Recombinant Thalassiosira pseudonana Cytochrome b559 subunit alpha (psbE)

What is Thalassiosira pseudonana and why is it an important model organism?

Thalassiosira pseudonana is a model diatom species used extensively in molecular biology and algal research. It has become genetically tractable through the development of transformation and gene editing techniques, making it as versatile as other model organisms like Nannochloropsis and Physcomitrella. The significance of T. pseudonana lies in advancing functional diatom biology, bionanotechnology, and biotechnological applications aimed at harnessing the metabolic potential of diatoms . As a diploid photosynthetic organism, T. pseudonana provides insights into marine primary production and carbon cycling processes that are crucial for understanding global biogeochemical cycles.

What is Cytochrome b559 subunit alpha (psbE) and what is its function in T. pseudonana?

Cytochrome b559 subunit alpha (psbE) is a protein component of the photosystem II (PSII) reaction center in T. pseudonana. Specifically, it is designated as "PSII reaction center subunit V" . The protein plays a critical role in the photosynthetic electron transport chain, contributing to the oxidation-reduction reactions that drive photosynthesis. The amino acid sequence of Cytochrome b559 subunit alpha consists of 84 amino acids with the sequence: MSGGSTGERPFSDIITSVRYWIIHTITIPSLFVSGWLFISTGLAYDVFGTPRPNEYFTQDRQQVPLVNDRFSAKQELEDLTKGL . The protein contains transmembrane regions that anchor it within the thylakoid membrane of the chloroplast, allowing it to function in electron transfer processes essential for photosynthesis.

How does psbE interact with other components of the photosynthetic apparatus?

Cytochrome b559 subunit alpha (psbE) functions as part of an integrated photosynthetic electron transport system in T. pseudonana. Proteome analysis of isolated plastids has confirmed the presence of this protein along with other core components of photosystem II. The protein works in concert with other photosystem components including PSI core proteins (PsaA, PsaB) and peripheral reaction center subunits (PsaC, PsaD, PsaE, PsaF, and PsaL) to enable electron flow to ferredoxin . Additionally, psbE operates in conjunction with the cytochrome b6f complex (consisting of PetA, PetB, PetC, and PetD subunits) and ATP synthase subunits to facilitate energy conversion during photosynthesis. This coordinated interaction is essential for the efficient capture and conversion of light energy into chemical energy in diatoms.

What are the recommended protocols for isolating plastids from T. pseudonana to study psbE in its native context?

The isolation of plastids from T. pseudonana requires a careful approach to maintain structural integrity while achieving good separation from other cellular components. The recommended protocol involves:

• Culture preparation: Grow T. pseudonana cultures to early-stationary phase (5-7 × 10^6 cells/ml) with an electrical volume diameter of 4.7-5.3 μm. The culture should be maintained at 20°C with constant light intensities of 40 μE m^-2 s^-1 on a 16-hour light/8-hour dark cycle .

• Cell disruption: Apply a consistent French Press working pressure of 90 MPa during cell rupture to preserve organelle structural integrity .

• Differential centrifugation: Separate organelles based on density using sequential centrifugation steps.

• Fraction verification: Confirm plastid isolation quality through chlorophyll autofluorescence monitoring and Western blot analysis using antibodies against plastid markers such as the D1 protein (PsbA) and Rubisco large subunit (RbcL) .

This protocol yields plastid fractions with intact thylakoid membrane organization, although most plastid envelopes are typically lost during isolation, and pyrenoids are not preserved in the isolated fraction. The effectiveness of plastid isolation can be verified by transmission electron microscopy (TEM) and protein analysis by immunoblotting .

What expression systems are recommended for producing recombinant T. pseudonana psbE protein?

For recombinant expression of T. pseudonana Cytochrome b559 subunit alpha (psbE), several approaches can be considered:

• Homologous expression in T. pseudonana: The development of CRISPR/Cas systems for T. pseudonana enables targeted gene replacement through homologous recombination with high efficiency (up to 85% positive colonies) . This approach allows for in situ tagging or modification of the native psbE gene.

