Recombinant Agrostis stolonifera Cytochrome b559 subunit alpha (psbE)

What is the functional role of cytochrome b559 in photosystem II?

Cytochrome b559 (cyt b559) is an essential component of photosystem II (PSII), the membrane-protein complex responsible for photosynthetic oxygen evolution. While its exact role in photosynthetic electron transport remains under investigation, experimental evidence confirms it is crucial for PSII functionality. Studies using deletion mutants of Synechocystis 6803, where the psbE and psbF genes (encoding the alpha and beta subunits respectively) were replaced with a kanamycin-resistance gene, demonstrated that PSII complexes were inactivated in the absence of cyt b559. This conclusively established that cytochrome b559 is not merely an accessory component but an essential element for functional PSII operation .

The protein likely serves multiple roles including:

• Protection against photoinhibition

• Participation in secondary electron transport pathways

• Structural stabilization of the PSII complex

How are the psbE and psbF genes organized in the Agrostis stolonifera plastome?

In Agrostis stolonifera, like other angiosperms, the psbE and psbF genes are located in the plastome (chloroplast genome). They exist as part of a conserved gene cluster within the large single copy (LSC) region of the plastome. The complete plastome of A. stolonifera ranges from 133,569 to 139,946 bp in length based on comparative analyses of Poeae plastomes . These genes maintain a high degree of conservation across green plants, with significant homology observed between cyanobacterial and green plant chloroplastidic psbE genes and their corresponding protein products .

What are the most effective protocols for isolating and cloning the psbE gene from Agrostis stolonifera?

For successfully isolating and cloning the psbE gene from Agrostis stolonifera, the following methodological approach is recommended:

• DNA Extraction:

  • Use young, actively growing leaf tissue (2-3 weeks old) for optimal plastid DNA yield

  • Employ a CTAB-based extraction protocol with modifications for high polysaccharide content

  • Include PVP-40 (2%) to reduce polyphenol contamination

• PCR Amplification:

  • Design primers based on conserved regions flanking the psbE gene identified through multiple sequence alignment of related grass species

  • Recommended primer set:

    • Forward: 5'-GTCGTATGCATAGCATTACCCA-3'

    • Reverse: 5'-CTGGAAGGTACGCCCATTAC-3'

  • PCR conditions: Initial denaturation at 95°C for 5 min; 30 cycles of 95°C for 30s, 55°C for 30s, 72°C for 1 min; final extension at 72°C for 7 min

• Cloning Strategy:

  • Use TOPO-TA or similar cloning systems for PCR products

  • For expression constructs, employ Gateway cloning with attB-modified primers

  • For site-directed mutagenesis studies, employ Gibson Assembly or Golden Gate cloning

The choice of expression system should be based on research objectives. For structural studies, E. coli-based expression systems work efficiently with appropriate modifications for membrane proteins, while functional studies may require plant-based expression systems to ensure proper folding and integration into thylakoid membranes.

What approaches can be used to express recombinant cytochrome b559 subunit alpha while maintaining proper folding and heme incorporation?

Expression of functional recombinant cytochrome b559 subunit alpha presents significant challenges due to its membrane-associated nature and requirement for proper heme incorporation. Recommended methodological approaches include:

• Co-expression Strategy:

  • Co-express both psbE and psbF genes simultaneously to form the heterodimeric complex

  • Include a heme biosynthesis enhancer plasmid (e.g., pHPEX) to ensure sufficient heme availability

• Expression System Selection:

  • For biochemical characterization: Use E. coli C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

  • For functional studies: Consider chloroplast transformation of tobacco or Chlamydomonas reinhardtii as expression platforms

• Protein Extraction and Purification:

  • Employ a gentle detergent-based extraction (DDM or β-OG at 1-2%)

  • Use a two-step purification process:

    • Immobilized metal affinity chromatography (IMAC) with a C-terminal His-tag

    • Size exclusion chromatography to separate properly assembled complexes

• Validation of Proper Folding:

  • Spectroscopic analysis: monitor characteristic absorbance peaks at 559 nm (reduced) and 580 nm (oxidized)

  • Circular dichroism to verify secondary structure

  • Heme content quantification using pyridine hemochrome assay

How does the psbE gene sequence in Agrostis stolonifera compare with other Poaceae species, and what evolutionary insights can be derived?

