Recombinant Physcomitrella patens subsp. patens Oxygen-evolving enhancer protein 2

What is Oxygen-evolving enhancer protein 2 (OEE2) in Physcomitrella patens and what is its function?

Oxygen-evolving enhancer protein 2 (OEE2), also called PsbQ2, is a nuclear-encoded chloroplast protein that binds to photosystem II (PSII) on the luminal side of the thylakoid membrane in Physcomitrella patens. OEE2 is one of three subunits of the oxygen-evolving complex (OEC), along with OEE1 (PsbO) and OEE3 (PsbQ) . This protein plays a crucial role in stabilizing the complex that catalyzes the photolysis of water, which is the first step in non-cyclic electron transport during photosynthesis . In this process, water molecules are oxidized to produce oxygen, protons, and electrons that enter the photosynthetic electron transport chain. Beyond its photosynthetic function, recent evidence suggests OEE2 may also function in stress responses, particularly in pathogen interactions .

How does OEE2 in P. patens differ from its counterparts in vascular plants?

While the photosynthetic machinery is largely conserved across plant lineages, there are several notable differences in OEE2 between P. patens and vascular plants:

P. patens, as an evolutionarily basal model system, offers unique insights into the ancestral functions of photosynthetic proteins like OEE2. Unlike vascular plants, P. patens responds to 12-Oxo-phytodienoic acid (OPDA) but not jasmonic acid (JA), suggesting different evolutionary trajectories for these signaling pathways .

How is recombinant P. patens OEE2 typically produced for research purposes?

Production of recombinant P. patens OEE2 typically follows these methodological steps:

• Gene isolation: The OEE2 coding sequence is amplified from P. patens genomic DNA or cDNA using PCR with specific primers designed based on the annotated gene sequence.

• Expression vector construction: The amplified OEE2 sequence is cloned into an appropriate expression vector, often containing tags (His, FLAG, etc.) for purification and detection purposes.

• Transformation: The construct can be expressed in various systems including:

  • E. coli bacterial expression systems

  • Yeast expression systems

  • Plant expression systems (including P. patens itself, which has high homologous recombination efficiency )

• Protein purification: Typically performed using affinity chromatography based on the fusion tag, followed by size exclusion chromatography.

When expressing the protein for functional studies, researchers should consider removing the native chloroplast transit peptide to improve expression efficiency while maintaining the functional domains.

What are the optimal conditions for expressing and purifying recombinant P. patens OEE2?

The optimal conditions for recombinant P. patens OEE2 expression and purification depend on the expression system, but generally follow these guidelines:

Bacterial Expression (E. coli):

• Expression strain: BL21(DE3) or Rosetta for improved eukaryotic codon usage

• Induction: 0.1-0.5 mM IPTG at 18-22°C overnight (to minimize inclusion body formation)

• Lysis buffer: 50 mM Tris-HCl (pH 7.5-8.0), 300 mM NaCl, 10% glycerol, 1 mM EDTA, protease inhibitors

• Purification: Ni-NTA chromatography for His-tagged proteins with increasing imidazole gradient

P. patens Expression System:

• Transformation: Utilize P. patens high homologous recombination efficiency (approximately 90%)

• Selection: Antibiotic resistance markers (hygromycin, G418)

• Extraction: Gentle lysis to preserve protein structure using non-ionic detergents

• Purification: Affinity chromatography followed by ion exchange chromatography

The purification strategy should be optimized to maintain the structural integrity of OEE2, particularly if downstream functional assays are planned. For proper folding, consider including stabilizing agents like glycerol (10-15%) and reducing agents like DTT or β-mercaptoethanol in purification buffers.

How can I verify the functionality of recombinant P. patens OEE2 protein in vitro?

Verifying the functionality of recombinant P. patens OEE2 requires assessing both its structural integrity and biological activity:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Size exclusion chromatography to verify oligomerization state

  • Thermal shift assays to assess protein stability

• Functional assays:

  • Oxygen evolution measurements using Clark-type electrodes with isolated thylakoid membranes

  • Reconstitution assays with PSII core complexes lacking OEE2

  • Binding assays to verify interaction with other PSII components

• Interaction studies:

  • Pull-down assays to confirm binding to known partners like other PSII components

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Bimolecular fluorescence complementation (BiFC) assays as demonstrated with LtGAPR1 and NbPsbQ2

A fully functional recombinant OEE2 should restore oxygen evolution activity in OEE2-depleted PSII preparations and demonstrate proper binding to its interaction partners.

