5g16590 function

The combination of these approaches provides multiple lines of evidence for gene function, allowing researchers to build a comprehensive understanding of At5g16590's role in plant biology.