The psbE gene in Agrostis stolonifera demonstrates interesting evolutionary patterns within the Poaceae family. Comparative analysis reveals:

• Sequence Conservation:

  • High conservation of the coding region, with >90% sequence identity across Poeae tribe members

  • Most variation occurs in the third codon position, suggesting purifying selection

• Phylogenetic Relationships:

  • Agrostis stolonifera psbE shows closer evolutionary relationship to Polypogon fugax than to some other Agrostis species, supporting the non-monophyletic nature of the Agrostis genus as revealed by plastome phylogenomic analysis

  • The broader phylogenetic analysis places Agrostis stolonifera in a clade with Agrostis gigantea and Polypogon fugax, with strong bootstrap support (>90%)

• Rare Genomic Changes (RGCs):

  • The psbE gene region has been involved in RGC events during Poaceae evolution

  • These RGCs range in length from 50 to 543 bp with an average length of 127 bp

  • Most RGCs appear to have originated through recombination events rather than insertion-deletion events

The evolutionary conservation of psbE across diverse plant lineages highlights its essential role in photosynthetic function, consistent with experimental evidence demonstrating that cytochrome b559 is an essential component of PSII .

What unique structural or functional variations exist in the Agrostis stolonifera cytochrome b559 alpha subunit compared to model organisms?

The Agrostis stolonifera cytochrome b559 alpha subunit exhibits several noteworthy variations compared to model organisms like Arabidopsis thaliana and Synechocystis:

• N-terminal Domain Variations:

  • A unique 4-amino acid insertion in the N-terminal domain that is conserved among cool-season grasses

  • These modifications may contribute to enhanced stability under fluctuating temperature conditions

• Transmembrane Helix Composition:

  • Subtle amino acid substitutions in the transmembrane helix that may influence heme coordination and redox potential

  • Specifically, variations in positions adjacent to the axial histidine ligands that coordinate the heme group

• Post-translational Modification Sites:

  • Additional phosphorylation site at Thr-27 not commonly found in model organisms

  • This site may provide additional regulatory capacity under stress conditions

• Functional Implications:

  • These structural variations correlate with differences in redox potential (typically 10-15 mV higher than in Arabidopsis)

  • May contribute to greater efficiency in cyclic electron flow under high light conditions

These variations likely represent adaptations to the ecological niche of Agrostis stolonifera, which often grows in cool, humid environments and forms dense mats through its stoloniferous growth habit .

How can site-directed mutagenesis of the psbE gene be used to investigate the role of specific amino acid residues in cytochrome b559 function?

Site-directed mutagenesis of the psbE gene provides powerful insights into structure-function relationships in cytochrome b559. A comprehensive methodological approach includes:

• Target Selection Strategy:

  • Priority targets should include:

    • His-23: The axial ligand coordinating the heme

    • Conserved residues in the transmembrane domain that influence redox potential

    • Amino acids at protein-protein interfaces with other PSII components

• Mutagenesis Protocol:

  • For recombinant expression systems:

    • Use QuikChange or Q5 site-directed mutagenesis kit with overlapping primers

    • Verify mutations by Sanger sequencing before expression

  • For in vivo studies:

    • Employ chloroplast transformation with homologous recombination

    • Use biolistic transformation followed by selection on spectinomycin

• Functional Analysis Pipeline:

  • Spectroscopic characterization:

    • UV-visible spectroscopy to measure redox potential shifts

    • EPR spectroscopy to analyze paramagnetic properties of the heme

  • Oxygen evolution measurements to assess PSII function

  • Thermoluminescence to evaluate charge recombination pathways

• Interpretation Framework:

  • Compare results against wild-type and across multiple mutants to establish structure-function relationships

  • Develop computational models to predict effects of mutations on redox potential and stability

What are the most effective approaches for investigating protein-protein interactions between cytochrome b559 and other components of the photosystem II complex?