• Heterologous expression systems: When purified recombinant protein is required, E. coli-based expression systems can be utilized with appropriate optimization of codon usage and consideration of post-translational modifications.

The choice of expression tag should be determined during the production process to optimize protein folding and function . For storage of expressed protein, a Tris-based buffer with 50% glycerol is recommended, with storage at -20°C for short-term use or -80°C for extended storage . Working aliquots can be maintained at 4°C for up to one week, avoiding repeated freeze-thaw cycles.

How can CRISPR/Cas techniques be applied to study psbE function in T. pseudonana?

CRISPR/Cas-mediated genome editing has proven highly efficient in T. pseudonana, making it an excellent tool for studying psbE function. The methodology involves:

• Construct design: Utilize Golden Gate cloning to assemble a CRISPR/Cas construct targeting the psbE locus .

• Homologous recombination strategy: Design a dsDNA donor matrix with homology arms flanking the psbE gene to enable precise gene replacement or modification .

• Transformation method: Co-transform the CRISPR/Cas construct with a selection plasmid (such as pTpfcpNAT) to enable selection of transformants .

• Screening approach: Screen NAT-resistant colonies using nested PCR to identify successful homologous recombination events .

• Validation: Confirm precise integration at the psbE locus using inverse PCR approaches and sequence verification .

This approach enables various experimental designs including:

• Complete knockout of psbE to assess its essentiality and impact on photosynthesis

• Introduction of point mutations to study structure-function relationships

• Addition of fluorescent or affinity tags for localization and interaction studies

How does psbE contribute to the structural integrity of pyrenoids in T. pseudonana?

Recent research has revealed that diatom pyrenoids, which are crucial for carbon fixation, are encased in a protein shell (PyShell) that enables efficient CO2 assimilation . While psbE is primarily associated with PSII function, its potential role in pyrenoid structure can be investigated in the context of thylakoid membrane arrangement.

Experimental evidence from pyrenoid mutants shows that disruption of the PyShell protein leads to fragmentation of pyrenoids and loss of the thylakoid membranes that normally traverse the Rubisco matrix . This suggests that proteins involved in thylakoid membrane organization, potentially including components like psbE, may indirectly affect pyrenoid architecture and function.

To investigate psbE's role in pyrenoid structure:

• Generate targeted mutations in psbE using CRISPR/Cas techniques

• Analyze pyrenoid structure by transmission electron microscopy

• Assess CO2 fixation efficiency in mutants using carbon isotope labeling experiments

The structural integrity of pyrenoids is essential for diatom carbon fixation, making this an important area for investigating potential roles of photosynthetic proteins like psbE in climate-relevant biological processes .

What is the relationship between psbE expression and stress responses in T. pseudonana?

The expression of photosynthetic proteins in diatoms, including components like psbE, can be modulated in response to environmental stressors. Proteome analyses have revealed that certain photosynthetic proteins show differential expression patterns under stress conditions compared to moderate growth conditions .

While some stress-responsive photosynthetic proteins were not detected in T. pseudonana grown under moderate conditions, these proteins appeared under stress conditions . This pattern suggests a hypothesis of stress-driven expression regulation for certain photosynthetic components.

Research approaches to investigate psbE stress response include:

Understanding how psbE expression responds to environmental stressors provides insights into the molecular mechanisms of diatom adaptation to changing oceanic conditions, which has implications for marine ecosystem dynamics and global carbon cycling.

How can recombinant psbE be used to investigate electron transport chain assembly in diatoms?

Recombinant Cytochrome b559 subunit alpha can serve as a valuable tool for investigating the assembly and function of the photosynthetic electron transport chain in diatoms. Key research approaches include:

• In vitro reconstitution studies: Purified recombinant psbE can be combined with other photosystem components to study the hierarchy and requirements for PSII assembly.

• Protein-protein interaction analysis: Using tagged recombinant psbE to identify interaction partners within the thylakoid membrane complex through techniques such as pull-down assays or cross-linking studies.

• Structural biology applications: Recombinant protein can contribute to structural determination efforts through crystallography or cryo-electron microscopy, providing insights into the arrangement of electron transport components.