What techniques are effective for studying OEE2 interactions with other proteins in P. patens?

Several complementary techniques have proven effective for studying OEE2 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Demonstrated successfully for identifying OEE2 interactions with proteins like LtGAPR1

  • Can be performed using antibodies against native OEE2 or epitope tags on recombinant proteins

  • Western blotting validates interactions with specific proteins of interest

• Bimolecular Fluorescence Complementation (BiFC):

  • Allows visualization of protein interactions in planta

  • Has been used to show OEE2 interactions at the periphery of chloroplasts

  • Requires fusion of split YFP fragments to potential interaction partners

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Powerful for identifying novel interaction partners

  • Successfully identified OEE2 as interacting with fungal effector proteins

  • Requires careful controls to eliminate false positives

• Yeast Two-Hybrid (Y2H):

  • Useful for initial screening of potential interactions

  • May require domain-specific constructs to avoid issues with transmembrane domains

  • Should be validated with in planta techniques

For optimal results, researchers should employ multiple complementary techniques, as each has specific strengths and limitations. When designing interaction studies, consider the subcellular localization of OEE2 at the periphery of the chloroplast when interpreting results .

How does OEE2 respond to stress conditions in P. patens, and how does this differ from vascular plants?

OEE2 in P. patens shows distinctive responses to stress conditions compared to vascular plants:

The unique aspect of P. patens stress response is that while OPDA (12-oxo-phytodienoic acid) is induced by wounding and pathogen infection, P. patens does not produce jasmonic acid (JA) in response to these stresses . This suggests OEE2 in P. patens operates under a more ancestral stress signaling network. Proteomic analysis reveals that when OPDA levels increase following wounding, OEE2 is one of the few proteins that shows increased abundance in protonemata , indicating its importance in the stress response pathway unique to this bryophyte.

What role does OEE2 play in the defense responses of P. patens against pathogens?

OEE2 plays a multifaceted role in P. patens defense responses against pathogens:

• Effector target: OEE2 (PsbQ2) has been identified as a target of pathogen effectors, such as LtGAPR1 from Lasiodiplodia theobromae . This suggests that pathogens may target OEE2 to manipulate host defense responses.

• ROS modulation: The interaction between pathogen effectors and OEE2 affects reactive oxygen species (ROS) production . Since OEE2 is involved in photosynthetic water splitting, changes in its function can directly impact ROS levels and signaling.

• OPDA-mediated responses: Upon pathogen infection, P. patens produces OPDA, which affects OEE2 abundance . This integration with OPDA signaling links OEE2 to broader defense response networks.

• Cell wall reinforcement: P. patens activates reinforcement of the cell wall after pathogen assault . OEE2's involvement in stress responses suggests it may contribute to this process, possibly through signaling pathways that activate phenolic compound incorporation into cell walls.

Research demonstrates that when PsbQ2 (OEE2) was overexpressed in Nicotiana benthamiana, it reduced susceptibility to L. theobromae, while silencing enhanced pathogen infection . This strongly suggests OEE2 plays a direct role in resistance against fungal pathogens, possibly through modulation of photosynthesis-derived signals or metabolites that influence defense responses.

How can genetic manipulation of OEE2 in P. patens inform our understanding of evolutionary aspects of photosynthesis?

Genetic manipulation of OEE2 in P. patens offers unique insights into the evolution of photosynthesis for several reasons:

• Evolutionary position: P. patens represents an evolutionarily basal land plant, providing a window into ancestral photosynthetic mechanisms . The P. patens genome has been completely sequenced, facilitating comparative genomic analyses .

• Homologous recombination efficiency: P. patens has a remarkably high frequency of homologous recombination compared to flowering plants , making it an excellent model for targeted gene disruption and functional studies.

• Methodological approaches for evolutionary research:

  a. Gene knockout/knockdown studies:

  • CRISPR/Cas9 system for precise gene editing

  • RNAi for specific knockdown

  • Homologous recombination for gene replacement

  b. Complementation experiments:

  • Expressing OEE2 from various evolutionary lineages in P. patens OEE2-deficient mutants

  • Analyzing functional conservation and divergence

  c. Domain swapping experiments:

  • Creating chimeric proteins combining domains from OEE2 of different species

  • Identifying functionally important regions that diverged during evolution

• Photosynthesis-defense signaling nexus: Studies manipulating OEE2 in P. patens can reveal how photosynthetic machinery became integrated with defense signaling pathways during land plant evolution. The unique OPDA-responsive (but JA-independent) signaling in P. patens represents an evolutionary intermediate that can be explored through OEE2 manipulation.