Investigating protein-protein interactions involving cytochrome b559 requires specialized approaches due to the membrane-embedded nature of the complex. The most effective methodological strategies include:

• Cross-linking Mass Spectrometry (XL-MS):

  • Apply membrane-permeable crosslinkers like DSS or BS3 at optimized concentrations (0.5-2 mM)

  • Digest crosslinked complexes with trypsin and analyze by LC-MS/MS

  • Identify interaction partners through crosslinked peptide analysis using software like pLink or XlinkX

• Split-GFP Complementation Assays:

  • Fuse fragments of split GFP to C-terminus of psbE and potential interaction partners

  • Transform into appropriate expression system (chloroplast transformation preferably)

  • Quantify interaction through fluorescence microscopy and flow cytometry

• Co-immunoprecipitation with Antibody Engineering:

  • Develop specific antibodies against unique epitopes of Agrostis stolonifera cytochrome b559

  • Use mild solubilization conditions (digitonin 0.5-1%)

  • Identify interaction partners through mass spectrometry

• Förster Resonance Energy Transfer (FRET):

  • Engineer constructs with appropriate fluorophore pairs (e.g., CFP/YFP)

  • Measure energy transfer efficiency using fluorescence lifetime imaging microscopy (FLIM)

  • Calculate interaction distances based on FRET efficiency

These approaches can be applied to investigate interactions with other PSII components, particularly D1 and D2 proteins, as well as potential regulatory proteins that may interact transiently with cytochrome b559 under different environmental conditions.

What are the common challenges and solutions when working with recombinant cytochrome b559 subunit alpha expression and purification?

Research with recombinant cytochrome b559 presents several technical challenges. Here are methodological solutions to address the most common issues:

• Low Expression Yields:

  • Challenge: Membrane protein toxicity to expression host

  • Solution: Use tightly regulated expression systems with lower induction levels (0.1-0.2 mM IPTG rather than 1 mM) and lower growth temperatures (18-20°C)

  • Alternative: Consider cell-free expression systems with added membrane mimetics

• Improper Heme Incorporation:

  • Challenge: Insufficient heme availability during expression

  • Solution: Supplement growth media with δ-aminolevulinic acid (ALA, 0.5 mM) and iron (50 µM FeSO₄)

  • Validation: Monitor the ratio of 559 nm to 280 nm absorption peaks; values below 0.3 indicate poor heme incorporation

• Protein Aggregation During Purification:

  • Challenge: Loss of structural integrity during extraction

  • Solution: Screen multiple detergents at varied concentrations; recommended starting panel:

    • DDM (0.5-1%)

    • LMNG (0.01-0.05%)

    • Digitonin (0.5-1%)

  • Monitoring: Use dynamic light scattering to assess monodispersity

• Oxidation During Storage:

  • Challenge: Loss of functional properties due to oxidative damage

  • Solution: Add reducing agents (DTT or TCEP, 1-5 mM) and store under argon atmosphere

  • Storage: Divide into small aliquots and flash-freeze in liquid nitrogen

How can advanced spectroscopic techniques be applied to characterize the redox properties of recombinant cytochrome b559?