When investigating electron transport, it's important to note that while ferredoxin-NADP+ reductase isoforms (Thaps3a_4586 and Thaps3a_4914) have been identified in T. pseudonana, neither ferredoxin (PetF) nor cytochrome c6 (PetJ) were found in proteome analyses . Instead, an uncharacterized ferredoxin protein containing a 2Fe-2S Rieske domain (Thaps3a_29842) was detected, suggesting unique aspects of the electron transport chain in this organism that warrant further investigation using recombinant components.

What phenotypes are expected when modifying psbE in T. pseudonana using CRISPR/Cas?

Modifications to psbE using CRISPR/Cas techniques would likely produce phenotypes related to photosynthetic efficiency and cellular energy metabolism. Based on known functions of Cytochrome b559 and results from similar modifications in other organisms, the following phenotypes might be expected:

• Complete knockout effects: Complete elimination of psbE function would likely impair PSII assembly and function, potentially leading to:

  • Reduced photosynthetic efficiency

  • Increased photosensitivity

  • Altered growth rates, particularly under high light conditions

  • Changes in cellular ultrastructure, especially thylakoid membrane organization

• Point mutation effects: Strategic amino acid substitutions could produce more subtle phenotypes:

  • Altered redox potential of the cytochrome

  • Modified interaction with other PSII components

  • Changed susceptibility to photoinhibition

For reference, previous CRISPR/Cas experiments in T. pseudonana targeting other genes have demonstrated clear phenotypic outcomes. For example, knockout of the silacidin gene caused a significant increase in cell size, confirming its role in cell-size regulation in centric diatoms . Similarly, knockouts of metabolic genes like nitrate reductase and urease impacted growth on specific nitrogen sources .

How can GFP fusion constructs be designed to study psbE localization and dynamics?

Fluorescent protein tagging of psbE can provide valuable insights into its localization, dynamics, and assembly into photosynthetic complexes. Based on successful approaches with other T. pseudonana proteins, the following methodology is recommended:

• Vector construction: Design a fusion construct linking the psbE coding sequence to GFP, considering both N-terminal and C-terminal fusions to determine which preserves protein function. Utilize established T. pseudonana expression vectors such as pTpFcpGFP .

• Promoter selection: For physiologically relevant expression, use the native psbE promoter or the fucoxanthin chlorophyll a/c binding protein (FCP) promoter for stronger expression .

• Transformation approach: Co-transform the GFP fusion construct with a selection plasmid (such as pTpfcpNAT) using methods described by Poulsen et al. (2006) .

• Screening strategy: Verify successful transformation using antibiotic selection and PCR confirmation, followed by fluorescence microscopy to confirm expression and localization.

A successful example of this approach is the TIG19 mutant line that expresses and targets GFP to mitochondria using the pre-sequence of mitochondrial triosephosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase (preTPI-GAPDH::GFP) . This approach allowed researchers to track mitochondria during organelle isolation through fluorescence microscopy.

What regulatory considerations should researchers be aware of when conducting recombinant DNA research with T. pseudonana?

When conducting recombinant DNA research with T. pseudonana, researchers should consider both formal regulatory requirements and scientific best practices:

• Institutional biosafety: All recombinant DNA work should be reviewed by institutional biosafety committees, following principles established since the early days of recombinant DNA technology .

• Containment procedures: Appropriate biological containment measures should be implemented to prevent unintended environmental release of genetically modified diatoms.

• Risk assessment: Researchers should carefully evaluate potential risks, particularly when introducing genes that might alter ecological fitness or produce bioactive compounds.

The history of recombinant DNA regulation provides important context for current practices. In the early 1970s, as concerns about potential risks grew, scientists like Maxine Singer advocated for prudent research practices that balanced scientific progress with public welfare . This led to the development of guidelines for recombinant DNA research that continue to inform current regulatory frameworks.

For researchers working with T. pseudonana, it's important to recognize that even though this organism is well-established as a laboratory model, engineering of photosynthetic components like psbE requires careful consideration of potential impacts on cellular physiology and adherence to established safety protocols.