Genetic manipulation experiments should be designed with consideration for the dual roles of OEE2 in photosynthesis and stress responses, potentially uncovering how these functions co-evolved during land plant diversification.

What proteomics approaches are most effective for studying OEE2 post-translational modifications in P. patens?

Several specialized proteomics approaches are particularly effective for studying post-translational modifications (PTMs) of OEE2 in P. patens:

• Enrichment strategies for specific PTMs:

  • Phosphorylation: Titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC)

  • Oxidation: Biotin-switch technique for redox modifications

  • Glycosylation: Lectin affinity chromatography or hydrazide chemistry

• Mass spectrometry methodologies:

  • High-resolution LC-MS/MS using Orbitrap or Q-TOF instruments

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for preserving labile PTMs

  • Parallel reaction monitoring (PRM) for targeted quantification of modified peptides

• Data analysis considerations:

  • Search against P. patens-specific protein databases

  • Include variable modifications relevant to stress conditions (oxidation, phosphorylation)

  • Apply appropriate false discovery rate controls

A comparative proteomics approach was effectively used in previous studies of P. patens, revealing that OPDA treatment affected protein abundance patterns . These gel-free/label-free proteomic techniques can be adapted specifically for OEE2 PTM analysis by incorporating enrichment steps and targeted data acquisition strategies.

How can I interpret contradictory results when studying OEE2 function across different developmental stages of P. patens?

Interpreting contradictory results regarding OEE2 function across different P. patens developmental stages requires systematic analysis:

• Stage-specific biology considerations:

  • Research has shown that OPDA affects different proteins in protonemata versus gametophores

  • OEE2 may have stage-specific roles and regulatory mechanisms

  • The impact of OPDA on rhizoid development varies from its effects on protonemata

• Methodological reconciliation framework:
  a. Assess experimental conditions:

  • Growth conditions (light intensity, media composition)

  • Tissue collection timing and procedures

  • Protein extraction methods optimized for each tissue type

  b. Validate with complementary techniques:

  • Combine proteomics with transcriptomics

  • Verify protein localization in different tissues

  • Perform developmental stage-specific genetic manipulations

  c. Control for developmental markers:

  • Include known stage-specific proteins as internal controls

  • Monitor key developmental regulators alongside OEE2

• Integrative data analysis approach:

  • Use statistical methods that account for stage-specific variance

  • Apply multivariate analysis to identify patterns across experimental datasets

  • Consider network analysis to place contradictory results in biological context

A recent study highlighted that "a subset of the physiological responses caused by OPDA is shown to differ between protonema and gametophore developmental stages" . This demonstrates that contradictory results may actually reflect genuine biological differences rather than experimental artifacts. Researchers should design experiments that specifically address these stage-dependent differences rather than attempting to resolve them as contradictions.

What techniques can detect subtle changes in OEE2 activity during early stress responses in P. patens?

Detecting subtle changes in OEE2 activity during early stress responses requires highly sensitive techniques:

• Real-time activity monitoring:

  • Polarographic oxygen measurements using Clark-type electrodes

  • Chlorophyll fluorescence imaging (PAM fluorometry) to detect PSII efficiency changes

  • Thylakoid membrane electrochromic shift (ECS) measurements for proton motive force analysis

• Rapid sampling approaches:

  • Flash-freezing tissues at precise time points (seconds to minutes after stress)

  • Microfluidic devices for controlled stress application and sampling

  • Single-cell analysis techniques for spatial resolution of responses

• Molecular probes and sensors:

  • Redox-sensitive fluorescent proteins fused to OEE2 or interacting partners

  • FRET-based biosensors for detecting conformational changes

  • Activity-based protein profiling for detecting functional state changes

• Data analysis considerations:

  • Kinetic modeling of time-course data

  • Signal deconvolution to separate overlapping responses

  • Machine learning approaches for pattern recognition in complex datasets

When studying early stress responses, researchers should consider that OPDA accumulation begins within 2 hours of stress application , with differential expression of stress-responsive genes like LOX and AOS occurring on a similar timescale. Experimental designs should include sampling points before and during this initial response window to capture the earliest changes in OEE2 activity or abundance.

How might CRISPR-Cas9 gene editing be optimized for studying OEE2 function in P. patens?