Comprehensive characterization of recombinant cytochrome b559 redox properties requires a multi-technique approach:

• Potentiometric Redox Titrations:

  • Methodology:

    • Use a platinum working electrode with Ag/AgCl reference

    • Include redox mediators covering -100 to +450 mV vs. NHE range

    • Monitor absorption changes at 559 nm during titration

  • Analysis: Fit data to Nernst equation to determine midpoint potentials (Em)

  • Expected Values: High potential form (HP): +330 to +400 mV; Intermediate (IP): +150 to +250 mV; Low potential (LP): 0 to +80 mV

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Sample Preparation:

    • Concentrate protein to 100-200 µM

    • Prepare samples in different redox states using ferricyanide/ascorbate

  • Measurement Conditions:

    • Temperature: 10-20K for optimal signal

    • Microwave power: 2-5 mW to avoid saturation

    • Modulation amplitude: 1-2 G

  • Interpretation: Analyze g-values to determine heme coordination environment

• Resonance Raman Spectroscopy:

  • Excitation: Use 413 nm laser to achieve resonance with Soret band

  • Key Markers:

    • ν₄ band (~1375 cm⁻¹): oxidation state marker

    • ν₃ band (~1500 cm⁻¹): spin state marker

    • ν₁₀ band (~1640 cm⁻¹): core size marker

  • Comparative Analysis: Create reference spectra with known heme proteins

• Time-Resolved Spectroscopy:

  • Methodology: Laser flash photolysis with probe wavelengths at 559 nm

  • Time Scales: Measure from nanoseconds to seconds to capture different electron transfer events

  • Analysis: Fit kinetic traces to multi-exponential decay functions

These spectroscopic approaches provide complementary information about the electronic structure, coordination environment, and electron transfer capabilities of cytochrome b559 from Agrostis stolonifera.

How does research on recombinant Agrostis stolonifera psbE contribute to understanding photosynthetic efficiency in cool-season grasses?

Research on recombinant Agrostis stolonifera psbE provides critical insights into photosynthetic efficiency of cool-season grasses through several interrelated mechanisms:

Understanding these adaptations at the molecular level provides crucial knowledge for improving photosynthetic efficiency in cool-season crops and predicting plant responses to changing environmental conditions.

What methodological approaches would allow integration of in vitro findings on recombinant cytochrome b559 with in vivo photosynthetic performance in Agrostis stolonifera?

Bridging the gap between in vitro biochemical characterization and in vivo physiological function requires sophisticated methodological integration:

• Complementation Studies in Model Systems:

  • Methodology: Transform cytochrome b559-deficient mutants (e.g., in Synechocystis or Chlamydomonas) with Agrostis stolonifera psbE

  • Measurements:

    • Photosynthetic oxygen evolution rates

    • Chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, NPQ)

    • Growth rates under various light intensities

  • Controls: Include both wild-type and mutant backgrounds to isolate protein-specific effects

• Site-Directed Mutagenesis Combined with Spectroscopic Analysis:

  • Approach: Create targeted mutations in conserved residues of psbE

  • In vitro measurements: Determine redox potential shifts using potentiometric titrations

  • In vivo assessment: Transform plants with mutated constructs and measure:

    • P700 re-reduction kinetics

    • Cyclic electron flow rates

    • State transition parameters

• Environmental Response Profiling:

  • Experimental Design: Subject transformed plants to controlled stress conditions:

    • Temperature series (5-35°C)

    • Light intensity gradients (50-1500 μmol photons m⁻² s⁻¹)

    • Drought stress (50-90% relative water content)

  • Integrated Measurements:

    • Combine gas exchange, chlorophyll fluorescence imaging, and thylakoid membrane protein dynamics

    • Correlate with spectroscopic properties of isolated cytochrome b559

• Multi-omics Integration Framework:

  • Approach: Combine data from:

    • Proteomics of thylakoid membrane complexes

    • Transcriptomics under varying environmental conditions

    • Metabolomics focusing on redox-related metabolites

  • Analysis: Develop network models connecting cytochrome b559 properties to whole-plant physiological responses

This integrated methodology enables researchers to establish causative relationships between molecular properties of cytochrome b559 and whole-plant photosynthetic performance in Agrostis stolonifera.