How conserved is psbE structure and function across different diatom species?

The Cytochrome b559 subunit alpha (psbE) shows significant conservation across diatom species, reflecting its fundamental role in photosynthesis. Comparative analysis reveals:

This high degree of conservation underscores the evolutionary constraints on this protein due to its essential function in photosystem II. The most highly conserved regions include the transmembrane domain and cofactor binding sites, while terminal regions show greater variability.

Despite this conservation, functional studies comparing psbE across diatom species have revealed species-specific adaptations that may relate to their ecological niches. For instance, differences in stress response patterns between T. pseudonana and P. tricornutum have been observed in proteome analyses, with certain photosynthetic proteins being detected under stress conditions in one species but not the other .

What unique features distinguish T. pseudonana psbE from its counterparts in other photosynthetic organisms?

T. pseudonana Cytochrome b559 subunit alpha (psbE) exhibits several distinctive features compared to its counterparts in other photosynthetic organisms:

• Sequence adaptations: The T. pseudonana psbE contains unique amino acid substitutions that may reflect adaptation to marine environments, particularly in regions exposed to the lumenal side of the thylakoid membrane.

• Interaction network: Diatom psbE appears to function within a somewhat modified PSII supercomplex architecture, as suggested by proteome analysis of plastid fractions .

• Regulatory patterns: Unlike some other photosynthetic organisms, T. pseudonana shows distinctive expression patterns for photosystem components including psbE, particularly under different growth and stress conditions .

• Association with pyrenoid structure: The potential involvement of thylakoid membrane proteins like psbE in maintaining pyrenoid structure represents a distinctive feature of diatom photosynthesis, as pyrenoids in diatoms are encased in a protein shell (PyShell) that enables efficient CO2 assimilation .

These unique features likely contribute to the remarkable efficiency of diatom photosynthesis, which has made these organisms responsible for approximately 20% of global primary production despite accounting for less than 1% of Earth's biomass.

What are the emerging techniques that could advance our understanding of psbE function in T. pseudonana?

Several cutting-edge methodologies hold promise for deepening our understanding of psbE function:

• Cryo-electron tomography: This technique could provide detailed structural information about psbE in its native membrane environment at near-atomic resolution, revealing how it interacts with other components of the photosynthetic apparatus.

• Single-molecule tracking: Advanced fluorescence techniques could allow tracking of individual psbE molecules within living T. pseudonana cells, providing insights into dynamic processes like photosystem assembly and repair.

• Rapid protein evolution approaches: Directed evolution techniques adapted for diatoms could generate psbE variants with modified properties, helping to map structure-function relationships.

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from wild-type and psbE-modified strains could reveal the cascading effects of psbE alterations on cellular physiology.

• Advanced microscopy of PyShell structures: Building on recent work that determined a 3.0 Å-resolution PyShell structure , investigating potential interactions between photosystem components and pyrenoid structures could reveal new functional relationships.

These approaches could significantly advance our understanding of how psbE contributes to the remarkable photosynthetic efficiency of diatoms and their adaptation to marine environments.

How might climate change impact psbE function and expression in marine diatoms?

Climate change presents multiple stressors that may affect psbE function and expression in marine diatoms like T. pseudonana:

• Ocean acidification: Decreasing pH may alter the proton gradient across thylakoid membranes, potentially affecting the function of membrane proteins like psbE.

• Temperature increases: Rising ocean temperatures could impact protein folding, stability, and turnover rates of photosynthetic components including psbE.

• Changes in light regimes: Altered stratification patterns may change light exposure, potentially requiring adjustments in photosystem stoichiometry and composition.

• Nutrient availability shifts: Changing nutrient patterns could affect resource allocation to photosynthetic machinery, including the expression and maintenance of psbE.

Research into these areas is crucial as diatoms are responsible for a significant portion of marine primary production, and their pyrenoid structures are described as being "on the front lines of climate change" . Understanding how key photosynthetic components like psbE respond to changing conditions will help predict future ocean productivity and carbon cycling.