CRISPR-Cas9 gene editing can be optimized for studying OEE2 function in P. patens through these methodological approaches:

• Guide RNA design considerations:

  • Target unique regions of OEE2 to prevent off-target effects

  • Design multiple gRNAs targeting different exons

  • Use P. patens-specific codon optimization for Cas9 expression

  • Consider targeting regulatory elements for expression modulation rather than complete knockout

• Delivery optimization:

  • Utilize PEG-mediated protoplast transformation, leveraging P. patens' high homologous recombination efficiency

  • Include appropriate selection markers (hygromycin, G418)

  • Optimize DNA concentration and protoplast regeneration conditions

  • Consider ribonucleoprotein (RNP) delivery to reduce off-target effects

• Specific editing strategies:

  • Knock-in approaches:

    • Insert fluorescent tags for live imaging

    • Introduce specific mutations to study PTM sites

    • Create domain swaps with other species' OEE2 orthologs

  • Promoter modifications:

    • Replace native promoter with inducible systems

    • Introduce tissue-specific promoters to study stage-specific functions

• Validation strategies:

  • PCR and sequencing to confirm edits

  • Proteomic verification of protein abundance changes

  • Functional assays as described in previous sections

  • Phenotypic analysis across multiple developmental stages

Given P. patens' unique position as a model bryophyte with efficient homologous recombination , CRISPR editing can be particularly effective when combined with homology-directed repair templates, allowing precise modifications that would be challenging in other plant systems.

What are the most promising approaches for studying the intersection of OEE2 function with OPDA signaling pathways?

The intersection of OEE2 function with OPDA signaling pathways represents a frontier in P. patens research, with several promising approaches:

• Genetic perturbation strategies:

  • Create OEE2 mutants with modified OPDA-responsive elements

  • Generate double mutants with both OEE2 and OPDA biosynthesis genes disrupted

  • Develop inducible OEE2 expression systems to temporally control its presence during OPDA responses

• Proximity-based interaction studies:

  • BioID or TurboID proximity labeling to identify proteins near OEE2 during OPDA treatment

  • In situ crosslinking to capture transient interactions during signaling events

  • Split ubiquitin systems to detect membrane-associated protein interactions

• Metabolic flux analysis:

  • Track isotope-labeled OPDA to identify metabolic changes dependent on OEE2

  • Measure changes in photosynthetic electron transport upon OPDA treatment in WT vs. OEE2 mutants

  • Analyze redox metabolite profiles to connect photosynthetic function with signaling

• Spatiotemporal signaling dynamics:

  • Real-time fluorescent reporters for OPDA-responsive promoters

  • Live-cell imaging of OEE2 localization during stress responses

  • Correlation of chloroplast ROS production with OPDA signaling events

Previous research has established that OPDA increases in P. patens tissues after fungal inoculation and that OPDA treatment specifically increases OEE2 abundance in protonemata . This suggests a direct link between OPDA signaling and OEE2 function, possibly representing an evolutionarily ancient stress response mechanism that predates JA signaling in vascular plants .

How can systems biology approaches integrate OEE2 function into broader photosynthetic and stress response networks in P. patens?

Systems biology approaches offer powerful frameworks for integrating OEE2 function into broader biological networks:

• Multi-omics data integration:

  • Combine proteomics, transcriptomics, and metabolomics data from OEE2 studies

  • Use correlation networks to identify genes/proteins with similar expression patterns

  • Develop P. patens-specific gene regulatory networks focusing on photosynthesis and stress responses

• Mathematical modeling approaches:

  • Develop kinetic models of PSII function incorporating OEE2 dynamics

  • Create Bayesian networks to predict stress response outcomes based on OEE2 status

  • Use flux balance analysis to connect photosynthetic output with defense metabolite production

• Network perturbation analysis:

  • Systematically disrupt network components to assess effects on OEE2 function

  • Apply network medicine approaches to identify critical nodes connecting photosynthesis to defense

  • Develop and validate predictive models through iterative experimentation

• Experimental validation strategies:

  • High-throughput phenotyping of OEE2 and related mutants under diverse stress conditions

  • Targeted metabolic engineering to test predicted network dependencies

  • Cross-species complementation studies to test network conservation

Research has shown that OEE2 connects multiple biological processes, including photosynthesis, pathogen defense , and responses to OPDA . Systems approaches could reveal how this protein serves as a nexus between these processes, potentially identifying evolutionary conserved regulatory mechanisms that could be leveraged for crop improvement.

By integrating these diverse data types, researchers can develop a comprehensive understanding of OEE2's multifaceted roles in P. patens biology.